Effects Of "Zhi Gan Prescription" On Mitochondrial Selective Autophagy And Degradation Mechanism Of NASH Hepatocytes

Objective: This subject aims to investigate the effect of Zhi Gan prescription on mitochondrial selective autophagy and its degradation mechanism in hepatocytes of NASH model rats.Methods: 80 SD mouse are randomly divided into 8 groups: ten 8w former group and ten 12 w former group.One of the former groups will be killed at the end of 8 weeks,and the other will be put to death at the end of 12 week,both of them are fed with standard diet.The remaining 60 rats were fed with high fat diet: 10 of the them are picked out randomly as 8W Model group and put to death After 8 weeks' high-fat diet.Then the rest rats were randomly divided into five groups: high-dose Zhi Gan Prescription group,low-dose Zhi Gan Prescription group,mid-dose Zhi Gan Prescription group,and Western medicine group and the 12 W model group.The rats were kept fed with high-fat diet at the same time.Make interventions during the 8th week to the 12 th week by give a gavage of Lipitor tablets in western medicine group,and Zhi Gan prescription in high,mid,low dose to the rats in Chinese Medicine group.These 50 rats will be kept fed with high-fat diet until 12 th week,the end of the experiment.At the end of 12 th week,blood samples and liver specimenswere taken for measuring the serum levels of AST,ALT,TG and TC by automatic biochemical analyzer after 12 hours of fasting,histopathological typing of hepatocytes was observed by microscope and ultrastructure of mitochondrial autophagy was observed by transmissionelectron microscopy.Western Blot detection protein expression levels of LC3-II,P62,Fis1 and Mfn1.Results: 1.The levels of serum TG,TC,AST and ALT in the model groups were significantly higher than those in the blank group(P <0.01).And the hepatocyte's inflammation and lipid deposition were more severe than those in the blank group.Indicating that high-fat feed NASH rats were successfully modeled and can be used in subsequent experiments.2.The degree of inflammatory cell infiltration,liver necrosis and hepatic steatosis in the 12 W model group were significantly higher than those in the8 W model group(P <0.05),and showed a large number of balloon-like changes,Mallory body and other liver tissue inflammatory response and collagen fiber deposition,while the 8W model group was less or did not show these pathological changes.Suggesting that the 8th week to 12 th week of high fat fed NASH model rats liver tissue lesions from NASLD to NASH stage progress.3.Compared with the 12 W model group,the histopathology of the treatment groups showed that the liver tissue fatty inflammation and liver lipid deposition were obviously reduced and improved,at the same time,serum TC,TG,ALT and AST levels were significantly reduced(P<0.05).Suggesting that the treatment of traditional Chinese medicine groups and Western medicine group have improved the role of NASH liver cell lesions,and the high-dose Zhi Gan Prescription the best effect.4.The mitochondria ultrastructure and autophagy of hepatocytes were observed under electron microscope.The results showed the number of damaged mitochondria in the model group was significantly higher than the control group,and the number of autophagy was significantly decreased.Suggesting that there is increased mitochondrial damage and autophagy reduced in NASH liver cells.5.Compared with the model group,the therapy groups' number of mitochondrial changes and the number of fragments were decreased,mitochondrial autophagy number increased significantly,of which the high-dose Zhi Gan Prescription group was the most obvious change.Suggesting that the treatment group have alleviated mitochondrial damage and enhanced cell autophagy to protect the survival of liver cells,including the high-dose Zhi Gan Prescription side of the best.6.Compared with the same period blank group,Western Blot results showed that the model group's expression of LC3-II protein was significantly decreased,the mitochondrial division-related protein Fis1 was significantly increased,and mitochondrial fusion-related protein Mfn1 was significantly decreased(P<0.05).Suggesting that The level of mitochondrial selective autophagy decreased,the mitochondrial division increased and the fusion appeared disorder in the NASH rat hepatocytes.Compared with the same period model group,the expression of P62 protein in the liver tissue of the treated group was significantly decreased,the expression of LC3-II protein was obviously increased,the Fis1 protein level was decreased,the Mfn1 protein level was significantly increased(P<0.01)The high-dose Zhi Gan Prescription group was superior to other treatment group(P<0.05)It suggested that intervention could improve the level of mitochondrial selective autophagy in hepatocytes of NASH rats,inhibit the mitochondrial damage and the apoptosis of hepatocytes,and the best effect of these intervention is the high-dose Zhi Gan Prescription.Conclusion: High fat diet can cause fat metabolism disorders in rats,liver index increased,liver cell pathological inflammation,mitochondrial damageand autophagy limited.Zhi Gan Prescription can remarkably improve the NASH model rats liver lipid metabolism,steatosis and pathological inflammation,And play a role in antagonizing the occurrence and development of NASH by regulating the mitochondrial selective autophagy,improving the mechanism of mitochondrial debris degradation and protecting the survival of hepatocytes.This experiment show that inhibition of mitochondrial damage and the promotion of mitochondrial selective autophagy may be one of the mechanisms of Zhi Gan Prescription in treatment of NASH.