基于AMPK/mTOR/ERRα信号通路探讨慈菇消脂方影响非酒精性脂肪性肝炎小鼠肝细胞自噬作用机制

Discussion on the Mechanism of Cigu Xiaozhi Prescription on Hepatocytes Autophagy in Nonalcoholic Steatohepatitis Mice Based on AMPK/mTOR/ERRαSignaling Pathway

目的基于AMPK/mTOR/ERRα信号通路探讨慈菇消脂方对非酒精性脂肪性肝炎(NASH)小鼠肝细胞自噬的调控机制。方法小鼠随机分为空白组、模型组、慈菇消脂方组和抑制剂组,每组10只。采用蛋氨酸胆碱缺乏饲料喂养构建NASH小鼠模型,造模成功后给药组予相应药物干预,连续4周。HE染色观察小鼠肝组织形态并进行非酒精性脂肪性肝病(NAFLD)活动度评分,电镜观察肝细胞超微结构,免疫组化检测肝组织Beclin1、LC3、p62蛋白表达,Western blot检测肝组织p-AMPK、p-mTOR、雌激素相关受体α(ERRα)蛋白表达。结果与空白组比较,模型组小鼠肝组织出现明显脂肪变性并可见大量炎性细胞浸润,肝细胞可见大量形态较规则的单层膜脂质结构,NAFLD活动度评分升高(P<0.01),肝组织Beclin1、LC3、p-AMPK蛋白表达降低(P<0.01),p62、p-mTOR、ERRα蛋白表达升高(P<0.01);与模型组比较,各给药组小鼠肝组织脂肪变性减少,炎性细胞浸润改善,可见少量自噬体,NAFLD活动度评分降低,肝组织Beclin1、LC3、p-AMPK蛋白表达升高(P<0.01,P<0.05),p62、p-mTOR、ERRα蛋白表达降低(P<0.01)。结论慈菇消脂方可能通过调控AMPK/mTOR/ERRα信号通路相关蛋白表达介导自噬,延缓NASH进展。

Objective To explore the mechanism of Cigu Xiaozhi Prescription on hepatocytes autophagy in nonalcoholic steatohepatitis(NASH)mice based on AMPK/mTOR/ERRαsignaling pathway.Methods The mice were randomly divided into blank group,model group,Cigu Xiaozhi Prescription group and inhibitor group,with 10 mice in each group.A mouse model of NASH was constructed using methionine-choline-deficient chow,and drug interventions were administered after successful modelling for consecutive 4 weeks.Histological changes in liver tissue was observed by HE staining,and the NAFLD activity score was evaluated,electron microscopy was used to observe the ultrastructure of hepatocytes,immunohistochemistry was used to detect the relative expressions of Beclin1,LC3 and p62 proteins in liver tissue,Western blot was used to detect the expressions of p-AMPK,p-mTOR and ERRαproteins.Results Compared with the blank group,the liver tissue of mice in the model group showed obvious steatosis and a large number of inflammatory cells infiltration,a large number of regularly shaped monolayer lipid structures could be observed in liver cells,and the NAFLD activity score significantly increased(P<0.01),the expressions of Beclin1,LC3,and p-AMPK proteins in liver tissue significantly decreased(P<0.01),while the protein expressions of p62,p-mTOR,and ERRαsignificantly increased(P<0.01).Compared with the model group,the steatosis in liver tissue of mice in each administration group reduced,inflammatory cell infiltration was improved,a small amount of autophagosomes were visible,and the NAFLD activity score was reduced,the expressions of Beclin1,LC3,and p-AMPK proteins in liver tissue significantly increased(P<0.01,P<0.05),while the protein expressions of p62,p-mTOR,and ERRαsignificantly decreased(P<0.01).Conclusion Cigu Xiaozhi Prescription may regulate related protein expressions of AMPK/mTOR/ERRαsignaling pathway to mediate autophagy and delay the progression of NASH.