Targeted PPARγ Activation Prevents Non-alcoholic Fibrotic Steatohepatitis In Mice

Objective: Non-alcoholic fatty liver disease (NAFLD) is a common liver disease. NAFLD and related metabolic disorder are increasing in recent years. As a hepatic manifestion of metabolic syndrome, NAFLD is closely associated with multiple factors such as obesity, hyperlipidemia, type 2 diabetes mullitus and hypertension. Nonalcoholic steatohepatitis (NASH), including hepatic steatosis, necrotic inflammation and fibrosis, is the key stage from simple steatosis to liver cirrhosis. NAFLD has actually been shown to be one of the etiologies in those patients with cryptogenic cirrhosis. Up to now, the mechanism of NASH was unclear. Peroxisome proliferator activated receptor gamma (PPARγ), one of nuclear receptor superfamily of transcription factors, is a key regulator of adipogenesis, inflammatory infiltration and fibrogenesis. This study aimed to elucidate the effect of targeted genetic modulation of PPARγon nutritional fibrotic steatohepatitis in mice.Methods: Forty two healthy 7 weeks old male C57BL/6J mice were fed with methionine-choline supplemented diet for one week. Then they were randomly divided into seven groups: control group were fed with methionine-choline supplemented diet (MCD+) for eight weeks. Model group were fed a methionine-choline deficient (MCD) diet for eight weeks to induce fibrotic steatohepatitis. PPARγagonist rosiglitazone (50mg/kg/d) orally (n=6) and/or adenovirus-PPARγ(1×1010v.p, three time/week, p.i.), adenovirus-LacZ (1×1010vp,three time/week, p.i.), antagonist 2-chloro-5-nitrobenzaniliden (GW9662) (1mg/kg,three time/week, p.i.) were supplement simultaneous with MCD diet for 8 weeks, respectively. After 8 weeks, all rats were sacrificed after 12h of fasting. Blood samples were collected for biochemical assays. The liver was removed, and sampled for histological study and RNA extraction. Liver weight, body weight, liver index, serum activitis of alanine aminotransferase (ALT) and liver histology of mice of all groups were assayed. 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. Effect of PPARγregulation was assessed by comparing severity of hepatic fibrosis in the liver sections, the mRNA and protein expression of hepatic inflammatory and fibrogenetic factors,α-SMA, TGFβ1, and MMP2.Results:1 The common behavior of mice: control mice were active, whose hair was bright and weight increased gradually. Body weight of model and intervention mice decreased remarkably compared to control group. The liver weight in model group and treatment group were notedly decreased compared with control group (both P<0.05). There was no significant difference in liver indexs among the seven groups (P>0.05). (Table2)2 Measurement of serum ALT and triglyceride: mice given the MCD diet and adenovirus-LacZ group showed significantly higher serum ALT level and triglyceride content compared with control mice, indicating hepatic injury, and a significant reduction was noticed after rosiglitazone and/or adenovirus-PPARγtreatment, especially in combinated rosiglitazone and adenovirus-PPARγtreatment group. Conversely, the GW9662 group showed the highest ALT level. However, serum triglyceride and cholesterol level was on significant altered except the control group (Table3).3 Liver histopathology showed that MCD fed mice and adenovirus-LacZ injected mice presented severe hepatic macrovesicular steatosis, inflammatory infiltratory and fibrosis. Severer liver injury was presented in giving GW9662 animals. Ad-PPARγor rosiglitazone administration could ameliorate hepatic steatosis, necrotic inflammation and progressive fibrosis. Further more, combination of rosiglitazone and Ad-PPARγshowed an additive effect on Ad-PPARγup-regulation and protection against liver injury.4 Effect of PPARγgene modulation on expression of inflammatory and fibrotic factors: PPARγmRNA and protein expression were up-regulated by given rosiglitazone and adenovirus-PPARγ, further enhanced by combinated rosiglitazone plus adenovirus-PPARγ, which companied with blunting of the pro-inflammatory and pro-fibrosis factors TNF-α,α-SMA, TGFβ1 and MMP2 mRNA and protein expression. Contrarily, PPARγexpression was down-regulated by given GW9662 (P<0.05). By immunochemical staning, the expression of TNFαwas located in inflammatory area and endotheliocyte of hepatic sinus.α-SMA, TGFβ1, MMP2 was expressed in inflammatory and fibrogenesis area.Conclusions: The present study provided a clear morphological and molecular biological evidence for the protective role of up-regulated PPARγin ameliorating heptic steatosis, inflammation and fibrosis in experimental nutritional steatohepatitis and fibrosis. Targeted genetic modulation of PPARγmight serve as a new approach for therapy of fibrotic steatohepatitis.