| Objective: Nonalcoholic fatty liver disease(NAFLD)is a risk factor affecting human health in modern society,and its pathogenesis has not been fully elucidated.This study was designed to explore the mechanism of catalpol on the treatment of nonalcoholic fatty liver.Palmitic acid(PA)treated HepG2 cells were used as NAFLD's in vitro model,then investigate the inhibitory effect of catalpol on PA treated HepG2 via inhibit lipid accumulation,cell apoptosis and oxidative stress.And whether catalpol was achieved by up regulating miR-96-5p on p66 Shc / cyto C pathway.Methods: In this study we need to verify the following questions.First,whether catalpol can improve the lipid accumulation and apoptosis in PA treated HepG2 cells.Divided cells into three groups: normal HepG2 cells as control group,PA treated HepG2 cells as model group,PA treated HepG2 cells and catalpol as treatment group.Then Nile red staining,TUNEL staining and DNPI staining were used to determine lipid accumulation and apoptosis in cells.Second,whether catalpa can inhibit oxidative stress in PA treated HepG2 cells.We divided all cells into six groups: normal HepG2 cells as control group,catalpol treated HepG2 cells as catalpol group,PA treated HepG2 cells as model group,PA treated HepG2 cells and catalpol as treatment group,in this group,5,20,and 80 ?M of catalpol were added,and then divided them into low-concentration group,medium-concentration group,and high-concentration group.Then the expression levels of P66 Shc and Cyto C in each group were detected by Western blot to verify the inhibitory effect of catalpol on oxidative stress in vitro model,and to investigate whether the inhibitory effect was correlated with catalpol concentration.Thirdly,we want to know if catalpol can improve lipid accumulation,cell apoptosis and oxidative stress through the miR-96-5p /P66Shc/Cyto C pathway.Then,the experiment was divided into four groups: normal HepG2 cells as control group,PA treated HepG2 cells as model group,PA treated HepG2 cells and agomir-96-5p as upregulate group,PA treated HepG2 cells and catalpol as treatment group.The levels of lipid accumulation,apoptosis and oxidative stress were detected by Nile red,TUNEL,DNPI,Western blot and q PCR.From these detections we can verified that whether catalpol can treat NAFLD via miR-96-5p/P66Shc/Cyto C pathway.Results: Under fluorescence microscope,lipid accumulation and apoptosis were significantly increased in the model group compared with the control group,while lipid accumulation and apoptosis were significantly decreased in the treatment group after adding catalpa.Western blot results showed that the expression of p66 Shc and Cyto C in the model group was significantly higher than that in the control group(P < 0.05),while the expression of p66 Shc and Cyto C in the treatment group with catalpol was significantly inhibited(P < 0.05).However,compared with the low concentration group,the medium concentration group and the high concentration group,there was no positive correlation between the concentration of catalpol and its inhibitory effect.After using agomir-96-5p to up regulate the expression of mir-96-5p,compared with the model group,the up-regulated group showed a significant decrease in lipid accumulation and apoptosis,and Western blot showed that the expression of p66 Shc and cyto C in the up-regulated group was significantly inhibited(P < 0.05).There was no significant difference between the up-regulated group and the catalpol group in the inhibitory effect on lipid accumulation,cell apoptosis,and expression of p66 shc and Cyto C.Conclusion: Catalpol can inhibit lipid accumulation,apoptosis and oxidative stress in PA treated HepG2 cells model,and miR-96-5p/p66shc/cytochrome C cascade may be a potential target.Catalpol can significantly increase the level of miR-96-5p and reduce the expression of p66 Shc and cyto C in PA treated HepG2 cells via miR-96-5p / p66 Shc / cyto C pathway. |
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