The Study On The Effect And Mechanism Of Ptf-5 In The Treatment Of Non-alcoholic Fatty Liver Disease

Objective:Our previous in vivo study have shown that protective effect of Pingtang Recipe 5(PTF-5)in alleviating lipid accumμlation in liver,however,the underlying mechanism remained unclear.To date,mounting studies have revealed the involvement of autophagy in the pathogenesis and treatment of non-alcoholic fatty liver disease.Therefore,the present study aimed to investigate whether protective effect of PTF-5 in treating non-alcoholic fatty liver disease was mediated by autophagy using both in vitro and in vivo study.Methods:In vitro study: HepG2 cells were introduced and treated with 0.25 mM palmitic acid(PA)for 72 hours to mimic NAFLD in vitro.Sprague Dawley Rats was given PTF-5 or saline as control by gastric gavage for 7 continuous days.Serum containing PTF-5 and control serum was collected and applied to PA-stimμlated HepG2 to measure protective effect of PTF-5.Involvements of autophagy were determined by further addition of specific agonist(Rapamycin)or inhibitor(3-MA).Oil red O staining and triglyceride kit was applied to detect the accumμlation of triglycerides;ROS level was determined by detection kits of MDA,GSH and SOD;Inflammation was determined by the mRNA level of iNOS,TNF-α,and IL-1β.Autophagy was determined by P62,Beclin-1 and LC3 at both mRNA by qPCR and protein level by Western Blot.Transmission electron microscope was applied to confirm autophagy in each group.In vivo study: After a week of adaptive feeding,the C57BL/6J mice were randomly divided into the control group,the model group and the PTF-5 treated group.The control group was given the ordinary chow.Both model group and the PTF-5treated group were given high fat diet(60%)for two weeks before PTF-5 treatment.The control group and the model group received saline as control and the treated group received PTF-5 by daily gastric gavage.After another 10 weeks,level of TG and TC in serum were detected by the kits;Inflammation was determined by the mRNA level of iNOS,TNF-α,COX-2 and IL-1β.Autophagy was determined by P62 and Beclin-1 at both mRNA by qPCR and protein level by Western Blot.Results:In vitro study: Compared with the control group,the content of triglyceride in the model group was increased accompanied with enhanced oxidative stress and inflammatory response.Autophagy was inhibited as indicated by P62,LC3 and Beclin-1.Compared with the model group,the lipid accumμlation,oxidative stress and inflammatory response was ameliorated by treatment with PTF-5 containing serum accompanied with activation of autophagy.The protective effect of PTF-5 containing serum was abrogated by 3-MA,the inhibitor of autophagy.In vivo study: Compared with the control group,the content of TG and TC in the serum of the model group was increased,inflammatory response was enhanced and the level of proteins and genes of autophagy was decreased;Compared with the model group,the content of TG and TC in the serum of the treatment group decreased significantly,inflammatory response was alleviated and the level of proteins and genes of autophagy increased.Conclusion:Taken these into together,it was suggested that PTF-5 coμld reduce the content of triglycerides in liver cells,alleviate oxidative stress and inflammatory response via activating autophagy.Therefore,targeting autophagy coμld be a potential approach for treatment of NAFLD.