上调GDF-15表达对H_2O_2诱导的H9C2心肌细胞生物学特性及PI3K/AKT信号通路的影响  

Effects of up regulation of GDF-15 expression on biological characteristics and PI3K/AKT signaling pathway in H9C2 cardiomyocytes induced by H_2O_2

作　　者：王军[1] 张松林[1] 和旭梅[1] 何璐[1] 范粉灵[1] WANG Jun;ZHANG Song-Lin;HE Xu-Mei;HE Lu;FAN Fen-Ling(Department of Structural Cardiology, the First Affiliated Hospital of Xi＇an Jiaotong University, Xi＇ an 710061, China)

机构地区：[1]西安交通大学第一附属医院结构性心脏病科,西安710061

出　　处：《中国免疫学杂志》2018年第5期658-664,669,共8页Chinese Journal of Immunology

基　　金：国家自然科学基金(No.81170294);西安交通大学第一附属医院课题基金(No.2016MS-09)资助

摘　　要：目的:探讨上调生长分化因子-15(GDF-15)的表达对H_2O_2诱导的H9C2心肌细胞增殖、凋亡及PI3K/AKT信号通路的影响。方法:CCK8法检测不同浓度的H_2O_2处理H9C2心肌细胞后的细胞增殖情况;H9C2心肌细胞分为Control组、NC组、H_2O_2组、GDF-15+H_2O_2组,各组细胞处理24 h后收集细胞,RT-PCR及Western blot分别检测各组细胞中GDF-15的mRNA及蛋白表达;CCK8法及流式细胞术分别检测细胞的增殖和凋亡情况;2',7'-二氯二氢荧光素黄二乙酸酯(DCFH-DA)探针检测细胞活性氧簇(ROS)水平;Western blot检测Ki67、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、PI3K、pAKT蛋白表达。10μmol/L的PI3K/AKT信号通路抑制剂LY294002处理H9C2心肌细胞,通过CCK8法及流式细胞术分别检测GDF-15+H_2O_2组及PI3K/AKT信号通路抑制剂组细胞活力及凋亡率,Western blot检测Ki67、Bcl-2、Bax、PI3K、p-AKT蛋白表达。结果:不同浓度H_2O_2处理H9C2心肌细胞后,细胞活力均受到抑制,且有浓度依赖性(P<0.05),由于200μmol/L的H_2O_2处理H9C2心肌细胞后可抑制将近一半的细胞增殖,选择200μmol/L的H_2O_2作为研究对象;与Control组比较,H_2O_2组GDF-15的mRNA及蛋白表达均显著升高,细胞增殖显著降低,凋亡率增加,ROS水平升高,Ki67、Bcl-2、PI3K、p-AKT蛋白表达降低,Bax蛋白表达升高(P<0.05);与H_2O_2组比较,GDF-15+H_2O_2组细胞GDF-15的mRNA及蛋白表达均显著升高,细胞增殖显著增加,凋亡率降低,ROS水平降低,Ki67、Bcl-2、PI3K、p-AKT蛋白表达升高,Bax蛋白表达降低(P<0.05)。PI3K/AKT信号抑制剂组细胞活力及Bcl-2、PI3K和p-AKT的蛋白表达均显著低于GDF-15+H_2O_2组,细胞凋亡率及Bax蛋白表达显著高于GDF-15+H_2O_2组(P<0.05)。结论:上调GDF-15表达可促进H_2O_2诱导的H9C2心肌细胞增殖,降低细胞凋亡,其机制可能与调节细胞中ROS水平,Ki67、Bcl-2、Bax表达及PI3K/AKT信号通路有关。Objective：To investigate the effect of up regulation of GDF-15 expression on the proliferation,apoptosis and PI3K/AKT signaling pathway of H9C2 cardiomyocytes induced by H2O2.Methods：CCK8 method was used to detect the proliferation of H9C2cardiomyocytes treated with different concentrations of H2O2;H9C2 cells were divided into Control group,NC group,H2O2group,GDF-15＋H2O2group,the cells were treated for 24 h,mRNA and protein expression of GDF-15 in each group were detected by RT-PCR and Western blot;the proliferation and apoptosis of the cells were detected by CCK8 and flow cytometry respectively;DCFH-DA probe was used to detect the level of ROS;the expression of Ki67,Bcl-2,Bax,PI3K and p-AKT protein was detected by Western blot.H9C2 cells were treated with 10μmol/L LY294002（a PI3K/AKT signal pathway inhibitor）,cell viability and apoptosis rate were detected by CCK8 assay and flow cytometryin espectively.Ki67,Bcl-2,Bax,PI3K and p-AKT protein expression were detected by Western blot.Results：Cell viability was inhibited after different concentrations H2O2treated H9C2 myocardial cells,which was concentration dependent（P〈0.05）,due to H9C2 cardiomyocytes treated with 200μmol/L H2O2inhibited nearly half of cell proliferation,and were chosen as subjects.Compared with control group,mRNA and protein expression of GDF-15 in H2O2group were significantly increased,cell proliferation was decreased significantly,the apoptosis rate was increased,ROS level was increased,the expression of Ki67,Bcl-2,PI3K,p-AKT protein were decreased,Bax protein expression was increased（P〈0.05）.Compared with H2O2group,mRNA and protein expression of GDF-15 in GDF-15＋H2O2group were significantly increased,cell proliferation was significantly increased,the apoptosis rate was decreased,ROS level was decreased,the expression of Ki67,Bcl-2,PI3K,p-AKT protein were increased,and Bax protein expression was decreased（P〈0.05）.The cell viability and protein expression of Bcl-2,PI3K and p-AKT in PI3K/AKT inhibitor group were si

关 键 词：GDF-15 H2O2 心肌细胞 增殖 凋亡 PI3K/AKT信号通路 

分 类 号：R542.2[医药卫生—心血管疾病]