重组人红细胞生成素对肾小管上皮细胞缺血再灌注损伤的保护作用

Effect of Recombinant Human Erythropoietin Pretreatment on PI3K- Akt- GSK- 3 β 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（PI3K）-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以及重组人红细胞生成素（rHuEPO）对其保护作用.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,20 μmol/L）、rHuEPO干预组（20 U/L）、rHuE-PO＋LY294002干预组、rHuEPO＋LiCl干预组.Western印迹法检测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干预组与IR组相比,细胞凋亡率下降（11.10%±1.62%）、Akt活性水平升高,GSK-3β及caspase-3酶活性下调,差异有统计学意义（P＜0.05）.与rHuEPO干预组相比,rHuEPO＋LY294002干预组细胞凋亡率升高（13.40%±1.94%）、Akt活性水平下降,GSK-3β及caspase-3酶活性上调,rHuEPO＋LiCl双干预细胞凋亡率下调（7.50%±1.31%）、Akt活性水平上升,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损伤可引起肾小管上皮细胞凋亡,Akt活性降低,GSK-3B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,减轻细胞凋亡,对HK-2IR损伤有一定的保护作用.

Objective To study the role of PI3K -Akt- GSK -3βsigualing in the cells apoptosis 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 received serum, ischemia - reperfusion （IR） group, LY294002 group received LY294002 （ AKT inhibitor） 10μmol/L 30 minutes before IR treated, LiC1 group received LiCI（ GSK -3β inhibitor） 20 μmol/L 30 minutes before IR treated, rHuEPO group received EPO 20 U/ml 30 minutes before IR treated, rHuEPO ＋ LY294002 group received EPO 20 U/ml and in the presence of LY294002 （ 10 μmoL/L） 30 minutes before IR treated, rHuEPO ＋ LiC1 group received EPO 20 U/m1 and in the presence of LiC1 （20 μmol/L） 30 minutes before IR treated. Akt,GSK -313 and caspase - 3 activation were measured by western blotting. The apoptotic ratio of HK - 2 cells was monitored by flow cytometry. Cell viability was monitored by MTF. Results In comparison with the control group, the apoptotic ratio raised up to （15.20% ± 1.43% ）, the expression of Akt activity decreased, GSK- 3βactivity and Caspase- 3 activity markedly elevated in IR group （P 〈 0.05 ）. LY29dO02 group up - regulated the apoptotic ratio （ 18.20% ±2.06% ）, decreasedthe expression of Akt activity and increased GSK -3βactivity and Caspase -3 activity. However, LiC1 group downregulated the apoptotic ratio （ 12. 30%± 0. 85% ）, increased the expression of Akt activity and decreased GSK 3βactivity 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 and decreased GSK- 3βactiyity and caspase- 3 activity compared with IR group （P 〈 0.05 ）. rHuEPO ＋ LY29dO02 group elevated the apoptotic ratio （ 13.40% ±1.94% ） ,decreased the expression of Akt activity and increased GSK - 3βactivity and caspase - 3 activity. Meanwhile, rHuEPO ＋ LiC1 group down - regulated the apoptotic ratio （ 7.50% ± 1.31% ）, increased the expression of Akt activity and decreased GSK - 3βactivity and caspase - 3 activity compared with rHuEPO group （ P 〈 0. 05 ）. Conclusion PI3 K -Akt -GSK -3βsignaling pathway is involved in HK -2 cells apoptosis induced by ischemia -reperfusion injury and rHuEPO may be used as a new therapy.