Résumé
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.
Résumé
Objective To investigate the effect of cysteine-rich protein 61 (Cyr61) on proliferation and cell cycle in human renal tubular epithelial cells (HK-2).Methods Cyr61 cDNA was cloned into pEGFP-N2,then HK-2 cells were transfected with the recombinant plasmid pEGFP-N2-Cyr61 by Lipofectamine.The cell proliferation was measured by MTT.The expression level of Cyr61,p-FAK and cyclin dependent cyclin-dependent kinase 2 (CDK2) protein were detected by Western blotting.The cell cycle and cell apoptosis were analyzed by flow cytometry.Results The recombinant plasmid pEGFP-N,-Cyr61 could be transfected into HK-2 efficiently.After transfection,the proliferative activity was significantly increased,the proportion of HK-2 cells in G1 phase decreased and in S-phase increased significantly,the level of cell apoptosis decreased markedly (all P ＜ 0.01).The expressions of Cyr61,p-FAK and CDK2 in Cyr61-transfected group were all amplified significantly (all P ＜ 0.01).Conclusions Cyr61 protein over-expressed in HK-2 cells can increase CDK2 expression throngh FAK pathway,resulting in the promotion of HK-2 cells entering into S phase,cell proliferation and the reduction of cell apoptosis.