Abstract:

Objective To investigate the protective role and molecular mechanism of recombinant human erythropoietin（rhEPO）in preventing hepatocytes against simulated ischemia-reperfusion injury. Methods BRL3Acells were purchased from cell bank of chinese academy of sciences in Shanghai and cultured in 96-well plates with 2 000（100 μl·well），H2O2 was added to induce cell oxidative damage at a concentration of 200 μmol/L for 2 h. Cultured BRL3A cells were divided into 4 groups ：① normal control group ；② H2O2 group ；③ H2O2 +10 U/ml rhEPO group ；④ H2O2 + 20 U/ml rhEPO. Continued culture for 4 h，CCK-8 was performed to test cell survival rate.The aspartate aminotransferase（AST）and alanine aminotransferase（ALT）was measured with chemical colorimetry.Acridine orange staining was used to detect the formation of autophagosome in hepatocytes. The protein expressionof LC3- Ⅰ，LC3- Ⅱ，p62，and the phosphorylation level of PI3K/AKT signal pathway were detected by western-blot. Results Treated with H2O2，the cell survival rate of BRL3A cells in vitro decreased significantly. The release of AST and ALT was significantly increased，and the formation of autophagosomes was significantly increased，LC3- Ⅱ expression was increased，p62 protein was decreased，and the expression of p-p85 and p-AKT was also decreased. After rhEPO treatment，the situation above was significantly improved. Compared with the control group，the cell survival rate increased，AST and ALT release decreased，autophagosome decreased，LC3- Ⅱ expression decreased，and p62，p-p85 and p-AKT increased. Conclusion The rhEPO treatment can mitigate simulated ischemia-reperfusion injury in hepatocytes via increasing phosphorylation of PI3K/AKT and decreasing autophagic cell death.