| Objective: Nonalcoholic steatohepatitis (NASH) is the basis of fibrosing NASH or NASH associated with fibrosis, which may be complicated by progressive fibrosis and cirrhosis. In the progression of fibrosing NASH, many fibrogenesis factors take part in the activation of hepatic stellate cells (HSCs) that can product excessive extracellular matrix (ECM). At present, the pathogenesis of fibrosing NASH is not adequately explored; there is growing evidence that the activation and phenotype transdifferentiation of hepatic stellate cells (HSCs) were the key event in the formation and progression of hepatic fibrosis. Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone receptor superfamily of transcription factors, whose activation is regulated by the high affinity binding of small lipophilic ligands. Recent studies revealed that PPARγis a crucial cytokine which paly an important role in maintenance the quiescent phenotype of HSCs. The hypothesis is that the activation of PPARγmay delay or inhibit the formation of hepatic fibrosis. Rosiglitazone have been developed as agonist that binds to the PPARγ. The role and pathogenesis of rosiglitazone in hepatic fibrosis has not been well addressed. In this study, experimental hepatic fibrosis models were established by feeding male C57BL6/J mice with high fat, methionine-choline deficient (MCD) diet, and use rosiglitazone as treatment group. The activation of HSCs was tested by detectingα-smooth muscular actin (α-SMA) using immunohistochemistry. Expression of PPARγand TGFβ1 was analyzed by RT-PCR and Western blot. Our objective was to investigate the expression and possible roles of PPARγand TGFβ1 in liver of mice with fibrosing NASH, and to evaluate the role and its pathogenesis of rosiglitazone in treatment of NASH associated with fibrosis induced by MCD diet.Methods: Experimental models of fibrosing nonalcoholic steatohepatitis were established by feeding mice with MCD diet. Thirty healthy male C57BL6/J mice were fed with methionine- choline supplemented diet for one week, then were randomly divided into three groups: control group (MCD+ 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 8 weeks. Serum alanine aminotransferase (ALT) and triglyceride (TG) were tested by enzymic method with automatic biochemistry analyzer. Hepatic steatosis was observed by SudanⅣstaining; liver inflammation and fibrosis were graded under HE staining, and liver fibrosis by Masson staining. The activation of HSCs was examined by detectingα-smooth muscular actin (α-SMA) using immunohistochemistry. The expression of PPARγand TGFβ1 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 38.00±0.60 g,17.60±0.89 g,18.25±0.50 g,1.37±0.35 g,0.70±0.07 g,0.73±0.05 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 0.036±0.008,0.040±0.002,0.040±0.020,there was no difference among the three groups (P >0.05).2 The examination of biochemical markers of serum: ALT level was markedly elevated in MCD diet mice compared with control mice (49.67±7.64 U/L vs. 591.50±71.60 U/L,P<0.01), and a significant reduction was noticed after rosiglitazone treatment (591.50±71.60 U/L vs. 270.70±85.05 U/L, P<0.01). There was no statistically difference among the three groups'serum TG content, control: 0.76±0.29 mmol/L, models: 0.34±0.81 mmol/L, treated group: 0.47±0.18 mmol/L, P >0.05.3 Routine pathologic examinations: The liver histology is normal in control mice. However, severe steatohepatitis and fibrosis was developed in mice fed with MCD diet, the hepatocytes presented heavy steatosis with the inflammation of interlobular areas. In addition to the macrovesicular steatosis mainly prominent in zone 3 near the central vein, large areas of mixed inflammatory infiltration with lymphocytes and poly- morphonuclear neutrophil necroinflammation can be seen throughout the hepatic lobular. The extent of steatohepatitis and fibrosis in liver of rosiglitazone treatment group is dramatically decreased.4α-SMA expresses mostly in artery wall and activated HSCs. Positive expression was only seen in artery wall of the control mice and overexpression ofα-SMA, which marked HSCs activation in Disse space, was induced by fed MCD diet (0.49±0.05 vs. 1.57±0.05, P <0.05). However, compared with model group, the expression ofα-SMA decreased by using rosiglitazone (1.57±0.05 vs. 1.07±0.06, P <0.05).5 The expression of PPARγmRNA was dramatically decreased in model animals compared with the control (0.73±0.17 vs. 0.53±0.08, P <0.05), whereas which was dramatically improved in treated animals (0.53±0.08 vs. 0.71±0.14, P <0.05).6 The expression of PPARγprotein was decreased in model animals compared with control group (0.39±0.01 vs. 0.27±0.01, P <0.05), whereas which was dramatically improved in treated mice (0.27±0.01 vs. 0.57±0.01, P <0.05).7 The expression of TGFβ1 mRNA was up-regulated in MCD- group (0.32±0.01 vs. 0.89±0.01, P <0.01) and could be down-regulated by rosiglitazone (0.89±0.01 vs. 0.46±0.05, P <0.05).8 The expression of TGFβ1 protein was up-regulated in model animals (0.55±0.01 vs. 1.15±0.00, P <0.01) and could be down-regulated by rosiglitazone in treatment group (1.15±0.00 vs. 0.93±0.01, P <0.05).Conclusion:1 The fibrosing NASH models could be established successfully by feeding mice with high fat, methionine-choline deficient diet for 8 weeks. This model is characteristed by rapidly formation, easily performed and consistent with the pathological features of patients of fibrosing NASH.2 The activated HSCs were increased in model group, which take part in the pathogenesis of hepatic fibrosis. The expression of PPARγwas decreased, which couldn't maintain the quiescent phenotype of HSCs. TGFβ1 was increased in model animals, all of the above indicate that HSCs and TGFβ1 could contribute to the development of NASH associated with fibrosis.3 Rosiglitazone, as PPARγagonist, could ameliorate experimental hepatic fibrosis. The increased expression of PPARγmight reverse activated HSCs into quiescent phenotype and reduce the expression of TGFβ1 to delay and inhibit the progression of fibrosing NASH. It provided the theory that rosiglitazone might be used to treat fibrosing NASH.
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