上调GDF-15 表达对H2O2 诱导的H9C2 心肌细胞生物学特性及PI3K/ AKT 信号通路的影响

目的：探讨上调生长分化因子-15（GDF-15）的表达对H₂O₂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H₂O₂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、H₂O₂组、GDF-15+H₂O₂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、p-AKT蛋白表达。10 μmol/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+H₂O₂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H₂O₂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200 μmol/L的H₂O₂处理H9C2心肌细胞后可抑制将近一半的细胞增殖，选择200 μmol/L的H₂O2作为研究对象；与Control组比较，H2O2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H₂O₂组比较，GDF-15+H₂O₂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+H₂O₂组，细胞凋亡率及Bax蛋白表达显著高于GDF-15+H₂O₂组（P<0.05）。结论：上调GDF-15表达可促进H₂O₂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。

Effects of up regulation of GDF-15 expression on biological characteristics and PI3K/AKT signaling pathway in H9C2 cardiomyocytes induced byH2O2

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 H9C2 cardiomyocytes treated with different concentrations of H₂O₂;H9C2 cells were divided into Control group,NC group,H₂O₂ group,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 10 μmol/L LY294002(a PI3K/AKT signal pathway inhibitor),cell viability and apoptosis rate were detected by CCK8 assay and flow cytometry in espectively.Ki67,Bcl-2,Bax,PI3K and p-AKT protein expression were detected by Western blot.Results:Cell viability was inhibited after different concentrations H₂O₂ treated H9C2 myocardial cells,which was concentration dependent (P<0.05),due to H9C2 cardiomyocytes treated with 200 μmol/L H₂O₂inhibited nearly half of cell proliferation,and were chosen as subjects.Compared with control group,mRNA and protein expression of GDF-15 in H₂O₂ group 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+H₂O₂ group 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 significantly lower than those in GDF-15+H₂O₂ group,and the apoptosis rate and Bax protein expression were significantly higher than those in GDF-15+H2O2 group (P<0.05).Conclusion:Up regulation of GDF-15 expression promote the proliferation of H9C2 cardiomyocytes induced by H₂O₂and reduce apoptosis,and the mechanism may be related to the regulation of ROS levels,Ki67,Bcl-2,Bax expression and PI3K/AKT signaling pathway in cells.