[关键词]
[摘要]
目的 探讨艾司洛尔对缺氧复氧诱导的心肌细胞凋亡的影响,并初步研究其作用机制。方法 体外培养大鼠心肌细胞(H9c2),并建立缺氧/复氧(I/R)损伤模型,随机分为对照组、H/R组和艾司洛尔低、中、高剂量(0.2、5.0、25.0 μmol/L)组;噻唑蓝(MTT)法检测各组H9c2细胞存活率变化情况;膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)法检测各组H9c2细胞凋亡情况;蛋白印迹分析法检测各组H9c2细胞Bcl-2相关X蛋白(Bax)、半胱天冬氨酸酶-3(Caspase-3)、Bcl-2蛋白及腺苷单磷酸激活的蛋白激酶(AMPK)、糖原合酶激酶3β(GSK-3β)磷酸化水平;实时荧光定量PCR(qRT-PCR)法检测各组H9c2细胞AMPK、GSK-3β mRNA表达情况。结果 与对照组相比,H/R组H9c2细胞存活率、Bcl-2蛋白表达水平、AMPK、GSK-3β蛋白磷酸化水平显著降低(P<0.05),细胞凋亡率、Bax、Caspase-3蛋白表达水平显著升高(P<0.05);与H/R组相比,艾司洛尔低、中、高剂量组H9c2细胞存活率、Bcl-2蛋白表达水平、AMPK、GSK-3β蛋白磷酸化水平依次升高(P<0.05),细胞凋亡率、H9c2细胞Bax、Caspase-3蛋白表达水平依次降低(P<0.05),均呈剂量依赖性。结论 艾司洛尔能够抑制I/R诱导下心肌细胞的凋亡,可能与激活AMPK/GSK-3β磷酸化水平相关。
[Key word]
[Abstract]
Objective To study the effect of esmolol on cardiomyocyte apoptosis induced by hypoxia/reoxygenation and its mechanism. Methods The rat cardiomyocytes (H9c2) were cultured in vitro, and the model of hypoxia/reoxygenation (I/R) injury was established. H9c2 cells was divided into control group, H/R group, esmolol low dose group, esmolol medium dose group and esmolol high dose group (0.2、5.0、25.0 μmol/L). The survival rate of H9c2 cells was measured by methyl thiazolyl tetrazolium (MTT) method. The apoptosis of H9c2 cells was detected by Annexin V-FITC/PI method. The phosphorylation levels of Bcl-2 related X protein (Bax), Caspase-3, Bcl-2 protein, adenosine monophosphate-activated protein kinase (AMPK) and glycosylase kinase-3β (GSK-3β) in H9c2 cells were detected by Western blotting. The expression of AMPK and GSK-3β mRNAs in H9c2 cells were detected by real-time fluorescent quantitative PCR (qRT-PCR). Results Compared with the control group, the survival rate, Bcl-2 protein expression level, AMPK and GSK-3β protein phosphorylation levels of H9c2 cells in H/R group were significantly lower (P<0.05), and the apoptosis rate, Bax and caspase-3 protein expression levels were significantly higher (P<0.05). Compared with H/R group, the survival rate, Bcl-2 protein expression level, AMPK and GSK-3β protein phosphorylation levels of H9c2 cells in esmolol low, middle and high dose groups were increased in turn (P<0.05), and the apoptosis rate, Bax and caspase-3 protein expression levels of H9c2 cells were decreased in turn (P<0.05). Meanwhile, all of them were dose-dependent. Conclusion Esmolol can inhibit the apoptosis of cardiomyocytes induced by I/R, which may be related to the activation of AMPK/GSK-3β phosphorylation.
[中图分类号]
R966
[基金项目]
河南省中医药科学研究专项课题(2019ZY2139)