Mechanism Of Mitochondrial Selective Autophagy Mediated By PINK/parkin In NASH Hepatocytes And Intervention Effect Of Zhigan Recipe

Objective:Objective to investigate the intervening effect of PINK/parkin mediated mitochondrial autophagy and fragmentation mechanism in non-alcoholic steatohepatitis（NASH）rats.Method:After 80 SD rats were adaptively fed for one week,10 rats were randomly chosen as a 8-week blank group(to be killed at the end of 8^th week)and another 10 rats were chosen as a 12-week blank group(to be killed at the end of 12^th week)and as a normal control group,which was fed with ordinary fodder all along.The remaining 60 rats were fed with high-fat fodder to build a rat model,respectively,8-week model group（to be killed at the end of 8^thh week）,Zhi Gan Fang low-dose group,Zhi Gan Fang medium-dose group,Zhi Gan Fang high-dose group,western medicine group（gavaged with lipitor）and 12^th-week model group,with 10 rats for each group severally.The abovementioned five groups of rat were fed until the 12^th week and then killed at the end of that week,and their blood specimens and liver specimen were collected to detect the level of Alanine aminotransferase（ALT）,Aspartate aminotransferase（AST）,triglyceride（TG）and total cholesterol（TC）in rats with automatic biochemical apparatus.The expression of Mfn1,Mfn2,Opa1and LC3 in the downstream of PINK/parkin signal pathway was detected by Western Bolt,and the expression of PINK and parkin was detected by immunohistochemistry.Results:1.The rats in high-Fat Model group had a significantly elevated level of ALT,AST,TG and TC in their serum as compared with the ones in the blank control group（p<0.01）,pathologically,the infiltration of inflammatory cells,lipid deposition and necrosis of liver cells in the model group were more serious than those in the same blank group,indicating that the model was successful.2.The structure of hepatic cellular mitochondrion and autophagosome was observed with electron microscope,it was found that,the normal group was found to have no autophagosome,the model group and Western Medicine group were found to have a small amount of autophagosomes,and the Chinese medicine group was found to have obviously increased number of autophagosomes,of which the structure was typical and covered with mitochondria fragment and other organelles,and the Chinese medicine high-dose Group was found to have the most obvious increase of all.3.12 weeks model group,the emergence of a large number of hepatic steatosis,ballooning,lobular inflammation and liver lipid deposition and inflammation phenomenon,and significantly higher than the model group 8weeks and 8 weeks,model group is a rare occurrence in these pathological changes,suggesting that the 8 to 12 weeks of high fat diet does induce liver tissue lesion of rats from NAFLD to NASH.4.Western Blot results indicated that the expression level of mitochondrial fusion protein Mfn1 in the model group was significantly lower than it was in the corresponding period（p<0.01）,which showed that the level of its mitochondrial autophagy decreased,and the fusion was blocked,and the expression level of optic atrophy protein Opa1 in mitochondrial inner membrane decreased significantly（p<0.01）.decrease the expression of autophagy specific protein LC3（P<0.01）,the expression of Mfn2 increased（P<0.01）,decreased,the mitochondrial autophagy fusion barrier.After a drug intervention was performed,mitochondrial fusion protein Mfn1 expression level increased,optic nerve atrophy Opa1 protein expression level increased significantly,the autophagy specific protein LC3 expression levels（P<0.05）,Mfn2（P<0.05）,which indicated that the drug intervention could improve the expression level of mitochondrial selective autophagy and reduce the apoptosis of mitochondria,among which,the Zhi Gan Fang high-dose group was found to have the most obvious increase of all.5.The results of immunohistochemistry showed that:PINK1 in the normal group,the positive expression rate is low,the tissue space is normal;in the model group,significantly increased the rate of positive expression showed brown staining,tissue clearance increased;after drug intervention,the positive expression of the traditional Chinese medicine group was slightly higher than that of normal group,mild interstitial increased,the positive expression of the western medicine group the rate is slightly higher than that of the traditional Chinese medicine group,interstitial mild increase.Parkin in the normal group,the positive expression rate is higher,showed brown staining,normal tissue space;in the model group,the positive expression rate was significantly decreased,the positive expression of tissue gap increases;the traditional Chinese medicine group was higher than that of the model group,the tissue space increased slightly;Western Medicine group and Chinese medicine group than the positive expression rate decreased,but still higher than the model group,the tissue space increased slightly.Conclusion:High fat diet can induce the formation of nonalcoholic steatohepatitis in rats,liver cell inflammation,mitochondrial damage,and autophagy restriction.Fatty liver can significantly improve hepatic steatosis and lipid deposition in NASH rats,and alleviate the liver inflammation and inhibit the apoptosis of hepatocytes by mediating the expression of PINK/parkin pathway and its downstream proteins Mfn1,Mfn2,Opa1 and Lc3.It has been proved that Zhi Gan Fang has a certain effect on alleviating the inflammatory reaction and anti apoptosis of liver,and it is possible to mediate the mechanism of mitochondrial selective autophagy mediated by PINK/parkin pathway.