PKCε信号通路介导甘青铁线莲活性成分APG抗心肌缺血再灌注损伤的机制研究

摘要

目的 研究不同浓度甘青铁线莲活性成分APG对H9C2大鼠心肌细胞缺血再灌注损伤的保护作用及其可能的机制.方法 体外培养H9C2大鼠心肌细胞,经2 μM/L和4 μM/L的APG预处理24 h后,建立缺氧复氧损伤模型(I/R)(缺氧45 min,复氧3 h).采用CCK-8法检测细胞活力,检测细胞培养液乳酸脱氢酶(LDH)的释放量、丙二醛(MDA)的释放量和细胞内超氧化物歧化酶(SOD)的活力;采用Western Blot法检测细胞内蛋白激酶Cε(PKCε)、Caspase-3、Bax和Bcl-2表达情况,使用特异性PKCε抑制剂CHE观察PKCε信号通路在此过程中的作用.结果 I/R处理可抑制H9C2细胞活性,细胞培养基中LDH、MDA含量升高,SOD活力降低(与Control组比较,P<0.05).抑制PKCε表达,上调Caspase-3表达,下调Bcl-2/Bax比例(与Control组比较,P<0.05).2 μM/L和4 μM/L的APG预处理均可发挥细胞保护作用,降低LDH与MDA释放量,提高SOD活力(与I/R组比较,P<0.05).此外,APG处理对抗了I/R损伤引起的PKCε表达下调,抑制Caspase-3表达,提高了Bcl-2/Bax比例(与I/R组比较,P<0.05).CHE处理后细胞活力下降,Caspase-3表达上调,Bcl-2/Bax比例下降,凋亡增加(与APG+I/R组比较,P<0.05).结论 甘青铁线莲中活性成分APG可减轻心肌缺血再灌注损伤,其机制可能是激活PKCε相关信号通路,最终抑制心肌细胞凋亡.%Objective To investigate the protective effects of APG on myocardial ischemia/reperfusion (I/R) injury (IRI) and its possible mechanism.Methods The cultured H9C2 cells were treated with APG for 24 h,and subjected to IRI (I 45 min,R 3 h).After the 3 h reperfusion,cell viability,serum LDH and serum MDA,cell SOD activity,apoptotic index,PKCε expression,Caspase-3 expression,Bax expression and Bcl-2 expression were detected,then the specific inhibitor of PKCε CHE was applied to evaluate the roles of PKCε in this process.Results I/R treatment reduced cell viability significantly,decreased SOD activity and the Bcl-2/Bax ratio,down-regulated PKCε expression,up-regulated LDH levels,MDA levels and Caspase-3 expression (vs the control group,P<0.05).Moreover,APG treatment (both 2 μM/L and 4 μM/L) increased the cell viability,SOD activity and the Bcl-2/Bax ratio,down-regulated LDH levels,MDA levels and Caspase-3 expression,up-regulated PKCε expression in a dose dependent manner (vs the I/R group,P<0.05).In addition,compared with the APG+IR group,CHE treatment reduced the cell viability,up-regulated Caspase-3 expression,decreased the Bcl-2/Bax ratio (vs the APG+I/R group,P<0.05).Conclusion APG treatment ameliorates I/R injury via activating PKCε pathway and inhibiting apoptosis.