Elabela对同型半胱氨酸诱导的H9C2细胞凋亡的影响与机制

Effect of Elabela on H9C2 cell apoptosis induced by homocysteine and its mechanism

目的探究多肽Elabela(ELA)对同型半胱氨酸(homocysteine,Hcy)诱导的H9C2细胞凋亡的作用及机制。方法采用CCK-8检测细胞活性,根据试验结果选择Elabela和Hcy的适宜浓度建立细胞实验模型。随机将细胞分成NC组(正常对照组)、ELA组、Hcy组、Hcy+ELA组。使用试剂盒检测ROS含量;利用TUNEL荧光染色和流式细胞术检测细胞的凋亡程度;通过Western印迹法检测H9C2细胞中PARP、Bcl-2、Bax、cleaved-Caspase3蛋白以及MAPK信号通路相关蛋白的表达水平。使用ERK特异性抑制剂U0126验证相关信号通路作用,检测细胞中凋亡相关蛋白的表达水平。结果与NC组相比,1 mmol/L的Hcy处理使H9C2细胞活性明显下降(P<0.001),而10μmol/L的Elabela显著逆转了Hcy诱导的H9C2细胞活性下降(P<0.001)。与Hcy处理组相比,Elabela的干预使细胞内ROS的产生减少,凋亡率下降,促凋亡蛋白cleaved-PARP、Bax、cleaved-Caspase3蛋白的表达水平下调,而Bcl-2、p-ERK蛋白的表达水平上调,p-P38、p-JUNK蛋白的表达水平下调(均P<0.05)。与Hcy+ELA组相比,U0126的干预抑制了Elabela对H9C2细胞的保护作用,使细胞促凋亡相关蛋白的表达水平上调,而Bcl-2的表达水平下调(均P<0.05)。结论多肽Elabela可能通过调节ERK/MAPK信号通路抑制Hcy诱导的H9C2细胞的凋亡。

Objective To explore the role and mechanism of Elabela(ELA)on apoptosis of rat cardiomyocyte H9C2 cell induced by homocysteine.Methods H9C2 cell viability was assessed by cell counting kit-8(CCK-8)to determine the appropriate concentration of ElA and homocysteine.H9C2 cells were randomly divided into normal control group(NC group),ELA group,Hcy group,Hcy+ELA group.The content of reactive oxygen species(ROS)in H9C2 was detected by ROS kit.The apoptosis level of H9C2 was analyzed by TUNEL fluorescence and flow cytometry.The expressions of apoptosis-related proteins PARP,Bcl-2,Bax,cleaved-Caspase3 and MAPK signaling pathway related proteins were analyzed by Western blotting.After ERK/MAPK signaling pathway was inhibited by U0126,the expressions of apoptosis-related proteins were also analyzed.Results Compared with the NC group,the treatment of 1 mmol/L homocysteine reduced the cell viability of H9C2(P<0.01),however,10μmol/L Elabela significantly reversed the reduction on cell viability(P<0.01).Compared with the Hcy group,the intervention of Elabela reduced the generation of ROS and the apoptosis in H9C2,downregulated the expressions of pro-apoptotic proteins including cleaved-PARP,Bax and cleaved-Caspase3,upregulated the expressions of Bcl-2,p-ERK,and downregulated the expressions of p-P38,p-JUNK(P<0.05).Compared with the Hcy+ELA group,U0126 reduced the protective effect of Elabela in H9C2,upregulated the expressions of cleaved-PARP,Bax and cleaved-Caspase3,downregulated the expression of Bcl-2(P<0.05).Conclusion Elabela can inhibit homocysteine-induced apoptosis of H9C2 cells through the ERK/MAPK signaling pathway.