越鞠丸通过AMPK/mTOR信号通路促进肝星状细胞自噬缓解肝纤维化的分子机制研究OA

Objective It is to explore the molecular mechanisms of Yueju pill(YJP)in alleviating hepatic fibrosis.Methods Eighty male C57BL/6J mice(aged6-7 weeks)were randomly divided into normal group,model group,YJP group,YJP+AMPK inhibitor group,YJP+solvent group.The mice of all groups except for the normal group were fed with choline deficiency and L-amino acid restriction diet to establish models of liver fibrosis.From the 14th week of the modeling process,the YJP group was given YJP aqueous solution 1 mg/mL by gavage,the YJP+AMPK inhibitor group was given YJP aqueous solution 1 mg/mL by gavage and AMPK inhibitor 2 μg by subcutaneous injection,the YJP+solvent group was given YJP aqueous solution 1 mg/mL by gavage and sterile normal saline by subcutaneous injection,all once daily,contin-uously treated for 8 weeks.After the last intervention,8 mice were randomly selected from each group,their body weights and liver masses were measured,the contents of alanine aminotransferase in serum and hydroxyproline in liver were meas-ured by ELISA,the pathology and fibrosis of liver tissue were observed by HE staining and Masson staining.The remaining mice in each group were sacrificed and the primary hepatic stellate cells(HSCs)were isolated from their livers.The mR-NA expressions of type I collagen α1(COL1A1)and type I collagen α2(COL1A2)in the HSCs were detected by qPCR,the protein expressions of desmin,p62,α-SMA,Bcl-2,Bax,cleaved Caspase-3,Caspase-3,phosphorylated AMPKα(p-AMPKα),AMPKα,phosphorylated mTOR(p-mTOR),mTOR and the ratio of LC3-Ⅱ/LC3-Ⅰ in the HSCs were detec-ted by Western blot.Results Compared with the normal group,the body weight,liver weight,levels of alanine aminotrans-ferase in serum and hydroxyproline in liver of mice in the model group and YJP+AMPK inhibitor group were obviously in-creased(all P<0.05);the liver cells of the mice were enlarged,with visible intracellular lipid droplets and large amount of fibrotic tissue in the intercellular spaces;the relative expressions of COL1A1 and COL1A2 mRNA,as well as the relative protein expressions of desmin,α-SMA,Bax,cleaved Caspase-3,p-mTOR proteins,and the LC3-Ⅱ/LC3-Ⅰ ratio in pri-mary HSCs were all significantly increased(all P<0.05),while the relative protein expressions of p62,Bcl-2 and p-AMPKα were all significantly decreased(all P<0.05).Compared with the model group and YJP+AMPK inhibitor group,the body weight,liver weight,levels of alanine aminotransferase in serum and hydroxyproline in liver of mice in the YJP group and YJP+solvent group were obviously decreased(all P<0.05);the pathologic changes in liver tissue were allevia-ted;the relative expressions of COL1A1 and COL1A2 mRNA,as well as the relative protein expressions of desmin,α-SMA,Bax,cleaved Caspase-3,p-mTOR proteins,and the LC3-Ⅱ/LC3-Ⅰ ratio in primary HSCs were all significantly de-creased(all P<0.05),while the relative protein expressions of p62,Bcl-2 and p-AMPKα were all significantly increased(all P<0.05).Conclusion YJP may promote HSCs autophagy and inhibit their apoptosis by activating AMPK and inhibi-ting mTOR,thereby alleviating liver fibrosis.