Protective role of melatonin in early‐stage and end‐stage liver cirrhosis

Abstract The liver is composed of hepatocytes, cholangiocytes, Kupffer cells, sinusoidal endothelial cells, hepatic stellate cells (HSCs) and dendritic cells; all these functional and interstitial cells contribute to the synthesis and secretion functions of liver tissue. However, various hepatotoxic factors including infection, chemicals, high‐fat diet consumption, surgical procedures and genetic mutations, as well as biliary tract diseases such as sclerosing cholangitis and bile duct ligation, ultimately progress into liver cirrhosis after activation of fibrogenesis. Melatonin (MT), a special hormone isolated from the pineal gland, participates in regulating multiple physiological functions including sleep promotion, circadian rhythms and neuroendocrine processes. Current evidence shows that MT protects against liver injury by inhibiting oxidation, inflammation, HSC proliferation and hepatocyte apoptosis, thereby inhibiting the progression of liver cirrhosis. In this review, we summarize the circadian rhythm of liver cirrhosis and its potential mechanisms as well as the therapeutic effects of MT on liver cirrhosis and earlier‐stage liver diseases including liver steatosis, nonalcoholic fatty liver disease and liver fibrosis. Given that MT is an antioxidative and anti‐inflammatory agent that is effective in eliminating liver injury, it is a potential agent with which to reverse liver cirrhosis in its early stage.

collagen deposition process gives rise to uncontrolled wound-healing pathophysiology and irreversible formation of intrahepatic scar tissue. 3,4 After liver fibrosis progresses into the end stage, multiple complications including acute or chronic liver failure, portal hypertension and hepatocarcinoma will follow.
In liver tissue, superoxide anions, hydrogen peroxide and hydroxyl radicals can be converted into stable reactive oxygen species (ROS) with strong toxicity, such as nitric oxide and peroxynitrites. 5 Thus, therapies targeting ROS inhibition are in great demand for inhibiting injury caused by oxidative stress and improving the prognosis of liver cirrhosis. Melatonin (MT), also known as N-acetyl-5-methoxytryptamine, is isolated from the pineal gland and participates in regulating multiple physiological functions including sleep promotion, circadian rhythms and neuroendocrine processes. [6][7][8] In the pineal gland, tryptophan is hydroxylated by tryptophan-5-hydroxylase to generate 5-hydroxytryptophan, which is then decarboxylated into 5-hydroxytryptamine (serotonin) by l-aromatic amino acid decarboxylase. After that, the serotonin is acetylated to generate N-acetylserotonin, which is finally converted to MT. 9 The hydroxylated MT generated by hepatic cytochrome P 450 mono-oxygenases is conjugated with sulphate to generate active 6-sulfatoxymelatonin. 10 MT not only exerts a strong antioxidant effect to protect cells and tissues from radical damage 11 but also inhibits proinflammatory cytokines including tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 b decreasing NF-κB during the development of hepatic fibrosis. 12,13 MT dramatically inhibits the activation of leucocyte, macrophage, mononuclear cells, mast cell and neutrophil infiltration in animal models of liver fibrosis. 14 Most importantly, MT contributes to a reduction in the amount of ECM deposition and significantly reduces histopathological changes in liver tissue. 15 This review summarizes the circadian rhythm of liver cirrhosis and its potential mechanisms, as well as the therapeutic effects of MT on liver cirrhosis and earlier-stage liver diseases including liver steatosis, nonalcoholic fatty liver disease (NAFLD) and liver fibrosis ( Figure 1). We describe how MT protects against liver injury against liver injury by inhibiting oxidation, inflammation, HSC proliferation and hepatocyte apoptosis, thereby inhibiting progression of liver cirrhosis. When the optimal dose, time-point, route and duration of administration are determined for MT to target oxidative liver damage in animal models and human beings, it may become a standard agent for liver disease treatment.
F I G U R E 1 Melatonin reduces oxidative stress and inflammation to eliminate collagen deposition and prevent fibrosis progression

| THE P OTENTIAL MECHANIS MS OF MT TARG E TING LIVER CIRRHOS IS
Multiple fibrogenic cytokines, chemokines, growth factors and lipid mediators as well as ROS production promote the transdifferentiation of quiescent HSCs into a myofibroblastic phenotype, and the interaction between apoptotic hepatocytes and Toll-like receptor 9 also induces HSCs to transdifferentiate into myofibroblasts. 16,17 On the other hand, the capillarization of sinusoidal endothelial cells upregulates transforming growth factor (TGF)-β levels, transdifferentiates HSCs into activated myofibroblasts, and triggers hepatocyte apoptosis. 18,19 TGF-β interacts with the type II and type I receptors to activate small mothers against decapentaplegic (SMAD)2/3 and initiate collagen deposition in liver tissue. 20 In addition, activated HSCs produce tissue inhibitors of metalloproteinases (TIMPs) and induce ECM deposition in liver tissue via up-regulation of IL-1β and TNF-α. 21 Then, the harsh microenvironment up-regulates the expression of inflammatory cytokines including TNF-α, the TGF family and platelet-derived growth factor (PDGF), activating KCs and aggravating the progression of liver fibrosis. 20 The PDGF-β signalling pathway subsequently activates other signalling pathways including the MAPK, p-AKT/PKB and PKC pathways to enhance the proliferation and fibrogenesis of HSCs. 22 In addition to HSCs and KCs, circulating fibrocytes, portal fibroblasts and bone marrow-derived cells also take part in ECM deposition during liver fibrosis. 23,24 Although excessive ROS has been demonstrated to up-regulate the cell death rate of HSCs, nontoxic ROS is able to promote the activation, proliferation and collagen production of HSCs. 25 After the balance of matrix metalloproteinases (MMPs) and TIMPs is damaged, the deposition of scar tissue will result in irreversible liver fibrosis even if the stimulating factors are withdrawn. 26 Therapeutic approaches targeting elimination of liver fibrosis should interrupt each step, such as inflammation, hepatocyte apoptosis, cholangiocyte proliferation, myofibroblast activation and ECM deposition. 27 As MT exhibits strong antioxidant activity and inhibits inflammation, HSC proliferation and hepatocyte apoptosis, interventions targeting the pathogenetic factors or pathways in this manner will help to block the initiation and progression of liver cirrhosis ( Figure 2).

| CIRC AD IAN RHY THM IN PATIENTS WITH LIVER CIRRHOS IS AND HEPATI C EN CEPHALOPATHY
Melatonin is generally regulated by light and darkness in both diurnal and nocturnal animals, as light eliminates MT synthesis, whereas darkness, peaking in the middle of the night, permits MT synthesis.
The central MT synthesis pattern has been shown to be disturbed in patients with hepatic cirrhosis and is correlated with the severity of liver injury. Patients with liver cirrhosis had markedly elevated MT levels during daytime hours, and both the onset time of the MT increase and the time of the peak MT concentration were consistently and significantly delayed in these patients. 28 The elevated daytime MT levels in patients with liver cirrhosis are probably related to a decreased metabolic clearance rate, decreased liver blood flow, lowered activity of 6-β-hydroxylase and competition with bilirubin in the intrahepatic transport system. 29 The disturbed circadian 6sulfatoxymelatonin rhythm impairs the quality of nighttime sleep F I G U R E 2 Several pathogenetic factors and pathways participate in the initiation and progression of liver cirrhosis and increases daytime sleepiness in patients with liver cirrhosis, and bright light therapy has been demonstrated to exert no beneficial effects on them, perhaps due to the severity of central circadian disruption at baseline. 30 However, there is a controversy about the relationship between circadian abnormalities and impaired sleep quality.
Montagnese et al found that although patients with liver cirrhosis demonstrated delayed peak serum MT, the urinary 6-sulfatoxymelatonin levels of patients and healthy volunteers did not differ. They concluded that there was no association between circadian abnormalities and impaired sleep quality. 31 Liver cirrhosis induces a type of brain dysfunction, namely, hepatic encephalopathy (HE), which includes multiple symptoms ranging from subclinical alterations to coma with or without neurological or psychiatric abnormalities. 32 Early in 1954, Sherlock found that there existed a so-called sleep-wake inversion, which combined restless nights and excessive daytime sleepiness in patients with HE induced by liver cirrhosis. 33  including daytime sleepiness and fatigue. 35 Chojnacki et al enrolled 75 patients with alcohol-induced liver cirrhosis and 25 healthy control individuals; they found that the levels of serotonin, urinary 5-hydroxyindoleacetic acid and 6-sulfatoxymelatonin were lowest while the level of MT was highest in patients with Child-Pugh C grade, and the disturbance of serotonin and MT homeostasis in patients with liver cirrhosis may be associated with advanced HE. 36 However, although ammonia level is highest in patients with grade 3 HE, there is no correlation between MT and ammonia levels in these patients. 37

| MT APPLI C ATI ON IN CLINI C AL TRIAL S
The fasting and postprandial plasma MT levels and portal hypertension rose significantly after treatment with MT or tryptophan, particularly in liver cirrhosis patients, which is attributable to portal systemic shunting and decreased liver degradation. 38 Nonalcoholic steatohepatitis (NASH) patients underwent treatment with Essentiale Forte and tryptophan or MT for 4 weeks demonstrated reduced expression levels of γ-glutamyl transpeptidase (GGTP), triglycerides and proinflammatory cytokines including IL-1, IL-6 and TNF-α, while these patients showed no significant alteration in alanine transaminase (ALT) level. 39 In addition, NAFLD patients underwent treatment with Essentiale Forte and tryptophan or MT for 14 months demonstrated reduced expression levels of GGTP, triglycerides, low-density lipoprotein cholesterol and proinflammatory cytokines including IL-1, IL-6 and TNF-α, while these patients demonstrated no significant difference in ALT level and other biochemical parameters. 40 A 12-week course of MT can not only reduce levels of liver enzymes during the treatment period but also maintain the alterations after discontinuation in patients with NASH. 41 Pakravan et al enrolled 100 patients with NAFLD for 3 months of MT treatment and found that the levels of diastolic blood pressure, aspartate aminotransferase (AST) and high-sensitivity C-reactive protein were significantly lower and the liver grades better in patients with NAFLD who received MT than in those received placebo. 42  and RAR-related orphan receptor-α (RORα) to maintain the circadian clock machinery in CCl 4 -treated mice. 44 MT treatment significantly abolished the activation of HSCs and increased the expression of nuclear factor erythroid-2-related factor 2 (Nrf2) while inhibiting the expression of profibrogenic genes such as MMP-9, collagens I and III, TGF-β, PDGF, connective tissue growth factor, amphiregulin and phospho-SMAD3 in mice with CCl 4 -induced liver fibrosis. 45 In addition, the levels of NF-κB in liver tissue and the levels of proinflammatory cytokines such as TNF-α and IL-1β released from KCs were down-regulated in rats with CCl 4 -induced fibrosis. 13 An important pathway, the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) axis, can also be inhibited by MT during the development of liver fibrogenesis. 46   deterioration of liver cirrhosis. 47    liver MDA levels and liver GSH/GSSG ratios in BDL rats, only a high dose of MT (1000 µg/kg/d) was able to improve the spatial performance of rats with BDL-induced liver fibrosis. 70

| NAFLD and NASH
Nonalcoholic fatty liver disease, a state ranging from simple steatosis to steatohepatitis, advanced fibrosis and cirrhosis, is attributed to specific dietary habits and lifestyles and results in liver dysfunction and end-stage liver cirrhosis. In particular, NASH, which is currently recognized as a serious condition, may also progress to liver cirrhosis because of pathophysiological mechanisms including oxidative stress, lipid peroxidation and excessive hepatocyte apoptosis. 12 Lipid infiltration results in mitochondrial fission and mitochondrial fragments in hepatocytes by disrupting the interaction of SIRT1 and An HFD significantly induced oxidative stress and up-regulated the levels of serum ALT, serum AST, total liver cholesterol and liver triglycerides in NAFLD rats. 73 Application of MT significantly reduces the pathogenetic changes in animal models with NAFLD and NASH according to current evidence (Table 3). Although application with MT did not alter the levels of lipids among HFD rats, MT is demonstrated to effectively reduce liver weight, the ratio of liver weight to bodyweight, portal vein pressure, the expression of HFD-induced plasma protein related to liver steatosis, the necrosis rate of liver cells and the extent of liver damage in animal models. 74  of hepatocytes in vivo. 79 Pioglitazone (an insulin sensitizer), pentoxifylline (a TNF-α inhibitor) and MT (an antioxidant) alone or in combination reduced severe hepatic steatosis, inflammation and fibrosis in rats with NAFLD. Pentoxifylline was demonstrated to decrease serum TNF-α levels, while pioglitazone and MT significantly reduced serum total cholesterol and triglycerides by down-regulating MDA levels and up-regulating GSH in animal models. 80

| Alcohol-induced liver fibrosis
Alcohol abuse leads to asymptomatic and reversible alcoholic liver disease, and sustained alcohol consumption also directly results in irreversible liver fibrosis. After ingestion, alcohol is metabolized into the intermediate product acetaldehyde, which participates in the development of hepatic fibrogenesis via activation of the TGF-β/SMAD signalling pathway, 84 which then activates HSCs by inhibiting autophagy and ER stress. In addition, cytochrome P450 2E1 completes a crucial step in alcohol-induced fibrogenesis by promoting overexpression of ROS in the liver, 85 ultimately leading to ECM deposition and alcoholic liver cirrhosis. 86 Ethanol can significantly up-regulate NF-κB translocation and MMP-9 expression but down-regulate TIMP-1 expression, while MT prevents all these changes in addition to alcohol-induced liver injury in mice. 87 Given that alcohol-induced liver cirrhosis is highly dependent on an ROSrelated mechanism, MT may serve as a potential agent to inhibit the alcohol-induced pathogenetic process, including elevation of aminotransferases, triglyceride and hepatic steatosis, by up-regulating phosphorylation of AMPK and SOD in alcohol-treated rats. 88   are conducted in animal models rather than human beings, leaving the therapeutic effects of MT on human liver fibrosis currently undefined. As we have discussed, MT is an effective antioxidative and anti-inflammatory agent for eliminating cirrhotic liver injury and has great potential for application in the pharmaceutical industry.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.