| Objective: Non-alcoholic steatohepatitis (NASH) was characterized with steatosis, periportal and lobular inflammation. The definition of non-alcoholic fatty liver disease (NAFLD) is that hepatocyte steatosis, necrosis inflammation, fibrosis and liver cirrhosis and no history of excessive alcohol consumption. The spectrum of NAFLD is from generally benign, simple steatosis, steatohepatitis and steatosis releted cirrhosis. Further it may develop as hepatocyte carcinoma. Clinically, NASH ranges from asymptomatic abnormal enzyme to cirrhosis and hepatocellular carcinoma presented liver failure. By the far, the mechanism of NASH remains not fully understanding. There is growing evidence that oxidative stress play an important role in the development of NASH, antioxidation stress may prevent the disease progress. 1-aminobenzotriazole(ABT)and vitamin E (Vit E), as antioxidants, can significantly reduce oxidative stress reaction The cytochrome P450 (CYP) in human body is a group of mixed oxidation enzyme on the surface of smooth endoplasmic reticulum (SER) and is one of the most important enzymes of liver metabolism. As the CYP antagonist, if ABT can inhibit NASH progression and its mechanism is not yet clear. Vitamin E is a kind of fat-soluble vitamin, it can remove free radicals and repress lipid peroxidation, which can prevent the process of chain reaction and protect cells from damage. This study used high-fat, methionine-choline deficient (MCD) diet to establish a mouse model of NASH, and treat the mice with CYP inhibitor ABT. Bax and Cyt C of the experimental mice were detected with reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot method to explore the relation of oxidative stress and the process of NASH, and investigate the role of ABT on inhibition of oxidative stress and NASH progression and its molecular mechanism. By the experiment, the noval method treating NASH might be found.Methods: Twenty five healthy male C57BL6/J mice were divided into five groups. Mice fed with high fat, methionine- choline deficient diet (MCD); The control group given choline and methionine adequate diet (MCD+); ABT intervention group fed with MCD diet and ABT intraperitoneal injection (100 mg/kg/d); vitamin E intervention group fed with high fat, methionine and choline difficient diet and vitamin E (100mg/kg chow); ABT+ vitamin E intervention group fed with high fat, methionine and choline difficient diet and vitamin E (100mg/kg chow)+ABT intraperitoneal injection (100mg/kg/d), feeding three weeks. Serum levels of triglyceride (TG) and alanine amino-transferase (ALT) and aspartate amino-transferase (AST) were detected. Pathological changes in liver tissue stained by SudanⅣand hematoxylineosin were observed; Liver tissue Cyt C mRNA and Bax mRNA and protein expression were analyzed by RT-PCR and Western blot.Results: 1 Regular behavior of mice: Control mice were active, their hairs were bright and body weight increased gradually. Body weight of model mice decreased remarkably compared with the control. The body weight of all the experimental mice, MCD-+Vit E group, MCD-+ABT group and MCD-+Vit E+ABT group, was in the order respectively: 16.96±1.38g,27.23±1.10g,17.84±0.86g,17.27±0.92g,17.82±0.86g ,0.70±0.15g,1.12±0.16g,0.74±0.11g,0.76±0.17g,0.78±0.19g,P<0.05. There no significant difference of the liver index among all the group, they are 4.17±0.26,3.91±0.34,3.98±0.28,4.11±0.15,4.13±0.21 as the above group order, P>0.05.2 The biochemical markers of serum: The levels of serum ALT of mice were following as order: model group, MCD-+Vit E group, MCD-+ABT group, MCD+group, MCD-+Vit E+ABT group (245.33±8.02 U/L,184.67±18.23 U/L,161.00±13.89 U/L,151.33±12.86 U/L,129.33±13.65 U/L,P<0.01); and the serum AST levels were decreasing: model group, MCD-+ Vit E group, MCD-+ABT group, MCD+ group, MCD-+ Vit E +ABT group (330.67±15.04 U/L,201.67±28.04 U/L,197.67±20.03 U/L,135.00±8.72 U/L,125.67±4.51 U/L,P<0.05); the serum TG in model group, control group, MCD-+ABT intervention group, MCD-+Vit E intervention group and MCD-+Vit E+ABT intervention group is following: 0.72±0.04 mmol/L,0.56±0.07 mmol/L,0.38±0.03 mmol/L,0.36±0.04 mmol/L,0.33±0.03 mmol/L,respectively, P<0.05. 3 Pathological observing by HE Staining: the liver is normal in control mice. However, the livers, in mice fed with MCD, were decreased significantly campared with the control, and moderate steatosis with inflammatory infiltration was found in the liver sections. Vit E and ABT supplement prevented steatohepatitis progression, particularly in combination of Vit E +ABT.4 Hepatic cytochrome C mRNA expression: cytochrome C mRNA expression in the livers were in order: model group, control group, Vit E intervention group, ABT intervention group, ABT+vit E intervention group, they are 1.26±0.04,1.14±0.08,1.04±0.03,1.00±0.02,0.99±0.01,respectively, P<0.01).5 Hepatic cytochrome C expression in protein level: the liver cytochrome C protein expression was following according to the order: the model group, control group, Vit E intervention group, ABT intervention group and ABT+vit E intervention group, (1.06±0.01,0.96±0.01,0.85±0.02,0.84±0.02,0.80±0.02,P<0.01).6 Hepatic Bax mRNA expression: Bax mRNA expression in the liver tissue was decreasing: model group, control group, Vitamin E intervention group, ABT intervention group, and ABT + vitamin E intervention group (0.83±0.03,0.77±0.02,0.71±0.01,0.73±0.03,0.70±0.02,P<0.01).7 Bax expression in protein level in the liver tissue: Bax protein expression were also decreasing and consistent with Bax mRNA expression: model group, control group, Vit E intervention group, ABT intervention group and ABT+vit E intervention group (0.55±0.02, 0.42±0.01, 0.40±0.02, 0.40±0.01, 0.37±0.01,P<0.01).Conclusion:1 The NASH model could be established successfully by feeding mice with high fat, choline-methionine deficiency diet for 3 weeks, in which moderate steatosis and inflammatory infiltration was developed in the liver. The liver injure in MCD induced NASH model is consistent with the pathological features of human.2 Bax and Cytochrome C were involved in non-alcoholic steatohepatitis induced with MCD diet. The mechanism of the diseae may be associated with hepatocyte apoptosis caused by Bax and Cytochrome C and down stream apoptotic signal transduction pathway.3 Bax and Cytochrome C expression could be modulated by CYP inhibitor ABT and (or) antioxidatent vitamine E, which ameliorated hepatic steatosis, inflammation and further inhibited NASH progrssion. Thus, antioxidation may serve as an effective approach for the NASH therapy.
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