重组人红细胞生成素预处理对肾小管上皮细胞缺血再灌注损伤中PI3K-Akt-GSK-3β通路的调控作用

Effect of recombinant human erythropoietin pretreatment on PI3K-Akt-GSK-3β signaling pathway in human renal tubular epithelial cells apoptosis induced by ischemia-reperfusion injury

目的 探讨磷脂酰肌醇-3激酶（P13K）-蛋白激酶B（Akt）-糖原合成酶激酶3β（GSK-3β）通路对人肾小管上皮细胞（HK-2）缺血再灌注（IR）损伤过程中细胞凋亡的调控及重组人红细胞生成素（rHuEPO）的保护作用.方法 正常培养的HK-2细胞,分为7组：正常对照组、IR组、LY294002干预组（P13K-Akt阻断剂,10 μmol/L）、LiCl干预组（GSK-3β阻断剂,20μmol/L）、rHuEPO干预组（20 U/L）、rHuEPO＋LY294002干预组、rHuEPO＋LiCl干预组.Westem印迹法榆测Akt（Ser473）、GSK-3β（Set9）及半胱氨酸天冬氨酸蛋白酶（caspase-3）活性;MTT法检测细胞活力;Annexin V和PI染色结合流式细胞仪技术检测细胞凋亡.结果 IR损伤诱导HK-2细胞凋亡率上调（15.20%±1.43%）、Akt活性水平下降、GSK-3β及caspase-3酶活性水平上调,与正常对照组相比,差异有统计学意义（P〈0.05）.与IR组相比,LY294002干预使细胞凋亡率进一步上调（18.20%＋2.06%）、Akt活性水平下调、GSK-3β及caspase-3酶活性上调,LiCl干预使细胞凋亡率下调（12.30%±0.85%）、Akt活性水平上调、GSK-3β及caspase-3酶活性下调,差异均有统计学意义（P〈0.05）. rHuEPO干预与IR组相比,细胞凋亡率下降（11.10%±1.62%）、Akt活性水平升高而GSK-3β及caspase-3酶活性下调,差异有统计学意义（P〈0.05）.与rHuEPO干预相比,rHuEPO＋LY294002双干预细胞凋亡率升高（13.40%±1.94%）、Akt活性水平下降而 GSK-3β及caspase-3酶活性上调,rHuEPO＋LiCI双干预细胞凋亡率下调（7.50%±1.31%）、Akt活性水平上升而GSK-3β及caspase-3酶活性下降,差异均有统计学意义（P〈0.05）.结论 IR损伤可引起肾小管卜皮细胞凋亡,Akt活性降低及GSK-3β活性升高,影响caspase-3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caspase-3酶活性,从而减轻细胞凋亡,对HK-2 IR损伤有一定的保护作用.

Objective To study the role of PI3K-Akt-GSK-3β signaling in the apoptosis of renal tubular cells after ischemia-reperfusion injury and the protective mechanism of recombinant human erythropoietin（rHuEPO）. Methods The human kidney tubular epithelial cells（HK-2）were cultured in vitro in different conditions as control group with serum, ischemia-reperfusion（IR）group, LY294002 group with LY294002（AKT inhibitor）10 μmol/L 30 minutes before IR treatment, LiCl group with LiCl（GSK-33 inhibitor）20 μtmol/L 30 minutes before IR treatment, rHuKPO group with EPO 20 U/ml 30 minutes before IR treatment, rHuEPO ＋ LY294002 group with EPO 20 U/ml and in the presence of LY294002（10 μmol/L）30 minutes before IR treatment, rHuEPO ＋LiCl group with EPO 20 U/ml and in the presence of LiCl（20 μmol/L）30 minutes before IR treatment. Akt, GSK-33 and caspase-3 activation were measured by Western blotting. The apoptotic ratio of HK-2 cells was measured by flow cytometry. Cell viability was detected by MTT. Results In comparison with the control group, the apoptotic ratio raised up to 15.20%±1.43%, the expression of Akt activity decreased, GSK-33 activity and caspase-3 activity markedly elevated in IR group（P〈0.05）. LY294002 group up-regulated the apoptotic ratio（18.20%±2.06%）, decreased the expression of Akt activity, increased GSK-33 activity and caspase-3 activity, however, LiCl group down-regulated the apoptotic ratio（12.30%±0.85%）, increased the expression of Akt activity, decreased GSK-33 activity and caspase-3 activity compared with IR group（P〈0.05）. rHuEPO group remarkably decreased the apoptotic ratio（11.10%±1.62%）, increased the expression of Akt activity, decreased GSK-33 activity and caspase-3 activity compared with IR group（P〈0.05）. rHuEPO＋LY294002 group elevated the apoptotic ratio（13.40%±1.94%）, decreased the expression of Akt activity, increased GSK-33 activity and caspase-3 activity, meanwhile, rHuEPO ＋LiCl group down-regulated the apoptotic ratio（7.50%±1.31%）, increased the expression of Akt activity, decreased GSK-33 activity and caspase-3 activity compared with rHuEPO group（P〈0.05）. Conclusions PI3K-Akt-GSK-3β signaling pathway is involved in HK-2 cells apoptosis induced by ischemia-reperfusion injury and rHuEPO may be used as a new therapy.