| Nonalcoholic fatty liver disease (NAFLD) is a clinical and pathological syndrome who is similar with alcoholic liver disease in pathologic histology but in the absence of significant alcohol consumption. The spectrum of it includes simple liver steatosis, nonalcoholic steatohepatitis (NASH) and fibrosis/cirrhosis in order. Even it can progress to hepatocellular carcinoma. So it is considered as a great threat against people's health. The prevalence of NAFLD is increasing year by year. NASH, as a key process from simple steatosis to fibrosis/cirrhosis, has not been clearly known in its pathogenesis, so an efficient approach has not been found by now. Adiponectin, a gelatin-binding hormone secreted by adipose tissue, is the only cytokine who is negatively correlated with lipid level in blood serum. Adiponectin plays an important role in regulating lipid metabolism and glycometabolism, ameliorating inflammation. Two adiponectin receptors have been found so far: AipoR1 and AdipoR2. AdipoR2 mostly expressis in liver. So it is supposed that adiponectin may participate in the formation and development of NASH. Thiazolidinediones (rosigliazone) is the specific agonist of peroxisome proliferator-activated receptor gamma (PPARγ). Our previous research showed that rosigliazone could inhibite the hepatic steatosis, inflammation and fibrosis/cirrhosis of mice induced with methionine-choline deficient (MCD) diet. But what is the role of adiponectin in the model is not clear. In this study was to investigate effect of adiponectin in MCD feeding mice and rosigliazone treated mice, which may provide a new approach of NASH therapy through gene modulation.Objective: In this study, experimental nonalcoholic steatohepatitis models were established by feeding male C57BL6/J mice with methionine-choline deficient (MCD) diet. The treatment group mice were administrated with MCD diet combined with rosigliazone. The expression of adiponectin, tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) were detected to clarify role and mechanism of PPARγagonist, rosigliazone in NASH. This study will provide an theoretical basis for target gene theropy on NASH.Methods: Experimental models of NASH were established by feeding mice with MCD diet. Thirty healthy male C57BL6/J mice were fed with methionine-choline supplemented diet for one week, and then were randomly divided into three groups: control group, were fed with methionine-choline supplemented diet, model group (MCD group), were given MCD diet, treatment group (MCD+R group), used MCD diet combined with rosiglitazone (50mg/kg/d) for 3 weeks. Serum alanine aminotransferase (ALT) and triglyceride (TG) were tested by enzymic method with automatic biochemistry analyzer. The grade of hepatic steatosis, inflammation and fibrosi were observed by Hematoxylin and eosin, and Masson trichromatism stained paraffin-embedded liver tissues isolated from mice. Adiponectin expression in the liver was examined by immunohistochemistry. The expression of TNF-αand IL-6 mRNA and protein were analyzed by RT-PCR. and western blot, respectively.Results:1 The common behavior of mice: control mice were active, their hair was bright and weight increased gradually. Body weight of mice in model group and rosiglitazone group decreased remarkably. Body weight and liver weight in control group, model group and treatment group were respectively :26.00±1.00 g 14.14±0.68 g, 14.31±0.81 g,0.89±0.12 g 0.53±0.10 g 0.56±0.10 g. Body weight and liver weight in model group and treatment group were notedly decreased compared with control group (both P value <0.05). The liver indexs of control group, model group and treatment group was respectively 0.034±0.005, 0.038±0.006, 0.039±0.008, there was no difference among the three groups (P >0.05).2 The test of biochemical markers of serum: ALT level was markedly elevated in MCD diet mice compared with control mice (50.78±10.03 U/L vs. 161.56±28.23 U/L,P<0.01), and a significant reduction was noticed after rosiglitazone treatment (161.56±28.23 U/L vs. 83.56±12.27 U/L, P<0.01). So does the three groups'serum TG content, control: 0.54±0.29 mmol/L, models: 0.97±0.81 mmol/L, treated group: 0.76±0.13 mmol/L, P <0.05.3 Liver histopathology : macroscopic observation: Livers in control group were in reddish-brown, with bright gloss. Livers in model group were much smaller than in control group. The whole livers were in light yellow and adhered with other tissues. The cut surface looked reddish-brown without gloss. Livers in treat group were little smaller than in control group and looked reddish-brown with gloss. light microscope observation: Histology of the livers were normal in control mice. Hepatic plates looked well. However, severe steatohepatitis was developed in mice fed with MCD diet, the hepatocytes presented severe macrovesicular steatosis and lacated mainly in zone 3 near the central veins. Spotty necrosis and confluent necrosis can be seen in hepatic lobulars, where mixed inflammatory infiltration with lymphocytes and polymorphonuclear may take place. There were not obvious hepatic fibrosis in perisinusoidal and portal areas (F2-3G2-3S0-1). The degree of hepatic steatosis and inflammation is dramatically ameliorated by administrated rosiglitazone in MCD fed mice (F1G1-2S0-1).4 The expression of TNF-αmRNA was dramatically improved in MCD fed animals compared with the control group (0.82±0.17 vs. 1.26±0.33,P <0.05), whereas which was dramatically decreased in rosiglitazone treated animals (1.26±0.33 vs. 0.94±0.14,P<0.05).5 The expression of TNF-αprotein was increased in MCD mice compared with control group (0.47±0.06 vs. 1.21±0.05,P<0.05), whereas which was dramatically decreased in rosiglitazone treated mice (1.21±0.05 vs. 0.83±0.03,P<0.05).6 The expression of IL-6 mRNA was up-regulated in MCD group (0.34±0.01 vs. 0.89±0.01 , P<0.01) and could be down-regulated by rosiglitazone (0.89±0.01 vs. 0.57±0.03,P<0.05). 7 The expression of IL-6 protein was up-regulated in model animals (0.46±0.01 vs. 0.91±0.01 , P<0.01) and could be down-regulated by rosiglitazone in treatment group (0.91±0.01 vs. 0.79±0.02,P<0.05).8 Hepatic adiponectin expression: Positive expression was presented around central veins and in endochylema of perisinusoidal cells in the livers of control group. But in model group positive expressions are dramatically enhanced and mainly express in the endochylema of steatosis hepatocytes, kupffer cells in inflammatory infiltration areas and perisinusoidal cells. (1.46±0.27 vs. 15.24±0.36,P<0.01). Compared with model group, the expression of adiponectin was decreased by using rosiglitazone. (15.24±0.36 vs. 9.46±0.74,P< 0.05).Conclusion:1 The expressions of adiponectin is dramatically enhanced in livers of the MCD fed mice, which was consistent with expressions of TNF-αand IL-6, which indicates that adiponectin through compensated up-regulation might prevent expression of inflammatory factors and inhibit NASH progression.2 Rosiglitazone could up-regulate PPARγexpression, further up-regulate adiponectin expression, down-regulate expression of TNF-αand IL-6. Thus, rosiglitazone can inhibit hepatic steatosis, inflammation and fibrosis induced by MCD diet by modulating PPARγ, adiponectin and related inflammatory factor expression.
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