中文摘要：

目的 观察富含半胱氨酸蛋白61（Cyr61）对缺氧后人肾小管上皮细胞（HK-2）氧化应激的影响,探讨Cyr61对HK-2的保护机制.方法 将HK-2细胞分为5组：空白组、Cyr61处理组、MAPK抑制剂组（Cyr61＋ PD98059预处理）、p38抑制剂组（Cyr61 ＋SB203580预处理）、PI3K抑制剂组[Cyr61＋渥曼青霉素（Wortmannin）预处理].各组细胞预处理12h后进行缺氧培养.采用四甲基偶氮唑盐（MTT）法检测缺氧前后细胞存活率,流式细胞术检测细胞凋亡率,双氯荧光黄乙酸乙酯（DCFH-DA）染色检测细胞内活性氧簇（ROS）的生成量,比色法检测细胞中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性,Western印迹法检测细胞磷酸化（p）Akt及细胞核内Nrf2蛋白表达水平. 结果 缺氧培养后空白组HK-2细胞内ROS含量和Nrf2蛋白表达增加,SOD、CAT活性下降,同时细胞存活率下降,凋亡率增高（均P＜0.05）.Cyr61处理组缺氧后存活率、细胞内SOD、CAT活性和p-Akt、Nrf2蛋白显著高于空白组,同时其凋亡率及ROS含量明显低于空白组（均P＜ 0.05）.与Cyr61处理组相比,缺氧后PI3K抑制剂组HK-2细胞存活率、细胞内SOD、CAT活性和p-Akt、Nrf2蛋白显著下降,凋亡率及ROS含量显著增加（均P＜0.05）;而MAPK抑制剂组和p38抑制剂组HK-2细胞的各项指标同Cyr61处理组相比差异无统计学意义（均P＞ 0.05）.结论 Cyr61可通过PI3K途径促进Nrf2表达,增强抗氧化物质SOD、CAT产生,减少ROS生成,在缺氧诱导的HK-2细胞氧化应激损伤中发挥保护作用.

英文摘要：

Objective To investigate the effect and mechanism of cysteine-rich protein 61 （Cyr61） on oxidative stress in human kidney tubular epithelial cell line after anoxia.Methods Human kidney tubular epithelial cell line （HK-2 cells） were divided into 5 groups：control group,Cyr61 group,MAPK inhibitor group （Cyr61 ＋PD98059）,p38 inhibitor group （Cyr61 ＋SB203580） and PI3K inhibitor group （Cyr61＋Wortmannin）.Each group was pretreated for 12 h and then injured by anoxia.The cell viability was determined by MTT assay and the apoptosis rate of HK-2 cells was determined by flow-cytometry.The cellular ROS level was measured by spectro-fluorometry.The cellular superoxide dismutase （SOD） and catalase （CAT） were measured by nephelometry test.The expression of Nrf2 in HK-2 cells was detected by Western blotting.Results Anoxia enhanced the expression of ROS and Nrf2,decreased the expression of SOD and CAT significantly,meanwhile decreased HK-2 viability and increased HK-2 apoptosis （all P 〈 0.05）.Cyr61 increased the expression of p-Akt,Nrf2,SOD and CAT in HK-2,and decreased the expression of ROS,at the same time increased HK-2 viability and decreased HK-2 apoptosis （all P 〈 0.05）.Wortmannin inhibited the expression of p-Akt,Nrf2,SOD and CAT,meanwhile decreased HK-2 viability and increased HK-2 apoptosis （P 〈 0.05）.PD98059 and SB203580 had no affect on HK-2 compared to Cyr61 group （P〉0.05）.Conclusions Cyr61 promotes the expression of Nrf2 through PI3K pathway in HK-2,which enhances the expression of SOD and CAT,and decreases the expression of ROS.Cyr61 exhibits protective effects on HK-2 cells injured by oxidative stress after anoxia.