Protective Effects Of Melatonin On Hepatic Fibrosis In Rats And Its Inhibitory Effects On NF-kappaB Activation

BACKGROUND/AIMS Hepatic fibrosis, a common pathological process of chronic hepatic disease, can lead to irreversible cirrhosis, and involves multiple cellular and molecular events that ultimately result in accumulation of collagen and extra cellular matrix protein in space of Disse. Hepatic fibrosis generally considered reversible conditions. If treated properly at fibrosis stage, cirrhosis can be prevented. However, no effective antifibrosis drugs are available at present. Therefore , one of important trends focuses on investigation the mechanisms of hepatic fibrosis and exploration new effective drugs without ill effects. Several lines of evidence suggest that oxidative stress plays an important role in the etiopathogenesis of hepatic fibrosis.Melatonin (N-acetyl-5-metyoxytryptamine), a secretory product of the pineal gland, is a powerful endogenous antioxidant, regulates circadian rhythms, sleep and immune system activity, behaves as a free radical scavenger, eliminates oxygen free radicals and reactive intermediates. Both in vitro and in vivo experiments have shown that melatonin can protect cells, tissues, and organs against oxidative damage induced by a variety of free-radical-generating agents and processes, such as safrole, lipopolysaccharide (LPS), carbon tetrachloride (CCl4), ischemia-reperfusion, amyloid-protein, and ionizing radiation. In addition, melatonin also has an indirect antioxidant effect by enhancing the levels of potential antioxidants such as glutathione peroxidase (GPx), superoxide dismutase (SOD), and glutathione (GSH). Recent studies showed that melatonin reduces fibroblast proliferation and collagen synthesis.Nuclear factor-kappaB (NF-кB) is a family of dimeric transcription factors that regulate inflammation, innate and adaptive immunity, wound healing respones, and cell fate and function. Physical stress , oxidative stress ,and exposure to certain chemicals can lead to activation of NF-кB , which may function as a critical mediator of stress responses. Excessive activation of NF-кB relates to many diseases.Emerging attention has been focused on the regulation and function of NF-кB during liver injury. NF-кB activation have been implicated as mediators of liver injury and fibrogenesis. The present study was designed to investigate the protective effects of melatonin on carbon tetrachloride (CCl4)-induced hepatic fibrosis in experimental rats and its possible mechanisms. Meanwhile, we evaluated the role of NF-кB on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats and investigated whether treatment with melatonin was able to modulate NF-kappaB activation.METHODS All rats were randomly divided into normal control group, model control group treated with CCl4 for 12 wk, CCl4 + NAC group treated with CCl4 + NAC (100 mg/kg, i.p.) for 12 wk, CCl4 + MEL-1 group treated with CCl4 + melatonin (2.5 mg/kg) for 12 wk, CCl4 + MEL-2 group treated with CCl4 + melatonin (5.0 mg/kg) for 12 wk, and CCl4 + MEL-3 group treated with CCl4 + melatonin (10 mg/kg) for 12 wk. Rats in the treatment groups were injected subcutaneously with sterile CCl4 (3 mL/kg, body weight) in a ratio of 2:3 with arachis oil twice a week. Rats in normal control group received hypodermic injection of arachis oil at the same dose and frequency as those in treatment groups. At the end of experiment, rats in each group were anesthetized and sacrificed. Hematoxylin and eosin (HE) staining and Van Gieson staining were used to examine changes in liver pathology. Serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and protein concentration were measured with routine laboratory methods using an autoanalyzer. Hydroxyproline (Hyp) content in liver and malondialdehyde (MDA) and glutathione peroxidase (GPx) levels in liver homogenates were assayed by spectrophotometry. Serum hyaluronic acid (HA), laminin (LN), and procollagenⅢN-terminal peptide (PⅢNP) were determined by radioimmunoassay. Expression of NF-кB P50 ,NF-кB P65, TNF-αand ICAM-1 in liver tissue was detected by immunohistochemistry and expression of procollagen typeⅠmRNA in liver tissue was detected by semiquantified RT-PCR. NF-kappaB binding activity in liver tissue was determined by electrophoretic mobility shift assay (EMSA). HSCs are isolated from the liver of SD rat through in situ perfusion with pronase and collagenas and they were identified by immunohistochemistry stain with desmin antibody. With LPS and various concentrations of MEL, the inhibition of HSC was determined by MTT method.RESULTS1. Protective effects of MEL on chemical hepatic fibrosis induced by CCl4 in ratThe hepatic fibrosis models in rats were successfully induced by CCl4. Pathologic grading showed that the fibrogenesis was much less severe in CCl4 + MEL3 group than in model control group (u = 2.172, P < 0.05), indicating that melatonin (10 mg/kg) can significantly ameliorate CCl4-induced hepatic fibrotic changes. The serum levels of ALT and AST were markedly lower in CCl4 + MEL treatment groups (5, 10 mg/kg) than in model control group. Similarly, melatonin (10 mg/kg) not only decreased hydroxyproline (Hyp) contents in liver , but also decreased the elevated level of laminin (LN) and hyaluronic acid (HA) in serum, which indicate that melatonin decrease the ECM production of hepatic fibrosis in rats. Moreover, melatonin also ameliorated the oxidative stress state of hepatic fibrosis rats. Treatment with melatonin (5, 10 mg/kg) significantly reduced the MDA content and increased the GPx activity in liver homogenates compared with model control group.2. Effects of MEL on LPS-mediated HSC proliferationHSCs are successfully isolated from the liver of SD rat through in situ perfusion with pronase and collagenase, crude cell suspensions were purified to 70-80% homogeneity by density gradient centrifugation. The cell viability was more than 90 %. The harvest rate of HSCs was 2×107cells per liver. HSC was identified by immunohistochemistry stain with desmin antibody. The desmin postive cell rate was more than 90 % in subculture.Compared with the control group, LPS (10ng/ml) significantly promoted the proliferation of rat HSCs. Compared with the LPS group, MEL obviously inhibited the proliferation of rat HSCs at the concentration of 0.1mmol/ml.3. Effect of MEL on hepatic mRNA expressions of procollagen typeⅠCompared with normal control group, the expression of procollagen typeⅠm RNA in liver tissue in CCl4-treated groups was significantly increased. Compared with model control group , the expression of procollagen typeⅠmRNA in liver tissue in CCl4 + MEL group(10 mg/kg) and CCl4+NAC group was significantly decreased .4. The role of NF-кB on hepatic fibrosis induced by CCl4 in rats and the effect of MEL on the NF-кB signaling pathwayImmunohistochemical analysis of liver tissue sections revealed that there were few positive expressions of NF-кB P65, NF-кB P50, TNF-αand ICAM-1 in the normal rat liver. NF-кB P65 and NF-кB P50 expression, as brown or yellow granulae in cytoplasm and nuleus, was apparently increased in response to 12-week subcutaneous injection of CCl4. MEL(5, 10 mg/kg) and NAC significantly reduced NF-кB P65 and NF-кB P50 expression. Meanwhile, expression of TNF-αand ICAM-1 whose transcription are regulated by NF-кB was also apparently increased in response to 12-week subcutaneous injection of CCl4. MEL(10 mg/kg)and NAC significantly reduced TNF-αand ICAM-1 expression. Furthermore, the NF-kappaB-DNA binding activity was confirmed by EMSA. The activation of NF-кB in the normal control group was weak. It was elevated significantly in the model control group. Treatment with melatonin significantly suppressed the NF-кB binding activity in a dose-dependent manner to some degree.CONCLUSIONS1. In this study, hepatic fibrosis was successfully induced by subcutaneous injection of sterile CCl4 twice weekly for 12 wk. Through this hepatic fibrosis model, the effects of melatonin on hepatic fibrosis induced by CCl4 in rats were examined.2. Melatonin may have beneficial effects on hepatic fibrosis induced by CCl4 in rats. The protective effect of melatonin on hepatic fibrosis may be related to its antioxidant activities.3. NF-кB activation are implicated as mediator of fibrogenesis. NF-кB plays an important role on hepatic fibrosis induced by CCl4 in rats.4. Melatonin may exert an effect on the inhibition of oxidation stress via downregulation of NF-кB signaling pathway to prevent the progression of hepatic fibrosis.All above-mentioned research results may provide the experimental basis for melatonin being developed to new drug treating hepatic fibrosis.