Gratitude, far from being a sentimental notion, has emerged as a scientifically supported force capable of transforming the human brain and improving overall mental health.

Neuroscientific studies have shown that regularly practicing gratitude activates brain regions associated with moral cognition, emotional regulation, and reward, particularly the medial prefrontal cortex and anterior cingulate cortex (Zahn et al., 2009).

Notably, Dr. Alex Korb, in his book The Upward Spiral, describes how gratitude stimulates the release of dopamine and serotonin—two neurotransmitters vital for mood stabilization and happiness —making it a powerful natural antidepressant.

A growing body of evidence confirms that this effect is not fleeting: a study by the University of California, Berkeley, led by Joel Wong and Joshua Brown (2017), found that individuals who wrote gratitude letters showed significantly better mental health outcomes even 12 weeks after the exercise, regardless of whether the letters were sent.

In parallel, Emmons and McCullough (2003) demonstrated that individuals who kept a gratitude journal for just 10 weeks reported increased optimism, better sleep, and more physical activity.

Furthermore, gratitude has been shown to enhance the neural modulation of the prefrontal cortex, which reduces symptoms of depression and anxiety by strengthening pathways that suppress negative emotions.

According to McCraty & Childre (2004), gratitude also reduces cortisol levels—the primary stress hormone—improving cardiovascular health and emotional resilience. At the structural level, researchers like Zahn et al. (2014) have found that individuals who frequently feel gratitude show increased gray matter volume in the right inferior temporal gyrus, which plays a key role in interpreting social signals and emotional meaning.

As UCLA’s Mindfulness Awareness Research Center puts it, “Gratitude changes the neural structures of the brain,” reshaping how we perceive and engage with the world. ”

Ultimately, gratitude doesn’t just feel good—it rewires the brain toward greater emotional intelligence, social connection, and resilience, offering a neuropsychological foundation for a healthier and more fulfilling life.

NOT JUST SCIENCE, BIBLICAL AS WELL. ONE DAY SCIENCE WILL ALIGN ITSELF FULLY WITH CREATION!

Gratitude Transforms Anxiety into Peace Bible verses to help us through our daily life and life in General

Philippians 4:6-7
“Do not be anxious about anything, but in every situation, by prayer and petition, with thanksgiving, present your requests to God. And the peace of God, which transcends all understanding, will guard your hearts and your minds in Christ Jesus.”

Gratitude Shifts Perspective and Brings Joy

1 Thessalonians 5:16-18
“Rejoice always, pray continually, give thanks in all circumstances; for this is God’s will for you in Christ Jesus.”

Gratitude Leads to God’s Presence

Psalm 100:4
“Enter his gates with thanksgiving and his courts with praise; give thanks to him and praise his name.”

Gratitude Transforms Hardship into Growth

James 1:2-4
“Consider it pure joy, my brothers and sisters, whenever you face trials of many kinds, because you know that the testing of your faith produces perseverance. Let perseverance finish its work so that you may be mature and complete, not lacking anything.”

Gratitude Deepens Our Relationship with God

Colossians 3:16-17
“Let the message of Christ dwell among you richly as you teach and admonish one another with all wisdom through psalms, hymns, and songs from the Spirit, singing to God with gratitude in your hearts. And whatever you do, whether in word or deed, do it all in the name of the Lord Jesus, giving thanks to God the Father through him.”

Gratitude Opens Our Eyes to God’s Blessings

Ephesians 5:20
“Always giving thanks to God the Father for everything, in the name of our Lord Jesus Christ.”

Gratitude Is an Act of Worship

Hebrews 12:28
“Therefore, since we are receiving a kingdom that cannot be shaken, let us be thankful, and so worship God acceptably with reverence and awe.”

Gratitude Leads to Wholeness and Healing

Luke 17:15-19 (The healing of the ten lepers)
“One of them, when he saw he was healed, came back, praising God in a loud voice. He threw himself at Jesus’ feet and thanked him… Then he said to him, ‘Rise and go; your faith has made you well.'”

Gratitude in Scripture is often a catalyst for profound change. Below, the examples are organized by the type of transformation experienced – Emotional, Spiritual, Physical, and Social – with each verse or passage quoted and explained.

Emotional Transformation (Anxiety to Peace, Sadness to Joy)

• Philippians 4:6–7 – “Do not be anxious about anything, but in everything by prayer and supplication with thanksgiving let your requests be made known to Godbiblehub.com. And the peace of God, which surpasses all understanding, will guard your hearts and your minds in Christ Jesus”biblehub.com. Transformation: A thankful posture in prayer turns anxiety into peace. Paul teaches that when we present our worries to God with gratitude, God’s transcendent peace will calm our hearts and mindsgotquestions.org. The act of thanking God, even as we petition Him, shifts our focus from troubles to trust, resulting in inner peace replacing fear.

• Psalm 30:11–12 – “You have turned for me my mourning into dancing; you have loosed my sackcloth and clothed me with gladness… O Lord my God, I will give thanks to you forever”biblehub.combiblehub.com. Transformation: David expresses that God transformed his sadness into joy (“mourning into dancing”). In response, David’s heart overflows with thanksgiving. Gratitude here is both a reaction to God’s deliverance and a means of fully embracing joy. By vowing to thank God forever, David shows how thankfulness sustains the joy that replaced his sorrow. His praise-filled gratitude cements the emotional transformation from grief to rejoicing.

Spiritual Transformation (Deeper Faith and Relationship with God)

• Luke 17:15–19 – One of the ten lepers, after being healed, “turned back, and with a loud voice glorified God, and fell down on his face at Jesus’ feet, giving Him thanks”biblehub.com. In response, “Jesus said to him, ‘Rise and go; your faith has made you well’”biblehub.com. Transformation: Gratitude was central to this man’s spiritual healing. All ten were cured physically, but only the thankful Samaritan received Jesus’ affirmation of saving faith. His thankful return to praise Jesus indicated a heart change and deeper faith. Jesus implied that this man gained something more – a spiritual wholeness: “There was an extra healing for this tenth leper… The other lepers had whole bodies, but sick hearts.”enduringword.com In other words, his grateful faith not only cleansed his body but also brought salvation and a restored relationship with God, whereas the others missed that deeper transformation.

• Colossians 2:6–7 – “So then, just as you received Christ Jesus as Lord, continue to live your lives in Him, rooted and built up in Him, strengthened in the faith as you were taught, and overflowing with thankfulness.”askaboutmyfaith.com Transformation: Thankfulness is portrayed as a byproduct of and catalyst for spiritual growth. As believers deepen their roots in Christ, their faith grows stronger and is marked by abundant gratitudeaskaboutmyfaith.com. This suggests that practicing thankfulness reinforces our faith. A grateful heart keeps us mindful of God’s goodness and sovereignty, drawing us into a closer walk with God. Thus, gratitude leads to a transformed spiritual life – one of deeper faith, steadiness in Christ, and continual awareness of God’s presence.

• Psalm 50:23 – “The one who offers thanksgiving as his sacrifice glorifies Me; to one who orders his way rightly I will show the salvation of God!”biblehub.com. Transformation: Here God promises that a thankful worshiper will see salvation. Offering thanks honors God and “prepares his way so that I will show God’s salvation”biblehub.com. This implies that gratitude opens the heart to God’s saving work. In a spiritual sense, a grateful attitude “orders” our life correctly before God, leading to a greater experience of His salvation and deliverance. In sum, thankfulness paves the way for a transformed heart that knows God’s saving power more intimately.

Physical Transformation (Healing, Provision, Protection)

• John 6:11 – “Jesus then took the loaves, and when He had given thanks, He distributed them to those who were seated… so also the fish, as much as they wanted.”biblehub.com Transformation: Jesus demonstrates gratitude before the miracle of feeding the 5,000. His giving of thanks to God preceded the multiplication of the loaves and fish, providing abundant provision for the hungry crowd. This shows thankfulness leading to a tangible transformation of circumstances – from lack to plenty. The simple act of thanking God for the little they had invited God’s power to supply the physical needs of thousandsflbchurch.org. Our example is that faithful thanks, even in scarcity, can unleash God’s provision.

• Jonah 2:9–10 – “But I, with the voice of thanksgiving, will sacrifice to You… Salvation comes from the Lord. And the Lord commanded the fish, and it vomited Jonah onto dry land.”biblehub.com Transformation: Inside the great fish, Jonah offered a prayer of grateful praise, even before his rescue: “With shouts of grateful praise, I will sacrifice to you… Salvation comes from the Lord”biblehub.com. Immediately after this thanksgiving proclamation, God delivered Jonah, commanding the fish to release him. Jonah’s gratitude in the darkest place preceded a dramatic physical deliverance from death. This illustrates how an attitude of thankfulness can invite God’s saving power into dire circumstances – turning near tragedy into rescue.

• Daniel 6:10, 22 – “[Daniel] got down on his knees three times a day and prayed and gave thanks before his God, as he had done previously.” (Daniel 6:10) Despite a royal ban on prayer, Daniel continued his habitual gratitude toward God. Later, after being thrown into the lions’ den, he emerged unharmed, declaring, “My God sent His angel and shut the lions’ mouths, and they have not harmed me” (Daniel 6:22). Transformation: Daniel’s unwavering thankfulness brought about divine protection. His grateful prayers demonstrated trust in God above all, and God miraculously saved him from deadly lions. Even the pagan king recognized God’s power: “He rescues and He saves”thewriteconversation.blogspot.com. Thus, in Daniel’s story, thankfulness under trial led to a powerful transformation of impending death into deliverance – a testimony to God’s protective intervention.

• Acts 16:25–26 – “About midnight Paul and Silas were praying and singing hymns to God… and suddenly there was a great earthquake, so that the foundations of the prison were shaken. And immediately all the doors were opened and everyone’s bonds were unfastened.”flbchurch.org Transformation: In this instance of praise (an expression of gratitude) amid suffering, God intervened with a physical miracle. Paul and Silas’s thankful worship turned a prison into a place of freedom – literally opening doors and breaking chains. Their gratitude to God despite wounds and confinement led to an earthquake that transformed their situation from captivity to liberty. This not only freed them physically but also set the stage for the jailer’s conversion (a spiritual change), showing how thankfulness can trigger God’s power to heal, protect, and save.

Social Transformation (Reconciliation, Favor, Influence)

• Acts 27:35–36 – Facing a storm at sea, “Paul took bread and gave thanks to God in the presence of all… then he broke it and began to eat. Then they all were encouraged and ate some food themselves.”biblehub.com Transformation: Paul’s public expression of gratitude during a crisis had a profound influence on those around him. His calm thankfulness to God for the meal (despite the impending shipwreck) lifted the others from despair to hope. All 276 passengers “took heart” and followed his example of eatingbiblehub.com. The social atmosphere on the ship shifted from panic to encouragement. Paul’s thankfulness instilled confidence that God would deliver them, demonstrating how one person’s grateful faith can positively transform a whole group’s outlook and unity.

• Acts 2:46–47 – In the early church, believers met together “with glad and sincere hearts, praising God,” and as a result they were “enjoying the favor of all the people. And the Lord added to their number daily those who were being saved.”biblehub.com Transformation: Corporate gratitude and praise produced a social impact in the community. The first Christians’ joyful, thankful worship fostered unity among themselves and earned them favor with outsiders. Their gratitude to God was contagious, drawing others to them. Scripture notes that this environment of praise and thankfulness led to continual growth of the church, as more people were being saved dailybiblehub.combiblehub.com. In other words, a culture of thankfulness in the community helped bring about reconciliation between individuals and God, and also gave the believers a positive reputation, transforming their social standing and influence in society.

• 2 Chronicles 20:21–22 – “Jehoshaphat appointed those who were to sing to the Lord and praise Him… As they began to sing and praise, the Lord set ambushes against the [enemy]… and they were defeated.” Transformation: Here praise and thanks became a “weapon” that led to a national victory and peace. Judah’s army marched into battle thanking God for His love, and God turned the enemies against each otherflbchurch.orgbiblehub.com. This brought deliverance without Judah fighting at all. The social/national transformation was twofold: fear gave way to faith among God’s people, and neighboring nations stood in awe. This example shows gratitude to God helping reconcile and unite a people, while also granting them favor and influence over surrounding nations who witnessed God’s power.

Each of these examples underscores a principle: thankfulness is often the key that unlocks God’s transformative work. Emotional turmoil gives way to joy and peace, faith is strengthened, physical needs are met or miracles occur, and even social situations are changed – all when people choose to “give thanks in all circumstances” as God’s will for us (1 Thessalonians 5:18). The Bible consistently demonstrates that a grateful heart isn’t just an appropriate response to God’s grace, but a powerful force that God uses to change lives and situations for the better.

• Releasing Negative Emotions: Holding onto anger, resentment, and bitterness creates a heavy burden that hinders inner peace. Forgiveness is an active process of consciously releasing these negative feelings.
• Breaking the Cycle of Pain: Forgiveness can interrupt the cycle of anger and hurt, leading to a state of peace.
• Personal Growth and Healing: By choosing to forgive, you make room for healing and personal growth, enabling you to move forward without being defined by past pain.
• Reducing Stress: Research shows a strong link between forgiveness and reduced stress levels. This, in turn, can improve mental health outcomes like anxiety and depression.
• Improving Relationships: Forgiveness can help rebuild trust and open communication, fostering stronger and more supportive relationships. 
• The benefits of forgiveness:Forgiveness can lead to reduced stress, improved mental and physical health, stronger relationships, and a greater sense of inner peace and happiness. 
• Forgiveness and letting go: Forgiveness often involves letting go of the past and its associated pain, allowing one to focus on the present and future. 
• Forgiveness and personal growth: It can be a powerful catalyst for personal growth and transformation, helping individuals to heal, evolve, and move forward with a more positive outlook.

• Forgiveness does not mean forgetting or condoning the offense. It’s about letting go of the negative emotional and psychological impact of the offense on yourself.
• Forgiveness is a process that may require time and reflection. 

Forgiveness is for us, not for them. For us to heal and move on but I really cant seem to do it. I get so angry every time I see my mom and I stay angry for a while. I hate the feelings it brings out of me.

Now my parents weren’t physically abusive but they were so unhappy in their marriage and their lives, they accidentally created shit childhoods for us.

I never felt loved or seen, there was only judgement and a sense of burdening them. Mom was angry at everything I did, she constantly complained about everything. She was a SAHM who did nothing to better herself or take care of her kids but she was judging the entire universe.

I see the impact of her choices on me and I see how much her own insecurities affected me and I cant help but think – why, why didn’t you better yourself? You had a lifetime and nothing else to do

If you have felt a similar way, what were the things that helped you forgive your parents?

Forgiveness Is the Key to Unlocking Your True Peace

Everyone experiences hurt – an inconsiderate friend, a betrayal, or worse – and the weight of grudges can be heavy. Increasingly, psychologists and even neuroscientists are finding truth in an age-old wisdom: forgiveness is the key to inner peace. Letting go of anger and resentment isn’t just a moral ideal celebrated by spiritual traditions; it’s a practical strategy for emotional freedom, better health, and resilience. “Forgive us our debts, as we forgive our debtors,” an ancient prayer intones, recognizing that forgiving others is crucial for our own well-being. Modern research agrees. Holding onto bitterness elevates stress, blood pressure, and anxiety, while choosing to forgive can lower stress hormones, improve mental clarity, and even rewire the brain for the better. This article explores forgiveness from a broad perspective – its psychological and physical benefits, its reflection in both secular and spiritual wisdom, and how real people have achieved personal peace by forgiving. By the end, you’ll see why turning the key of forgiveness unlocks true peace in your mind and life.

Why Letting Go is Good for You (Understanding Forgiveness)

Forgiveness is often misunderstood. It does not mean forgetting what happened, condoning the harm, or reconciling with an abuser. Rather, psychologists define forgiveness as a conscious choice to release feelings of anger, resentment, or vengeance toward someone who hurt you, whether they deserve it or not. It’s “replacing ill will toward an offender with goodwill,” as one Harvard expert puts ithealth.harvard.edu. In essence, you decide to cancel the “debt” of the offense so that it no longer controls your life. This act is fundamentally for your benefit – the other person may never even know you forgave them.

Why go through this difficult process? Because refusing to forgive keeps you stuck in a cycle of stress and pain. Ruminating over past wrongs triggers the body’s stress response each time: heart rate and blood pressure spike, muscles tense, and emotions churn with anger or sadness. Over time, this state of unforgiveness can take a serious toll on mental and physical health. In fact, remaining in a hostile, resentful state has been likened to a wound that never heals. “Feeling stuck in hostility can torment us mentally,” causing “intense rumination – a looping of negative thoughts”health.harvard.edu. We relive the hurt repeatedly, which impedes our ability to move forward. On the flip side, forgiveness allows us to finally exhale that toxic anger. It’s an emotional release – a way of saying “I’m not going to let this grievance dominate my life anymore.” As the Greater Good Science Center reports, forgiving someone engages brain regions that “empower you to step beyond painful experiences in an energized, motivated, and connected way.” It actually builds resilience and strengthgreatergood.berkeley.edu. In choosing to forgive, you free yourself from the grip of the past. The result is often a profound sense of peace and freedom, as countless individuals attest.

Psychological Benefits of Forgiveness (Healing the Mind)

Modern psychology has amassed substantial evidence that forgiveness benefits mental health. Whether through formal forgiveness training or personal practice, letting go of grudges has been shown to produce positive changes in mood and mindset. A 2023 multi-country study led by Harvard researchers found that when people actively learned how to forgive, they saw significant reductions in depression and anxiety symptoms compared to a control grouphealth.harvard.eduwashingtonpost.com. In fact, forgiving was so impactful that researchers suggested it as a public health intervention for improving mental well-beingwashingtonpost.com. Here are some key psychological benefits highlighted by recent studies and experts:

• Less anxiety and depression: Across five countries, those who practiced forgiveness reported feeling less anxious and less depressed than beforehealth.harvard.edu. By releasing resentment, they alleviated emotional burdens that fuel mental distress. Other research concurs that forgiveness therapy leads to fewer symptoms of depression and anxiety and greater feelings of hope for the futurepmc.ncbi.nlm.nih.gov.

• Reduced anger and stress: It’s no surprise that forgiving someone reduces anger – you’re letting go of the grudge, after all. But importantly, this also lowers chronic stress. Psychologists note that holding onto anger puts your body in a state of tension. When you forgive, you switch off that “fight or flight” response, often experiencing immediate relief and calm. Over time, this can break the cycle of rumination (repeatedly revisiting the hurt), which frees your mind to focus on more positive things.

• Improved self-esteem and mental clarity: Remarkably, studies have found that as people forgive, their self-esteem tends to risepmc.ncbi.nlm.nih.gov. Why? Forgiveness empowers you – you’re no longer a victim of the offense, but a victor over the negative emotions associated with it. This positive shift can boost confidence and clarity. Many report that after forgiving, they feel lighter, as if a mental fog has lifted. Decisions come easier, and they regain a sense of control over their emotions rather than feeling controlled by past pain.

• Greater resilience: Forgiveness doesn’t just help you heal from this hurt; it can build resilience to face future challenges. Brain imaging studies reveal that forgiving engages brain pathways associated with problem-solving, empathy, and emotional regulationgreatergood.berkeley.edu. Essentially, practicing forgiveness is like a workout for your brain’s “resilience muscles,” teaching you how to cope with adversity in a healthy way. Over time, people who cultivate forgiveness often bounce back faster from setbacks and exhibit more optimism in difficult situations.

In sum, forgiveness is therapeutic. As one researcher noted, “When we learn to forgive others, we simultaneously heal ourselves.”pmc.ncbi.nlm.nih.gov By cleaning the wound of old anger, we prevent further infection of our psyche. What’s left is a mind at peace, better equipped to experience joy and engage fully with life.

Physical Health Benefits of Forgiveness (Healing the Body)

The body responds dramatically to our emotional state. Harboring grudges has been linked to physical stress ailments, while forgiving can trigger physiological healing. Science is now catching up to this wisdom. According to a review of studies, forgiveness contributes to lower blood pressure, better sleep quality, and even a stronger immune systemwashingtonpost.com. Here are some notable physical health benefits:

• Heart health and blood pressure: The act of forgiveness has a calming effect on the cardiovascular system. In moments of anger or recalling a grudge, your blood pressure spikes and heart beats faster. Chronic unforgiveness keeps you in this state, which over time can damage arteries and strain the heart. Forgiving, however, can “lower blood pressure and heart rate,” effectively reducing cardiovascular strainhealth.harvard.edu. Some studies found that people who let go of grudges showed improvements in hypertension (high blood pressure) readings. Simply put, a peaceful heart tends to be a healthier heart.

• Stress reduction and immunity: Emotional stress from resentment can suppress your immune function, making you more vulnerable to illness. By releasing resentment, you dial down the body’s stress hormones (like cortisol). Lower stress can improve everything from digestion to immune response. Many individuals report that after forgiving, they feel fewer physical symptoms of stress – for example, tension headaches or muscle pain might diminish as the body relaxes. Over the long term, this means fewer stress-related ailments. In fact, researchers in one study noted that people stuck in unforgiveness showed higher physiological stress responses, whereas those practicing forgiveness had more normalized stress levelshealth.harvard.edu.

• Better sleep and energy: Ever lain awake stewing over how someone wronged you? Grudges are exhausting. Forgiveness, on the other hand, is associated with better sleep. A 2019 study in Psychology & Health found that those who scored higher on forgiveness measures tended to have better sleep quality and less fatigue than those who held grudges. By resolving inner conflicts, forgiveness quiets the mind at night, leading to more restful sleep. In turn, improved sleep boosts your daytime energy and concentration. It’s a virtuous cycle: peace of mind yields peaceful sleep, which yields a healthier, more energetic you.

• Longer lifespan and overall wellness: While research is ongoing, some evidence suggests that forgiving might even contribute to longevity. Chronic anger and hostility have been identified as risk factors for health conditions that shorten life (like heart disease). Conversely, people who forgive more readily often have lower inflammation levels and healthier lifestyles (since they may cope through positive means rather than substance abuse or overeating out of stress). All these factors can add up to a longer, healthier life. As one health publication put it, forgiveness is “not just good for the soul,” it’s good for the body toohealth.harvard.edu.

The takeaway is clear: your body listens to your heart. By purging bitterness from your heart, you also purge toxins from your body. Medical clinics, including the renowned Mayo Clinic, now advocate forgiveness as part of a holistic approach to wellness, citing benefits like lower blood pressure, less anxiety, and reduced chronic pain. In summary, forgiveness is a mind-body medicine – free to use, with only positive side effects!

The Spiritual Dimension of Forgiveness (Universal Wisdom)

Even in a secular discussion, it’s worth noting that forgiveness has deep spiritual and cultural roots. Virtually every major religion and moral philosophy extols the virtue of forgiving others. This isn’t a coincidence; across time and culture, people observed that forgiveness leads to inner freedom and social harmony. For example, Christian scripture teaches, “Do not judge… do not condemn… Forgive, and you will be forgiven.” (Luke 6:37biblehub.com). In Judaism and Islam, forgiving others is highly honored, and withholding forgiveness is seen as corrosive to the soul. Buddhism advocates compassion and letting go of anger as a path to enlightenment. Secular humanist thinkers, too, recognize the transformative power of forgiveness in conflict resolution and personal growth.

What all these traditions share is the understanding that holding hate only hurts the hater. Forgiveness, by contrast, is often described as liberation. It frees your spirit from the burden of past injuries. Many who forgive describe feeling lighter or as if “a weight was lifted” off them – imagery common to spiritual rebirth. In a broader sense, forgiveness can also mend the social fabric. When we forgive, we break cycles of retaliation and revenge that cause endless suffering. This is why leaders like Martin Luther King Jr. and Mahatma Gandhi, though leading secular movements, spoke frequently about forgiveness and love as powerful forces for change.

Importantly, you don’t have to follow a particular faith to appreciate the inner transformation forgiveness brings. However, for some, spiritual beliefs can provide strength in the forgiveness journey. They might find it helpful to reflect on their own imperfections and how they’d want to be forgiven (echoing the Golden Rule). Others draw on concepts like karma or divine grace to release their grudges. Even without religious context, one might frame forgiveness as aligning with your highest self – choosing empathy and peace over anger. Ultimately, whether one views it as a divine command or simply wise living, forgiving others is a profound act of self-care and virtue recognized worldwide. It connects us to something larger – be it community, humanity, or a sense of higher purpose – by dissolving the walls that resentment builds between us.

Real-Life Story: Finding Personal Peace Through Forgiveness

To see how forgiveness can unlock true peace, consider the remarkable story of Mary Johnson. Mary is a woman from Minneapolis whose only son was tragically murdered in 1993 by a teen named Oshea. Understandably, Mary was consumed by grief and bitterness after her son’s death. “I was angry and resentful,” she admitted – the thought of forgiving the killer seemed impossible at first. Oshea went to prison, but Mary remained a prisoner of her own rage for over a decade. Seventeen years later, still without peace, Mary felt led to meet Oshea face-to-face. She says her faith and desire to move forward pushed her to do this. The meeting, held in the prison visiting room, was life-changing. Oshea sincerely apologized for what he had done and, with tears, asked Mary for forgiveness. In that pivotal moment, Mary chose to let go of the hatred she’d harbored. As they cautiously embraced, she recalls, “I just hugged the man that murdered my son… and I instantly knew that all anger and animosity… was over.”prisonfellowship.org The burden she’d carried for years melted away in an instant of mercy.

Mary Johnson’s act of forgiveness not only brought her inner peace – it also sparked an unlikely friendship. After Oshea served his sentence, Mary welcomed him back to society. In fact, he eventually moved into the apartment next door to Mary, with her blessingprisonfellowship.org. She began referring to Oshea as her “spiritual son,” saying, “I forgave him and gained a son”. In 2010, when Mary remarried, Oshea was even the one who walked her down the aisleprisonfellowship.orgprisonfellowship.org. The two now frequently share their story together at community events, showing the world that reconciliation and healing are possible. Mary went on to found an organization called From Death to Life, which helps others impacted by violence to find healing through forgivenessprisonfellowship.org.

Mary’s journey illustrates that forgiveness is not about the offender deserving it – it’s about you deserving peace. By forgiving the young man who took her son’s life, she released herself from a prison of bitterness. The transformation was tangible: her blood pressure dropped, her depression lifted, and she smiled again. “Forgiveness freed both the one forgiven and the one forgiving from the bondage of the past,” as one article noted about Mary’s experienceprisonfellowship.org. Indeed, both she and Oshea were able to move forward and find purpose beyond the tragedy. Not every story of forgiveness will end in such a beautiful friendship, but Mary’s example shows that even the deepest wounds can be healed. The key was her willingness to let go for her own sake, to trade rage for serenity. In doing so, she unlocked a life of peace that had been sealed away by resentment.

Forgiveness is the key – whether viewed through the lens of science, psychology, or spirituality, this truth stands. Letting go of anger might be hard, but it is one of the most liberating choices we can make. It clears out the negativity occupying our hearts and minds, making room for peace, joy, and connection. As you consider your own life, ask: is there a past hurt that still locks up a part of you? By turning the key of forgiveness, you open the door to freedom. The path may not be easy, but on the other side lies the true peace we all deserve. Embrace forgiveness – for your health, your happiness, and your future. The sooner you do, the sooner you’ll step into the lightness and relief that comes from a heart unburdened, finally at peace. pureflix.comprisonfellowship.org

Although this skin disease is prevalent, many people are still unaware of its impact. Unfortunately, there are many misconceptions about the disease; for example, that it is contagious.

What is Psoriasis?

Psoriasis isn’t just a skin disease; it is actually am autoimmune condition that has the potential to cause widespread systemic effects. These widespread systemic effects are most commonly described as effects on the skin, joints and heart. There are different forms of Psoriasis and some are more common than others.

Psoriasis is a chronic autoimmune disease that causes skin cells to multiply up to 10 times faster than normal. This makes the skin build up into bumpy red patches covered with white scales that can grow anywhere, but typically appear on the scalp, elbows, knees, and lower back. Psoriasis is not contagious nor is it caused or worsened by poor personal hygiene. Psoriasis may be inherited and can range from a very mild, hardly noticeable rash to a severe eruption that covers large areas of the body. Affiliated Dermatology’s Dr. Andrew Newman shares some facts about psoriasis:

“Although psoriasis is typically thought to be a condition that only affects the skin, it affects the ENTIRE body.  In fact, joint disease, heart disease, and depression are common features in psoriasis. It’s caused by many factors including genetic predisposition, certain medications, and some infections such as strep throat. People with psoriasis most often are regularly taken care of by a dermatologist.”

In some patients, psoriasis causes nail changes and joint pain (psoriatic arthritis). The first episode usually strikes between the ages of 15 and 35. This chronic condition will then cycle through flare-ups and remissions throughout the rest of the patient’s life.

What are the different forms of Psoriasis?

The most common form of the disease, plaque psoriasis, appears as raised, red patches covered with an accumulation of white dead skin cells. Other areas affected by the different types of psoriasis include the face, skin folds, hands, feet, genitals, and nails.

Most individuals will be afflicted with one form of Psoriasis at a time, there are known treatments for Psoriasis, but currently there is no known cure. Occasionally, when one form of Psoriasis clears up and symptoms reside, another form may appear due to exposure to a trigger. Triggers include but are not limited to; skin injury, stress, certain medications, infections, weather, diet and allergies.

1. Plaque Psoriasis – Plaque Psoriasis is the most common type of Psoriasis; it can also be called ‘Psoriasis Vulgaris’. This type of Psoriasis appears as red, inflamed patches of skin covered with a white or silvery buildup of dead skin cells (known as plaque). It can cause the skin to feel painful to the touch and itchy and typically effects the knees, elbows, scalp or lower back, however … it can occur anywhere on the body.

2. Inverse Psoriasis – Inverse psoriasis makes bright red, shiny lesions that appear in skin folds, such as the armpits, groin, and under the breasts.

Inverse Psoriasis appears as areas od shiny, red and inflamed skin. This type of Psoriasis is typically located in the folds of the body; under the arm pits or breasts, behind the knees, around the groin or even the skin folds that surround the genitals.

3. Guttate Psoriasis – Guttate psoriasis often starts in childhood or young adulthood, causes small, red spots, mainly on the torso and limbs. Triggers may be respiratory infections, strep throat, tonsillitis, stress, injury to the skin, and taking antimalarial and beta-blocker medications.

Guttate Psoriasis will more commonly present in childhood or amongst young adults. Symptoms appear as small pinkish-red spots or lesions, typically on the arms, legs and torso.

4. Erythrodermic Psoriasis – Erythrodermic psoriasis causes fiery redness of the skin and shedding of scales in sheets. It’s triggered by severe sunburn, infections, certain medications, and stopping some kinds of psoriasis treatment. It needs to be treated immediately because it can lead to severe illness.

This is one of the least common types of Psoriasis but it one of the most serious. More severe symptoms include severe burning, itching and peeling of the skin, changes in body temperature and a faster heart rate. If you believe you are suffering from this type of Psoriasis, see your doctor immediately, it can cause severe illness.

5. Pustular Psoriasis –  Pustular psoriasis causes red and scaly skin with tiny pustules on the palms of the hands and soles of the feet.  Pustular Psoriasis is another typically uncommon type of Psoriasis that mainly appears in older adults. Symptoms include pus-filled bumps, known as pustules, the surrounding skin can appear red and inflamed, oftentimes looking infectious (however, it is not). This type of Psoriasis can appear mainly on the hands and feet but can appear on other parts of the body as well. Symptoms of Pustular Psoriasis can include; nausea, fever, chills, muscle weakness, and rapid heart rate.

6. Psoriatic Arthritis – Psoriatic Arthritis is a variant of the condition where the individual has both arthritis (joint inflammation) and psoriasis. Typically, this condition appears years after the onset of Psoriasis symptoms. Symptoms can include; warm or discolored joints, swelling of the joints – fingers and toes, and stiff, painful joints that are worse after rest or in the mornings.

7. Nail Psoriasis – Nail Psoriasis is another variant of the condition and it more commonly affects those who are afflicted by Psoriatic Arthritis. Symptoms can include; painful, tender nails, color changes to the nails, a white chalk-like material under your nails, pitting of your nails, separation of the nail from the nail bed.

8. Scalp psoriasis can cause dandruff-like itching and flaking. Psoriasis happens when the immune system triggers too many skin cells to grow on various parts of the body. That can include your scalp. People with psoriasis may be more likely to get dandruff, but psoriasis is not dandruff.

Living with Psoriasis can affect your quality of life; however, certain treatments are available. You can work with your doctor to develop a plan of care and guide you in figuring out what your environmental triggers are or other lifestyle factors. Triggers and lifestyle factors could be the culprit behind flare-ups.

What causes Psoriasis?

Although psoriasis appears on the skin, it is an immune system disease that is not caused or worsened by poor personal hygiene. People with the disease have a genetic tendency to develop it. There are certain things that can trigger flare-ups including skin injury, stress, hormonal changes, infection, and medications. Most people with the disease experience cycles of clear skin and outbreaks. Dr. Dustin Mullens of Affiliated Dermatology spoke on how Psoriasis starts:

“The nervous system and stress affect a multitude of skin conditions in humans. There are many types of cells in the skin affected such as immune cells and endothelial cells, both can be regulated by neuropeptides and neurotransmitters, which are chemicals released by the skin’s nerve endings. Stress can result in the skin’s nerve endings releasing an increased level of these chemicals and when this occurs, it can lead to inflammation of the skin. This is why people often experience a flare-up of their inflammatory skin conditions such as psoriasis during times of stress.”Things that can trigger an outbreak of psoriasis include:

• Cuts, scrapes, or surgery
• Emotional stress
• Strep infections
• Medications, including
• Blood pressure medications (like beta-blockers)
• Hydroxychloroquine, antimalarial medication

Symptoms of Psoriasis

The truth is that there are many people with psoriasis who don’t even know they have it! Skin rashes are not uncommon so dermatologists need to rule out a list of other possible causes like an allergy to food/medication and viruses. Careful visual inspection is needed for diagnosing psoriasis, but sometimes there is a need for a skin biopsy.

Is infection a possibility? Infections are actually quite rare due to the fact that psoriasis itself is due to an overactive immune system. That being said, repeated scratching and excoriation can disrupt the skin barrier and facilitate bacterial invasion and is thus strongly discouraged. All patients with psoriasis should be seen at the very least annually by a dermatologist and when treatment and medications are ineffective at controlling disease severity and flares. Patients requiring systemic treatment should be seen every 3 months for check-ups while on these more sophisticated/complex medications.

How to Treat Psoriasis?

There’s currently no cure for this chronic autoimmune condition, but caring for psoriasis can slow down the growth of skin cells and relieve pain, itching, and discomfort. Treatment of psoriasis depends on a patient’s overall health, presence of joint pain, and severity of skin involvement. When asked about treatment for psoriasis, Dr. Newman shares,

“The type of treatment used depends on the total body surface area involved and severity, etc. In mild psoriasis, I think natural medicines work well. Some people find benefit from taking the natural anti-inflammatories quercetin and curcumin. Additionally, they may find that applying aloe vera gel to the skin does wonders. Lastly, sunlight also helps with mild psoriasis. That’s right, the UV rays of the sun decrease the skin inflammation in psoriasis! In fact, this explains why my colleagues and I see less psoriasis where we practice in the sun-rich Phoenix, Arizona, compared to areas like the midwest.”

In mild cases, topical corticosteroids and medications are prescribed. Psoriasis is not curable, but it is controllable. No single approach works for everyone. Therapy is individually tailored and based on your health, goals, and a careful assessment of potential risks and benefits of treatment. Treatments can be divided into four main types:

• Topical treatments
• Light therapy
• Systemic medications
• Biologics

Dr. Newman goes on to say, “For more serious psoriasis, it will be almost impossible to successfully manage the disease without sophisticated prescription medicines. Usually, this will entail potent topical corticosteroids and/or certain oral or injectable medicines that help regulate the body’s immune system (which has gone haywire in psoriasis). Importantly, if you have psoriasis (mild or severe), you should discuss the use of both natural and prescription medicines with your primary care doctor and your dermatologist.”

Find Relief for Psoriasis

The best treatment varies by individual, taking into consideration the type of psoriasis you have, where it is on your body and the possible side effects of medications. Another AffDerm dermatologist, Dr. Mitchell Manway, gave us some extra tips on what to do when you have psoriasis.

Moisturizers: which kind are the best? “In general, the thicker or greasier the moisturizer, the better. Creams and ointments that come in a tub or jar are more effective at restoring the skin barrier than lotions or products that come in pump-dispensers. Products containing petrolatum or ceramides can be particularly effective or preferred,” says Dr. Manway.

Scale softening products? What ingredients work best? Dr. Manway advises, “Products that contain lactic acid (Amlactin/Lac Hydrin), salicylic acid (Salex), or urea are more effective at removing scale and improving skin texture.”

Cold showers/cold packs or warm baths/heating pads? According to Dr. Manway, “Ice-packs and heat may be effective at treating symptoms of itch by distracting nerve receptors, but I would avoid exposure to showers or bathing as this may promote further water-loss and drying of the skin.”

Stress relief options like meditation, acupuncture, etc? “Studies directly involving acupuncture and treatment of psoriasis are still inconclusive, with some proposing benefit and others with no significant results. However, anything that can promote stress relief may be helpful at preventing and controlling flare-ups as stress can be a major contributor for worsening of the disease,” said Dr. Manway

Exercise? “Daily or weekly exercise can stimulate and regulate the immune system and decrease stress levels, and thus is an important part of disease management.”

Over-the-counter remedies like calamine lotion? “In my experience calamine lotion is not very effective at reducing itch or pain. Topical preparations that contain pramoxine (Sarna Sensitive) or menthol (Sarna) are preferred.  Surprisingly, brief periods of exposure to sunlight and UV rays can also benefit psoriasis, but limited exposure should be stressed due to the increased risk of skin cancer associated with chronic UVA and UVB damage,” said Dr. Manway.

Prescription medications? Dr. Manway agrees, “Rx medications are by far the most effective topical treatment approach available and help to decrease inflammation at the site of disease. Potent topical steroids such as clobetasol or betamethasone are the most common medications prescribed, but other mechanisms such as vitamin D analogues and calcineurin inhibitors can provide significant and adjunct benefits towards the reduction of psoriatic plaques with less risk of long-term local side-effects. When local disease can not be maintained on topical medications or development of psoriatic arthritis is present, systemic oral medications or biologic therapy/injections are necessary.”

When should you see your dermatologist for psoriasis? Look out for any suspicious changes such as lesions that show signs of persistent flaking, scaling, roughness, redness, scabbing, bleeding, or otherwise non-healing areas. These symptoms are uncomfortable and could be an indication of something more serious.

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Genetics of Generalized Pustular Psoriasis: Current Understanding and Implications for Future Therapeutics

1. Introduction

Psoriasis is a common chronic inflammatory skin disease with a variety of clinical manifestations [1]. Psoriasis may be classified into non-pustular and pustular forms. Pustular psoriasis may be further stratified into localized and generalized forms [2]. It is believed that both environmental and genetic factors participate in the immune mechanisms of psoriasis [3]. Current studies have demonstrated genetic susceptibility to psoriasis involving components of both innate and adaptive immune systems [1]. Prolonged inflammation results in dysregulated keratinocyte proliferation and differentiation, and the keratinocytes participate in both the initiation and maintenance phases of psoriasis [4].

Psoriasis vulgaris (PV) is known to be the most common subtype of psoriasis. Both immune and genetic studies have identified interleukin (IL)-23 and IL-17 as the main drivers of psoriasis vulgaris [5,6]. It is characterized by relatively stable and localized erythematous scaly plaques. On the other hand, pustular psoriasis (PP) is rarer but potentially life-threatening and is associated with innate immune system overactivation. It may present with erythematous, scaly skin, including pustules and systemic neutrophilia. Pustular psoriasis can present in various forms, including localized pustules, as in acrodermatitis continua of Hallopeau (ACH) or palmoplantar pustulosis (PPP), or diffuse, non-acral pustules with systemic inflammation, as in generalized pustular psoriasis (GPP) [2].

GPP is a severe type of psoriatic disease. It is characterized by the onset of widespread, macroscopically visible pustules on non-acral skin with or without systemic symptoms such as fever, neutrophilia, and elevated serum levels of C-reactive protein [7]. The extent of systemic symptoms varies among patients as well as between flares within the same patient.

Clarifying the immune mechanisms behind GPP helps to develop potential therapeutic targets for this disease. Meanwhile, we should also keep in mind that the age of onset and the frequency of genetic mutations vary significantly among different subtypes [8].

In 2017, Akiyama et al. first proposed the term “autoinflammatory keratinization diseases” (AiKDs) to describe the inflammatory keratinization of the skin due to genetic autoinflammatory pathomechanisms [9]. As the pathogenic mechanism of AiKD becomes elucidated, there will be more appropriate treatment methods and precision medicines available [10]. This novel concept also sheds light on the development of therapeutic agents for pustular psoriasis.

Recent studies of the molecular pathomechanisms of pustular psoriasis suggest that the inhibition of specific cytokines, including the IL-36 axis, is a potential therapeutic strategy to control the disease activity of pustular psoriasis [11].

Autoimmunity is characterized by the activation of the adaptive immune system, including T and B cells, while autoinflammatory responses are driven by endogenous danger signals as well as inflammatory mediators and cytokines. In complex inflammatory conditions such as psoriasis, these two processes frequently coexist and can influence and trigger each other. This review will discuss the mechanism of psoriasis based on the autoimmune and autoinflammatory processes that are activated. We also aim to provide an up-to-date elucidation of the genetic mutations associated with different subtypes of pustular psoriasis and, ultimately, focus on biological treatments available for GPP.

2. Genetics of Pustular Psoriasis

Although the first GPP case was reported a century ago, its etiology and detailed pathogenesis have only been discussed within the last ten years (Table 1). It was not until 2011 that IL36RN was initially discovered as a gene responsible for causing GPP [12,13]. Since then, a growing number of genetic mutations such as CARD14, AP1S3, MPO, and the SERPIN family have been identified as associated with GPP. However, not all GPP patients carry mutations of these genes, suggesting that there are still other genetic factors to be discovered. These disease-causing genes may participate in common or similar pathogenic molecular pathways [14].

Table 1. Genetic mutations associated with generalized pustular psoriasis and their proposed effects.

Ethnic differences in GPP should also take into consideration. For example, pathogenic mutations of AP1S3 have been reported in individuals of European origin but not in Malaysian populations [15,16], while MPO and SERPINA3 variants were identified in patients of European descent [17,18]. Associations with other ethnic groups remain to be elucidated.

The cases of pustular psoriasis are classified into GPP, PPP, and ACH according to the ERASPEN criteria [2]. Assan et al. suggested that PPP and ACH might be separate diseases while still maintaining some overlap [19]. Accordingly, there are prospective phenotype–genotype and multi-omics studies to better recognize the mechanisms of each subgroup. Another study conducted in Italy in a real-life setting revealed the concomitant rate of plaque psoriasis, which was the greatest in GPP and the least in ACH [20]. To distinguish GPP alone from those with PV is quite important since the selection of treatment is based on the disease mechanism and the clinical phenotype, which can include GPP alone, ACH alone, predominate ACH, ACH evolving into GPP, and ACH with GPP.

Adult-onset immunodeficiency syndrome (AOID) is known as an AIDS-like illness with abnormal interferon-γ (IFN-γ)/IL12 signaling. It is associated with high-titer neutralizing antibodies to IFN-γ, the controller of numerous pathogens [21]. The majority of cases exhibit skin-related symptoms, such as reactive skin conditions (82%) and infectious skin diseases (45%), with neutrophilic dermatoses being the most common among them [21,22]. A recent study conducted by Piranit et al. supports that both GPP and AOID involving pustular reactions are diseases caused by dysregulated proteolytic and apoptotic processes [23]. Clinically and genetically, GPP and AOID are likely to share some common pathogenetic mechanisms. To date, there have been no reports of AOID and GPP occurring in the same individuals or within the same families. However, genetic research has found heterozygous variants in the SERPINA3 and SERPINA1 genes in patients with AOID and GPP, respectively [24,25].

2.1. IL36RN

IL-36 cytokines are relatively novel and belong to the IL-1 family, which has members that are produced by many sources, such as epithelial cells, myeloid dendritic cells, and monocytes. IL36RN encodes for IL-36Ra, which inhibits the pro-inflammatory effects of IL-36 cytokines by binding their receptors, then preventing the release of mediators that stimulate the pustule formation seen in GPP [26].

Onoufriadis et al. reported that IL-36RN mutations can cause sporadic GPP, and according to their study, IL-36 mutations underline sporadic European GPP, as well as Tunisian autosomal recessive GPP [12]. Additionally, the first Asian case of GPP associated with IL36RN mutations was reported in 2012, therefore indicating that IL36RN mutations are common in some GPP cases worldwide [27]. The prevalence of IL36RN mutations among pustular psoriasis subtypes is different; patients with GPP have the highest prevalence of these mutations (23.7%). This is followed by ACH, which has the second-highest prevalence (17.4%), and lastly, PPP demonstrates the lowest prevalence of these mutations (5.1%) [8].

Hence, in order to ascertain if IL36RN alleles are the crucial determinants of pustular psoriasis across various disease subtypes, a regression analysis was carried out, incorporating clinical diagnosis as a covariate [28]. Individuals with homozygous mutations of IL36RN tend to experience more severe disease manifestations compared to those with heterozygous mutations, and these mutations are inherited through an autosomal recessive pattern [29]. Another study indicated that IL36RN mutations are almost not seen in individuals with both PPP and GPP [30]. Accordingly, this finding suggests that a large proportion of cases of GPP alone are caused by homozygous or compound heterozygous mutations of IL36RN.

On the other hand, the presence of IL36RN disease alleles demonstrated a dose-dependent influence on the age of onset across all types of pustular psoriasis [28]. According to genetic analyses, the frequency of IL36 mutations plays a role in differentiating pustular psoriasis subtypes [8]. Sophie et al. found that the percentage of individuals carrying IL36RN disease-associated alleles was higher in those with GPP and ACH. Individuals with GPP and ACH were more likely to have biallelic mutations compared to those affected by PPP.

2.2. CARD14

Caspase recruitment domain family member 14 (CARD14) is a gene located in the psoriasis susceptibility locus 2 (PSORS2). CARD is a protein-binding molecule that facilitates the formation of complexes containing CARD proteins, which are involved in apoptosis and NF-κB signaling pathways. Among them, CARD14 is found to be specifically expressed in diseases of the skin and is primarily localized in the basal and suprabasal epidermal layers [31]. Some CARD proteins are related to chronic inflammatory skin diseases, such as early-onset sarcoidosis or amyopathic dermatomyositis [32]. The role of CARD14 mutations as either causal factors or disease susceptibility factors for PV, GPP, or pityriasis rubra pilaris may depend on the specific mutation or variant position within the CARD14 gene. [28].

Differences in ethnical groups and geographic areas affect the outcome to some extent. A study revealed that the carrier rate of the CARD14 variant in Japanese individuals is higher than in Europeans. Therefore, we can consider CARD14 an important predisposing factor for GPP with PV in the Japanese population [33,34].

2.3. AP1S3

The AP1S3 gene, which encodes adaptor protein complex 1 (AP-1), plays a crucial role in stabilizing AP-1 heterotetramers that participate in vesicular trafficking between the trans-Golgi network and endosomes [35]. Cells with mutations in AP1S3 have decreased autophagosome formation in keratinocytes, leading to p62 build-up and resulting in enhanced NF-κB signaling [16]. Loss-of-function mutations of the AP1S3 gene were found relevant in GPP, which implies pustular psoriasis as an autoinflammatory manifestation resulting from impaired vesicular trafficking [15].

The pathogenic variants are distributed mainly in Europeans and rarely in East Asians and Africans. The variant frequency of AP1S3 in GPP patients of European ancestry is about 10.8% [15]. Suppressing AP1S3 expression in human keratinocytes and HEK293 cells eliminates endosomal activation by polyinosinic-polycytidylic acid, a TLR3 agonist involved in responding to viral infections. Researchers suggested that abnormalities in vesicular trafficking could be a significant pathological basis for the autoinflammatory process in pustular psoriasis [15].

Another study investigating genetic variations in patients with pustular psoriasis found that AP1S3 mutations were in fewer GPP cases than IL36RN, and patients with AP1S3 disease alleles were mainly female [8].

2.4. MPO

Deficiencies in MPO, a heme-containing peroxidase secreted by neutrophil granulocytes that catalyzes the formation of reactive oxygen species (ROS), have just been identified in association with GPP [14]. The association between MPO deficiency and pustular skin disease was first recognized by Vergnano et al. with phenome-wide association studies [36], and in vitro functional studies showed that mutations in the MPO gene lead to elevated neutrophil accumulation and activity, suggesting a role of MPO mutations in the pathogenesis of GPP [37].

The quantity of mutant MPO alleles was positively correlated with a younger age of onset, which is similar to the genotype-phenotype correlation of the IL36RN gene and further validates the genetic correlation of GPP [17]. The discovery that the MPO gene plays a pathogenic role in GPP provides perspectives on understanding GPP pathogenesis.

2.5. SERPINA1, SERPINA3

SERPINA1 and SERPINA3 are inhibitors of cathepsin G, the primary serine protease involved in cleaving and activating IL-36 precursors. The loss of function of these protease inhibitors may induce severe inflammatory effects [25]. Additionally, heterozygous loss-of-function mutations in both SERPINA1 and SERPINA3 were identified in individuals with GPP, and decreased protease inhibitor activity may result in enhanced IL-36 activation [18].

A study conducted by Piranit et al. reinforced the concept that the biological functions of SERPINB3 involve inhibiting cysteine proteases when mutated, and the subsequent overactivation of proteases leads to an intensified inflammatory reaction accompanied by heightened neutrophil recruitment [23]. Patients carrying SERPINB3 mutations exhibit aberrant SERPINB3 expression. The accumulation of misfolded SERPINB3 proteins causes the overactivation of cathepsin L, followed by the inactivation of SERPINA1, finally evolves into AOID with pustular reactions [38,39].

2.6. BTN3A3

BTN3A3 belongs to the human butyrophilin (BTN) 3 family, which has the ability to activate the NF-κB pathway, resulting in an excessive inflammatory response by suppressing the expression of IL-36Ra. To investigate the molecular pathogenesis of GPP, Q. Zhang et al. conducted a whole-exome sequencing study in the Chinese Han population [40]. However, the result found only two loci identified with exome-wide significance: the strongest one was in the IL36RN gene, and the other was located within the MHC region. A subsequent gene burden test demonstrated a correlation between BTN3A3 and GPP. Subtype analysis revealed that both IL36RN and BTN3A3 were markedly linked to GPP alone and GPP with PV. The BTN3A3 gene carried two LOF mutations with the most significant association. As a previously unreported determinant of GPP, BTN3A3 acted as a key regulator of cell proliferation, and its expression was associated with inflammatory imbalance.

2.7. TGFBR2

TGF-β signaling is recognized for its inhibitory effects on cell proliferation and immune system suppression [41]. Thus, the hyperproliferation of keratinocytes in the psoriatic epidermis is consistent with disrupted TGF-β signaling because of heterozygous loss-of-function TGFBR2 mutations. Concomitant with the overexpression of KRT17, there is an increase in keratinocyte proliferation and subsequent recruitment of neutrophils [42]. The overexpression of KRT17 is thus in line with a potential role for diminished TGFBR2 function in both GPP and AOID. Whole-exome sequencing (WES) was carried out on a total of 53 patients, comprising 32 individuals exhibiting pustular psoriasis phenotypes and 21 individuals with AOID presenting with pustular skin reactions [43]. The result showed that 4 Thai patients displaying similar pustular phenotypes, including two diagnosed with GPP and two with AOID, were found to carry the same rare TGFBR2 frameshift mutation. It is concluded that AOID might share pathogenic mechanisms with GPP.

Mechanistically, TGFBR1 and TGFBR2 are transmembrane serine/threonine kinases [44]. TGFBR2 expression is remarkably reduced or absent in psoriatic skin. As a result, it has been suggested that genetic variations in TGFBR2 could enhance susceptibility to GPP and AOID in some patients.

3. Current and Potential Therapeutic Agents Targeting Immune Mediators in Generalized Pustular Psoriasis

The phenotype and pathogenesis of different psoriasis subtypes are on a spectrum. On the one hand, plaque psoriasis is associated with the overactivation of the adaptive immune system, including T and B cells, and is thought to involve self-perpetuating inflammatory mechanisms through the IL-23/Th17 axis [45]. On the other end, pustular psoriasis has been associated with the stimulation of innate immune responses and the activation of IL-36 cytokine pathways [46]. Based on the pathomechanism, therapeutic agents for patients who have plaque psoriasis and GPP at the same time need to target not only the adaptive immune pathways but also the innate immune axis [47].

IL-36 cytokines are members of the IL-1 superfamily, and the IL-1/IL-36–chemokine–neutrophil axis plays a significant role in driving disease pathology in GPP. The first pathogenic variant found to be linked with GPP was a homozygous mutation of the IL36RN gene [48], and further studies have looked into the distribution over different populations [48,49].

Progress in understanding the relationship between autoinflammation and clinical phenotypes has contributed to the development of highly efficacious targeted treatments such as TNF-α, IL-17, IL-23, IL-1α/β, or IL-36 inhibitors or receptor blockers, as well as small molecule drugs such as PDE4 inhibitors, JAK inhibitors, and ROR-γt inhibitors.

Well-established treatment guidelines for GPP are currently lacking, and multiple biologic and non-biologic treatments exist. Considering the variety of comorbidities and severity associated with GPP, personalized treatments should be tailored. Figure 1 shows a graphical abstract of current and emerging biologic agents for GPP.

Figure 1. Graphical abstract of mechanisms of current and potential biologic agents for generalized pustular psoriasis.

3.1. IL-36 Pathway Inhibitors

Anti-IL-36 receptor antibodies can be employed to block the signaling pathway responsible for GPP flares and can be effective for patients with mutant IL36RN [48].

At present, only a single GPP-specific treatment, spesolimab, an interleukin-36 receptor antagonist, has received approval for use in the United States. With the experience of GPP complete remission after two doses of spesolimab [50], spesolimab was then approved by European Commission in adult GPP flares [51]. Spesolimab has been demonstrated to reduce the levels of relevant serum biomarkers and cellular populations in the skin lesions of patients with GPP, such as CD3+ T, CD11c+, and IL-36γ+ cells and lipocalin-2-expressing cells.

In patients with GPP, spesolimab has been observed to induce rapid changes in commonly disrupted molecular pathways in both GPP and PPP, suggesting that it may have the potential to improve clinical outcomes [52]. The results of a randomized controlled trial indicated that a 900 mg intravenous infusion of spesolimab led to greater lesion resolution in a patient group experiencing active GPP flare-ups after one week [53]. The improvement of the condition was evaluated using the GPPGA, which is a standardized assessment of a subject’s skin status based on three factors: erythema, pustules, and scaling/crusting [54]. After one week, there were almost four times the number patients who received spesolimab and achieved a GPPGA total score of 0 or 1 compared to control patients. Furthermore, it was found that spesolimab may relate to a higher incidence of infection, though neither opportunistic nor severe [55]. Long-term management options also were assessed; patient-reported outcomes were improved, and markers of systemic inflammation were normalized [56]. Recent research also indicates that spesolimab is effective for patients with GPP without IL36RN mutations [57].

Additional potential therapies targeting the IL-36 pathway for GPP are currently under development. Imsidolimab, an IL-36 inhibitor, recently passed through a phase 3 clinical trial to evaluate its efficacy and safety [58]. Patients received 750 mg of IV imsidolimab on day 1 and added 100 mg of subcutaneous imsidolimab every 4 weeks until day 85. Imsidolimab exhibited a rapid and sustained alleviation of symptoms and pustular eruptions in patients with GPP.

There are currently efforts underway to develop small molecule inhibitors of IL-36γ, which could have the potential to treat GPP. A-552 was identified as a potent inhibitor of IL-36γ in humans [59]. Phenotypic analysis of individuals without the IL-36R-encoding gene disclosed that they do not exhibit severe immunodeficiency, further supporting that the IL-36 pathway is a promising therapeutic target with minimal side effects [60].

3.2. IL-1RAcP

Interleukin-1 receptor accessory protein (IL-1RAcP) antibodies represent another feasible treatment alternative for GPP patients. IL-1RAcP, a member of the immunoglobulin superfamily proteins, has a crucial function in the signaling of the IL-1 family cytokines, such as IL-1, IL-33, and IL-36. Blocking IL-1RAcP’s ability to form a dimer with IL-36R could prevent the overactivation of the IL-36 pathway and subsequent inflammation [61]. Zarezadeh et al. indicated IL-1RAcP as a potential therapeutic target for inflammatory and autoimmune diseases [62]. However, the long-term safety and effectiveness of IL-1RAcP antibodies need to be determined since IL-1RAcP is expressed in a wide range of cell types, and excessive suppression may result in multiple toxic effects [63].

3.3. TNF-α Inhibitors

TNF-α, produced by activated plasmacytoid dendritic cells (DCs) and damaged keratinocytes, can stimulate the IL-36 pathway. TNF-α inhibitors indirectly suppress the expression of IL-36γ, resulting in reduced activation of the pro-inflammatory IL-36 pathway [64]. Adalimumab, infliximab, and certolizumab pegol are TNF-α inhibitors that have been approved for GPP treatment in Japan [65]. Cases with rapid and sustained resolution of skin lesions after infliximab used were reported in Poland [66]. A retrospective study showed the treatment efficacy rate of pustule clearance, which was 100% in the adalimumab + acitretin group [67].

However, paradoxical GPP is a potential adverse effect of TNF-α inhibitors. A study conducted in Turkey involving 156 GPP patients revealed that TNF-α inhibitors were the only biologic that triggered paradoxical GPP [68]. It is estimated that 0.6%-5.3% of patients receiving TNF-α inhibitors developed paradoxical GPP, with infliximab being the most frequently associated biologic with this condition [69].

3.4. IL-17 Inhibitors

Secukinumab, ixekizumab, and brodalumab are biologics that have been proven to manage GPP patients in Japan [70,71,72]. A retrospective study in Germany compared the rate of excellent response to GPP patients, with 60.0% in the secukinumab group and 50.0% in the ixekizumab group [73]. A phase IV, multicenter, open-label randomized control trial in Japan demonstrated that skin lesions mostly resolved in GPP patients under ixekizumab treatment, and there were no side effects reported [74].

However, there was a case of a Japanese individual that developed increased serum levels of liver enzymes during treatment with brodalumab for generalized pustular psoriasis [75]. The relationship between brodalumab and autoimmune hepatitis (AIH)/primary biliary cholangitis (PBC) overlap syndrome should be noted.

These IL-17 inhibitors mentioned above have been shown to be effective in controlling flares in the acute phase or as maintenance therapy in adult patients with GPP.

3.5. IL-23 Inhibitors

IL-23 regulates the production of IL-17, which subsequently stimulates the synthesis of pro-inflammatory IL-36R agonists, leading to the overactivation of the IL-36 pathway. The IL-23 inhibitors risankizumab and guselkumab are indicated for the treatment of GPP in Japan [76,77]. Ustekinumab, as an IL-12/23 antagonist, has been introduced to GPP patients, who achieved complete remission after its dose was titrated [78]. Additionally, both newly diagnosed ACH cases and already known therapy-refractory ACH cases had satisfactory and sustained therapy responses to guselkumab and risankizumab [79].

This suggests that IL-23 inhibitors may control flares in the acute phase or as maintenance therapy in adult patients with GPP.

3.6. Additional Biological Therapy and Non-Biologic Options

While the TNF-α/IL-17/IL-23 axis is predominantly targeted in plaque psoriasis, the IL-1/IL-36–chemokine–neutrophil axis shows greater potential as a therapeutic target in GPP. Previous studies have explored the use of IL-1 targeting biologics, such as the IL-1α receptor antagonist anakinra, as well as the IL-1ββmonoclonal antibodies gevokizumab and canakinumab, in GPP patients [80]. Anakinra is a successful treatment in patients with GPP carrying mutant IL36RN genes, while gevokizumab and canakinumab are effective in blocking the pro-inflammatory cytokine IL-1β [81].

As for non-biologic immunomodulatory management, methotrexate, cyclosporine, apremilast, and retinoids have been used for the treatment of GPP, but the efficacy is only based on case reports and non-randomized studies. Japanese guidelines have suggested the use of topical treatments as maintenance therapy following flares or as a supplementary therapy to address psoriasis-like symptoms [65].

4. Conclusions

GPP is a severe inflammatory disease distinct from PV [82]. Recent genetic observations and investigations provided us with insight into the disease. We found specific genes that are associated with pustular skin disease, including IL36RN, CARD14, AP1S3, MPO, SERPINA1, SERPINA3, BTN3A3, and TGFBR2. The immunologic pathway implicates IL-36 as a central node cytokine. That is, GPP constitutes a large IL-36-dominated keratinocyte cytokine storm and epidermal neutrophil aggregation.

The advances in our comprehension of GPP and its treatment options have the potential to improve patient care. It is known that the IL-36 pathway is the main inflammatory pathway implicated in GPP, but it is neither necessary nor sufficient to cause the disease. Aside from genes that play a role in the regulation of IL-36 signaling, there are IL36RN-negative GPP cases that have been noted. Due to the rarity of GPP, it has been challenging to identify additional disease-causing genes in the past. However, by combining whole-exome sequence data from various centers and targeting cases that are more prone to be monogenic in origin, progress could be achieved.

Progressive biologic therapies that target different chemokine receptors show efficacy, but there are also safety considerations. As more relevant and efficacious treatment options become available, patient outcomes and quality of life will improve. We should also keep in mind that immediate treatment goals during GPP flares are to alleviate skin inflammation and minimize the impact of systemic symptoms to avoid complications such as cardiovascular aseptic shock, heart failure, acute respiratory distress syndrome, prerenal kidney failure, neutrophilic cholangitis, uveitis, and severe infections [80,83]. Prevention of flare-ups of GPP is another treatment goal, and further clinical studies are indicated to evaluate the efficacy of the prevention of GPP flares. Additionally, the prevalence of genetic mutations of GPP varies in different countries and ethnic groups. It is important to investigate if patients with different genetic mutations of GPP have different short-term and long-term treatment responses.

Author Contributions

Conceptualization, S.-F.Y., M.-H.L., P.-C.C., S.-K.H., S.-Y.S., H.-S.Y. and S.Y.; literature review, S.-F.Y., M.-H.L., P.-C.C., S.-K.H., S.-Y.S. and S.Y.; writing—original draft preparation, S.-F.Y.; writing—review and editing, S.-F.Y., M.-H.L., P.-C.C., S.-K.H., S.-Y.S., H.-S.Y. and S.Y.; supervision, H.-S.Y. and S.Y.; project administration, S.Y.; funding acquisition, S.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by grants from the Taiwan National Science and Technology Council (MOST-110-2628-B-037-007 and NSTC-111-2314-B-037-042) to S.Y. and grants from Kaohsiung Medical University Hospital (KMUH110-0R61 and KMUH111-1R59) to S.Y.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Acknowledgments

This study is supported partially by Kaohsiung Medical University Research Center Grant (KMU-TC111B02).

Conflicts of Interest

S.Y. a guest editor of the Special Issue: Genetics of Complex Cutaneous Disorders, had no role in the peer review process or decision to publish this article.

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Generalized Pustular Psoriasis: Divergence of Innate and Adaptive Immunity

Abstract

Generalized pustular psoriasis (GPP) is a severe, relapsing, immune-mediated disease characterized by the presence of multiple sterile pustules all over the body. The exact pathomechanisms behind GPP remain elusive, although increased interest in the genetic basis and immunological disturbances have provided some revealing insights into the underlying signaling pathways and their mutual interaction. The genetic background of GPP has been thoroughly investigated over the past few years. The conducted studies have identified genetic variants that predispose to pustular forms of psoriasis. The loss-of-function mutation of the interleukin 36 receptor antagonist gene, along with rare gain-of-function mutations in the gene that encodes the keratinocyte signaling molecule (CARD14), are examples of the uncovered abnormalities. Interleukin 36 (IL-36), along with neutrophils, is now considered a central cytokine in GPP pathogenesis, with IL-36 signaling providing a link between innate and adaptive immune responses. More recently, a new concept of inflammation, caused by a predominantly genetically determined abnormal activation of innate immune response and leading to inflammatory keratinization, has arisen. GPP is currently considered a representative of this novel group of skin conditions, called autoinflammatory keratinization diseases. As no therapeutic agents have been approved for GPP to date in the United States and Europe, the novel anti-IL-36R antibodies are particularly promising and may revolutionize management of the disease.

Keywords:

generalized pustular psoriasis; von Zumbusch; IL-36; autoinflammation; innate immunity; genetics

 

1. Introduction

Generalized pustular psoriasis (GPP) is a rare, chronic, highly inflammatory, and potentially life-threatening variant of psoriasis [1,2,3]. GPP is more prevalent in Asians than Caucasians (annual prevalence of 7.46 cases/million people in Japan in contrast to 1.76 cases/million in France) and accounts for about 1% of all psoriasis cases [4,5,6,7]. GPP is approximately twice as common in women than in men, as was reported in both European and Asian cohort studies [8,9]. The mean age of onset of GPP is 31 years, which is lower than that of palmoplantar pustulosis or acrodermatitis continua Hallopeau [8]. Epidemiological data on GPP are in contrast to those on plaque psoriasis, which is reported to be equally prevalent among men and women and to occur most frequently between the ages of 15–20 years, with a second smaller peak occurring at 55–60 years [10]. GPP is characterized by recurrent episodes of widespread neutrophilic aseptic pustular eruptions, with accompanying symptoms of systemic inflammation [11]. The acute onset of GPP is usually associated with one or several general symptoms, such as pyrexia, malaise, and fatigue, and extracutaneous manifestations including arthritis, uveitis, acute respiratory distress syndrome, cardiovascular shock, and neutrophilic cholangitis [3,12,13]. Typical laboratory abnormalities include elevated C-reactive protein, leukocytosis, neutrophilia, and elevated liver function tests [3,13,14]. Acute GPP flares are associated with significant morbidity and mortality, if inadequately treated [2,15]. GPP may either be associated with pre-existing plaque psoriasis or can develop independently [16]. In a minority of cases, typical plaque-type psoriasis lesions arise after GPP has appeared [1]. Due to its low prevalence, GPP is regarded as an orphan disease (ORPHA:247353) [3,4,5,15]. GPP has a relapsing–remitting course with a highly variable clinical phenotype and pattern of flares. In some patients, the skin is entirely cleared between episodic acute flares, whereas in others a more persistent course is characterized by sharply defined localized or widespread erythematous plaques, with or without pustules [17]. GPP flares are idiopathic in most cases, although elicitation by certain endogenous and exogenous trigger factors, including infection, pregnancy, withdrawal of corticosteroids, and certain medications (e.g., ustekinumab, infliximab) is not uncommon [3,15,18,19]. Histologically, GPP is characterized by spongiform pustules of Kogoj and Munro’s subcorneal microabscesses, with the presence of an excessive amount of infiltrating neutrophils [20]. The most important clinical and histopathological differential diagnosis of GPP is acute generalized exanthematous pustulosis (AGEP), a rare and severe pustular skin reaction. Clinically, AGEP has a more abrupt onset, shorter duration, usually does not recur, and the patients do not have a personal or family history of psoriasis [21]. Moreover, AGEP has been strongly linked to certain drugs, such as ampicillin/amoxicillin, fluoroquinolones, sulfonamides, terbinafine, and diltiazem [21]. Although the microscopic features of these two pustular eruptions can be very similar, in most cases it is possible to differentiate them based on clinicopathological features [20].

GPP is traditionally classified as a variant of psoriasis. However, the distinct clinical, histological, and genetic features of the former suggest that these two diseases have, at least partially, different pathogenic mechanisms. It has been thus suggested that GPP should be regarded as a separate entity and that it requires a different therapeutic approach [4,16,22,23,24]. To date, no standard treatment guidelines exist for GPP in the United States and Europe; however, both conventional and biological agents used for plaque psoriasis have been incorporated into the therapeutic regime. Non-biological systemic therapy in adult patients typically includes acitretin, cyclosporine A, and methotrexate [25,26]. Only in Japan, several biologics have been approved for the treatment of GPP in patients who had an inadequate response to conventional therapy, including monoclonal antibodies against interleukin (IL)-17 (secukinumab and ixekizumab) or its receptor (brodalumab) and against IL-23 (risankizumab and guselkumab) [27,28,29,30,31,32,33,34]. Since the adaptive immune system plays a critical role in the pathogenesis of plaque psoriasis, agents specifically targeting elements of adaptive immunity are highly efficacious for the treatment of chronic plaque psoriasis [35]. It is worth noting that these therapies are generally less effective in the management of GPP than plaque psoriasis. This again suggests a divergent underlying pathogenic mechanism in the pustular variants of psoriasis [36]. It also needs to be pointed out that a paradoxical induction of GPP has been reported with biological agents [18,19,37,38]. Case reports, case series, and small open-label clinical trials have been published on novel biologics that target the cytokines involved in GPP pathogenesis. Recent gene expression analyses have demonstrated that the transcriptome of GPP shares some common features with that of plaque psoriasis. However, it is dominated by innate immune system activation and autoinflammation, whereas adaptive immune responses predominate in plaque psoriasis [39,40].

This article aims to elucidate and discuss the intricate interaction between the innate and adaptive immune mechanisms in the autoinflammatory pathogenesis of GPP. It also summarizes the up-to-date knowledge on the genetic background of this disease, discussing the clinical significance of the uncovered mutations. Moreover, it provides an overview of the current options for targeted therapies for GPP, including data from the most recent clinical trials.

2. Gene Mutations in GPP

The first indication that genetic abnormalities may lead to pustular dermatitis was the identification of homozygous mutations in IL-1 receptor antagonist (IL-1Ra) gene (IL1RN) in six families with a deficiency of IL-1Ra (DIRA) [41]. The absence of IL-1Ra allows the unopposed action of pro-inflammatory cytokines IL-1α and IL-1β, which results in life-threatening systemic inflammation with skin and bone involvement. This was first described in nine children harboring mutations that lead to the synthesis of a truncated non-functional form of IL-1Ra. All but one of those patients suffered from pustular skin disease of varied severity, ranging from localized pustules to generalized severe pustulosis [41]. Similar cases involving acute pustular rash with severe systemic symptoms have been reported by several other groups [42,43,44,45].

Although the first patient with GPP was described in 1910, it was not until over 100 years later that the etiology and detailed pathogenesis were elucidated. The high severity of inflammation seen in GPP patients and the existence of numerous familial cases led to the hypothesis of a monogenic inheritance pattern. This hypothesis was proved by the identification of homozygous and composite heterozygous loss-of-function mutations of IL-36 receptor antagonist gene (IL36RN) in 2011. The acronym DITRA (deficiency of interleukin thirty-six-receptor antagonist) is often used for those cases of GPP in which IL36RN mutation is detected [46]. Pathogenic IL36RN mutations were originally identified in consanguineous GPP pedigrees of Tunisian origin and in five isolated cases from the UK [46,47]. The knockout of the IL-36 receptor (IL-36R) in a murine model of deficiency of IL-36R antagonist led to the dramatic resolution of skin inflammation, making the blockade of IL-36R signaling a novel and promising therapeutic approach for patients with pustular variants of psoriasis [48,49]. Other important mutations that underlie the enhanced inflammatory cascade and the recruitment of neutrophils and macrophages have also been described in different groups of GPP patients. These include mutations in the CARD14 gene that encodes caspase-activating recruitment domain member 14 and in the AP1S3 gene that encodes adaptor protein complex 1 subunit sigma 3 [24,47,50,51,52]. Additional disease-associated variants in CARD14 and/or AP1S3 were identified in 15% of IL36RN mutation carriers, indicating an oligogenic instead of monogenic inheritance pattern [53].

2.1. Mutations of IL-36 Receptor Antagonist

The IL-36 family is a relatively novel group of cytokines that belongs to the IL-1 superfamily and consists of three pro-inflammatory agonists, IL-36α, IL-36β, and IL-36γ, and two antagonists, IL-36 receptor antagonist (IL-36Ra) and IL-38. These IL-36 cytokines are expressed in epithelial and immune cells and function through a shared receptor (IL-36R) to modulate innate and adaptive immune responses [54]. IL-36 cytokines can induce the downstream pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways via an intracellular signaling cascade by binding to IL-36R. Subsequently, the release of inflammatory mediators and chemotaxis that promote activation of neutrophils, macrophages, dendritic cells, and T cells is induced, ultimately causing the amplification of inflammatory responses [55].

IL36RN encodes the IL-36Ra, which suppresses the pro-inflammatory effects of IL-36 cytokines (namely IL-36α, IL-36β, and IL-36γ) by binding their receptor, interleukin-1 receptor-like 2 (IL-1RL2), and preventing the release of chemokines that stimulate the activation of neutrophils, macrophages, dendritic cells, and T cells; inducing neutrophil chemokine expression, infiltration, and pustule formation in GPP [56,57]. In vitro and ex vivo observations revealed that GPP alleles abolish the antagonistic effect of IL-36Ra; thus, IL-36 stimulation of patients’ cells results in enhanced production of pro-inflammatory cytokines such as IL-1, IL-6, and IL-8 [46,47]. Mutations in IL36RN, which were first described in 2009 in two families with severe pustular psoriasis, lead to functional impairment of IL-36Ra and subsequent amplification of the downstream inflammatory responses [46,47]. Such mutations in IL36RN gene were initially identified in north-African families suffering from autosomal recessive GPP. They were homozygous missense mutations, with the substitution of proline for leucine at position 27 (p.Leu27Pro) [46]. In another pioneering study of five European cases of GPP, three individuals were found to have mutations in IL36RN, including a novel homozygous missense mutation (p.Ser113Leu) and one compound heterozygote carrier (p.Ser113Leu and p.Arg48Trp) [47]. IL36RN mutations do not contribute to the risk of plaque psoriasis. In fact, most IL36RN mutations are identified in patients with GPP that do not suffer from concurrent plaque psoriasis [58]. This observation was confirmed by Sugiura et al., who first screened for IL36RN gene within two subgroups of patients with GPP (GPP alone and GPP with concurrent psoriasis vulgaris). They showed that all GPP patients without psoriasis vulgaris carried homozygous or compound heterozygous mutations in the IL36RN gene, whereas only 2 out of 20 cases of GPP with psoriasis vulgaris harbored compound heterozygous mutations [24]. Based on these results, it was suggested that GPP alone may represent a distinct subtype of GPP that is etiologically distinguishable from GPP occurring with psoriasis vulgaris [24].

Several types of IL36RN mutations, including substitution, frameshift, and splicing defects, have been reported as the causative genetic background in numerous GPP cases, in various geographical regions [8,24,46,47,53,59,60,61,62,63]. In addition, Hussain et al. demonstrated that IL36RN mutation carriers exhibit a more severe clinical phenotype (e.g., earlier age of disease onset, increased risk of systemic manifestations) and the absence of co-existing plaque psoriasis, when compared to individuals without IL36RN mutation [64]. The most recent analysis, which included a cohort of 251 unrelated patients with GPP from multiple countries, also showed that IL36RN gene mutations were associated with an early age of onset, prevalence of psoriasis vulgaris, and high recurrence rate of GPP [8]. On the basis of the findings of their study, the authors recommended that patients who present with GPP before the age of 30 should be screened for IL36RN mutations [8]. Overall, the prevalence of IL36RN mutations in patients with GPP has ranged between 10% and 82%, and was significantly lower in cases with associated plaque psoriasis than in those linked to GPP alone [23,65,66]. Biallelic IL36RN mutations are known to be disease-causing or disease-contributing in 21–41% of patients with GPP [24,46,47,53,64].

2.2. CARD14 Mutations/Variants

Rare gain-of-function mutations in the gene that encodes the keratinocyte signaling molecule (CARD14) were found to be causative of familial psoriasis vulgaris and familial pityriasis rubra pilaris in 2012 [67]. CARD14, expressed and localized predominantly in keratinocytes, is a scaffold protein that mediates NF-κB signal transduction, thus contributing to inflammatory responses within the epidermis [52,67,68,69,70]. Interestingly, CARD14 expression is essentially confined to the basal layer of epidermis in unaffected skin. However, it is upregulated in the granular layers in the skin of patients with GPP [69]. In 2019, Shao et al. reported that neutrophils isolated from patients with GPP induced the upregulated expression of inflammatory genes, including IL-1b, IL-36G, IL-18, tumor necrosis factor alpha (TNF-α), and C-X-C motif chemokine ligands in keratinocytes, and more than normal neutrophils. Moreover, neutrophils from patients with GPP secreted more exosomes than the controls. These neutrophils were then rapidly internalized by keratinocytes, which increased the expression of these inflammatory molecules by activating the NF-κB and MAPK signaling pathways [71]. Two independent groups reported that variants of the CARD14 gene are associated with GPP and palmoplantar pustular psoriasis [52,72]. Moreover, the first autosomal dominant familial pedigree of GPP associated with CARD14 mutations was described in [73]. Mutations in CARD14 gene account for only a small proportion of cases of GPP; in most cases they are present in GPP patients with concomitant psoriasis vulgaris, but were only rarely identified in GPP alone [8]. No mutations of the CARD14 gene that are specific to patients suffering from psoriasis vulgaris and GPP have yet been found. Therefore, the correlation between CARD14 gene mutations and the onset of GPP remains to be further elucidated.

2.3. AP1S3 Mutations

Adaptor-related protein complex 1 (AP-1) is a highly-conserved heterotetramer that plays a pivotal role in vesicular trafficking between the trans-Golgi network and endosomes [36]. In 2014, mutations in AP1S3, the gene encoding AP-1 complex subunit sigma 3, were found in unrelated individuals with severe pustular psoriasis, including patients with GPP not harboring IL36RN and CARD14 mutations [50]. In addition, Mahil et al. reported that knockout of AP1S3, which is highly expressed in keratinocytes, disrupted keratinocyte autophagy in several cell lines. This alteration results in the abnormal accumulation of p62, an adaptor protein mediating NF-κB activation, and thereby upregulation of IL-1 signaling and overexpression of IL-36α among other cytokines [51]. To date, there are fewer mutational reports on AP1S3 than on IL36RN or CARD14, as they only account for approximately 11% of GPP cases in Europe and are rarely found in East Asians [32,50].

2.4. TNIP1 Mutations

Three cytokine signaling pathways important in GPP pathogenesis (including angiopoietin signaling, NF-κB signaling, and retinoic acid receptor activation) were significantly associated with the TNIP1 gene encoding TNF-alpha induced protein 3-interacting protein 1 (TNIP1). This led to the designation of TNIP1 as a potential candidate susceptibility gene for GPP [74,75]. In a study of 73 patients with GPP in a Han Chinese population, six polymorphisms were identified in TNIP1 gene locus; however, they were shown to be only weakly associated with GPP [76].

2.5. SERPINA3 Mutations

SERPINA3 (Serpin Family A Member 3) encodes serine protease inhibitor A3 (serpin A3, also known as α1-antichymotrypsin), which specifically inhibits several proteases [77]. More recently, a new candidate gene for GPP was proposed in a publication by Frey et al. They detected a novel, rare loss-of-function variant in SERPINA3 in 2 out of 25 independent patients via whole exome sequencing [78]. SERPINA3 strongly inhibits the neutrophil protease cathepsin G (CTSG), which has been shown to process full-length secreted IL-36 cytokines to their more active forms, thereby increasing their pro-inflammatory activity ~500-fold [23,79,80].

2.6. MPO Mutation

The MPO gene encodes myeloperoxidase (MPO), an essential component of neutrophil azurophil granules [81]. Although the relationship between MPO deficiency and pustular psoriasis was first described in 1996 in an individual case report, it was only recently that a mutation in MPO gene was recognized as a background for GPP [82,83]. Vergnano et al. performed a whole-exome sequencing of 19 unrelated individuals with GPP and identified a subject harboring a homozygous splice-site mutation in MPO. MPO screening in diseases phenotypically related to GPP uncovered further disease alleles in one patient with acral pustular psoriasis and in two subjects with AGEP [83]. Importantly, all three MPO gene variants that were observed in that study have a well-established impact on protein function, as they have been repeatedly observed in individuals with MPO deficiency [84,85]. Moreover, the phenotypic effects of MPO mutations were explored using a phenome-wide association study (PheWAS), which allowed identification of important relationships between genetic variants and a wide range of phenotypes. In vitro functional analysis revealed that mutations in the MPO gene cause an increase in neutrophil accumulation and activity, as well as a reduction in the number of apoptotic neutrophils. This observation further supported the role of this gene in neutrophil hemostasis and indicated its role in GPP pathogenesis [83]. These important findings regarding the significance of MPO gene variants in GPP were further confirmed by Haskamp et al., who discovered that 15 out of 74 patients affected by GPP carried eight variants in MPO gene that were all validated as loss-of-function mutations. They also performed a downstream analysis, which subsequently found that the activity of neutrophil elastase (NE), CTSG, and proteinase 3 (PR3), serine proteases that cleave IL-36 precursors into very active pro-inflammatory IL-36 cytokines, inversely correlated with MPO activity. This observation demonstrated that MPO deficiency was strongly linked to IL-36 pathway activation. Moreover, MPO deficiency caused defective formation of neutrophil extracellular traps (NETs) in the phorbol myristate acetate-induced pathway and reduced phagocytosis of neutrophils by monocytes (efferocytosis), thereby contributing to the prolonged persistence of harmful neutrophils and the reduced ability to resolve skin inflammation in GPP. Notably, a genotype–phenotype relationship similar to that of IL36RN gene was found in the abovementioned study, as the dosage of abnormal alleles of MPO gene negatively correlated with the age of disease onset [86]. Considering that the results of these studies implicated MPO as an important modulating enzyme of inflammation, MPO itself or MPO-related pathways represent attractive targets for anti-inflammatory therapies in GPP.

The above described mutations underlying GPP and their significance are depicted in Table 1.

Table 1. Summary of mutations associated with generalized pustular psoriasis. (ACH—acrodermatitis continua Hallopeau, GPP—generalized pustular psoriasis, IL-36—interleukin 36, NF-ƙB—nuclear factor kappa-light-chain-enhancer of activated B cells, PPP—palmoplantar pustulosis, PsV—psoriasis vulgaris).

3. Immunopathogenesis

3.1. Autoinflammation and Autoimmunity in GPP

Overexpression of IL-36 inflammatory cytokines in cutaneous lesions and loss-of-function mutations in IL36RN gene, as well as mutations in other genes related to the IL-36 pathway (e.g., CARD14, AP1S3, SERPINA3), have been identified in some patients; indicating that the IL-36 signaling pathway may be pivotal in the pathogenesis of GPP [46,50,52]. It has been discovered that IL36RN, CARD14, and AP1S3 gene mutations activate pro-inflammatory signaling pathways via NF-κB, which further results in an increased expression of CXCL1-3, IL-1, IL-8, and IL-36 pro-inflammatory cytokines. In addition, MPO gene deficiency also promotes the activation of IL-36 signaling by regulating the activity of NE, CTSG, and PR3 serine proteases [32]. In addition, data from gene expression analyses have revealed that the transcriptome of GPP shares many similarities with that of plaque psoriasis, but it is inclined more towards innate immune mechanisms [23]. Thereby, subtypes of psoriasis are thought to exist within a continuum, wherein plaque psoriasis is characterized by an adaptive immunity involving a cluster of differentiation four-positive (CD4+) and CD8+ T cells and the key role of the IL-17/IL-23 immune pathway. Oppositely, in pustular variants of psoriasis, it is the innate immune responses involving IL-36 activation, neutrophil infiltration, and autoinflammation that are central to the pathogenesis [63].

Recent research on the interplay between IL-17- and IL-36-driven inflammation has shed a new light on how individual mediators may modify the spectrum of psoriasis via shifting innate to adaptive immunity or vice versa. The pathogenesis of GPP partly overlaps with the typical pathways of psoriasis vulgaris but exerts a more pronounced activation of the innate immune system. Therefore, cytokines such as IL-17A, IL-22, IL-23, and TNF-α were found to be elevated in both psoriasis vulgaris and GPP; however, GPP lesions yielded significantly higher IL-1 and IL-36, and lower IL-17A and interferon-gamma (IFN-γ) messenger RNA (mRNA) expressions, than plaque psoriasis lesions [23].

The discovery of the IL36RN mutation in GPP provided a rationale for blocking inflammasome, thus inhibiting autoinflammation. Antibodies targeting the IL-1–/IL-36–chemokine–neutrophil axis, including the recombinant IL-1 receptor antagonist anakinra and the anti-IL-1β monoclonal antibodies, canakinumab and gevokizumab, were beneficial in GPP, but the efficacy data comes only from isolated case reports and small case series [87,88,89,90]. More recently, as a result of better understanding of the immunopathogenesis of GPP, specific therapies targeting IL-36 have been developed. Two monoclonal antibodies targeting IL-36R, spesolimab (BI 655130) and ANB019, have shown promising initial results in GPP and have proceeded to phase II clinical trials [91,92,93,94].

3.2. GPP as an Autoinflammatory Keratinization Disorder

The term “autoinflammatory diseases” emerged in 1999, when germline mutations in tumor necrosis factor receptor superfamily 1A (TNFRSF1A) were reported as causative in tumor necrosis factor receptor-associated periodic syndrome (TRAPS) [95]. Autoinflammatory diseases, which are usually monogenic disorders with a systemic inflammatory component, are caused by genetic mutations in the molecules and signaling pathways involved in innate immune responses [95,96]. In order to highlight the major cutaneous manifestations of various autoinflammatory diseases, Akiyama et al. proposed a new term to encompass inflammatory keratinization diseases with a prominent autoinflammatory component, namely autoinflammatory keratinization disorders (AiKDs) [60]. AiKDs involve significant genetic factors causing the hyper-activation of innate immunity, primarily within the epidermis and the superficial dermis, which results in abnormally up-regulated keratinization [60]. Importantly, since AiKDs include conditions with mixed pathological mechanisms of autoinflammation and autoimmunity, they are unique, and in many ways different, from classic autoinflammatory diseases. Initially, AiKDs comprised pustular psoriasis and related entities, including GPP, impetigo herpetiformis, and acrodermatitis continua Hallopeau due to mutations in IL36RN, GPP and palmoplantar pustular psoriasis due to CARD14 variants [72], and pityriasis rubra pilaris caused by CARD14 mutations/variants [73]; the AiKDs spectrum has since been extended and now includes several entities [61,62].

3.3. IL-1/IL-36 Inflammatory Axis

IL36-chemokine–neutrophil axis appears to be central to the pathogenesis of GPP. The most prominent inflammatory response in pustular forms of psoriasis involves activation of IL-1 and IL-36 signaling [23]. IL-36 cytokines are part of the IL-1 family, which consists of 11 members: IL-1 (IL-1α, IL-1β, IL-1RA), IL-18, IL-33, IL-36 (IL-36α, IL-36β, IL-36γ, IL-36RA), IL-37, and IL-38 [97]. IL-36 signals to keratinocytes in an autocrine fashion, inducing the expression and enhancing the synthesis of more IL-36 cytokines. This further promotes the release of pro-inflammatory cytokines, antimicrobial peptides, and neutrophil chemokines, such as the chemokine (C-X-C) motif ligand 1 (CXCL1), CXCL2, and CXCL8, acting through six-transmembrane epithelial antigens of prostate (STEAP)1 and STEAP4 metalloreductases, and hence creating a feedback inflammatory loop in the epidermis that drives the disease [23,39,98,99]. To underline the important contrast between psoriasis vulgaris and pustular variants of psoriasis, STEAP1 and STEAP4 are only upregulated in the latter. This fact further confirms that neutrophil recruitment is preferentially active in pustular psoriasis, whereas plaque-type psoriasis is predominantly characterized by IL-17/IL-23 immune responses [26,100,101,102]. IL-36 acts on both naïve CD4+ T cells and dendritic cells [103]. With respect to dendritic cells, IL-36 activation promotes maturation and increases the expression of major histocompatibility complex class II molecules, along with the co-stimulatory molecules B7-1 (CD80) and B7-2 (CD86), in addition to promoting the secretion of such pro-inflammatory cytokines as IL-1, IL-23, TNF-α, and IL-6 [63,104]. IL-36 leads to the induction of IFN-γ, IL-4, and IL-17 by T cells and has also been shown to promote clonal CD4+ T cell expansion, T-helper type 17 (Th17) cells differentiation, and IL-17A production in GPP [105]. This activation, as well as the contribution of both T cells and dendritic cells in IL-36 responses, may be a justification for the good treatment response to anti-TNF-α, anti-IL-17A, and anti-IL-23 biologics that has been achieved in many patients with GPP [27,30,106].

3.4. IL-17/IL-36 Axis as a Bridge between Innate and Adaptive Immunity

IL-17 is one of the main cytokines produced by Th17/Th1 cells, which play a pivotal role in the immunopathogenesis of plaque psoriasis [107,108]. There are two highly homologous members of the IL-17 protein family, IL-17A and IL-17F [109]. Even though IL-36 is the main pathogenic cytokine in GPP, a strong expression of IL-17A is observed among patients with GPP. Nevertheless, the levels of its expression in the lesional skin of GPP patients are significantly lower than in patients with plaque psoriasis [23]. Due to the IL-36 pathway intertwining with the TNF-α/IL-23/IL-17/IL-22 axis, a positive inflammatory feedback loop is created, as explained above [110,111]. IL-17A promotes the chemotaxis and accumulation of inflammatory cells, such as neutrophils, at the sites of inflammation. However, it is believed that Th17 cells might not be solely responsible for IL-17 overexpression in GPP, with neutrophils being an additional source of IL-17 [26,112,113]. As mentioned previously, the CD4+ T cells, mainly CD4+ Th17 cells, secrete IL-17. Interestingly, the augmented proliferation of IL-17 producing CD4+ T cells is promoted via IL-36 signaling, as was first observed by Arakawa et al. [105]. This interlinking between innate and adaptive immune systems has unexpected consequences and links the IL-17 and IL-36 pathways in GPP pathogenesis (Figure 1) [105].

Figure 1. Pathogenesis of generalized pustular psoriasis and plaque psoriasis—a cross-talk between innate and adaptive immunity (modified from [63]). In GPP, skin injury causes dead keratinocytes to release cathelicidin LL-37, a protein that stimulates surrounding keratinocytes to release IL-36, which further enhances the production of different chemokines and recruitment of neutrophils, T cells, dendritic cells, and monocytes. IL-36 expression is induced by other pro-inflammatory cytokines, such as IL-1, TNF-α, and IL-17A. Additionally, neutrophil proteases process and activate IL-36 family cytokines that escalate the inflammatory process. The serine protease inhibitors SERPINA1 and SERPINA3 can inhibit neutrophil proteases, which have been shown to process full-length secreted IL-36 cytokines to their more active forms, thereby increasing their pro-inflammatory activity. The mutation of the IL36RN gene can lead to IL36Ra deficiency, aggravating the inflammatory response and triggering GPP. Other genes (CARD14, AP1S3, TNIP1) are also known to predispose to GPP. In plaque psoriasis, various triggers can cause activation of keratinocytes and the release of self-nucleic acids and antimicrobial peptides (e.g., cathelicidin LL-37), which, along with type I interferons (e.g., IFN-α and IFN-β), activate plasmacytoid and myeloid dendritic cells. Activated dendritic cells promote differentiation of naïve CD4+ cells into Th1, Th17, and Th22 cells. Cytokines produced by these T cells, such as IFNγ, IL-17, and IL-22, act on keratinocytes and cause hyperproliferation. Keratinocytes release chemokines and attract neutrophils and other leukocytes. In plaque psoriasis, a different cytokine pathway than in GPP subsequently results in the same pathophysiological outcome via chemokine and cytokine secretions from keratinocytes and both IL-17 and IL-22, promoting neutrophil infiltration. (AP1S3—adaptor related protein complex 1 subunit sigma 3, CARD14—caspase recruitment domain-containing protein 14, CD4+—cluster of differentiation four-positive, CXCL1—chemokine (C-X-C) motif ligand 1, CXCL2—chemokine (C-X-C) motif ligand 2, CXCL8—chemokine (C-X-C) motif ligand 8, DC—dendritic cell, IFN-α interferon-alpha, IFN-β—interferon-beta, IFN-γ—interferon-gamma, IL-1—interleukin 1, IL-8—interleukin 8, IL-17—interleukin 17, IL-17A—interleukin 17A, IL-17C—interleukin 17C, IL-17R—interleukin 17 receptor, IL-22—interleukin 22, IL-23—interleukin 23, IL-36—interleukin 36, IL-36R—interleukin 36 receptor, IL-36Ra—interleukin 36 receptor antagonist, MAPK—mitogen-activated protein kinase, mRNA—messenger RNA, NF-ƙB—nuclear factor kappa-light-chain-enhancer of activated B cells, SERPINA3—serpin family A member 3, STAT3—signal transducer and activator of transcription 3, Th1—T-helper 1 cells, Th17—T-helper 17 cells, Th22—T-helper 22 cells, TNF-α—tumor necrosis factor alpha, TNIP1—TNFAIP3 interacting protein 1). Parts of the figure were drawn by using pictures from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/), accessed on 1 Jun 2021.

4. Biologic Therapeutics for GPP in the Light of Novel Genetic and Immunological Findings

Recently published findings of a survey regarding dermatologists’ opinions on the treatment efficacy in GPP revealed interesting and somewhat paradoxical results. While most physicians indicated that GPP flare treatments were adequate, they also stated that the response was slow and that many patients suffered from residual post-flare symptoms. It was indicated that the use of plaque psoriasis medications usually provides some benefits for GPP patients, but unmet needs clearly remain. The better utilization of the currently available therapies and the development of novel molecules will ensure safe long-term flare control [114].

TNF-α inhibitors (infliximab, adalimumab, and etanercept) were the first biologic agents to be used as an off-label treatment of GPP; therefore, the available data comprise a considerable number of GPP patients treated with those drugs [26,115]. The administration of those biologics results in rapid neutralization of TNF-α, which is also upregulated in GPP skin lesions [23]. Infliximab, the most-studied TNF-α blocking agent in GPP, showed a good response rate in 58% of patients and partial response in 28%. Notably, a quick onset of action was observed (pustule clearance in 1-3 days) [26]. Case report data also showed that infliximab can effectively treat juvenile GPP [116,117,118,119]. Treatment with TNF-α blockers was also highly effective in patients having IL-36Ra deficiency [120,121]. Interestingly, adalimumab has been shown to be a potential alternative treatment option in patients who fail infliximab, as Matsumoto et al. demonstrated significant improvement of GPP lesions in all four of their patients who had previously failed numerous systemic treatments, including infliximab, prior to switching to adalimumab [122]. It needs to be noted that most studies of TNF-α blocking agents in GPP are case reports. Therefore, further phase II and III clinical trials are necessary to evaluate the benefits and safety of these biologics in this indication.

Considering the upregulation of IL-17 and the pronounced neutrophilic infiltration in the skin of GPP patients, anti-IL-17 treatment appeared to be a very promising option [33]. Three IL-17 inhibitors (secukinumab, ixekizumab, and brodalumab) are currently licensed and approved for the treatment of moderate-to-severe plaque psoriasis [123]. All of the mentioned agents were used in GPP patients, including three open-label phase III clinical trials. Overall, a complete response was demonstrated in approximately two thirds of treated individuals, whereas only one in ten patients exhibited weak to no response [26]. The promising efficacy data for each of those compounds resulted in their approval for the treatment of GPP in Japan.

Since IL-23 plays a significant role in the pathogenesis of GPP, ustekinumab, an anti-IL-12/23 p40 monoclonal antibody, has also been successfully utilized in the management of GPP [124]. Out of a total of seven described patients, complete remission has been achieved in six individuals; however, all but one of them were IL36RN-negative [26,124,125,126]. Risankizumab and guselkumab are both highly effective and safe inhibitors of the IL-23 p19 subunit, and which are approved for the treatment of moderate-to-severe plaque psoriasis [127,128]. Guselkumab was assessed in a phase III open-label study in GPP and was less efficient when compared to IL-17 inhibitors [30]. A phase III clinical trial to evaluate the efficacy and safety of risankizumab in Japanese patients with GPP has been completed but detailed results have to date not been published [129]

Even though blocking of the TNF-α/IL-17/IL-23 axis has resulted in some degree of success in GPP, the IL-1/IL-36-chemokine–neutrophil axis appears to be a more promising therapeutic target, especially in the context of the aforementioned immunopathogenetic findings [23].

IL-1 targeting with biologics has been previously performed in GPP patients using the IL-1α receptor antagonist (IL-1-RA) anakinra and the IL-1β monoclonal antibodies gevokizumab and canakinumab. Anakinra, a recombinant IL-1 receptor antagonist, frequently used in the treatment of other autoinflammatory diseases, was also documented to be successfully used in GPP, including a juvenile case [88,130]. However, further randomized control trials are needed to evaluate the efficacy and safety of anakinra in GPP. Gevokizumab is a monoclonal antibody blocking the pro-inflammatory cytokine IL-1β and its signal transduction in inflammatory cells [131]. Mansouri et al. reported a 79 and 65% reduction in GPP area and severity index scores at weeks 4 and 12 after treatment with gevokizumab in two patients with severe, recalcitrant GPP [90]. Another IL-1β antagonist, canakinumab, induced the complete and long-term clearance of GPP lesions in a patient in whom anakinra had been withdrawn due to hypersensitivity reactions [89].

The novel monoclonal antibody spesolimab (formerly BI 655130), targeting IL-36R, can effectively block the IL-36 signaling pathway, to alleviate inflammatory response in GPP patients [132]. Recently, a phase I clinical trial evaluated the safety and efficacy of this molecule in seven biologic-naïve adult patients with moderate GPP flare. The results showed that all patients carrying a homozygous IL36RN mutation (n = 3) or heterozygous mutation in CARD14 (n = 1) or wild-type alleles (n = 4) significantly responded to a single intravenous dose at week 4 [91,93]. None of these patients, nor any of the 124 healthy volunteers who participated in this study, experienced severe adverse effects [93]. This finding suggested that IL-36R inhibition with a single dose of spesolimab can effectively alleviate the severity of GPP, regardless of the presence of a disease-causing gene mutation, and has great potential for the future clinical treatment of GPP

Results of a healthy volunteer phase I study of another anti-IL-36R drug, imsidolimab (formerly ANB019), also suggested a favorable side effect profile of inhibiting the function of the IL-36 pathway, which supported the advancement of imsidolimab into a phase II trial (GALLOP) [94]. Preliminary results were encouraging, as six out of eight patients treated with imsidolimab monotherapy achieved the primary endpoint of improvement in the clinical global impression scale after 28 days of treatment. Imsidolimab was generally well-tolerated, and most treatment-emergent adverse events were mild to moderate in severity and resolved without sequelae. No infusion or injection site reactions were observed. Detailed information on the identified gene mutations in those patients were not disclosed [133]. More detailed characteristics and data on the efficacy of the abovementioned therapies are summarized in Table 2.

Table 2. Targeted therapies in generalized pustular psoriasis. (CD25—cluster of differentiation 25, CGI-I—clinical global impression of improvement, Fab’—humanized antigen-binding fragment, GPP—generalized pustular psoriasis, IFN-γ—interferon-gamma, IgG—immunoglobulin G, IgG1—immunoglobulin G1, IgG1κ—immunoglobulin G1 kappa, IgG1λ—immunoglobulin G1 lambda, IgG2—immunoglobulin G2, IgG4—immunoglobulin G4, IL-1—interleukin 1, IL-1β—interleukin 1 beta, IL-1R—interleukin 1 receptor, IL-2—interleukin 2, IL-2Rα—interleukin 2 receptor alpha, IL-12—interleukin 12, IL-12/23 p40—p40 subunit of interleukin 12 and interleukin 23, IL-17—interleukin 17, IL-17A—interleukin 17A, IL-17RA—interleukin 17 receptor A, IL-23—interleukin 23, IL-23 p19—p19 subunit of interleukin 23, IL-36—interleukin 36, IL-36R—interleukin 36 receptor, IL36RN—IL-36 receptor antagonist gene, Th1—T-helper 1 cells, Th17—T-helper 17 cells, TNF-α—tumor necrosis factor alpha).

5. Conclusions

GPP is a serious and potentially life-threatening disease that is often difficult to treat. The past decade has witnessed enormous progress in the understanding of the molecular and immunologic basis of GPP. Arguably, one of the most important discoveries leading to a better understanding of the pathogenesis of this exceptional type of psoriasis was the report of the association between IL36RN and GPP, which was shortly followed by other significant genetic findings [70]. However, numerous studies found that a large number of patients with GPP did not carry any known variations in the above described genes, which implies that some novel variants located in introns or regulatory regions and other genetic factors may contribute to GPP’s pathogenesis [53]. Further screening and identification of other genes will therefore complement the current genetic map of GPP and is likely to greatly contribute to novel therapeutic approaches. The last few years have shed some new light on the immunological disturbances behind GPP. As shown by the recent studies, the TNF-α/IL-23/IL-17/IL-22 axis and IL-36 pathway intertwine in GPP pathogenesis [105]. This significant observation allowed the use of biologics, known for being effective in the treatment of plaque psoriasis, to be also used in GPP, regardless of IL36RN mutation status. However, the emerging need for more effective targeted therapies resulted in the development of a novel group of drugs that directly inhibits IL-36R [91].

Therapeutic intervention in GPP is a significant challenge. Given the rarity of GPP, the recruitment of a sufficient number of patients to conduct a large, randomized, controlled clinical trial, to adequately investigate the efficacy and safety of therapeutics, is the main difficulty. Moreover, the variable and unpredictable course of GPP makes it even more difficult to assess the efficacy of any intervention in this indication.

Author Contributions

Conceptualization, D.S., J.S., A.R.; Resources, D.S., J.S.; Writing—Original Draft Preparation, D.S., J.S.; Writing—Review & Editing, D.S., J.S., A.R.; Supervision, A.R. All authors contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

Funding

The publication fee was covered by the grant of the University of Rzeszow: “Analysis of clinical and molecular parameters and studies on new drugs in skin diseases” (Scientific Research of Institute of Medical Sciences University of Rzeszow, 500-3-60-601/2021).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data availability is not applicable to this article, as no new data were created or analyzed in this study.

Conflicts of Interest

The authors declare no conflict of interest.

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Demons concretely penetrated the body of the victim and occasionally possession cases had sexual connotations. In the didactic material, demonic presence was linked to women’s promiscuity, but ultimately the question was not one of women’s carnality or immorality, but of hierarchy—who was to define the proper religious practice. This chapter argues that disobedience towards clerical authorities was demonized; demonic sex and feminine lust served to propagate proper order, proper ritual practice, the position of the clergy, and the sanctity of a saint. For the victims, demons were a rhetorical resource revealing inner conflict, and turning one’s experiences into the language of the demonic may have been the only way to act out tribulations in a comprehensible manner. On a cultural level, cases with sexual undertones reveal the fears the ultimate uncontrollability of inner spirituality caused and show that chastity was a cultural sore point. source

The Demon of Lust

prith meaning

• 1) To extend.
• 2) To throw, cast.
• 3) To send, direct.

Sodom and Gomorrah are two ancient cities in the Bible’s Book of Genesis that were destroyed by God with fire due to their wickedness. The story of their destruction is told in Genesis 19 and parallels the story of the Genesis flood in its theme of God’s anger in response to human sin. The cities are traditionally associated with homosexual acts, rape, child sexual abuse, indecent assault, pride, arrogance, violence, and inhospitality. The Qurʾān also states that the people of Sodom and Gomorrah lusted after men instead of women, and engaged in highway robbery and other “evil deeds”. However, some modern scholars believe that it was the inhabitants’ inhospitality that offended God.

The story of Sodom and Gomorrah can be seen as a warning to consider one’s own actions, and to avoid economic egoism and inhospitality. It can also serve as a reminder to serve others in need, and to keep a balance between serving oneself and one’s community.

The story of Sodom and Gomorrah asks us to consider our own inhospitality

“This was the guilt of your sister Sodom: she and her daughters had pride, excess of food, and prosperous ease, but did not aid the poor and needy. They were haughty, and did abominable things before me; therefore I removed them when I saw it.” (Ezek. 16:49-50)

For much of my early life, I was under the impression that the destruction of Sodom and Gomorrah in Genesis 19 was an expression of God’s wrath against people like me: gay people. I was subtly encouraged to feel little for the wicked inhabitants of those cities, while inwardly I saw myself in them.

Consequently, I felt that I could never let anyone know what I was experiencing. I wondered if God was angry with me because of feelings I could not control or erase, no matter how much I tried. I wondered if God wanted to destroy me.

Vitally important lessons from the passage went over my head, because I had misunderstood it to be about gay people. But a deeper look at the story reveals a message that all of us desperately need to hear.

Years after coming out in college, I have discovered just how much the story has to teach us about God’s righteous anger towards arrogance and violence, and about about his abundant hospitality towards us despite our weakness and failure.

To see this more clearly, the passage must be examined in its larger context. The real beginning for this episode is in Chapter 18, when three angels in the form of three men appear to Abraham “as he sat at the entrance of his tent in the heat of the day” (Gen. 18:1).

Abraham’s response to the angels reveals him to be an exceptional host. He brings out water to wash their feet after their long journey, and rushes off to prepare a feast. He tells his wife, Sarah, to prepare cakes, slaughters a young calf and brings out milk and rich cheese.

Through the angels, God delivers a message to the elderly couple: Sarah will give birth to a son at the same time next year. The two struggle to believe—Sarah even fails to contain her laughter and is gently rebuked—but finally they accept the angelic message. Just before the guests leave, God reveals to Abraham his plans to destroy Sodom and Gomorrah, a city of extreme wickedness. An initial contrast is set up here between Abraham’s hospitality and the unnamed “sin of Sodom.”

Abraham is distraught over the fate of the cities, where he knows his nephew Lot is currently living; his care for the stranger extends to the inhabitants of the cities of the plain.

“Will you indeed sweep away the righteous with the wicked?” he asks God. “Suppose there are fifty righteous within the city; will you then sweep away the place and not forgive it for the fifty righteous who are in it? Far be it from you to do such a thing, to slay the righteous with the wicked, so that the righteous fare as the wicked!” (Gen. 18: 23-25)

God graciously agrees to relent for the sake of 50 righteous people. But Abraham does not stop there—he bargains with God until the number is reduced to a mere 10. However, the reader soon discovers that even the drastically reduced number is not met.

Two of the angels then travel directly to Sodom to rescue Lot, who greets them at the city gate. Attempting to be a good host himself, and aware of how dangerous the city could become at night, Lot urges them to stay at his own house, rather than in the city square.

He prepares a feast as well, but interestingly, the only food explicitly mentioned in the text is “unleavened bread,” perhaps indicating that the feast was not as generous as the one offered by his uncle Abraham.

Before any in the house lie down to sleep, a crowd arrives. “[T]he men of the city, young and old, all people to the last man” arrive at the front door and demand to rape the honored guests (Gen. 19:4-5). The fact that every single man from the city makes this demand suggests that this is not a group of gay men. What kind of city, in the ancient world or in any time and place, would have had an entirely gay population?

The emphasis is clearly on violent humiliation, not sexual intimacy or pleasure. The men of Sodom wish to brutalize the angels, not seduce them. What could be the motivation for wanting to humiliate the angels in this way?

Lot, for all his attempts at being a good host, utterly fails as a father when he offers the men his own young daughters instead. “Look, I have two daughters who have not known a man; let me bring them out to you, and do to them as you please; only do nothing to these men, for they have come under the shelter of my roof” (Gen. 19:8).

Lot not only struggles to be hospitable for guests, he fails to make his house safe for his own children. We get further insight into the character and motivation of these men in their response to Lot’s offer. They deride him for being a foreigner in their city and threaten to rape him. These are not gay men looking for a good time with out-of-towners. This is a hateful, violent mob.

The angels miraculously disperse the crowd with a blinding flash of light and command Lot to gather his family and flee first thing in the morning. Lot attempts to round them up that night, and only his wife and daughters wish to go with him. But even after the dawn breaks, Lot fails to act decisively. Graciously, the angels grab them all by the hand and drag them to safety outside the city gate. Lot seems maddeningly ambivalent about leaving, but God rescues him and his family from destruction.

Before leaving, the angels enjoin them to flee to the hills without looking back, and in an echo of Abraham’s bargaining with God to spare Sodom and Gomorrah, Lot bargains with the angels to allow him and his family to flee to the nearby town of Zoar instead.

The comparison is striking: Abraham bargains for mercy upon inhabitants of the cities and Lot bargains for mercy for himself. Abraham bargains for God to relent while Lot bargains for God to ease his escape. As they flee, Lot’s wife, perhaps identifying with the wicked cities, turns back one final time, and turns into a pillar of salt.

The episode concludes by returning to Abraham:

Abraham went early in the morning to the place where he had stood before the Lord; and he looked down toward Sodom and Gomorrah and toward all the land of the Plain and saw the smoke of the land going up like the smoke of a furnace. So it was that, when God destroyed the cities of the Plain, God remembered Abraham, and sent Lot out of the midst of the overthrow, when he overthrew the cities in which Lot had settled (Gen. 19:27-29).

With this full context in mind, the meaning of the destruction of Sodom and Gomorrah is, after all, summed up by the prophet Ezekiel: they “had pride, excess of food, and prosperous ease, but did not aid the poor and needy,” including the angels, who should have been honored guests. “They were haughty, and did abominable things before me,” arriving in a violent mob to gang rape visitors. “Therefore, I removed them when I saw it,” the inhospitality and disregard for foreign guests.

As followers of Christ, we know that by practicing hospitality, “many have entertained angels without knowing it,” as the author of Hebrews tells us (Heb. 13:2). John writes that “we love because he first loved us” (1 Jn. 4:19). Likewise, we invite others into our family because we have been invited into the family of God. Jesus makes it clear that what is done for “the least of these [his] siblings” is in a real sense done for him (Mt 25:40).

Our faith demands hospitality of us, especially toward the poor, the needy and the stranger.

Using the story of Sodom and Gomorrah to suggest God’s wrath against gay people makes it rather easy to avoid its actual demands to open up our wallets, homes and hearts to people in need. How many times has misunderstanding, empowered by hatred of the other, led some to use this passage to sanction everything that made Sodom guilty: kicking children of their homes, refusing to serve certain people, discriminating in housing and jobs, breaking families apart, fighting against civil rights?

If one were trying to relieve oneself of this passage’s radical demands of hospitality, twisting the story to focus on gay people would be a convenient strategy.

But as important as that message is, stopping there would keep us from the other, deeper, point of the story: God’s gracious hospitality toward us. God is obviously under no obligation to bargain with human beings, but he allows Abraham to haggle with him to save Sodom and Gomorrah. When even a handful are not found, God sets out to save Lot and his family from destruction. When Lot, who struggles as a host and fails as a father, hesitates instead of taking decisive action, God drags them all to safety, and even puts up with Lot’s complaining after the fact.

God’s call for us to show hospitality to the poor, the needy and the stranger is naturally connected to seeing ourselves as poor, needy strangers to whom God displays hospitality, despite our weakness and even our resistance. “We love because [God] first loved us” (1 Jn 4:19).

We show hospitality to others because God has shown immense hospitality to us. Our motivation for opening our wallets, homes and hearts to the poor, the needy and the stranger must not merely be a sense of guilt. We offer hospitality out of an overflow of the riches we have already been given by God.

I invite you to see yourself in the story of the destruction of Sodom and Gomorrah. I invite you to see yourself as an inhabitant of the cities, not because the story is about some divine wrath against gay people, but because a propensity toward inhospitality, pride, violence and selfishness is within all of us.

But I also invite you to see yourself in Lot, an inhabitant of the city who was saved. I invite you to consider that even when we fall short of the demands of hospitality, even when we fail in loving the poor and needy, God is gracious and hospitable, willing and able to drag us to safety and invite us into his family. source

God does love all of us, but we must submit and turn away from our sin, whatever sins they may be, we must accept obedience!

Scientists find link between mouthwash use and raised blood pressure

The researchers used 16S rRNA gene sequencing and analysis to examine whether using chlorhexidine antiseptic mouthwash twice a day for one week would change the oral bacterial communities and blood pressure levels in 26 healthy individuals. They collected samples of the participants’ saliva and tongue scrapings and measured their blood pressure at baseline as well as seven, ten and 14 days later.

The results indicated that using chlorhexidine twice a day was associated with a significant increase in systolic blood pressure and that recovery from use resulted in an enhancement in nitrate-reducing bacteria on the tongue. Individuals with relatively high levels of bacterial nitrite reductases had lower resting systolic blood pressure.

“The demonstration that the presence of NO-producing bacteria in the oral cavity can help maintain normal blood pressure gives us another target to help the more than 100 million Americans living with high blood pressure,” said lead researcher Dr. Nathan S. Bryan, an adjunct professor in the Department of Molecular and Human Genetics at the Baylor College of Medicine in Houston. “Two out of three patients prescribed high blood pressure medication do not have their blood pressure adequately managed,” he added. “None of the [current] drugs for management of hypertension are targeted towards these NO-producing bacteria.”

According to Bryan, owing to the widespread nature of the molecule, oral bacteria may have other profound effects on human health besides regulating blood pressure. “We know one cannot be well without an adequate amount of NO circulating throughout the body. Yet, the very first thing over 200 million Americans do each day is use an antiseptic mouthwash, which destroys the ‘good bacteria’ that help to create the NO. These once thought good habits may be doing more harm than good,” he said.

The study, titled “Frequency of tongue cleaning impacts the human tongue microbiome composition and enterosalivary circulation of nitrate,” was published online on March 1, 2019, in Frontiers in Cellular and Infection Microbiology. source

Can mouthwash raise your blood pressure?

New research, published in the journal Frontiers in Cellular and Infection Microbiology, shows that an antiseptic compound found in mouthwash destroys “friendly” oral bacteria that help maintain normal blood pressure levels.

Scientists know that the bacteria in our guts influence overall health, but perhaps less obvious is the connection between oral bacteria and a variety of health conditions.

For instance, Medical News Today recently reported on a range of studies that linked gum disease and the buildup of certain bacteria in the mouth with Alzheimer’s disease, cardiovascular disease, and respiratory conditions.

Another recent article showed how a specific oral bacterium could speed up the progression of colorectal cancer and make the disease more aggressive.

These studies focused on bacteria that cause disease, but, just like our guts, our mouths also contain “friendly” bacteria, which are necessary for maintaining good health.

An oral microbiome with a good balance between these different kinds of bacteria can keep disease at bay. StudiesTrusted Source have found that when this balance is upset it “contributes to oral and whole-body systematic diseases” as diverse as inflammatory bowel disease, Alzheimer’s, rheumatoid arthritis, obesity, atherosclerosis, and diabetes.

New research points out that a balanced oral microbiome helps maintain good cardiovascular health by helping the conversion of dietary nitrateTrusted Source into nitric oxide (NO) — a signaling molecule that helps maintain normal blood pressure.

Worryingly, however, the new study shows that chlorhexidine, an antiseptic substance in mouthwash, may kill NO-producing bacteria, which in turn, may raise systolic blood pressure.

Nathan Bryan, Ph.D., from the Department of Molecular and Human Genetics at Baylor College of Medicine in Houston, TX, led the new research.

Bryan and colleagues used “16S rRNA gene sequencing and analysis” to examine whether using chlorhexidine antiseptic mouthwash twice a day for 1 week changed the oral bacterial communities and blood pressure levels in 26 healthy individuals.

After 1 week, the 26 study volunteers went back to their usual oral hygiene practices.

The researchers collected samples of the participants’ saliva and tongue scrapings and measured their blood pressure at four different points throughout the study: at baseline, then 7, 10, and 14 days later.

Bryan and colleagues report that “twice-daily chlorhexidine usage was associated with a significant increase in systolic blood pressure after 1 week of use and recovery from use resulted in an enrichment in nitrate-reducing bacteria on the tongue.”

“The demonstration that the presence of NO-producing bacteria in the oral cavity can help maintain normal blood pressure gives us another target to help the more than 100 million Americans living with high blood pressure,” comments the study’s senior author.

“Two out of three patients prescribed high blood pressure medication do not have their blood pressure adequately managed,” he adds, and “this may provide an explanation as to why. None of the [current] drugs for management of hypertension are targeted towards these NO-producing bacteria.”

The researcher continues to explain the mechanisms underlying the findings, saying that NO “is one of the most important signaling molecules produced in the human body.”

Because of the “ubiquitous” nature of this molecule, “the systemic effects of orally produced bacteria may have other significant effects on human health beyond maintenance of blood pressure,” Bryan says. source

“We know one cannot be well without an adequate amount of NO circulating throughout the body. Yet, the very first thing over 200 million Americans do each day is use an antiseptic mouthwash, which destroys the ‘good bacteria’ that helps to create the NO. These once thought good habits may be doing more harm than good.”

Nathan Bryan, Ph.D.

Does mouthwash kill the mouth’s healthy bacteria?

WHETHER YOU’RE TRYING to get rid of garlic breath, prevent cavities or stave off gum disease, you may be concerned that swishing mouthwash will upset the balance of bacteria/”bugs” in your mouth and cause health issues.

The chance is very low, especially if you use it on a temporary basis. Mouthwash acts as a barrier, reducing the bacteria from attaching to the teeth. And, because it has such a short amount of contact with those bugs, there isn’t much concern that it could wreak havoc in your mouth. Read on for answers to more common questions about mouthwash and the mouth’s microbiome.

Is mouthwash safe to use?

Mouthwashes are cleared by the U.S. Food and Drug Administration, endorsed by the American Dental Association and, in general, safe to use. But keep in mind that they are used for prevention of oral health issues and will not treat those issues.

What is the microbiome in the mouth, and how does mouthwash affect it?

The microbiome is the balance of healthy and unhealthy bacteria in the mouth. Mouthwash can change the proportion, but there’s not much evidence to show this is cause for concern, especially when it’s used on a temporary basis.

What are benefits of mouthwash?

Mouthwash can help reduce the chance of getting gingivitis, which causes inflammation of the gums, the soft tissue around the teeth. Gingivitis can lead to worsening gum disease that ultimately involves losing jawbones around teeth (periodontitis).

Mouthwash also can be useful on a temporary basis to help prevent cavities for people wearing braces or who have recently had gum surgery and have difficulty brushing their teeth.

It’s important to emphasize that you need to use mouthwash before problems develop. Once you progress to gum disease or jawbone loss, or once bacteria become calcified (calculus), mouthwash won’t help.

What are some drawbacks of using mouthwash?

Sometimes mouthwash can stain teeth after a few weeks of use, because certain formulas attach to the tooth. In some people, the chemicals can affect taste.

Also, many mouthwashes contain alcohol and can be detrimental to people who are sensitive to alcohol. Mouthwashes with alcohol also should be avoided by children and pregnant people.

Is there a link between the oral microbiome and high blood pressure?

There is some correlation between oral hygiene and heart health. Patients with poor oral hygiene habits have a higher chance of developing cardiovascular complications when compared with patients with good oral hygiene habits. However, there could be other factors at play. For example, patients with poor oral hygiene habits may tend to have other habits that contribute to heart disease.

There also is some preliminary data that shows bacteria that’s usually found in the oral cavity also being found in the heart of a patient who suffered from heart complications. That’s indirect evidence that somehow oral bacteria may migrate, but it’s not strong evidence.

However, good oral hygiene is simple to achieve, costs very little, and there are many possible benefits. So, if there’s potential to improve your heart health by taking care of your teeth, you should brush and floss regularly and use mouthwash if you choose.

Is there a benefit to probiotic mouthwash?

The concept of probiotic mouthwashes is to supplement the user’s good bacteria. But currently, there’s no strong evidence to show that probiotic mouthwashes are better than standard mouthwashes.

How do I choose a mouthwash?

Choose a mouthwash tailored to what you’re trying to prevent. If you have gingivitis or some initial gum problems, choose one that targets gum inflammation. If you want to prevent decay from occurring, you could choose one that contains fluoride. Consult with your dentist about this.

If you’re pregnant, have children in the home or are susceptible to alcohol, choose one that’s alcohol-free.

Does mouthwash help with bad breath?

The causes of bad breath are gum disease, stomach issues, sleep problems, stress and specific types of bacteria in your mouth. Sometimes mouthwash helps. Sometimes it doesn’t. But it’s just a temporary fix. If you have chronic bad breath, you need to determine the root cause and address it.

How would I know if I have an imbalance of mouth bacteria?

It’s hard to know if the bacteria in your mouth are imbalanced unless you do a bacterial test, which looks at the profile in your saliva. It’s possible you may have symptoms. Some bacteria cause cavities and some cause gum disease. source

You might find it surprising, but there are important things you need to know about the potential harm caused by mouthwash. In this post, I’ll delve into the reasons why mouthwash can cause disease and suggest alternative ways to improve your oral health.

Check out my video on mouthwash to hear what I really think of it!

Did you know that mouthwash can…

• Disrupt your gut microbiome
• Reduce your body’s ability to make nitric oxide
• Increase your risk for cardiovascular disease and diabetes
• Increase systemic inflammation
• Raise your blood pressure

These are all scientifically-proven facts. Let’s talk about how mouthwash can promote disease and what to do instead.

The Cascading Effect of a Disrupted Oral Microbiome

You’ve probably heard about the importance of the gut microbiome, and if you’ve been around RIFM more than a minute, you definitely have! Research shows that our microbiome is a major key to our health. I’ve discussed on the blog how poor gut health is directly related to chronic disease, cardiovascular disease, and mental health problems.

But crucial bacteria are not limited to just our gut. They also exist in our mouth, on our skin, in our sinuses, and even on our genitalia. Each of these areas has its own unique microbiome or bacterial colonies, and when they are in harmony and balance, they play a beneficial role in maintaining our overall health.

Did you know your gut microbiome is actually seeded by bacteria in your mouth? Maintaining a balanced pH and fostering a healthy bacteria population in your mouth directly influences your gut health.

Now that we know that a disruption in the oral microbiome can directly disrupt the gut microbiome, it’s clear to see how that seemingly innocent bottle of mouthwash on your bathroom counter could be very harmful to your health.

How Mouthwash Affects Nitric Oxide

Ever heard of nitric oxide? It’s the compound responsible for the functioning of medications like Viagra and nitroglycerin. Nitric oxide plays a pivotal role in dilating blood vessels, lowering blood pressure, preventing heart attacks, and promoting healthy sexual function. To ensure its presence in the arteries, it’s essential to consume nitrates found in foods like beets and leafy greens.

The surprising element in the nitric oxide story is that we need specific mouth bacteria to help release these nitrates from foods so that our body can absorb them. What could kill these good bacteria in the mouth, preventing this critical release of nitrates? You guessed it – mouthwash. So now we can see how mouthwash can also prevent the absorption of these critical nutrients from our food.

Without the right bacteria in the mouth – because of mouthwash – nitric oxide fails to reach your system, impacting brain function, blood pressure regulation, and other vital processes.

Oral Health Out of Balance: It’s Not Just About Pretty Teeth

One noticeable sign of imbalance in the mouth is the presence of tartar. If you frequently experience tartar buildup or scaling, it indicates the formation of a biofilm that harbors harmful bacteria in your mouth. This condition is linked to various oral problems, such as periodontal disease, gum recession, and cavities, all of which can have a significant impact on your overall oral health. But the effects of an imbalanced oral microbiome extend beyond just the mouth, affecting the entire body.

During my medical career, I’ve observed interesting connections between oral health and other conditions. It’s remarkable how often I encounter diabetic patients or individuals with severe gut issues who also have poor dentition and oral hygiene. New research is showing associations between periodontitis (chronic gum infections) and conditions like Alzheimer’s disease, along with other disease states. We are also seeing a link between the bacteria in your mouth and vascular health.

While I don’t recommend using mouthwash, I always recommend that my patients take their oral health seriously because of its intricate relationship with our overall well-being.

Ways to Promote Oral Health Without Using Mouthwash

There are several simple steps you can take to promote a healthy mouth and oral microbiome. If you’re experiencing things like gum inflammation, tartar buildup, foul-smelling breath, or a white tongue, it’s important to take some steps to address your oral health.

1. One effective technique is called oil pulling, which involves swishing coconut oil or olive oil in your mouth for about five minutes, twice a day. This practice helps combat harmful bacteria and promotes a healthier balance of microbes in the mouth.
2. Another option is to incorporate oral probiotics into your routine. These specially formulated probiotics are chewed so that they are lodged on the gums, contributing to a healthier balance of bacteria. Some toothpaste brands even offer probiotic-infused options, providing an additional way to support oral health. See below for the oral probiotic that my family uses.
3. There are new mouthwashes containing essential oils can be beneficial in controlling bacteria in both your mouth and gut. These mouthwashes have shown positive results in putting periodontal disease into remission, reducing tartar buildup, and enhancing overall oral health.

Beyond Fresh Breath: The Far-Reaching Effects of Imbalanced Oral Microbiome

As you can see, maintaining a healthy oral microbiome is vital for your overall well-being. The bacteria in your mouth have a significant impact on your gut health and can even affect processes like nitric oxide production, blood pressure regulation, blood sugar balance, sexual health, and brain function. Since mouthwash disrupts our microbiome, it’s easy to see how it could be very harmful to your health.

There are simple steps you can take to improve your oral health without relying on mouthwash. Prioritizing your oral health is a great place to start to improve your overall health trajectory and ensure that you get the most benefit from the nutritious foods that you eat. source

The role of dietary nitrate and the oral microbiome on blood pressure and vascular tone

Abstract

Over-the-counter mouthwash use, nitric oxide and hypertension risk

References

 

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1. What is fatty liver disease?

Fatty liver is a common disease with an increasing incidence. Fatty liver disease, if not detected and treated early, can lead to hepatitis, cirrhosis, and even liver cancer. Therefore, it is best to prevent disease with foods that help balance fatty liver.

2. Fatty liver eat what?

3. Lifestyle changes in fatty liver disease

The BEST Drink for a Fatty Liver

Dandelion root tea is also believed by some to help detoxify the liver and relieve symptoms of liver disease. To make dandelion tea, you can steep one tablespoon of dandelion roots or flowers in five ounces of boiling water for 30 minutes, then strain or drink the mixture.

However, you should avoid dandelion tea if you are pregnant or breastfeeding, taking diuretics, or taking antibiotics like Cipro or Levaquin. Dandelion also has natural diuretic properties, so it may interfere with the action of lithium and similar medications. If you are being treated for liver or kidney issues, you should also avoid dandelion or dandelion tea unless your doctor says it’s okay.

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Easy Liver Cleanse Recipe: Detox Naturally with Lemon and Olive Oil

Are you looking for a quick and easy way to cleanse your liver and improve your overall health?

You only need to look at simple liver cleanse recipes that combine the flavorful combination of lemon and olive oil. This cleansing method supports liver function and promotes general well-being by utilizing the natural detoxifying qualities of these herbs in a gentle yet effective way.

Olive oil and lemon are the main ingredients in the main cuisine of European restaurants. Many people say that the combination of lemon juice and olive oil can treat many diseases like gallbladder stones.

Furthermore, research has looked at the possible health benefits of the components in lemon juice and olive oil independently.

This article will talk about the fact that studies support the purported health benefits of combining lemon juice with olive oil.

We’ll also review the olive oil lemon juice liver cleanse recipe.

Discover the Mutual Impact of Olive Oil and Lemon on liver 

There we will learn about the combination of lemon juice and olive oil. Lemon and olive oil make an extremely unique combination. It is open to all, committed, and productive for the skin, liver, and even blood flow.

Let’s first explore the two fruits independently: The ability of lemon to act as an acidifying agent is its power.

The human body can become acidified if its pH level is out of balance, which can have major negative effects on health. Hyperacidity is countered and the pH value is adjusted during alkalizing. Therefore, sick cells typically lose their strength as well as their breeding ground.

There are many liver cleansing recipes with lemon juice and olive oil. Because these are the most easily used liver detox. Lemon Liver detox works properly to clean liver stones and many other harmful small agents.

Olive oil and Lemon Juice Liver Cleanse Recipe

Ingredients:

• 1/2 cup of extra virgin olive oil
• 1/4 cup of freshly squeezed lemon juice
• 1-2 cloves of minced garlic

Instructions:

• Before beginning the cleanse, fast for a minimum of 12 hours to enable your liver to get ready for detoxification.
• In a bowl, thoroughly mix the olive oil, lemon juice, and minced garlic.
• During one to two hours, take one tiny serving (about one to two tablespoons) of the olive oil mixture every fifteen minutes.
• After you’ve finished the olive oil mixture, lie on your right side with your knees pulled up to your chest for half an hour.
• Spend the rest of the evening sleeping so that the cleanse has time to work.

How do you know that this combination is best for you?

Some research shows that eating lemon juice and olive oil together has health benefits. Many claim that they can aid in weight loss, treat and prevent gallstones, and perform cleanses and detoxes.

Let’s look at each of these claims properly.

Cleansing and Detoxification Ideas

A fast internet search will bring up a variety of recipes claiming to use lemon juice, olive oil, or both to detox and cleanse. It is said that waste and toxins that have accumulated in your body over time are taken out by cleanses and detoxes.

On the other hand, research on the potential cleansing and detoxifying effects of lemon juice and olive oil does not seem to be very extensive.

 

When compared to those who consumed other plant oils, the researchers discovered that those who consumed olive oil during the study period had greater levels of HDL (good) cholesterol and lower levels of LDL (bad) cholesterol in their blood.

In this article, we will give you the natural liver detox recipe for your best hygiene.

However, the polyphenols and antioxidants in lemon juice and olive oil could be referred to as “cleansing” because they “clean up” or neutralize dangerous free radicals. Which otherwise damages cells and may be a factor in sickness and disease.

Lemon and olive oil are recommended as the best quick liver detox. The human body uses a variety of biological processes to get rid of pollutants and keep itself operating at its best.

I suggest eating a diet that is rich in fruits, vegetables, whole grains, legumes, nuts, seeds, and lean protein sources to support your body’s optimal functioning.

Weight Loss 

Vitamin C is rich in lemon juice. 38.7 mg, or 43% of the Recommended Dietary Allowance (RDA) for men and 52% of the RDA for women, is present in a 3-ounce (100-gram) serving. Vitamin C plays a crucial role in the body’s synthesis of carnitine.

A substance called carnitine is responsible for moving fat molecules into cells so they may be broken down and used as an energy source. Thus, a low vitamin C consumption could result in less fat being broken down. For

weight loss, many quick liver detox drinks are also available in the market.

 

Health Benefits of using Liver oil and Lemon Cleanse 

What health advantages are associated with gallbladder cleansing?

To prevent gallstones, certain supporters of alternative healthcare advise liver cleansing. They maintain that the liver releases the gallstones as a result of the liver cleaning.

The gallstones should then ideally pass via the stool. This would mean that the patient would have fewer gallstones left to produce uncomfortable symptoms and might be able to avoid surgery.

There are various kinds of liver cleanses. Alternative medicine practitioners provide many ” liver detox recipes” and traditional treatments online.

• Olive oil and lemon juice. Using this method, you will fast for 12 hours during the day and then drink 4 tablespoons of olive oil and 1 tablespoon of lemon juice, eight times every 15 minutes, starting at 7 p.m.
• Vegetables and apple juice. Using this method, all you consume till five o’clock in the evening is apple and vegetable juice. Every fifteen minutes after 5 p.m., ingest eight ounces of olive oil by consuming 18 milliliters (ml) of olive oil mixed with 9 ml of lemon juice.

In these points, we tell you about the easy liver detox remedy that you can use at home easily.

How might a liver cleanse cause side effects?

Depending on the “recipe” one follows, cleansing may have different adverse effects. For example, a lot of individuals cleanse their livers using olive oil. When consumed in excess, this may have a laxative effect.

The following symptoms are possible for some people to experience after a gallbladder cleanse:

• Diarrhea vomiting nausea
• Depending on the herbs or other things someone utilizes in their cleanse, there could be additional negative consequences.

 

Furthermore, a person may perform a gallbladder cleansing yet still not be able to get rid of their gallstones. To prevent their symptoms from getting worse or an infection in their gallbladder. They probably need to have surgery at that point.

How to liver cleanse at home?

To support your liver’s natural detoxification activities and enhance general well-being. Liver cleansing at home might be a quick and easy solution. A possible strategy is to consume fewer processed meals, alcoholic beverages, and added sugars while increasing your intake of foods high in fiber, such as fruits, vegetables, and whole grains.

 

Furthermore, drinking lots of water throughout the day to stay hydrated promotes liver function and the removal of toxins. Herbal teas that you can drink regularly, like milk thistle or dandelion root, are also well-known for their liver-cleansing qualities. Different liver detox tea recipes are also present on the internet.

How much olive oil for detox?

Depending on things like tolerance levels, individual health status, and particular detox procedures, there are differences in the suggested quantity of olive oil for detoxification. In general, it’s important to balance safety and efficacy while using olive oil in a detox program.

As part of a detox treatment, it is generally recommended to ingest 1-2 tablespoons of extra virgin olive oil daily.

The body’s natural detoxification processes are supported by this moderate amount of beneficial substances, which do not overburden the system. These components include antioxidants and monounsaturated fats.

Conclusion

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Cleanse Your Liver

In addition to lifestyle changes, nutrition can have an impact on liver health. Check out these beverages that can aid in improving liver function.

Your liver plays many important roles in your physical health. It aids in digestion and metabolism and acts as a filter for the blood, breaking down harmful substances into waste that is expelled from the body through urine and stool.

According to the Mayo Clinic, symptoms of liver dysfunction include:

• Skin and eyes that appear yellowish (jaundice)
• Abdominal pain and swelling
• Swelling in the legs and ankles
• Itchy skin
• Dark urine color
• Pale stool color
• Chronic fatigue
• Nausea or vomiting
• Loss of appetite
• Tendency to bruise easily

 

Liver disease can be genetic. Other factors like viral infections, age, obesity, or excessive alcohol use may also cause liver damage or dysfunction. If left untreated, this damage can be fatal.

The good news is the liver is the only organ in the body with the ability to regenerate new cells and repair damaged ones. Repairing liver damage can be challenging; however, it is possible through consistent diet and lifestyle changes. These include:

• Maintaining a healthy weight
• Eating nutritious food and having a balanced diet
• Getting regular exercise
• Avoiding harmful additives and chemicals
• Avoiding excessive or continued alcohol or illegal drug use (damage from alcohol abuse can cause irreversible cirrhosis of the liver)

Also, many beverages, such as water, tea, and grapefruit juice, can be beneficial for your overall health and may aid detoxification of the body and liver.

Water

Staying properly hydrated is an important factor in maintaining a healthy liver. Dehydration can greatly affect liver function, especially the ability to detoxify blood. On average, you should drink eight to ten glasses of water a day; those with health conditions may need to increase their water intake beyond the recommended amount.

Teas

There are a few natural teas that may assist in liver function. Several popular and possibly beneficial teas for liver health include:

• Lemon Ginger Tea – reduces the risk of liver disease
• Peppermint Tea – improves digestion and detoxifying functions of the liver
• Green Tea – reduces the accumulation of lipids in the liver and contains antioxidants

Research the health effects and benefits of specific teas or discuss recommendations with your health care provider to ensure safe recommended use.

Grapefruit Juice

Grapefruit juice contains specific antioxidants that stimulate the liver and help filter and excrete chemicals from the body. Grapefruit also contains flavonoids naringin and naringenin, which have anti-inflammatory and antioxidant properties that may help protect the liver. However, it is recommended to not consume more than six ounces of grapefruit juice per day. Grapefruit juice also interacts with many prescription medications, so please check with your doctor or pharmacist and read your medication warning labels before adding it to your diet.

Turmeric Water

Turmeric is a commonly used supplement that may decrease inflammation and assist with liver repair, due to its ability to help flush out harmful toxins while decreasing fat buildup in the liver. For safe use, medical studies recommend mixing one to three grams of dried turmeric root in hot water each day for up to three months.

Lemon Water

Many citrus fruits, including lemon, can be added to water to help stimulate and flush out the liver. Lemons are high in nutrients like vitamin C and antioxidants. To help prevent liver disease, enjoy four to six tablespoons of lemon juice mixed with water each day.

Ginger Water

Ginger helps protect your liver and reduces inflammation in the body. It may also boost immunity and improve digestive health. The recommended consumption is less than four grams of ginger per day, mixed with warm or cold water.

As with any change in nutritional intake or supplement use, please use caution. Some people may experience unwanted results or side effects, and some herbs and foods may interact with certain medications. Also, the information presented above is based on adult studies and should not be used for children. Research any supplements and consult a health care professional before making abrupt changes to your diet or lifestyle. source

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DRINK 1 CUP PER DAY to Remove Fat from Your Liver – Dr. Berg

Many people have a fatty liver and don’t even know it.

Today I want to help you better understand the liver and how to remove fat from the liver. The purpose of the liver is to break down toxins into harmless particles. A fatty liver can become heavy and enlarged. This can lead to fullness under the right rib cage and shoulder pain.

Important functions of the liver:

• It makes bile
• It helps convert thyroid hormones
• It buffers excess sex hormones

Bile is made by the liver, and it helps you break down fat. A lack of bile can lead to a lot of various health problems. Bile is also very important to keep fat out of the liver. Anything you can do to help increase bile reserves will help you remove liver fat. You may even want to take purified bile salts. Keep in mind that the ketogenic diet and intermittent fasting can help remove fat from the liver. I believe this is essential if you have a fatty liver.

A great shake to keep fat off the liver: Ingredients (organic if possible):

• 2 cups kale (frozen)
• 1 cup blueberries (frozen)
• 1 cup water
• 1 cup plain whole-milk kefir (organic and grass-fed if possible)

Instructions: Blend all of the ingredients in a blender for a couple of minutes. Drink once a day in combination with a Healthy Keto diet and intermittent fasting.

 

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Protective Effects of Lemon Juice on Alcohol-Induced Liver Injury in Mice

Abstract

Chronic excessive alcohol consumption (more than 40-80 g/day for males and more than 20-40 g/day for females) could induce serious liver injury. In this study, effects of lemon juice on chronic alcohol-induced liver injury in mice were evaluated. The serum biochemical profiles and hepatic lipid peroxidation levels, triacylglycerol (TG) contents, antioxidant enzyme activities, and histopathological changes were examined for evaluating the hepatoprotective effects of lemon juice in mice. In addition, the in vitro antioxidant capacities of lemon juice were determined. The results showed that lemon juice significantly inhibited alcohol-induced increase of alanine transaminase (ALT), aspartate transaminase (AST), hepatic TG, and lipid peroxidation levels in a dose-dependent manner. Histopathological changes induced by alcohol were also remarkably improved by lemon juice treatment. These findings suggest that lemon juice has protective effects on alcohol-induced liver injury in mice. The protective effects might be related to the antioxidant capacity of lemon juice because lemon juice showed in vitro antioxidant capacity.

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1. Introduction

Alcohol abuse and alcoholism could lead to serious health and socioeconomic problems worldwide. Chronic excessive alcohol consumption (more than 40–80 g/day for males and more than 20–40 g/day for females) could lead to several illnesses, such as gastrointestinal damage, pancreatitis, alcoholic liver disease, neurologic disorders, diabetes mellitus, and cancer [1, 2]. Among these diseases, alcoholic liver disease has attracted more attention due to its high morbidity and mortality. Alcoholic liver disease is a major type of chronic liver disease throughout the world and can progress to liver cirrhosis and liver cancer.

Chronic alcohol consumption can generate abundant reactive oxygen species (ROS), including superoxide anion radical (O₂^−•), hydroxyl radical (OH^•), and hydrogen peroxide (H₂O₂). The ROS can react with most cellular macromolecules and subsequently cause cellular damage [3]. Therefore, the excessive ROS induced by alcohol is regarded as an important factor in the development of alcohol-induced liver injury. Various enzymatic and nonenzymatic antioxidants are related to protecting cells against ROS. Antioxidant enzymes include catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), and nonenzymatic antioxidants include glutathione (GSH), vitamin E, ascorbate, vitamin A, and ubiquinone [4]. Nonenzymatic antioxidants can be enhanced by antioxidant intake. In recent years, many natural products that have abundant antioxidants were reported to possess the effect of scavenging free radicals and protecting the liver from oxidative damage [4, 5].

Lemon is a popular fruit consumed as juice and contains high contents of vitamins and polyphenols (mainly flavonoids), such as hesperidin, eriocitrin, naringin, neohesperidin, rutin quercetin, chlorogenic acid, luteolin, and kaempferol [6]. The in vivo and in vitro experiments have shown that lemon has various health benefits, such as anticancer effect, antimicrobial effect, lipid-lowering effect, and protective effect against cardiovascular diseases [6]. In addition, lemon is used to treat liver ailments in tribal medicine [7]. However, effects of lemon juice on chronic alcohol-induced liver injury have not been reported in the literature. The objective of this study is to investigate the effects of lemon juice on chronic alcohol-induced liver injury in mice. In addition, the in vitro antioxidant capacities of lemon juice were evaluated. The results of this study could supply valuable information for the general public to reduce harm of alcohol consumption.

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2. Materials and Methods

2.1. Chemicals and Reagents

The compounds 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tri(2-pyridyl)- S-triazine (TPTZ), quercetin, gallic acid, and Folin–Ciocalteu’s phenol reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Assay kits for the determination of SOD, lipid peroxidation, CAT, and TG contents were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Other chemicals were of analytical grade.

2.2. Materials

Lemon was obtained from markets in Guangzhou, China. The fruit was cleaned with deionized water. The edible portion was weighed precisely and mixed with deionized water (1 : 1, m/v), and the mixture was ground into a homogenate with a homogenizer. Then, the homogenate was centrifuged at 5,000g for 10 min, and the supernatant was obtained. The supernatant was used for the measurement of antioxidant capacity, total phenolic contents (TPC), and total flavonoid contents (TFC) and for animal experiments. Moreover, in animal experiments, the original supernatant and the diluted supernatant (1 : 5 and 1 : 10, m/v) were used as the high, medium, and low dose, respectively. The lemon juice was freshly prepared before gavage every time.

2.3. Animal Study

Male C57BL/6 mice (20–25 g) were employed in this study. Thirty mice were randomly divided into 5 groups, each group containing 6 mice. They were maintained in a SPF laboratory animal room, which kept a 12 h light/dark cycle at 22 ± 0.5°C with 40%–60% relative humidity. The animal study was performed according to the “Principles of Laboratory Animal Care” and approved by the Institutional Animal Ethics Committee of Sun Yat-sen University. The model group was treated daily with ethanol and distilled water (10 mL/kg) at the same time; the lemon juice treatment groups were treated daily with different concentrations (high dose 1 : 1 (m/v), medium dose 1 : 5, and low dose 1 : 10) of lemon juice (10 mL/kg) and ethanol simultaneously; the control group was treated daily with isometric distilled water. The model group and the lemon juice treatment groups were given ethanol according to the following ways: 35% ethanol (v/v) at a dose of 3 g/kg body weight for 7 days, 40% ethanol (v/v) at a dose of 4 g/kg body weight for the next 7 days, and 52% ethanol (v/v) at a dose of 5 g/kg body weight on the 15th day [8]. All the intervention methods were intragastric administration. The blood and liver were collected from mice 9 h after the last ethanol administration. The blood sample was centrifuged at 4,000g for 10 min and the serum was collected. The obtained serums were stored at −22°C before determination. A piece of tissue was taken from liver and fixed in 4% paraformaldehyde, and then the remaining liver tissue was stored at −22°C until use.

2.4. Measurement of Biochemical Parameters in the Serum

The levels of ALT, AST, and TG in serum were determined by a Hitachi-7180 automated biochemistry analyzer (Hitachi, Japan) with the corresponding reagent kit.

2.5. Measurement of TG and Antioxidant Enzyme Activities in the Liver

The levels of TG, SOD, and CAT in liver tissue were measured using the commercial detection kits according to the manufacturer’s instructions.

2.6. Measurement of Lipid Peroxidation Levels in the Liver

The levels of lipid peroxidation in liver tissue were measured by thiobarbituric acid (TBA) method using the commercial detection kits according to the manufacturer’s instructions. The reference standard was malondialdehyde (MDA), and the results were expressed as nmol MDA equivalent/mg prot.

2.7. Liver Histopathological Assessment

The liver tissue fixed in 4% paraformaldehyde was embedded in paraffin, sectioned into 5 μm thickness, and stained with hematoxylin-eosin (H&E) for evaluation of histopathological changes. The histopathological changes of stained liver slices were observed under a bright-field microscope.

2.8. Ferric-Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was performed based on the method described in the literature [9]. In brief, the FRAP reagent was prepared from 10 mmol/L TPTZ solution, 20 mmol/L iron(III) chloride solution, and 300 mmol/L sodium acetate buffer solution (pH 3.6) in a volume ratio of 1 : 1 : 10, respectively. 100 μL of the diluted sample was added to 3 mL of the FRAP reagent and the mixture was measured after 4 min at 593 nm. The standard curve was established using FeSO₄ solution, and the results were expressed as μmol Fe(II)/g dry weight of lemon.

2.9. Trolox Equivalent Antioxidant Capacity (TEAC) Assay

The TEAC assay was carried out according to the procedure in the literature [10]. Briefly, the ABTS^•+ stock solution was prepared from 2.45 mmol/L potassium persulfate and 7 mmol/L ABTS solution in a volume ratio of 1 : 1 and then placed in the dark for 16 h at room temperature. The ABTS^•+ working solution was prepared by diluting the stock solution, and the absorbance of ABTS^•+ working solution was 0.710 ± 0.05 at 734 nm. 100 μL of the diluted sample was mixed with 3.8 mL ABTS^•+ working solution, and the absorbance of the mixture was measured at 734 nm after 6 min, and the percent of inhibition of absorbance at 734 nm was calculated. The reference standard was Trolox, and the results were expressed as μmol Trolox/g dry weight of lemon.

2.10. Determination of TPC

TPC were measured according to the literature [11]. Briefly, 0.50 mL of the diluted sample was added to 2.5 mL of 0.2 mmol/L Folin–Ciocalteu reagent. After 4 min, 2 mL of saturated sodium carbonate solution was added. After incubation for 2 h at room temperature, the absorbance of the mixture was measured at 760 nm. The reference standard was gallic acid, and the results were expressed as mg gallic acid equivalent (GAE)/g dry weight of lemon.

2.11. Determination of TFC

TFC were measured according to the literature [12]. In brief, 0.50 mL of the sample was mixed with 1.5 mL of 95% ethanol (v/v), 0.1 mL of 10% aluminum chloride (w/v), 0.1 mL of 1 mol/L potassium acetate, and 2.8 mL of water. After incubation for 30 min at room temperature, the absorbance of the mixture was determined at 415 nm. The reference standard was quercetin, and the results were expressed as mg of quercetin equivalent (QE)/g dry weight of lemon.

2.12. Statistical Analysis

Statistical analysis was carried out by one-way analysis of variance (ANOVA) with post hoc LSD test using SPSS 13.0 software. p < 0.05 was regarded as significant.

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3. Results

3.1. Effects of Lemon Juice on the Levels of ALT and AST in Serum

As shown in Figure 1, the administration of alcohol led to a significant (p < 0.05) elevation of alanine transaminase (ALT) and aspartate transaminase (AST) levels in serum of the model group compared with that of the control group. The administration of low and medium concentration of lemon juice slightly prevented the elevation of serum level of AST, while a high dose of lemon juice significantly (p < 0.05) decreased it. At the same time, the prevention of the elevation of serum levels of ALT was observed significantly (p < 0.05) in medium and high concentration of lemon juice group and displayed a dose-effect relationship.

Figure 1

Effects of lemon juice on the levels of AST (a) and ALT (b) in serum of mice. Control: normal group; Model: alcohol group; LL: alcohol and low dose of lemon juice group; LM: alcohol and medium dose of lemon juice group; LH: alcohol and high dose of lemon juice group. ∗ means the levels of parameters in the model group were significantly (p < 0.05) different from those of the control group. # means the levels of parameters in the treatment group were significantly (p < 0.05) different from those of the model group.

3.2. Effects of Lemon Juice on the Levels of TG in Serum and Liver

Triacylglycerol (TG) content in serum was significantly (p < 0.05) increased in the model group compared with that in the control group (Figure 2(a)). Administration of lemon juice reduced the accumulation of TG in a dose-dependent manner, especially in high concentration of lemon juice group (p < 0.05). In addition, hepatic TG content was significantly (p < 0.05) increased in model group compared with that in the control group (Figure 2(b)). Administration of medium and high concentration of lemon juice significantly (p < 0.05) reduced the accumulation of hepatic TG in a dose-dependent manner.

Figure 2

Effects of lemon juice on TG contents in serum (a) and liver (b). Control: normal group; Model: alcohol group; LL: alcohol and low dose of lemon juice group; LM: alcohol and medium dose of lemon juice group; LH: alcohol and high dose of lemon juice group. ∗ means the levels of parameters in the model group were significantly (p < 0.05) different from those of the control group. # means the levels of the parameters in the treatment group were significantly (p < 0.05) different from those of the model group.

3.3. Effects of Lemon Juice on Liver Lipid Peroxidation Levels

The lipid peroxidation levels in liver tissue are shown in Figure 3. Compared with that of the control group, there was a significant (p < 0.05) increase in the lipid peroxidation level of the model group. The administration of lemon juice significantly (p < 0.05) decreased the level of lipid peroxidation in a dose-dependent manner.

Figure 3

Effects of lemon juice on hepatic lipid peroxidation level in mice. Control: normal group; Model: alcohol group; LL: alcohol and low dose of lemon juice group; LM: alcohol and medium dose of lemon juice group; LH: alcohol and high dose of lemon juice group. ∗ means the levels of the parameters in the model group were significantly (p < 0.05) different from those of the control group. # means the levels of the parameters in the treatment group were significantly (p < 0.05) different from those of the model group.

3.4. Effects of Lemon Juice on Liver Antioxidant Enzyme Activities

Figure 4 represents the results of hepatic antioxidant enzyme activities in five groups. The SOD level in the liver increased significantly (p < 0.05) in the model group compared with that in the control group. The CAT level in the liver decreased only slightly (p > 0.05) in the model group compared with the control group in this study. However, treatment with lemon juice significantly (p < 0.05) decreased the levels of SOD and CAT compared with those of the model group. In addition, all the biochemical parameters are summarized in Table 1.

Figure 4

Effects of lemon juice on the activities of SOD (a) and CAT (b) in liver. Control: normal group; Model: alcohol group; LL: alcohol and low dose of lemon juice group; LM: alcohol and medium dose of lemon juice group; LH: alcohol and high dose of lemon juice group. ∗ means the levels of the parameters in the model group were significantly (p < 0.05) different from those of the control group. # means the levels of the parameters in the treatment group were significantly (p < 0.05) different from those of the model group.

Table 1

Effects of lemon juice on the levels of several biochemical parameters.

Parameters	Control	Model	LL	LM	LH
AST (U/L)	103 ± 10.45	136.53 ± 19.94∗	117.88 ± 15.37	113.5 ± 7.7	98.85 ± 10.94#
ALT (U/L)	40.5 ± 3.89	54.32 ± 4.76∗	54.05 ± 7.18	41.32 ± 6.25#	34.68 ± 2.71#
Serum TG (nmol/L)	0.4 ± 0.06	1.01 ± 0.12∗	1.09 ± 0.04	1.03 ± 0.05	0.82 ± 0.08#
Liver TG (mmol/g prot)	0.07 ± 0.01	0.1 ± 0.02∗	0.09 ± 0.01	0.07 ± 0.01#	0.06 ± 0.01#
Lipid peroxidation (nmol MDA equivalent/mg prot)	0.64 ± 0.14	1.26 ± 0.22∗	0.88 ± 0.12#	0.84 ± 0.15#	0.72 ± 0.13#
SOD (U/mg prot)	89.6 ± 3.42	97.51 ± 3.96∗	85.27 ± 5.57#	83 ± 9.28#	81.03 ± 6.65#
CAT (U/mg prot)	6.55 ± 0.41	6.29 ± 0.39	5.55 ± 0.64#	5.47 ± 0.28#	5.17 ± 0.51#

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Note. Control: normal group; Model: alcohol group; LL: alcohol and low dose of lemon juice group; LM: alcohol and medium dose of lemon juice group; LH: alcohol and high dose of lemon juice group. ∗ means the levels of the parameters in the model group were significantly (p < 0.05) different from that of the control group. # means the levels of the parameters in the treatment group were significantly (p < 0.05) different from that of the model group.

3.5. Histopathological Evaluation

Histopathology assessment of the liver was carried out for all groups (Figure 5). There was no pathological abnormality observed in the liver of the control group with preserved cytoplasm and distinct nucleus as shown in Figure 5(a). In Figure 5(b), it was observed in the model group that ethanol induced necrosis changes and substantial small fat droplets changes in liver section. However, livers of mice in all lemon juice treated groups showed noticeable recovery from ethanol induced liver damage with fewer small fat droplets changes and hepatocytes necrosis features.

Figure 5

The photomicrographs of liver sections taken from mice. (a) Normal group; (b) alcohol group; (c) alcohol and low dose of lemon juice group; (d) alcohol and medium dose of lemon juice group; (e) alcohol and high dose of lemon juice group. Arrow indicates a condition of small fat droplets changes, and the circle indicates hepatocytes necrosis, which mainly occurs in alcohol model group.

3.6. The In Vitro Antioxidant Activity, Total Phenolic Contents (TPC), and Total Flavonoid Contents (TFC) of Lemon Juice

The in vitro antioxidant activities of lemon were evaluated using ferric-reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays. The FRAP and TEAC values were 50.82 ± 2.70 μmol Fe(II)/g dry weight (DW) and 19.88 ± 0.66 μmol Trolox/g DW, respectively. The total phenolic contents (TPC) and total flavonoid contents (TFC) of lemon were 6.21 ± 0.28 mg GAE/g DW and 0.30 ± 0.03 mg QE/g DW, respectively.

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4. Discussion

Alcohol use disorder causes substantial diseases, and the liver is the most adversely affected organ. In the present study, the effects of lemon juice on chronic alcohol-induced liver injury in mice were investigated. Ethanol induced impairment of liver in mice was evidenced by increased AST and ALT levels. Treatment with lemon juice lowered the increased levels of AST and ALT in serum. The return of the activities of aminotransferases (AST or ALT) in serum to normal indicates the regeneration of hepatocytes and the healing of hepatic parenchyma; therefore, lemon juice had a protective effect on alcohol-induced liver injury. The results were in agreement with previous reports that showed lemon possessing a hepatoprotective effect on liver injury induced by carbon tetrachloride and acute exercise [7, 13]. In addition, the chronic alcohol-induced liver damage was further confirmed by liver histopathological changes in the present study, and treatment with lemon juice also remarkably improved the liver histopathological changes, which further confirmed the hepatoprotective activity of lemon juice on alcohol-induced liver injury in mice.

Various factors and mechanisms are associated with the pathological progress of alcohol-induced liver injury, and oxidative stress was one of them [3]. ROS is one kind of prooxidants including hydroxyl radical, superoxide radical, and hydrogen peroxide, which are frequently generated spontaneously during metabolism. Normally produced ROS is rapidly eliminated by the antioxidant defense system. The antioxidant defense system is able to scavenge ROS and terminate chain reaction of free radicals in vivo. Alcoholic exposure can result in excessive accumulation of ROS and contribute to cellular damage. Excessive accumulation of ROS could cause lipid peroxidation of hepatocytes, which was regarded as the primary mechanism concerned with chronic alcohol-induced liver damage [8]. MDA, the product of lipid peroxidation induced by ROS, also accumulates in the alcohol-damaged liver and represents a good estimation of the total oxidative stress [3]. In the present study, alcohol significantly augmented lipid peroxidation levels, which was similar to the previous study that showed increased lipid peroxidation in alcoholic patients [14]. Treatment with lemon juice reduced the level of lipid peroxidation to a normal level, which showed a significant protective effect of lemon juice against alcohol-induced oxidative stress.

Liver steatosis is the earliest disease of the liver on account of chronic ethanol consumption, with the characteristic of fat accumulation. It is generally accepted that, in the development of hepatic steatosis, ethanol exposure increases the ratio of reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide in hepatocytes, which disturb mitochondrial fatty acid β-oxidation and induce steatosis further [15]. In this study, alcohol-induced occurrence of hepatic steatosis was confirmed by increased hepatic TG contents and histopathological changes. Treatment with lemon juice significantly lowered the hepatic TG contents and improved the damaged histopathological changes. In particular, the mice given high dose of lemon juice had almost completely recovered to normal.

The antioxidant enzymes, such as SOD and CAT, represent the defense response system to excessive ROS. SOD catalyzes the dismutation of two superoxide anions to hydrogen peroxide and oxygen, and then CAT degrades two hydrogen peroxide molecules to water and oxygen [16]. SOD is also considered as front line among antioxidant enzymes in defense against free radicals. In the literature, the effects of alcohol treatment on the levels of SOD/CAT are controversial. SOD showed an increase, no changes, or a decrease, depending on the model, diet, duration, and amount of alcohol consumption [17–19]. In addition, it was reported that CAT activity decreased upon chronic ethanol consumption in a study [20]. However, another study showed that CAT activity was increased in rat liver [18]. In our study, the alcohol treatment significantly increased the activity of SOD and slightly decreased the activity of CAT, while treatment with lemon juice decreased the activities of SOD and CAT. The increased activity of SOD reflects the activation of the compensatory mechanism which might be an attempt to counteract free radicals in the liver [21]. The treatment with lemon juice prevented ROS accumulation, and the compensatory effects were not available in the liver. Therefore, lemon juice decreased the activities of SOD and CAT. The results were similar to the report of Gasparotto et al. [22]. In addition, the in vitro antioxidant experiment of lemon also showed that lemon had medium in vitro antioxidant capacities, which contribute to the explanation of the in vivo free radical scavenging effect of lemon.

Lemon contains numerous beneficial bioactive compositions, including phenolic compounds (mainly flavonoids), vitamins, carotenoids, essential oils, minerals, and dietary fiber [6]. The hepatoprotective effect of lemon may be attributable to the presence of vitamins, flavonoids, essential oils, and pectin. Vitamin C, a water-soluble antioxidant in lemon, is in a unique position to scavenge aqueous peroxyl radicals and react with free radicals, thus preventing oxidative damage including lipid peroxidation [14]. Sometimes, vitamin C could exert prooxidative effects at low concentrations and in the existence of transition metal ions [23], which might aggravate oxidative stress. However, it is difficult for vitamin C to have prooxidative effects in vivo due to the presence of NADPH-dependent recycling systems and glutathione [24]. In addition, there were some literatures reporting that vitamin C supplementation alone could reduce oxidative stress induced by ethanol, and the hepatoprotective effect of vitamin C treatment was more effective than silymarin, quercetin, and thiamine [25, 26]. Flavonoids, a class of secondary plant phenolics, can interact with hydroxyl radicals, chelate metal catalysts, and inhibit oxidases [27]. In previous studies, lemon flavonoid was shown to possess a hepatoprotective effect on liver damage induced by carbon tetrachloride and acute exercise, and the mechanism of the protective effect was related to the antioxidant capacity [7, 13]. Lemon essential oils and pectin were found to have protective effects on stomach and intestine barrier function [28, 29]. Ethanol exposure can injure the defensive intestinal barrier and increase the permeability of the small intestine, which lead to bacterial endotoxins leakage [25]. The bacterial endotoxins leakage is an important factor in the pathogenesis of alcohol-induced liver injury [30]. Therefore, the lemon essential oils and pectin might protect the intestine barrier function, thus indirectly protecting against alcohol-induced liver injury.

In this study, lemon juice revealed a protective effect on chronic alcohol-induced liver injury. Due to the fact that lemon contains a variety of bioactive ingredients, the hepatoprotective effect might be the result of joint action of multiple mechanisms, and it is difficult to clarify the specific mechanism of effect. The medium in vitro antioxidant capacities of lemon and reduced in vivo MDA levels indicated that lemon might reduce the oxidative stress induced by ethanol, thus exerting hepatoprotective effects. This study has found that lemon juice has a strong hepatoprotective effect, which provides valuable information for the general public to reduce harm of alcohol consumption. In the future, active components in lemon juice should be separated and identified, and the mechanism of action of the purified compound should be explored, including the action on the small intestine.

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5. Conclusions

Chronic alcohol consumption could induce liver injury. Lemon juice is readily available as a widely consumed beverage. In this study, we found that treatment with lemon juice exerted hepatoprotective effects on alcohol-induced liver injury in mice through decreasing the levels of serum ALT and AST as well as hepatic TG and lipid peroxidation. In addition, the in vitro antioxidant experiment of lemon showed that lemon had medium in vitro antioxidant capacities. Therefore, we speculate that the hepatoprotective effects might be related to the antioxidant capacities of lemon juice. The results showed that lemon juice might be a potential dietary supplement for the prevention and treatment of liver injury related to chronic alcohol consumption.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (no. 81372976), Key Project of Guangdong Provincial Science and Technology Program (no. 2014B020205002), and the Hundred-Talents Scheme of Sun Yat-sen University.

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Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

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Authors’ Contributions

Tong Zhou and Yu-Jie Zhang contributed equally to this work.

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References

1. 1. Zhou Y., Zheng J., Li S., Zhou T., Zhang P., Li H.-B. Alcoholic beverage consumption and chronic diseases. International Journal of Environmental Research and Public Health. 2016;13(6, article 522) doi: 10.3390/ijerph13060522. – DOI – PMC – PubMed
2. 1. Arteel G., Marsano L., Mendez C., Bentley F., McClain C. J. Advances in alcoholic liver disease. Best Practice and Research in Clinical Gastroenterology. 2003;17(4):625–647. – PubMed
3. 1. Cao Y.-W., Jiang Y., Zhang D.-Y., et al. Protective effects of Penthorum Chinense Pursh against chronic ethanol-induced liver injury in mice. Journal of Ethnopharmacology. 2015;161:92–98. doi: 10.1016/j.jep.2014.12.013. – DOI – PubMed
4. 1. Li S., Tan H.-Y., Wang N., et al. The role of oxidative stress and antioxidants in liver diseases. International Journal of Molecular Sciences. 2015;16(11):26087–26124. doi: 10.3390/ijms161125942. – DOI – PMC – PubMed
5. 1. Wang F., Li Y., Zhang Y. J., Zhou Y., Li S., Li H. B. Natural products for the prevention and treatment of hangover and alcohol use disorder. Molecules. 2016;21(1, article 64) doi: 10.3390/molecules21010064. – DOI – PMC – PubMed

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Taraxacum official (dandelion) leaf extract alleviates high-fat diet-induced nonalcoholic fatty liver

Abstract

The purpose of this study is to determine the protective effect of Taraxacum official (dandelion) leaf extract (DLE) on high-fat-diet (HFD)-induced hepatic steatosis, and elucidate the molecular mechanisms behind its effects. To determine the hepatoprotective effect of DLE, we fed C57BL/6 mice with normal chow diet (NCD), high-fat diet (HFD), HFD supplemented with 2g/kg DLE DLE (DL), and HFD supplemented with 5 g/kg DLE (DH). We found that the HFD supplemented by DLE dramatically reduced hepatic lipid accumulation compared to HFD alone. Body and liver weights of the DL and DH groups were significantly lesser than those of the HFD group, and DLE supplementation dramatically suppressed triglyceride (TG), total cholesterol (TC), insulin, fasting glucose level in serum, and Homeostatic Model Assessment Insulin Resistance (HOMA-IR) induced by HFD. In addition, DLE treatment significantly increased activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) in liver and muscle protein. DLE significantly suppressed lipid accumulation in the liver, reduced insulin resistance, and lipid in HFD-fed C57BL/6 mice via the AMPK pathway. These results indicate that the DLE may represent a promising approach for the prevention and treatment of obesity-related nonalcoholic fatty liver disease.

Keywords: AMPK; Fatty liver; High-fat diet; Insulin resistance; Taraxacum official (dandelion).

Copyright © 2013 Elsevier Ltd. All rights reserved.

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Protective Effects of Taraxacum officinale L. (Dandelion) Root Extract in Experimental Acute on Chronic Liver Failure

Abstract

Background: Taraxacum officinale (TO) or dandelion has been frequently used to prevent or treat different liver diseases because of its rich composition in phytochemicals with demonstrated effect against hepatic injuries. This study aimed to investigate the possible preventing effect of ethanolic TO root extract (TOERE) on a rat experimental acute on chronic liver failure (ACLF) model. Methods: Chronic liver failure (CLF) was induced by human serum albumin, and ACLF was induced in CLF by D-galactosamine and lipopolysaccharide (D-Gal-LPS). Five groups (n = 5) of male Wistar rats (200–250 g) were used: ACLF, ACLF-silymarin (200 mg/kg b.w./day), three ACLF-TO administered in three doses (200 mg, 100 mg, 50 mg/kg b.w./day). Results: The in vivo results showed that treatment with TOERE administered in three chosen doses before ACLF induction reduced serum liver injury markers (AST, ALT, ALP, GGT, total bilirubin), renal tests (creatinine, urea), and oxidative stress tests (TOS, OSI, MDA, NO, 3NT). Histopathologically, TOERE diminished the level of liver tissue injury and 3NT immunoexpression. Conclusions: This paper indicated oxidative stress reduction as possible mechanisms for the hepatoprotective effect of TOERE in ACLF and provided evidence for the preventive treatment.

Keywords: acute on chronic liver failure, hepatoprotective, oxidative stress, Taraxacum officinale, 3-nitrotyrosine

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1. Introduction

Liver diseases are one of the major health problems in the world and became a general health care problem due to the high morbidity rate. They are associated with several risk factors such as inadequate nutrition, metabolic diseases, viral infection, ethanol, and drug use. Liver injury may trigger the onset of liver failure, a common medical condition with very high mortality [1]. Liver failure can progress as acute liver failure (ALF), as acute on chronic liver failure (ACLF), or as acute decompensation of end-stage liver disease [2]. ALF is defined as a severe liver injury in the absence of pre-existing liver disease. According to the World Gastroenterology Organization ACLF is defined as “a syndrome in patients with chronic liver disease with or without previously diagnosed cirrhosis, characterized by acute hepatic decompensation resulting in liver failure (jaundice and prolongation of the international normalized ratio) and one or more extrahepatic organ failures, associated with increased mortality up to three months” [2,3]. The prevalence of ACLF ranges from 24% to 40%, and it usually occurs in young or middle-aged patients [4] and it is potentially reversible [2].

The exact mechanism of ACLF is not fully elucidated but based on what was found the pathophysiology was described using a four-stage model: precipitating event, hepatic injury due to precipitating event, response to injury, and failure of other organs [4,5]. The precipitating event can be triggered by one or more factors, identified or unidentified, like infections, alcohol, gastrointestinal bleeding, reactivation of viral hepatitis B (HBV), superinfection with hepatitis A or E virus, acute episodes of autoimmune hepatitis, Wilson’s disease, or vascular liver disease [2,6,7]. During the propagation phase, the number of proinflammatory mediators increases, a systemic inflammatory response syndrome and a vascular endothelial dysfunction will be activated, with progression to organs failure. At the same time, liver macrophages release anti-inflammatory cytokines that will initiate a compensatory anti-inflammatory response syndrome, leading to an acquired immunodeficiency, a “paralysis of the immune response” [4,8]. Measurement of oxidative stress, inflammation, necrosis, and apoptosis biomarkers can define the risk profile of ACLF [4,8]. The presence of multiple organ failure is a requirement for the diagnosis of ACLF, and the number of affected systems has a prognostic value. The kidneys are the most commonly affected organs [2,3].

Thus, the therapy for chronic hepatic diseases needs to develop new prophylactic agents to prevent ACLF. With the extended studies upon the use of medicinal plants, phytotherapy became an important support for the treatment of many diseases. The use of medicinal plants in the treatment of liver diseases has a long history worldwide because many phytochemicals have hepatoprotective activity [1,9,10]. Only a few of the ethnomedicinal effects have been scientifically validated [11]. Considering that in ACLF inflammation and oxidative stress are important pathogenetic mechanisms, the herbal medicines that have anti-inflammatory and antioxidant effects could be a promising source of bioactive compounds [12].

The Taraxacum officinale F. H. Wigg. (TO) (dandelion) species belong to the Asteraceae family, includes 30–57 varieties, and are widely distributed in the warm-temperate zones of the Northern Hemisphere [13,14,15]. It is a plant used in folk medicine from ancient times as anti-inflammatory, antioxidant, diuretic, choleretic, laxative, and hepatoprotective. Because the phytochemical components may define the medicinal value of a plant, their identification and effects mechanism in disease prevention and treatment is a necessity [11]. Furthermore, it has to be considered that the chemical composition of the TO extracts depends on both the extraction protocol and the solvents used (ethanol, acetone, water, or methanol) [16], but also on which part of the plant has been used (whole plant, roots, stem, leaves, flowers).

TO is also frequently used in different food products, and dietary supplements [17]. These plants were found to be rich in polyphenolic compounds, vitamins, inositol, lecithin, and minerals, and to exhibit antioxidant, anti-inflammatory, antiallergic, anti-hyperglycemic, hypolipidemic, and anticoagulant activities [9,18,19], to protect against hepatic injuries, but the mechanisms of action are still incompletely investigated [20].

It was demonstrated that TO root extract may protect against some toxic hepatic injury [11,13,21,22], but there are no studies on the potential hepatoprotective effect of this extract in ACLF. Therefore, our study aimed to extend the characterization of the ethanolic TO root extract (TOERE) and evaluate the potential use as a preventive hepatoprotective agent in a rat d-galactosamine and lipopolysaccharide (D-Gal-LPS)-induced rat ACLF model.

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2. Materials and Methods

2.1. Chemicals

Phenolic compounds Sigma (St. Louis, MO, USA), Roth (Karlsruhe, Germany), Dalton (Toronto, ON, Canada); phytosterols Sigma (St. Louis, MO, USA); Folin–Ciocâlteu reagent, sulfanylamide (SULF), N- (1-Naphthyl) ethylenediamine dihydrochloric acid (NEDD), vanadium chloride (III) (VCl3), methanol, diethyl ether, xylenol orange [o-cresosulfonphthalein-3,3-bis (sodium methyliminodiacetate)], orthodianisidinedihydrochloric acid (3-3′-dimethoxybenzidine), ferrous ammonium sulfate, hydrogen peroxide (H2O2), sulfuric acid, hydrochloric acid, glycerol, trichloroacetic acid (TCA), ethylenediaminetetra-acetic acid, sodium dodecal, sulfate butylated hydroxytoluene, thiobarbituric acid, 1,1,3,3-tetraethoxypropane, 2,4-dinitrophenylhydrazine (DNPH), 5,5’-dithionitrobis 2-nitrobenzoic acid (DTNB), 1,1-diphenyl-2-picrilhydrazyl (DPPH), o-phthalaldehyde Merck (Darmstadt, Germany); Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) Alfa-Aesar (Karlsruhe, Germany); Freund’s adjuvant, d-galactosamine (D-Gal) and lipopolysaccharide (LPS) from Merck and Sigma-Aldrich (Taufkirchen, Germany); Human serum albumin (HSA) (Octapharma GmbH, Austria). All chemicals were of analytical grade. Aspartate aminotransferase, alanine aminotransferase, total bilirubin, alkaline phosphatase, gamma glutamate transferase, creatinine, and urea kits were purchased from Spinreact (Sant Esteve de Bas, Spain). ELISA kit for 3-nitrotyrosine (KA0445-ABNOVA EMBLEM, Heidelberg, Germany) and primary antibody to 3-Nitrotyrosine for immunohistochemistry (Code ALX-804-505-C050, Enzo Life Sciences) were also used.

2.2. Plant Material

Fresh T. officinale F.H. Wigg. roots from the Alexandru Borza Botanical Garden “Babes-Bolyai” University of Cluj-Napoca, Romania, were purchased in June 2020, deposited in “Alexandru Borza” Botanical Garden Herbarium (Voucher CL:669002), and plant extract was prepared as previously described [23]. The roots were dried in a shaded place, grounded in a coffee grinder (Argis, RC-21, Electroarges SA, Curtea de Arges, Romania) for 5 min, and then the powder was screened through a 200 μm Retsch sieve [24]. Fifty grams were weighed and extracted with 70% ethanol, twice for 30 min using the UltraTurrax extraction apparatus (T 18; IKA Labortechnik, Staufen, Germany) at room temperature. The samples were then centrifuged at 4000 rpm for 30 min, and the supernatant was recovered, and filtered through a 0.45 μm micropore membrane (PTFE, Waters, Milford, MA, USA). The solvent was evaporated at 40 °C using a rotary evaporator (Hei-VAP, Heidolph Instruments GmbH & Co., Schwabach, Germany). Further, the obtained extracts were lyophilized (Advantage 2.0, SP Scientific, Warminster, PA, USA) [24]. The extract powder was stored at room temperature in airtight bottles. The extraction yield was 15.2% (w/w).

2.3. Phytochemical Analysis

 

Identification and Quantification of Polyphenolic Compounds by HPLC-DAD-ESI MS

The phenolic compounds of the T. officinale extracts were determined as previously described [23,24] with some modifications. Prior to LC analysis, the lyophilized extract was dissolved in MeOH. Chlorogenic acid was used for phenolic acid quantification, and results were expressed as mg chlorogenic acid equiv./g of dry plant material (mg CA/g d.w.) [25].

2.4. Animals and Experimental Design

The experiments were carried out on adult male Albino Wistar rats (strain Crl: WI), weighing 200–250 g, bred in the Animal Facility of Iuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca as previously described [23]. Animals were randomly divided into 6 groups (n = 5): Control group with no disease and no treatment, acute on chronic liver failure (ACLF) group, ACLF with Silymarin pretreatment (ACLF-SYL) group (200 mg/kg b.w./day) [26], ACLF groups with TOERE pretreatment in three doses, respectively ACLF-TO200 (200 mg dry plant material/kg b.w./day), ACLF–TO100 (100 mg dry plant material/kg b.w./day), and ACLF-TO50 (50mg dry plant material/kg b.w./day). The daily dose of TOERE has been dissolved in corn oil (1ml/day/animal). All the procedures performed on laboratory animals, comply with the Directive 2010/63/EU, and Romanian national law 43/2014 for animal protection used for scientific purposes. The project was approved by the Veterinary Sanitary Direction and Food Safety Cluj-Napoca as previously described (no. 19/ 13.12.2016) [23].

The ACLF rat model was induced by human serum albumin (HSA), d-galactosamine (D-Gal), and lipopolysaccharide (LPS) as previously described [27,28]. Silymarin (SYL) or TO have been administrated per os (p.o.) by gavage for 7 days. The ACLF group was pretreated for 7 days with physiological saline (1 mL/day/animal). After completing the treatments, on day 8 in the ACLF, ACLF-TO200, ACLF-TO100, ACLF–TO50, and ACLF-SYL groups ACLF was induced by the intraperitoneal injection (i.p.) of d-galactosamine (D-Gal) (400 mg/kg b.w.) and lipopolysaccharide (LPS) (100 μg/kg b.w.) [27,29]. Six hours after ACLF induction the rats were anesthetized with ketamine (60 mg/kg b.w.) and xylazine (15 mg/kg b.w.), blood was withdrawn by retro-orbital puncture, serum was separated by centrifugation, and stored at −80 °C until use. At the end of the experiment, under general anesthesia animals were killed by cervical dislocation and liver biopsy was harvested from each animal. The experiments were performed in triplicate.

2.5. Biochemical Serum Analysis

The hepatic injury was evaluated with conventional serum liver markers: serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (BT), alkaline phosphatase (ALP), and gamma glutamate transferase (GGT) as previously described [23]. Oxidative stress associated with liver injury was evaluated by measuring serum total oxidative status (TOS), total antioxidant reactivity (TAR), oxidative stress index (OSI), malondialdehyde (MDA), total thiols (SH), total nitrites, and nitrates (NOx) and 3-nitrotyrosine (3NT) levels as previously described [23,30,31]. Renal failure induced by ACLF was diagnosed with creatinine and urea.

2.6. Histological Assessment

For the histological analysis two liver fragments were collected from the left lateral and right medial lobes [32], fixed in 10% phosphate-buffered formalin for 24 h, and routinely processed and embedded in paraffin wax. From each tissue fragment, two serial sections of 3 µm were stained with hematoxylin and eosin (H&E). The hepatic parenchyma was histologically assessed for intralobular and periportal degeneration/necrosis, portal inflammation, and fibrosis, and the Histological Activity Index (HAI) was calculated [33,34].

2.7. Immunohistochemical Analysis of 3-Nitrotyrosine

For the immunohistochemical analysis of 3NT, the paraffin sections were dewaxed in xylene, followed by rehydration in decreasing the concentration of alcohol. Sodium citrate buffer (pH = 6) was used for epitope retrieval and endogenous peroxidase was blocked with peroxidase for 5 min. The primary mouse monoclonal [clone 39B6] antibody to 3NT was diluted in 1% PBS-BSA (bovine serum albumin) at 1:200, and maintained overnight at 4 °C in a humid chamber, followed by placing the secondary antibody. The reaction was visualized using 3,3’-diaminobenzidine. Finally, the sections were counterstained with Mayer’s hematoxylin. The positive reaction was given by the brown labeling of the hepatocytes. Immunopositivity for 3NT was evaluated and scored, as follows: grade 0, no staining; grade 1, positive staining in less than 10% of hepatocytes/10 high power fields; grade 2, positive staining in more than 10% but less than 50% of hepatocytes/10 high power fields; grade 3, positive staining of more than 50% of hepatocytes/10 high power fields [35].

The sections were independently examined by two pathologists (MT and CT) using a light Olympus BX-41 microscope, and a multi-head microscope Zeiss Axio Scope A1 (Carl Zeiss Microscopy GmbH, Germany). When there was a divergence of opinion, an agreed diagnosis was reached by a simultaneous evaluation in a multi-head microscope Zeiss Axio Scope A1 (Carl Zeiss Microscopy GmbH, Germany). The photomicrographs were taken using an Olympus SP 350 digital camera and Stream Basic imaging software (Olympus Corporation, Tokyo, Japan).

2.8. Statistical Analysis

All results were expressed as mean ± standard deviation (SD) whenever data were normally distributed. Comparisons between the different experimental groups were performed using the one-way ANOVA test and the post hoc Bonferroni–Holm test. The correlations analysis was performed with the Pearson test. Values of p < 0.05 were considered statistically significant. The analysis was performed using IBM SPSS Statistics, version 20 (SPSS Inc. Chicago, IL, USA).

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3. Results

3.1. Phytochemical Analysis

In our study, HPLC-DAD-ESI MS identified significant concentrations of hydroxybenzoic, caffeic, and chicoric acids (Figure 1, Table 1).

Figure 1

Chromatogram obtained by HPLC-DAD-ESI MS analysis of a Taraxacum officinale root extract at 340 nm. For peak assignments, see Table 1.

Table 1

Identification and quantification of Taraxacum officinale root extract polyphenols from hydroxybenzoic and hydroxycinnamic acids groups.

No	Retention Time Rt (min)	UV λmax (nm)	[M+H]+ (m/z)	Tentative Identification	Concentration * mg CA/ g TOERE
1	2.95	270	138	Hydroxybenzoic acid	3.65 ± 0.15
2	13.62	320	181, 163	Caffeic acid	1.09 ± 0.02
3	14.19	322	475, 312	Chicoric acid	1.95 ± 0.15
4	15.50	322	369	Feruloylquinic acid	0.6 ± 0.08
5	19.93	322	516, 181,163	Dicaffeoylquinic acid	0.53 ± 0.04
6	20.12	322	516, 181,163	Dicaffeoylquinic acid isomer	0.4 ± 0.03

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* mg CA/g TOERE-chlorogenic acid equiv. mg/g Taraxacum officinale ethanolic root extract. Values are the mean ± SD (n = 3).

3.2. Biochemical Serum Analysis

The hepatic injury was evaluated by measuring liver markers (AST, ALT, ALP, GGT, TB) [36] (Table 2). ACLF induction by D-Gal-LPS caused a severe increase of the liver markers than in Control animals (p < 0.001). Administration for a week of three different doses of TOERE or SYL in ACLF animals significantly prevented severe ACLF-induced increase of the AST, ALT, ALP, GGT, and TB (p < 0.001). Furthermore, the TOERE effect was dose-dependent, with the 100 mg TOERE/kg b.w./day concentration having the best inhibitory effect. In ACLF-TO200 and ACLF-TO100 groups TOERE hepatoprotective effects were better than in ACLF-SYL animals (p < 0.01) (Table 2).

Table 2

Liver and renal screening tests of the study groups.

Groups	AST (U/L)	ALT (U/L)	TB (mg/dL)	ALP (mg/dL)	GGT (mg/dL)	Urea (mg/dL)	CR (mg/dL)
ACLF-TO200	81.12 a ± 5.27	71.64 a,b,c ± 11.32	2.27 a,b,c ± 0.37	328.45 a,b ± 14.72	60.42 a,b,c ± 9.20	67.14 a,b,c ± 4.21	1.75 a,b ± 0.21
ACLF-TO100	82.14 a,b,c ± 4.20	54.08 b,c ± 12.37	1.30 b,c ± 0.27	310.38 a,b,c ± 11.19	49.97 b,c ± 8.37	78.93 a,b ± 5.18	1.78 a,b ± 0.14
ACLF-TO50	84.24 a,b,c ± 8.06	144.93 a,b,c ± 19.79	2.02 a,b ± 0.51	329.61 a,b ± 37.89	107.34 a,b,c ± 18.33	110.30 a,b,c ± 7.89	2.15 a,b ± 0.40
ACLF-SYL	126.37 a,b ± 6.58	111.67 a,b ± 13.04	2.44 a,b ± 0.13	332.59 a ± 29.20	74.51 a,b ± 9.86	81.25 a,b ± 12.15	2.02 a,b ± 0.29
ACLF	222.65 a,c ± 11.08	174.08 a,c ± 15.16	3.74 a,c ± 0.53	358.94 a,c ± 13.55	117.71 a,c ± 15.47	255.49 a,c ± 19.48	3.53 a,c ± 0.28
Control	35.04 ± 6.63	47.55 ± 10.08	1.01 ± 0.11	263.75 ± 15.20	44.31 ± 4.58	39.16 ± 2.71	0.57 ± 0.04

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Results are expressed as mean ± SD. Values are expressed as mean ± SD (n = 5). ^a p ˂ 0.05, versus Control; ^b p ˂ 0.05, versus ACLF; ^c p ˂ 0.05, versus SYL. AST—aspartate aminotransferase; ALT—alanine aminotransferase; TB—total bilirubin; ALP—alkaline phosphatase; GGT—gamma-glutamyltransferase; CR—creatinine; ACLF-TO200- acute on chronic liver failure pretreated with 200 mg TOERE/kg b.w./day; ACLF-TO100—acute on chronic liver failure pretreated with 100mg TOERE/kg b.w./day; ACLF-TO50—acute on chronic liver failure pretreated with 50 mg TOERE/kg b.w./day; ACLF-SYL—acute on chronic liver failure pretreated with 200 mg silymarin/kg b.w./d; ACLF—acute on chronic liver failure; Control—negative control.

Considering that in ACLF kidneys are the most affected organs, in a study of a plant with possible hepatoprotective use in ACLF it is also important to determine the nephroprotective activity. ACLF induction by D-Gal- LPS caused a severe increase of creatinine and urea (p < 0.001). Renal dysfunction tests were positively correlated with the liver markers (r = 0.6–0.9) in ACLF animals. The treatments of ACLF rats with TOERE or SYL caused a smaller increase of serum creatinine and urea after ACLF induction (p < 0.001). SYL effect was comparable to that from ACLF-TO200 and ACLF-TO100 groups, but better than that from ACLF-TO50 animals (Table 2).

In our study systemic oxidative stress was also evaluated. Compared to the control, serum TOS, OSI, and MDA were elevated in ACLF animals (p < 0.001. The treatment with TOERE or SYL reduced TOS, OSI and MDA increase after ACLF induction (p < 0.01). TOERE effect on the oxidative stress was dose-dependent, the higher concentration having the best antioxidant effect (Table 3).

Table 3

Oxidative stress tests of the study groups.

Groups	TOS (µM H2O2/L)	TAR (mM TROLOX/L)	OSI	MDA (nM/L)	NOx (µM/L)	3NT (nmol/L)	SH (mM GSH/L)
ACLF-TO200	30.61 a,b,c ± 6.85	1.088 ± 0.001	31.57 a,b,c ± 6.13	3.05 a,b,c ± 0.28	21.92 b,c ± 3.74	769.36 a,b,c ± 78.46	0.48 a,b ± 0.03
ACLF-TO100	35.50 a,b,c ± 7.27	1.089 ± 0.001	31.17 a,b,c ± 4.84	3.67 b ± 0.59	25.76 a,b,c ± 4.50	768.66 a,b,c ± 69.75	0.48 a,b ± 0.08
ACLF-TO50	40.45 a,b,c ± 8.46	1.089 ± 0.001	31.82 a,b,c ± 9.39	3.95 a,b ± 0.47	30.52 a,b,c ± 7.60	820.20 a,b,c ± 48.43	0.48 a,b ± 0.02
ACLF-SYL	36.41 a,b ± 7.75	1.092 ± 0.003	37.03 a,b ± 8.27	3.83 a,b ± 0.34	36.56 a,b ± 6.76	971.07 a,b ± 68.34	0.52 a,b ± 0.02
ACLF	47.98 a,c ± 7.95	1.089 ± 0.001	40.40 a,c ± 8.60	5.37 a,c ± 0.08	51.49 a,c ± 7.32	1053.99 a,c ± 91.15	0.40 a,c ± 0.03
Control	21.18 ± 1.72	1.089 ± 0.001	21.59 ± 4.61	3.57 ± 0.36	19.98 ± 1.99	480.45 ± 56.62	0.59 ± 0.01

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Results are expressed as mean ± SD. ^a p ˂ 0.05, versus Control; ^b p ˂ 0.05, versus ACLF; ^c p ˂ 0.05, versus SYL. TOS—total oxidative status; TAR—total antioxidant reactivity; OSI—oxidative stress index; NOx—nitric oxide; 3NT—3-nitrotyrosine; MDA—malondialdehyde; SH—total thiols; ACLF-TO200—acute on chronic liver failure pretreated with 200 mg TOERE/kg b.w./day; ACLF-TO100—acute on chronic liver failure pretreated with 100 mg TOERE/kg b.w./day; ACLF-TO50—acute on chronic liver failure pretreated with 50 mg TOERE/kg b.w./day; ACLF-SYL—acute on chronic liver failure pretreated with 200 mg silymarin/kg b.w./d; ACLFacute on chronic liver failure; Control—negative control.

In ACLF rats NOx and 3NT were also increased (p < 0.001). TOERE pretreatments prevented NOx and 3NT elevation (p < 0.001) after ACLF induction in a dose-dependent way, with a higher concentration having a better inhibitory activity. SYL was also a good inhibitor of NO production and peroxidation in ACLF animals (p < 0.001), but the effect was smaller than that of TOERE (Table 3).

Additionally, serum antioxidative activity was evaluated by measuring TAR and SH. TAR was not influenced by ACLF induction (p > 0.05). A depletion in the level of SH was observed in ACLF rats (p < 0.01), and the treatment with TO or SYL prevent SH reduction (p < 0.05) after ACLF induction. SYL has a better effect than TO on SH (Table 3).

3.3. Histological Assessment

In the livers of the Control group, no significant structural changes were observed (Figure 2a).

Figure 2

Photomicrographs of the liver tissues from the control and experimental animals. H&E stain: (a). Control; (b). ACLF; (c). ACLF-SYL; (d). ACLF-TO200; (e). ACLF-TO100; (f). ACLF-TO50; Bar = 50 µm (a–c,f) and 20 µm (d,e). ACLF—acute on chronic liver failure; ACLF-SYL—acute on chronic liver failure pretreated with 200 mg silymarin/kg b.w./d; ACLF-TO200—acute on chronic liver failure pretreated with 200 mg TOERE/kg b.w./day; ACLF-TO100—acute on chronic liver failure pretreated with 100 mg TOERE/kg b.w./day; ACLF-TO50—acute on chronic liver failure pretreated with 50 mg TOERE/kg b.w./day.

The highest histological scores were identified in the livers of the ACLF group (p < 0.001) (Table 4). The changes were represented by congestion, hemorrhages, multifocal to coalescing areas of coagulative necrosis, randomly distributed within the hepatic lobules or centered on periportal regions and associated with severe and mixed inflammatory infiltrates. The portal spaces were also affected and expanded by fibrosis, bile duct hyperplasia, and large numbers of inflammatory cells, predominated by small lymphocytes and macrophages (Figure 2b). The microscopical examination of the livers from the group ACLF-SYL group (Figure 2c) revealed the lowest histological scores if compared to ACLF, ACLF-TO200, ACLF-TO100, and ACLF-TO50 (p < 0,001). Compared to the untreated ACLF group, in ACLF-TO200 (Figure 2d), ACLF-TO100 (Figure 2e), and ACLF-TO50 (Figure 2f) animals, the hepatic injuries were significantly reduced by the TOERE pretreatments (p < 0.001), with no important differences between different TOERE doses (p > 0.05). Liver necroinflammatory scores and serum liver tests were positively correlated.

Table 4

Histological and IHC scores of the liver biopsies.

Groups	Portal Inflammation	Periportal Degeneration/ Necrosis	Intralobular Degeneration/ Necrosis	Fibrosis	HAI	3NT
ACLF-TO200	1.60 a,b,c ± 0.89	2.20 a,b,c ± 0.10	1.00 a,b,c ± 0.01	1.20 a,c ± 0.10	5.80 a,c ± 1.92	1.40 a,b,c ± 0.55
ACLF-TO100	2.20 a,b,c ± 0.10	2.60 a,b,c ± 0.89	1.40 a,b,c ± 0.89	1.00 a,c ± 0.10	7.20 a,b,c ± 1.10	1.40 a,b,c ± 0.55
ACLF-TO50	2.60 a,b,c ± 0.89	2.20 a,b,c ± 1.10	2.20 a,b,c ± 1.10	1.00 a,c ± 0.10	8.00 a,b,c ± 1.41	1.80 a,b,c ± 0.45
ACLF-SYL	1.00 a,b ± 0.00	0.60 a,b ± 0.55	0.80 a,b ± 0.45	0.40 a,b ± 0.55	2.80 a,b ± 0.45	1.20 a,b ± 0.45
ACLF	3.60 a,c ± 0.55	4.80 a,c ± 0.84	3.60 a,b ± 0.55	1.00 a,b ± 0.10	12.80 a,b ± 1.64	2.40 a,b ± 0.55
Control	0.40 ± 0.55	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.20 ± 0.45	0.00 ± 0.00

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Results are expressed as mean ± SD. ^a p ˂ 0.05, versus Control; ^b p ˂ 0.05, versus ACLF; ^c p ˂ 0.05, versus SYL; ACLF-TO200—acute on chronic liver failure pretreated with 200 mg TOERE/kg b.w./day; ACLF-TO100—acute on chronic liver failure pretreated with 100mg TOERE/kg b.w./day; ACLF-TO50—acute on chronic liver failure pretreated with 50 mg TOERE/kg b.w./day; ACLF-SYL—acute on chronic liver failure pretreated with 200 mg silymarin/kg b.w./d; ACLF—acute on chronic liver failure; Control—negative control; HAI—histological activity index; 3NT—3-nitrotyrosine.

3.4. 3-Nitrityrosine Evaluation

3-NT immunoexpression was negative in the livers of the control group (Figure 3a). A marked hepatocellular immunoexpression of 3-NT with a diffuse or mediolobular pattern was found in all liver samples from the ACLF group (Figure 3b) (p < 0.01). In the group ACLF-SYL, the 3-NT expression was reduced compared to the ACLF group (p < 0.01), being mainly limited to hepatocytes near the portal spaces (Figure 3c) (Table 4).

Figure 3

Immunohistochemical expression of 3-nitrotyrosine (3-NT) in liver tissues from the control and experimental animals: (a). Control; (b). ACLF; (c). ACLF-SYL; (d). ACLF-TO200; (e). ACLF-TO100; (f). ACLF-TO50; Bar = 50 μm (a) and 20 μm (b–f). ACLF-TO200—acute on chronic liver failure pretreated with 200 mg TOERE/kg b.w./day; ACLF-TO100—acute on chronic liver failure pretreated with 100 mg TOERE/kg b.w./day; ACLF-TO50—acute on chronic liver failure pretreated with 50 mg TOERE/kg b.w./day; ACLF-SYL—acute on chronic liver failure pretreated with 200 mg silymarin/kg b.w./d.

As compared to the ACLF group, the expression of 3NT was lower in liver biopsies from TOERE treated animals, particularly in the ACLF-TO200 (Figure 3d) and ACLF-TO100 (Figure 3e) (p < 0.01) groups. The expression of 3NT in the ACLF-TO50 group (Figure 3f) was higher compared to the other treated groups. SYL effect on 3NT expression was better than that of TOERE (p < 0.05) (Table 4).

The correlation between the histological scores and biochemical tests were also analyzed. In ACLF, ACLF-TO200, ACLF-TO100, ACLF-TO50, and ACLF-SYL groups all histopathological scores were positively correlated with liver, renal, and oxidative stress markers.

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4. Discussion

In the current study, D-Gal-LPS-induced ACLF in rats with HAS-induced chronic liver failure triggered an immune-mediated liver injury with pathological serum liver marker tests and histological liver changes. The liver injuries were also associated with renal failure and systemic oxidative stress. A seven days pretreatment with TOERE reduced ACLF induced liver injury. The protecting effect of TOERE can be attributed, at least in part, to the reduction of the oxidative stress associated with immune liver injury in D-Gal-LPS-induced ACLF. Depending on the dose, the hepatoprotective effect of TOERE was similar or lower than that of SYL, an already used hepatoprotective drug.

By analyzing the TOERE extract, other studies identified sesquiterpenes, various triterpenes, phenolic compounds, and phytosterols. Our previous phytochemical analysis [23] showed that the tested TO root extract had a lower TPC than in Aremu et al.’s analysis of TO root extract (1.14 ± 0.01 mg/100 GAE/mg extract) and TO leaf extract (4.35 ± 0.15 mg GAE/mg extract) [37], but higher than in the TO aerial part extract (15.50 mg GAE/g d.w.) [38].

The HPLC-DAD-ESI MS analysis of our TO root extract identified, caffeic acid, chicoric acid, as previously described [23], plus feruloylquinic acid, dicaffeoylquinic acid, and dicaffeoylquinic acid isomer. All these compounds have anti-inflammatory and antioxidant properties [11,23,39,40,41]. The antioxidant activity of the TOERE measured by DPPH and FRAP tests was proved in our previous study [23].

Because the chemical composition correlates with the pharmacological effects, TO extracts from different plant parts had different activities. Several studies demonstrated that the TO roots extract reduces alcohol-induced oxidative stress, TO leaf extract alleviates high-fat diet-induced nonalcoholic fatty liver, and TO flower extract can scavenge reactive oxygen species [22]. Similar to other studies [16,42,43], and based on the evidence of the phytochemical analysis results, our TO root extract can be considered a good natural antioxidant candidate. These results encouraged us to continue by testing the in vivo hepatoprotective and antioxidant effects of TOERE in an experimental ACLF.

For ACLF experimental model, first HAS administration in rats caused an immune liver injury and fibrosis, and then LPS stimulated liver macrophages leading to hepatic necro-inflammatory change. D-Gal, an amino sugar metabolized selectively by the hepatocytes, in a few hours potentiated the hepatotoxic effect of LPS by inhibiting mRNA and protein synthesis, leading to acute hepatitis [28]. The hepatoprotective effect of the TOERE was evaluated by using serum liver markers and liver histological analysis [2,3]. AST and ALT elevation reflects generalized damage to hepatocytes, TB increase reflects liver metabolism, ALP and GGT elevation reflects cholestasis [20,43,44]. In ACLF rats, liver markers were consistent with hepatocytes injury, cholestasis and, lower liver metabolism, demonstrating that an ACLF model was successfully induced [27]. The preliminary tests evaluating the TOERE effect on negative control animals indicated that the product had no significant activity on the healthy liver and oxidative stress (see supplemental data). In ACLF TO pretreatment reduced liver markers, suggesting that TOERE may prevent severe ACLF and by that to reduce the mortality due to ACLF. ACLF-TO100 group had the best hepatoprotective effect. In a previous study, we also evaluated in the TO root extract some phytosterols with anti-inflammatory and antioxidative properties [23]. It was demonstrated that due to their structural similarity with cholesterol, phytosterols are prone to be oxidized and transformed into oxyphytosterols [45,46,47], and from antioxidants to become pro-oxidants. By lowering the dose of TO from 200 to 100mg dry plant material/kg b.w./day, phytosterol reduction may be involved in the better hepatoprotective effect of ACLF-TO100 than of ACLF-TO200.

Liver injury diagnosed by serum liver tests was further confirmed by histopathological characteristics. In a normal liver, there is a hypoimmune response, and in chronic liver inflammation there is a high cellular recruitment, extended tissue damage, and the repair process leads to tissue remodeling, fibrosis, and liver dysfunction [41,48,49]. Fibrosis represents a key characteristic of progression towards liver cirrhosis and hepatic failure. In the present work liver biopsy of the ACLF animals showed extended necro-inflammatory changes, fibrosis, and bile ducts hyperplasia. In ACLF rats, like previously observed, histopathological scores increased due to the ongoing inflammation activation and the direct cytotoxic effect of cell death products [50]. Pretreatment with our TOERE in ACLF had hepatoprotective activity by reducing liver necro-inflammatory changes, with no important effect on liver fibrosis.

Under physiological conditions, free radicals are scavenged by antioxidant mechanisms. If there is an excess of free radicals or if there is a deficiency of antioxidants, oxidative stress will build up and will cause oxidative damage of lipids, proteins, and DNA [2,43,49]. In D-Gal-LPS-induced ACLF, immune-induced liver injury triggered an important liver inflammatory response and systemic oxidative stress, with high serum TOS and OSI, along with increased production of MDA, NOx, and 3NT.

Many studies correlated the hepatoprotective activity of the medicinal plant extracts with the antioxidant compounds from these plants [51]. Moreover, other experimental studies demonstrated that the polyphenolic compounds isolated from TO extracts had a hepatoprotective effect by reducing oxidative stress through direct free radical scavenging activity, metal ions chelation, and regeneration of the membrane-bound antioxidants [40,49]. In the present work, TOERE decreased serum TOS, OSI, and MDA levels in a dose-dependent way. MDA reduction was relevant because recently lipid peroxidation was considered a vital process in chronic liver diseases [44]. TOERE did not affect TAR, and SH was just slightly increased, indicating that this extract reduced systemic oxidative stress mainly by scavenging the oxidants and less by increasing the antioxidant capacity.

In mammals there are three NO synthase (NOS) isoenzymes that are involved in NO synthesis: neuronal (nNOS/NOS-1), inducible (iNOS/NOS-2), and endothelial (eNOS/NOS-3). Inflammatory stimuli up-regulate iNOS, and excessively generate NO induces nitrosative and oxidative damage. In liver injury, NO can be produced by hepatocytes, Kupffer cells, hepatic stellate cells (HSCs), and hepatic sinusoidal endothelial cells. It was observed that NO may have a dichotomous effect on liver disease, respectively in chronic liver diseases NO can promote HSC apoptosis, but in acute liver diseases, NO may increase liver damage. In LPS-treated rats, the marked hepatocellular immunoexpression of 3NT indicated that the iNOS-ROS cycle augments liver injury [52]. In this study, we found that liver 3NT was down-regulated following treatment with TOERE, suggesting that TOERE may reduce liver inflammatory responses and oxidative stress by reducing NO production through the inhibition of iNOS gene expression. These properties of TOERE may be explained by the high content of antioxidant phytochemicals.

Only iNOS and eNOS were highly expressed in acute liver failure (ALF) liver tissue, causing plasma NO elevation, and in humans increased plasma NO levels were correlated to the clinical severity of ALF [52]. In D-Gal-LPS-induced ACLF elevation of serum NOx and 3NT confirmed excessive NO synthesis due to the severe liver injury and inflammation. The treatment with TOERE reduced the serum NOx and 3NT, indirectly indicating that TOERE had a significant inhibitory effect on systemic NO production.

Because systemic oxidative stress markers reduction was correlated with serum liver markers and liver histopathological scores improvement, we concluded that TOERE lowered liver injury by reducing oxidative stress. Like in other studies [10], it was found that the antioxidant effect of TOERE was dose-dependent, the higher extract concentration had better antioxidant activity due to the high concentration of antioxidant ingredients.

According to the World Gastroenterology Organization ACLF is characterized by acute liver failure and one or more extrahepatic organ failure because in ACLF liver inflammation may trigger systemic inflammation. The kidneys are the most commonly affected organs and renal failure range from acute kidney injury (AKI) to acute-on-chronic kidney failure [2,3]. Therefore, acute kidney injury (AKI) was tested as a major criterion in ACLF severity grading [53]. In ACLF two subgroups of secondary renal dysfunctions with different pathophysiology and prognosis can be associated. One is the hepatorenal syndrome-acute kidney injury (HRS-AKI), a reduction of kidney function without parenchymal damage caused by prerenal insults such as hypovolemia. The other one is the non–HRS-AKI, induced by a renal insult such as inflammatory tubular injury in sepsis, bile acid nephropathy, and drug-induced tubular damage [54]. In ACLF liver protein synthesis lowers and may cause complications like coagulopathy, hemodynamic instability, jaundice, hepatic encephalopathy, hepatorenal syndrome, and sepsis [50]. At the same time, the systemic inflammatory response may also cause an inflammatory kidney injury with anon–HRS-AKI. Moreover, in experimental ACLF proinflammatory cytokines and LPS can cause directly renal tubular injury with cell apoptosis. Intrahepatic cholestasis from ACLF with increased serum bilirubin and bile acids may induce renal injury due to the direct renal toxicity and by tubular obstruction [54]. In our study, in ACLF animals creatinine and urea reached AKI levels, and there was a positive correlation between serum creatinine and urea, liver biopsy scores, and serum liver test. TOERE and SYL pretreatments reduced serum creatinine and urea in a dose-dependent way, indicating that in ACLF animals TOERE hepatoprotective activity is associated with a nephroprotective effect.

Lately, SYL has been used as a hepatoprotective agent due to its antioxidant and anti-inflammatory effects [55]. A finding of the study was that in experimental ACLF TO roots extract effects on serum liver markers were better than those of SYL, and SYL caused a higher reduction of the liver histological scores and 3NT immunoexpression. These differences suggested that TOERE better prevented acute liver injury and SYL reduces more the chronic response to liver injury. Even when SYL can reduce oxidative stress by scavenging ROS, by inhibiting ROS production [56], and by activating antioxidant enzymes [55], in our study it had a lower systemic antioxidant activity than TOERE.

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5. Conclusions

This report highlights the hepatoprotective and nephroprotective effects of an ethanolic TO root extract on D-Gal-LPS-induced ACLF. The mechanism proposed is the antioxidant activity of the bioactive components of the TOERE. These findings suggest for the first time that TOERE may be a potential preventive therapeutic agent for the severe liver and renal inflammatory injury associated with ACLF. These observations are important considering that ACLF has a high mortality rate. Further studies and clinical trials are required to fully elucidate the beneficial effects of TO root extract supplementation to prevent ACLF.

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Author Contributions

Conceptualization, I.O.P. and A.E.P.; methodology, T.A., R.M.P., L.V., and A.U.; software, R.M.P. and A.U.; validation, I.O.P., A.E.P., and R.O.; formal analysis, D.T. and C.T.; investigation, T.A., R.M.P., L.V., A.U, and C.T.; resources, I.O.P. and M.T.; writing—original draft preparation, I.O.P., A.E.P., R.M.P., and M.T. All authors have read and agreed to the published version of the manuscript.

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Funding

This research received no external funding.

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Institutional Review Board Statement

The study was approved by the Institutional Review Board (or Ethics Committee) of “Iuliu Hațieganu University of Medicine and Pharmacy”, Cluj-Napoca (no. 19/ 13.12.2016).

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Informed Consent Statement

Not applicable.

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Data Availability Statement

Not applicable.

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Conflicts of Interest

The authors declare no conflict of interest.

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Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

 

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