PI3K/Akt/GSK-3β信号通路在肾小管上皮细胞缺血再灌注损伤中的调控作用及重组人红细胞生成素预保护效应

Effect of rHuEPO Pretreatment on PI3K/Akt/GSK-3β Signaling Pathway in Human Renal Tubular Epithelial Cells Apoptosis Induced by Ischemia-reperfusion Injury

目的探讨PI3K/Akt/GSK-3β通路对人肾小管上皮细胞(HK-2)缺血再灌注损伤过程中细胞凋亡的调控及重组人红细胞生成素(rHuEPO)的预保护作用。方法正常培养的HK-2细胞,分为7组,正常对照组、缺血再灌注(I/R)组、LY294002干预组(PI3K/Akt阻断剂10μmol/L)、LiCl干预组(GSK-3β阻断剂20μmol/L)、rHuEPO干预组(20U/L)、rHuEPO+LY294002双干预组、rHuEPO+LiCl双干预组。Western blot法检测蛋白激酶B(Akt)、AktSer473、糖原合成酶激酶3β(GSK-3β)、GSK-3βSer9及半胱氨酸天冬氨酸蛋白酶(Caspase)3活性;MTT法检测细胞活力;An-nexinⅤ/PI染色结合流式细胞仪技术检测细胞凋亡。结果缺血再灌注损伤诱导HK-2细胞凋亡率上调[(15.2±1.4)%]、Akt活性水平下降、GSK-3β及Caspase3酶活性水平上调,与正常对照组相比,差异均有统计学意义(均P<0.05)。与I/R组相比,LY294002干预使细胞凋亡率进一步上调[(18.2±2.1)%]、Akt活性水平下调、GSK-3β及Caspase3酶活性上调;LiCl干预使细胞凋亡率下调[(12.3±0.8)%]、Akt活性水平上调、GSK-3β及Caspase3酶活性下调,差异均有统计学意义(均P<0.05)。rHuEPO干预与I/R组相比,细胞凋亡率下降[(11.1±1.6)%]、Akt活性水平升高而GSK-3β及Caspase3酶活性下调,差异均有统计学意义(均P<0.05)。与rHuEPO干预组比较,rHuEPO+LY294002双干预组细胞凋亡率升高[(13.4±1.9)%]、Akt活性水平下降而GSK-3β及Caspase3酶活性上调;rHuEPO+LiCl双干预组细胞凋亡率下调[(7.5±1.3)%]、Akt活性水平上升而GSK-3β及Caspase3酶活性下降,差异均有统计学意义(均P<0.05)。结论缺血再灌注损伤可引起肾小管上皮细胞凋亡,Akt活性降低及GSK-3β活性升高,影响Caspase3依赖的外源性凋亡途径可能是其凋亡机制之一。rHuEPO可通过增强Akt活性,降低GSK-3β及Caspase3酶活性,减少细胞凋亡,对HK-2缺血再灌注损伤有一定的保护作用。

Objective To probe into the PI3K/Akt/GSK-3β signaling regulatory mechanism of HK-2 apoptosis induced by ischemia-reperfusion （I/R） injury and the protective effect mechanism of rHuEPO.Methods The human kidney tubular epithelial cells（HK-2）were cultured in vitro under different conditions as①control group receiving serum,②I/R group,③LY294002 group receiving LY294002（AKT inhibitor）10 μmol/L 30 min before I/R,④LiCl group receiving LiCl（GSK-3β inhibitor）20 μmol/L 30 min before I/R,⑤rHuEPO group receiving EPO 20 U/L 30 min before I/R,⑥rHuEPO＋LY294002 group receiving EPO 20 U/L and LY294002（10 μmol/L）30 min before I/R,and ⑦ rHuEPO＋LiCl group receiving EPO 20 U/L and LiCl （20 μmol/L）30 min before I/R.Akt,GSK-3β and Caspase 3 activities were measured by Western blot.The apoptotic ratio of HK-2 cells was examined by flow cytometry.Cell viability was measured by MTT.Results In comparison with the control group,the apoptotic ratio was increased up to （15.2±1.4）%,the expression of Akt was decreased,GSK-3β activity and Caspase 3 activity were remarkably elevated in I/R group（P〈0.05）.In LY294002 group,the apoptotic ratio was increased to （18.2±2.1）%,the expression of Akt was reduced,GSK-3β activity and Caspase-3 activity were increased.However,in LiCl group the apoptotic ratio was decreased to （12.3±0.8）%,the expression of Akt was increased,and the GSK-3β activity and Caspase 3 activity were decreased as compared with I/R group（P〈0.05）.In rHuEPO group,the apoptotic ratio was remarkably decreased to （11.1±1.6）%,the expression of Akt was increased,and the GSK-3β activity and Caspase 3 activity were reduced as compared with I/R group（P〈0.05）.In rHuEPO＋LY294002 group the apoptotic ratio was elevated to（13.4±1.9）%,the expression of Akt activity was decreased,and GSK-3β activity and Caspase 3 activity were increased,and in rHuEPO＋LiCl group the apoptotic ratio was reduced to（7.5±1.3）%,the expression of Akt was increased,and the GSK-3β activity and Caspase 3 activity were reduced as compared with rHuEPO group（P〈0.05）.Conclusion PI3K/Akt/GSK-3β signaling pathway is involved in HK-2 cells apoptosis induced by I/R injury and rHuEPO may exert the protective effect during the process above.