An Experiment On Metformin's Protection Effect In Alcoholic Liver Disease

Objective: Alcoholic liver disease (ALD) is a liver impairment caused by alcohol abuse. Histopathological features of ALD include adiposis hepatica, steatohepatitis, followed by fibrosis and cirrhosis. Now the etiopathogenesis of ALD remains obscure. In recent years, surveys presents with the hypothesis of"two hits"which is characteristic by center of oxidative stress and lipid peroxidation to account for the mechanism of alcoholic induced liver injury. The"first hit"causes lipid deposition in the hepatocytes which leads to adiposis hepatica. The"second hit"involves oxidative stress and lipid peroxidation. Insulin, an important metabolic regulator in vivo, has many biological effects. Insulin receptor (INSR), which are widely distributed in cell envelope all over the body apparatus, have an tyrosine phosphorylation of activation. When the combination of insulin and insulin receptor happened, a signal transduction which has two mainly signal transduction pathway is began. The two signal transduction pathway including PI-3K signal transduction pathway and Ras-MAPK signal transduction pathway. The PI-3K signal transduction pathway, however, is the most important one. Insulin resistance can cause fatty degeneration in the liver. AMP-activated protein kinase (AMPK), a part of serine/threonine protein kinase family members, exists in the mitochondria. They can promote ATP synthesis and inhibited ATP decomposition, and play an important role in increaseing fatty acid oxidation, insulin sensitivity and oxidative stress. Sterol regulatory element binding proteins (SREBP) are transcription factors, and they can regulat lipogenesis, raise fat synthesis in the liver. Currently, metformin, the first-line medication in treatment with diabetes, which can improve the state of metabolic disorders and insulin resistance, has been found to be an allosteric activator of AMPK. Aboveall, metformin, thus, may have a preventive and therapeutic effect in alcoholic liver disease. In this study, the model of rats with ALD was established by intergastric alcohol with or without metfornin to detect the expression of AMPK, SREBP and INSR in the liver of rats by immunohistochemistry staining and reverse transcription polymerase chain reaction (RT-PCR), to explore the mechanism of metformin in ALD, and to provide a potential way of the prevention and treatment of ALD.Methods: 30 male depuratory grade Wistar rats, weighting 200±20g, were acclimatized for 7 days and then they were divided into 3 groups with a random digits table: normal control group (N), modle group (M), intervention model group (I), n=10. M and I group were fed alcohol (30%-60%, 5-9g/kg/d); I group had a more aqueous solution of metformin (80mg/ml, 200mg/kg/d). At the end of 16th week, rats in the 3 groups with a number of 10, 8, 8 were sacrificed, and the serum and liver samples were collected respectively. The contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholinesterase (CHE), total cholesterol(TC), triglyeride (TG), very low density lipoprotein(VLDL), low density lipoprotein(LDL), high density lipoprotein (HDL), glucose(GLU) and insulin in serum were examined by OLYMPUS AU-600 automatic biochemical analyzer. Some of the hepatic tissue were made of frozen sections and stained by SudanⅣto observe the liver steatosis, others were fixed with 4% neutral formalin and embedded in paraffinum, and tissue sections of 5μm were cut and stained by hematoxylin- eosin(HE). Paraffin sections are also used for immuno- histochemical staining to examine the expression of AMPK, SREBP and INSR. The rest tissues of liver were frozen quickly in liquid nitrogen, then in -80℃ultra cold freezer, to detect the expression of AMPK, SREBP and INSR mRNA by RT-PCR.Result:1 Changes of weight and weight-related index: in model group, liver weight and liver index have statistical differences with the other two groups (p<0.05). There's no significant difference between normal group and intervention model group (p>0.05).2 Changes of biochemical indicator in serum: in model group the level of ALT, AST, TG, LDL, VLDL is higher than normal control group. The level of CHE, HDL, however, is lower than normal control group. All of them have significant differences (p<0.05). The level of ALT, CHE, TG, in intervention model group, has significant differences with model group (p<0.05). It has significant differences that the level of ALT, AST, VLDL, HDL between normal control group and intervention model group (p<0.05). The level of TC has no significant differences among those 3 groups (p>0.05).3 Changes of insulin in serum: the level of serum glucose and insulin is higher in modle group than in the two others. Insulin sensitivity index, in modle group, is lower in modle group than in the two others. All of them have significant differences (p<0.05).4 Histopathological changes of hepatic tissue: the rats in normal control group developed mild steatosis in pericentral to midzonal regions of hepatic lobules, while diffuse microvesicular adipose degeneration, fibrosis in liver sinus and fibrosis septa in the portal area were observed in hepatic tissue which belonging to normal control group. The frozen sections stained by SudanⅣshowed that nonsteatosis was observed in the hepatic tissue of normal control group, however, a large quantity of lipid droplets, in modle group, distributed in hepatocytes are observed.5 The immunohistochemistry of AMPK, SREBP and INSR protein in the liver of rats: metformin has no obvious improvement for the reduction of AMPK which maybe due to alcohol abuse; but has an effect of increasing expression of INSR. SREBP was significantly higher in modle group than the others.6 The expression of AMPK, SREBP and INSR mRNA in the liver of rats: the levels of AMPK, INSR are decreased, while the level of SREBP is increased in modle group. Metformin can improve this change. All of them have significance differences in modle group with the others.Conclusion:1 The model of rats with ALD can be established successfully by intragastric alcohol of increasing concentration gradually. The pathological changes, such as steatosis, inflammation and fibrosis, can reflect human ALD.2 Insulin resistance and metabolic disorders play an important role in the process of ALD.3 The change of the level of AMPK, SREBP, INSR mRNA could be a reason for the etiopathogenesis of ALD.4 Metformin may mitigate insulin resistance; reduce liver steatosis by changing the level of AMPK, SREBP, INSR mRNA.5 Metformin could be used to treat alcoholic liver disease.