| Objective: Investigate the correlation between PINK1 and parkin,P62 and LC3 proteins in hepatocyte mitochondria selective autophagy on non-alcoholic steatohepatitis(NASH)model rats and the interventional effect of zhiganfang on NASH.Methods: All 90 SD rats were adaptively fed for 1 week.20 of them were randomly selected as the normal control group and fed with the food according to a normal(regulated)diet,afterwards during the study;10 rats were sacrificed at the end of week 8 and 10 rats within the end of week 12.The remaining 70 rats were fed consumable food containing high amounts of carbohydrate and glucose according to a high-fat diet for 8 weeks to replicate the non-alcoholic fatty liver disease(NAFLD)model.10 rats were randomly assigned to the 8w model group(no drugs were administered to the test subjects for this study and the test subject rats were sacrificed at the end of the 8th week after high-fat feeding),and 10 were used as an elf-healing group(no medicine or supplemental treatment were given and rats were fed respectively according to a balanced high-fat diet and normal diet within the first 8 weeks and the 9th to 12 th week).The remaining 50 rats continued to befed with a high-fat diet for 12 weeks,these rats were randomly divided into a 12 w model group(no drugs were administered or orally given to these test subject rats either,they were also sacrificed at the end of the 12 th week after high-fat feeding).The groups organized according to medicinal studies were: zhiganfang high dosage group,zhiganfang medium dosage group,zhiganfang low dosage group,and Western medicine group(Lipitor).10 rats were contained within each group.50 rats(5 groups)were sacrificed at the end of the 12 th week and samples from their carcasses were collected.The levels of ALT,AST,TG and TC were detected by a medical instrument;the automatic biochemical analyzer.The interactions between PINK1 and parkin,P62 and LC3 protein were detected by immunoprecipitation.Focusing microscopy(photon method)was used to observe the localization of PINK1,parkin,P62,and LC3 proteins.Results: 1.Compared to the normal group during the same period;the levels of TC,TG,AST and ALT in the 8w model group and the 12 w model group increased significantly(P<0.01).With the increase of high-fat feeding time in the 12 w model group,the levels of TC and TG,AST and ALT also increased(P<0.01),thus indicating that the models were successful,and NAFLD progressed to NASH during the 8th week to 12 th week.That is an indication that the high-fat diet induces damage to the hepatocyte cells from the control liver(mimicking the effect of liver necrosis)in NAFLD rats whilst causing elevated blood lipids,which in the long-term;leads to liver lipid depositionand induces NSAH.Compared with the 12 w model group,levels of TC,TG,AST and ALT in each treatment group were significantly lower(P<0.01).Compared with the 8w model group,except for the self-healing group(P>0.05);the levels of TC,TG,AST and ALT within the study subjects of each treatment group were significantly lower(P<0.01).Compared against the western medicine group,the levels of TC,TG,AST and ALT in the zhiganfang high dose group were significantly lower(P<0.05,P<0.01),indicating the best efficacy,and there were no significant differences in the medium and low dose groups(P>0.05).These indicate that zhiganfang can improve liver lipid levels and liver function.2.Expressions of PINK1 and parkin were detected by Western Blot in each group before the co-immunoprecipitation experiment.Co-precipitation results showed that both PINK1 and parkin proteins could be detected in each sample when target proteins were precipitated by PINK1 antibodies and targeted precipitates detected by parkin antibody.And it found that all groups detected different degrees of precipitation phenomenon when parkin was used to precipitate PINK1 protein.PINK1 and parkin proteins interacted weakly in the normal group,the model group and the self-healing group,while stronger in the zhiganfang groups and the western medicine group.The expressions of P62 and LC3 were detected by Western Blot before the co-immunoprecipitation experiment.The results of coprecipitation(a simultaneous precipitation of more than one compound from a solution)showed that P62 and LC3 proteins could be detected in each sample when the target proteins were precipitated by P62 antibody and the target precipitates were detected by LC3 antibody.With further findings indicating that all groups could detect different degrees of precipitation when LC3 was used to precipitate P62 protein.P62 and LC3 proteins interacted rather weak in the self-healing group and relatively weak in the normal group,including the model group,while stronger in the zhiganfang groups and the western medicine group.3.Confocal microscopy revealed that the positive expression of PINK1 protein in the liver which was located in the cytoplasm,mainly concentrated around the central vein and the portal area of the subjects’ liver.Compared to the normal groups during the same period,the positive rates of PINK1 protein in the model groups were significantly lower(P<0.05).Comparisons to the 8w model group,except for the self-healing group and zhiganfang low dose group(P>0.05),results indicated the positive rates of PINK1 protein significantly increased in other treatment groups(P<0.05).In the comparison with the 12 w model group,the positive rates of PINK1 protein in each group significantly increased(P<0.05).Among the treatment groups,compared against the western medicine group,except the zhiganfang low dose group(P<0.05),the positive rates of PINK1 protein in each treatment group significantly increased(P<0.05).The positive expression of parkin protein in the liver located in the cytoplasm,nucleus or cell membrane respectively,mainly concentrated around the central vein and the portal area.Compared to the normal groups during the same period,the positive rates of parkin protein in the model groups were significantly lower(P<0.05).Compared with the 8w model group,except for the self-healing group(P>0.05),the positive rates of parkin protein in each treatment group significantly increased(P<0.05).Compared with the 12 w model group,the positive expressions of parkin protein in each group significantly increased(P<0.05).And the zhiganfang high dose group was significantly better/superior for rresulting in positive improvements of the liver cells than the western medicine group(P<0.05),while no significant difference between the zhiganfang low dose group and western medicine group(P>0.05).The positive expression of P62 protein in the liver located in the cytoplasm or nucleus mainly concentrated around the central vein and the portal area.Compared with the normal groups during the same period,the positive rates of P62 protein in the model groups significantly increased(P<0.05).Compared to the 8w model group,except for the self-healing group(P>0.05),the positive rates of P62 protein in each treatment group were significantly lower(P<0.05).Compared to the 12 w model group,the positive rates of P62 protein in each group were significantly lower(P<0.05).Compared with the western medicine group,except for the zhiganfang low dose group(P>0.05),the positive rates of P62 protein in other treatment groups obvious decreased(P<0.05).The positive expression of LC3 protein in the liver located in the cytoplasm or membranerespectively,mainly concentrated around the central vein and the portal area.Compared with the normal groups during the same period,the positive rates of LC3 protein in the model groups were significantly lower(P<0.05).Compared with the 8w model group,the positive rates of LC3 protein in each treatment group significantly elevated(P<0.05),except the zhiganfang low dose group and the self-healing group(P>0.05).Compared with the 12 w model group,the positive rates of LC3 protein in each group significantly increased(P<0.05).Among the treatment groups,compared against the western medicine group,except the zhiganfang low dose group(P>0.05),the positive rates of LC3 protein in other treatment groups significantly increased(P<0.05).Conclusions: High-fat intake in feeding can cause NAFLD and induce it to develop into NASH.Zhiganfang can significantly improve liver lipid levels and efficiency of liver function on NASH rats,and has an improved effect on NASH.There are interactions between PINK1 and parkin,P62 and LC3,and the interactions may affect the mechanism of mitochondria selective autophagy in NASH liver cells. |
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