The Influence Of Reduced Glutathione On Apoptosis Of L02Hepatocyte Which In The Process Of Fatty Degeneration

Objective To establish the cell model of NAFLD in vitro with palmitic acid, and observe the hepatocyte apoptosis and the expression of Smac/DIABLO dynamically; Then to explore the influence and possible mechanism of GSH on NAFLD.Method1. We choose palmitic acid to induce steatosis in L-02hepatocyte to establish NAFLD cell model in vitro, make the trials divided into normal control group and palmitic model group, and select24、48、72hours for experiments. Then, the accumulation of lipid droplets in the hepatocytes with Oil Red O staining was observed by optical microscopy, the apoptosis was evaluated by Annexin-V FITC/PI double staining combining Flow Cytometer(FCM), and the expression of Smac/DIABLO was detected by Immunocytochemistry(ICC).2. We chose the optimal concentration range of GSH by MTT assay and select72h for experiments, then make the trials divided into normal control group, palmitic model group, and reduced glutathione (low, medium and high concentration) intervention group. The apoptosis and the expression of Smac/DIABLO were detected by using the above methods.Result 1.(1) PA-induced hepatocytes steatosis was successfully established and verified by Oil Red O staining with lipid droplets accumulation inside the L-02hepatocytes cytoplasms. The lipid droplets accumulation became more prominent as PA treatment prolonged.(2) Annxin-V FITC/PI double staining combining FCM showed that the ratio of hepatic apoptosis in palmitic acid groups was gradually increased accompanied with the prolonging of the culture time in vitro, and it was significantly increased compared with the normal control groups (P<0.05).(3) ICC showed that there were weak positive expression of Smac/DIABLO in several hepatocytes at48h,72h in the normal control groups. The positive rate of Smac/DIABLO expression in palmitic acid groups was gradually increased accompanied with the prolonging of the culture time, and it was significantly increased compared with the normal control groups (P<0.05).2.(1) The result of MTT assay showed that different concentration of GSH produce different effect on L02cells. When the concentration of GSH was less than20μmol/L, the cells survival rate was more than80%; But When it was more than40μ mol/L, the cells survival rate decreased obviously, showed inhibition effect and had statistically significant differences compared with the normal control group. So we choose GSH5,10,20μ mol/L as the appropriate follow-up concentration.(2) After given GSH, we found that all GSH(low, medium and high concentration) intervention group can reduce the ratio of apoptosis and the positive rate of Smac/DIABLO, and it was significantly reduced compared with the palmitic acid group (P<0.05). The protection became more and more obvious along with the increasing concentration.Conclusion1. Palmitic acid can induce L02hepatic steatosis and establish NAFLD cell model in vitro.2. Smac/DIABLO, the proapoptosis protein of mitochondria, may be involved in palmitic acid-induced hepatocyte apoptosis in vitro of NAFLD.3. Reduced glutathione (GSH) inhibits hepatocyte apoptosis may be by down-regulating the expression of Smac/DIABLO protein.