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.

摘要：

目的 建立模拟缺血 / 再灌注损伤的肝细胞缺氧 / 复氧培养模型，从细胞水平探索重组人促红细胞生成素（recombinant human erythropoietin，rhEPO）的保护作用及分子机制。方法 大鼠肝细胞BRL3A 购于上海中科院细胞库，将复苏后呈指数生长的 BRL3A 细胞按照 2 000 个 /（100 μl·孔）的密度种植于 96 孔板，加入 H2O2（终浓度为 200 μmol/L）刺激细胞 2 h，取 2 ～ 3 代培养的 BRL3A 细胞随机分为4 组 ：① 正常对照组 ；② H2O2 处理组 ；③ H2O2 + 10 U/ml rhEPO 处理组 ；④ H2O2 + 20 U/ml rhEPO 处理组。将各组细胞继续培养 4 h 后，采用 CCK-8 测定细胞存活率，化学比色法检测培养上清中的天冬氨酸转氨酶（aspartate aminotransferase，AST）和丙氨酸转氨酶（alanine aminotransferase，ALT）含量，吖啶橙染色检测肝细胞自噬小体形成情况，Western Blot 检测细胞 LC3- Ⅰ，LC3- Ⅱ和 p62 表达情况，PI3K/AKT 蛋白磷酸化水平。结果 体外培养的 BRL3A 细胞经过 H2O2 刺激，细胞存活率明显下降，AST 和 ALT 的释放量明显增加，且细胞中酸性自噬小体形成明显增多，LC3- Ⅱ表达增加，同时 p62 蛋白减少，p-p85 和 p-AKT 表达亦减少。rhEPO 处理后上述情况得到明显改善，与对照组相比，细胞存活率增加，AST 和 ALT 释放减少，自噬小体减少，LC3- Ⅱ表达减少，且 p62、p-p85 和 p-AKT 均表达增加。结论 rhEPO 处理能显著减少肝细胞的模拟缺血 / 再灌注损伤，其机制可能与促进肝细胞 PI3K/AKT 蛋白磷酸化，降低自噬性细胞死亡有关。