ECE1过表达通过eNOS/NO通路促进Ang Ⅱ诱导的体外培养大鼠心肌细胞肥大和凋亡

Over-expression of ECE1 promotes AngⅡ-induced hypertrophy and apoptosis of rat cardiomyocytes in vitro through eNOS/NO signaling pathway

目的:探讨过表达内皮素转换酶1(ECE1)对血管紧张素Ⅱ(AngⅡ)诱导的体外培养大鼠H9C2心肌细胞肥大和凋亡的影响。方法:采用AngⅡ作用于体外培养的H9C2心肌细胞,建立心肌细胞肥大模型。将细胞分为空白对照组、AngⅡ组、AngⅡ+质粒空载体(AngⅡ+NC)组和AngⅡ+ECE1过表达组。CCK-8法检测细胞活力;RT-qPCR检测ECE-1及心脏肥大因子心房钠尿肽(ANP)和脑钠肽(BNP)的mRNA表达水平;流式细胞术检测心肌细胞凋亡;Western blot检测ECE1、Bcl-2、Bax、内皮型一氧化氮合酶(eNOS)和p-eNOS的蛋白水平;硝酸还原酶法检测细胞中一氧化氮(NO)含量。结果:与空白对照组相比,AngⅡ组中ECE1表达、ANP和BNP mRNA表达、Bax蛋白表达及细胞凋亡显著增加(P<0.05),而细胞活力、NO含量及Bcl-2和p-eNOS蛋白水平显著下降(P<0.05);与AngⅡ组相比,AngⅡ+ECE-1过表达组中ECE1表达、ANP和BNP的mRNA表达、Bax蛋白表达及细胞凋亡进一步增加(P<0.05),而细胞活力、NO含量及Bcl-2和p-eNOS蛋白水平进一步下降(P<0.05)。结论:ECE1过表达可能通过调控eNOS/NO通路而促进AngⅡ诱导的体外培养大鼠H9C2心肌细胞发生肥大和凋亡。

AIM:To explore the effect of endothelin-converting enzyme 1(ECE1)over-expression on angiotensinⅡ(AngⅡ)-induced hypertrophy and apoptosis of rat H9C2 cardiomyocytes in vitro.METHODS:The H9C2 cells were induced by AngⅡto establish cardiomyocyte hypertrophy model.The cells were divided into control group,AngⅡgroup,AngⅡ+empty vector(AngⅡ+NC)group and AngⅡ+ECE-1 over-expression group.Cell viability was measured by CCK-8 assay.The mRNA expression of ECE-1,and cardiac hypertrophy factors atrial natriuretic peptide(ANP)and brain natriuretic peptide(BNP)was detected by RT-qPCR.The apoptosis level was analyzed by flow cytometry.The protein levels of ECE1,Bcl-2,Bax,endothelial nitric oxide synthase(eNOS)and p-eNOS were determined by Western blot.Nitric oxide(NO)content in each group was detected by nitrate reductase method.RESULTS:Compared with control group,the expression of ECE1,the mRNA levels of ANP and BNP,the protein level of Bax and the apoptosis rate in AngⅡgroup were significantly increased(P<0.05),while the cell viability,the NO content and the protein levels of Bcl-2 and p-eNOS were significantly decreased(P<0.05).Compared with AngⅡgroup,the expression of ECE1,the mRNA levels of ANP and BNP,the protein level of Bax and the apoptosis rate in AngⅡ+ECE-1 over-expression group were further increased(P<0.05),while the cell viability,the NO content and the protein levels of Bcl-2 and p-eNOS were further decreased(P<0.05).CONCLUSION:Over-expression of ECE1 might promote AngⅡ-induced hypertrophy and apoptosis of H9C2 cardiomyocytes in vitro through regulating the eNOS/NO signaling pathway.