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

关键词:  Elabela  同型半胱氨酸  凋亡  H9C2细胞

Key words:  Elabela  homocysteine  apoptosis  H9C2 cells

摘要:

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，1mmol/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。

Abstract:

ObjectiveTo explore the role and mechanism of Elabela（ELA）on apoptosis of rat cardiomyocyte H9C2 cell induced by homocysteine. MethodsH9C2 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. ResultsCompared 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). ConclusionElabela can inhibit homocysteine-induced apoptosis of H9C2 cells through the ERK/MAPK signaling pathway.