Expression And Significance Of INOS In Rat Liver Of 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. nitric oxide synthase (NOS), a key enzyme of NO synthesis, has at least 3 subtypes: inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). iNOS and eNOS may play important roles in liver diseases. iNOS is little or no expressed in normal hepatocytes, but excessively expressed in the injured liver tissue. With mass reactive oxygen species, NO and superoxide anion (O2-) can react to form peroxynitrite (ONOO-), which is associated with energy expenditure, lipid metabolism, cell differentiation, and apoptosis by nitrifying tyrosine to nitro- tyrosine (NT). In this study, the model of rats with ALD was established by intergastric alcohol to detect the expression of iNOS and NT in the liver of rats by immunohistochemistry staining and the expression of iNOS by reverse transcription polymerase chain reaction (RT-PCR), to explore the mechanism of iNOS in ALD, and to provide a potential way of the prevention and treatment of ALD.Methods: 50 male depuratory grade Wistar rats, weighting 200±20g, were acclimatized for 7 days and then 10 rats were randomly assigned to the normal control group. Others were to develop the rats model of ALD by intragastric alcohol of increasing concentration gradually (30%-60%, 5-9g·kg-1·d-1), 8, 8, 8 and 9 rats were sacrificed randomly at the end of 4th, 8th , 12th and 16th week, and the serum, liver samples and liver homogenate (10%) were collected respectively. The contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and cholinesterase (CHE) in serum were examined by OLYMPUS AU-600 automatic biochemical analyzer and the concentrations of triglyceride (TG), oxygen free radical (OFR), malondialdehyde (MDA) and superoxide dismutase (SOD) in the liver homogenate were also measured by biochemical chromatometry. Some of the hepatic tissue were made of frozen sections and stained by SudanⅣand to observe the liver steatosis. Some part of hepatic tissue were fixed with 4% neutral formalin and embedded in paraffinum, and tissue secions of 5μm were cut and stained by hematoxylin-eosin (HE) and Sirius red to observe the ordinary pathologic changes and liver fibrosis. Paraffin sections are also used for immunohistochemical staining to examine the expression of iNOS and NT. The rest tissues of liver were frozen quickly in liquid nitrogen, then in -80℃frig , to detect the expression of iNOS mRNA by RT- PCR.Result:1 The change of biochemical index of serum: With the con- sumpion of ethanol increase, the level of ALT and AST in the serum increased while the CHE decreased gradually, compared with those of the normal control group P<0.05 and P<0.01.2 The examination of oxidative stress index of hepatic tissue homogenate: With the consumption of ethanol increase, the contents of TG, OFR and MDA in the hepatic tissue homogenate increased while the SOD decreased gradually, compared with those of the normal control group P<0.05 and P<0.01.3 Histopathological changes of hepatic tissue: At the 4th week, the rats developed mild steatosis in pericentral to midzonal regions of hepatic lobules. With the progress of ALD, hepato- cytes steatosis, necrosis and inflammation in liver lobules as well as fibroplasias became aggravated. At the 16th week, diffuse microvesicular adipose degeneration, fibrosis in liver sinus and fibrosis septa in the portal area were observed in hepatic tissue. The frozen sections stained by SudanⅣshowed that nonsteatosis was observed in the hepatic tissue of normal control group. At 4th week, there were a little lipid droplets in cytoplasm. With the process of ALD, the quantity of lipid droplets increased gradually, and at 16th week, a large quantity of lipid droplets distributed in hepatocytes. The frozen sections stained by sirius red showed that at 4th week, the fibrosis of the hepatic tissues were not obvious. At 12th week, fibrosis in liver sinus and fibrosis septa in the portal area were observed in hepatic tissue. And at 16th week, the fibrosis septa were obvious.4 The expression of iNOS and NT in the liver of rats: There was only a little expression of iNOS and NT in hepatic tissue of rats in normal control group. With the progress of ALD, the positive expression became more severe, and the main expression located in cytoplasm.5 The expression of iNOS mRNA in the liver of rats: There were only a little expression of iNOS in hepatic tissue of rats in normal control group. The expression of iNOS increased gradually with the progress of ALD, and compared with those of the normal control group P<0.05.6 The expression of NT was positively correlated with the relative iNOS mRNA expression (r=0.71, P<0.01).7 Pearson correlation analysis showed that the expression of NT was positively correlated with the level of AST (r=0.65, P<0.01), and negatively correlateed with the level of CHE (r=-0.54, P<0.01).8 The relative iNOS expression was positively correlated with the contents of OFR and MDA of the hepatic tissue homogenate (r=0.84, P<0.01; r=0.82, P<0.01), and negatively correlated with the contents of SOD (r=-0.69, P<0.01).9 Pearson correlation analysis showed that the relative iNOS expression was positively correlated with the level of AST (r=0.42, P<0.01), and negatively correlateed with the level of CHE (r=-0.52, P<0.01). 10 The relative iNOS expression was positively correlated with the contents of OFR and MDA of the hepatic tissue homogenate (r=0.57, P<0.01; r=0.66, P<0.01), and negatively correlated with the contents of SOD (r=-0.46, P<0.01).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 Nitrative stress caused by iNOS and oxidative stress play important roles in the etiopathogenesis of ALD.