| In recent years, with the continuous improvement of people’s living standard and the change of eating habits, there is an upward trend in the incidence of alcoholic liver disease (ALD). ALD is a chronic liver disease due to long-term heavy drinking, originally only performance for alcoholic fatty liver (AFL). It may develop advanced liver diseases such as alcoholic hepatitis, fibrosis, and even cirrhosis with the continuation of alcohol abuse. AFL is initial stage of ALD, also is the most common pathological changes, however, there is still lack of effective drugs for treatment. Therefore, to study the pathogenesis of AFL and efficient drugs for prevention and treatment of AFL has the vital significance.Melatonin is a endogenous active compound secreted by the pineal gland, has extensive physiological functions, including regulation of hormone secretion, control of biological rhythm, immune regulation, anti-aging, etc., and melatonin has the protective effects for cardiovascular, kidney and liver organs. In recent years, the studies found that melatonin played a role on the protection of nonalcoholic liver disease (NAFLD) by against oxidation damage and reducing oxidative stress and the release of inflammatory cytokines. But the mechanism of malatonin on hepatic steatosis induced by alcohol is still not fully clear.Aims:The purpose of this study was to evaluate the protective effects of melatonin on alcohol fatty liver (AFL) in rats and investigate the potential molecular mechanism, and establish vitro model to further explore the regulation of melatonin on lipid whether is related to its receptors.Methods:We conducted AFL model in rats fed with ethanol and high-fat diet and treated with melatonin. After 6 weeks blood samples were collected into tubes and serums were separated. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) were measured according to commercial analysis kits. The activity of hepatic SOD and the levels of hepatic MDA and TG were measured by liver homogenate. Paraffin sections of liver were prepared for histological examination. Western blot detected the expression of AMPK-a and p-AMPK-a.We established vitro model of hepatic lipid accumulation in HepG2 cells in the presence of oleic acid and alcohol. The vitro model was evaluated by oil red O (ORO) staining. The intracellular levels of TG and cAMP were measured according to commercial analysis kits. Western blot detected the expression of AMPK-a, p-AMPK-a and melatonin receptor 1 (MT1R).Results:1. The Protective effects of melatonin on AFL in SD ratsThis study built AFL model in rats fed with ethanol and high-fat diet and treated with melatoninatonin (10,20,40 mg/kg) by gavage, positive medicine group was given reduced glutathione (GSH,180 mg/kg), intraperitoneal injection, for 6 weeks. Pathology examination showed that melatonin and GSH can significantly improve the degree of liver steatosis. The body weight in model group was significantly lower than control group, melatonin has no obvious effects on body weight, but low, medium and high dose melatonin group can reduce liver index in different degree. Biological indicator test showed that melatonin can significantly reduce the levels of serum ALT and AST. The above results suggested melatonin has obvious protective effects on AFL rats.2. The effects of melatonin on the lipid metabolism in AFL ratsCompare with control group, ethanol and high-fat diet resulted in significant increase in the serum levels of TG, LDL-C and liver levels of TG, and significant decrease in the serum levels of HLD-C. Medium dose or high dose melatonin can significantly decrease the serum levels of TG, LDL-C and hepatic TG level, increase serum HLD-C level. Thus melatonin may is through regulating lipid metabolism and reducing lipid accumulation, plays a role on protection of AFL rats.3. The effects of melatonin on the oxidative metabolism in AFL ratsModel group showed a significant decrease in the activities of SOD and a remarkable increase in the level of MDA compared with control group. This suggests that alcohol and high-fat diet results in oxidation and antioxidant imbalance, reactive oxygen species (ROS) and free radicals increases, the lipid peroxidation in model group rats. After the treatment of melatonin, the liver SOD activity increased significantly, the level of MDA decreased significantly. Thus melatonin may be through against oxidative stress and reducing lipid peroxidation, plays a role on protection of AFL rats.4. The effects of melatonin on the expression of AMPK-a in AFL ratsAdenosine monophosphate activated protein kinase (AMPK) is a kind of protein kinase which can be AMP activated and play an important role on the regulation of lipid metabolism. Compared with control group, the phosphorylation levels of AMPK-a decreased significantly in liver of AFL rats, and increased after the treatment of melatonin. The phosphorylated of AMPK-a is its form of activation, melatonin increase hepatic AMPK-a activity. The results suggest that the effects of melatonin on regulating lipid metabolism may be associated with activated AMPK-a activity.5. The effects of melatonin on the lipid metabolism in HepG2 cells induced by oleic acid and alcoholTo further explore how melatonin regulates AMPK-a activity, we exposed alcohol and oleic acid in HepG2 cells to establish vitro model of lipid accumulation. ORO staining results show that control group only have light red lipid droplets in cells, alcohol and oleic acid group (O+A) group have more lipid droplets, after the treatment of melatonin lipid droplets decreased significantly. At the same time, ORO staining quantitative results also show that melatonin 10-8,10-7,10-6,10-5 M dose groups all can reduce intracellular lipid droplets in different degrees. TG measurement results showed that compared with control group, intracellular TG content increased significantly in O+A group, melatonin also can reduce the intracellular TG content. The above results show that we have established successful hepatic lipid accumulation model in vitro using ethanol and oleic acid, and melatonin can decrease lipid accumulation in the vitro model.6. The effects of melatonin on the activity of AMPK-α in HepG2 cells induced by oleic acid and alcoholCompared with control group, the expreesion of p-AMPK-a expression significantly decreased in O+A group, the phosphorylation levels of AMPK-a increased significantly after the treatment of melatonin, while melatonin receptor competitive antagonist Luzindole decreased significantly the phosphorylation levels of AMPK-a compared with melatonin treatment alone. The results show that the regulation of melatonin on AMPK-a activity in HepG2 cells may be mediated by its receptors.7. The effects of melatonin on the MT1R/cAMP signaling in HepG2 cells induced by oleic acid and alcoholCompared with control group, the expression of MT1R significantly decreased in O+A group, and significantly increased in MEL and Luzindole groups. cAMP measurement results showed that ethanol and oleic acid stimulated significantly the increase of intracellular cAMP levels, melatonin 10-7,10-6,10-5 M can significantly reduce the levels of cAMP. However, Luzindole (5uM) reversed the effects of melatonin on levels of cAMP in HepG2 cells. The results indicated the effects of melatonin on the regulation of AMPK-a activity may be involved in MT1R/cAMP signal pathway.Conclusion:1. Melatonin can protected AFL rats by decreasing the serum levels of ALT, AST, TG, LDL-C and hepatic levels of TG and MDA, increasing the serum level of HLD-C and the activity of SOD.2. Melatonin can improve lipid accumulation in HepG2 cells induced by alcohol and oleic acid by reducing the TG level and AMPK-a activity, which may be associated with the effects of melatonin on the regulation of MTlR/cAMP signal pathway. |
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