重组人促红细胞生成素对肝细胞模拟缺血/再灌注损伤的保护作用  

The role of recombinant human erythropoietin in protecting hepatocytes against simulated ischemia-reperfusion injury

作　　者：李龙 温陈 解维敏 刘文玉 刘波 陶开山 窦科峰 慕喜喜 Li Long;Wen Chen;Xie Weimin;Liu Wenyu;Liu Bo;Tao Kaishan;Dou Kefeng;Mu Xixi(Department of Hepatobiliary Surgery,Shenmu Hospital,Shenmu 719300,Shaanxi,China;Department of Hepatobiliary Surgery,Xijing Hospital,the Fourth Military Medical University,Xi’an 710032,Shaanxi,China;Department of General Surgery,Xi’an Central Hospital,Xi’an 710003,Shaanxi,China)

机构地区：[1]神木市医院肝胆外科,陕西神木719300 [2]空军军医大学西京医院肝胆外科,陕西西安710032 [3]西安市中心医院普通外科,陕西西安710003

出　　处：《实用器官移植电子杂志》2019年第3期198-202,共5页Practical Journal of Organ Transplantation(Electronic Version)

基　　金：国家重点研发计划资助项目(2017YFC1103703);国家973计划资助项目(2015CB554100)

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。Objective To investigate the protective role and molecular mechanism of recombinant human erythropoietin( rhEPO) in preventing hepatocytes against simulated ischemia-reperfusion injury. Methods BRL3A cells 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 expression of LC3-Ⅰ,LC3-Ⅱ, p62, and the phosphorylation level of PI3K/AKT signal pathway were detected by westernblot. 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 ischemiareperfusion injury in hepatocytes via increasing phosphorylation of PI3K/AKT and decreasing autophagic cell death.

关 键 词：重组人促红细胞生成素 肝细胞 氧化应激 自噬 

分 类 号：R657.3[医药卫生—外科学]