Chikusetsu Saponin Ⅳa Alleviated Nonalcoholic Steatohepatitis Induced By A High-fat Diet In Mice Through Regulating MiR-17-5p/MFN2 Signaling Pathway

Objective To investigate the pathogenesis and possible treatment methods of nonalcoholic steatohepatitis,and to specifically study the effect of Chikusetsu SaponinⅣa（CHS-Ⅳa）on mi R-17-5p/MFN2 signal pathway to improve nonalcoholic steatohepatitis induced by a high-fat diet,and discuss the possible molecular mechanisms.Methods（1）In vivo:High-fat diets were used to induce pathological changes in mice.Liver index was calculated by weighing liver weight and body weight.Serum was collected to detect serum biochemical indicators ALT,TG,and GLU.HE and Masson staining were used to detect hepatic steatosis and fibrosis.Real-time PCR was used to detect the expression levels of lipid metabolism-related genes FASN,ACC1,LXR,ch REBP,inflammatory factors IL-6,IL-1β,TNF-α,as well as the expression levels of mir-17-5p and MFN2 in liver tissues.Western blot was used to detect the expression levels of TNF-αand MFN2 proteins.（2）In vitro:Hep G2 cells were used as research objects,cells were treated with different combinations of palmitic acid,oleic acid and glucose,and the expression changes of mir-17-5p and MFN2 were detected by Real-time PCR and Western blot,and the cell model was constructed by selecting appropriate treatment methods.After intervention with CHS-Ⅳa,observe the effect of CHS-Ⅳa on mi R-17-5p and MFN2 expression.The wild-type and mutant luciferase reporter plasmids containing MFN2 3’-UTR were constructed to verify the targeted regulatory relationship between mi R-17-5p and MFN2.Results（1）Compared with the control group,the liver index in the HFD+CCl₄group was significantly increased（P<0.05）,and the contents of serum biochemical indicators such as ALT,TG and GLU were significantly increased（P<0.05）.HE and Masson staining results showed that significant steatosis and collagen fiber deposition appeared in the liver of the HFD+CCl₄ group.Real-time PCR results showed that the expression levels of liver lipid metabolism-related genes,inflammatory factors and mi R-17-5p in the HFD+CCl₄ group were significantly increased（P<0.05）,and the expression levels of MFN2 were decreased（P<0.05）.Western blot results showed that the protein levels of TNF-αexpression were increased（P<0.01）,while the protein levels of MFN2 were decreased（P<0.01）.After intervention by the CHS-Ⅳa,the content of serum biochemical indicators decreased（P<0.05）,HE and Masson staining showed a reduced hepatic steatosis and the collagen deposition was decreased in mice;Real-time PCR results showed that the expression levels of lipid metabolism-related genes,inflammatory factors and mi R-17-5p in liver tissues was decreased（P<0.05）,and the expression levels of MFN2was increased（P<0.05）.Western blot results showed that the protein level of TNF-αdecreased（P<0.01）and the protein level of MFN2 increased（P<0.05）.（2）In vitro cell culture was used to construct a cell inflammation model.The results showed that Hep G2cells treated with PA 100μM and GLU 100 m M could induce high expression of mi R-17-5p and low expression of MFN2（P<0.05）.After intervention by the CHS-Ⅳa,which can effectively reverse this phenomenon.The results of luciferase reporter gene assay showed that mi R-17-5p mimic could significantly reduce the activity of luciferase in wild-type plasmids（P<0.01）.Conclusion In the model of non-alcoholic steatohepatitis,a high-fat diet combined with intraperitoneal injection of CCl₄,the mi R-17-5p/MFN2 signaling pathway is involved in the occurrence and development of disease.The MFN2 signaling pathway plays a protective role in NASH and provides a new research idea for the drug treatment of non-alcoholic steatohepatitis.