黄芪甲苷对D-半乳糖诱导原代培养心肌细胞凋亡的保护作用及机制研究

摘要

目的:研究黄芪甲苷对D-半乳糖(D-gal)诱导原代培养心肌细胞凋亡的保护作用及机制.方法:分离和培养Wistar乳鼠原代心肌细胞,将细胞分为正常对照组(无血清DMEM高糖培养基)、D-gal组(含5 g/L D-gal的无血清DMEM高糖培养基)和黄芪甲苷低、中、高质量浓度组(分别先以含25、50、100μg/mL黄芪甲苷的无血清DMEM高糖培养基培养1 h,然后换为含有相应质量浓度黄芪甲苷和5 g/L D-gal的无血清DMEM高糖培养基).采用CCK-8法检测培养48 h后细胞活性,分别采用Hoechst染色法(培养24 h)和流式细胞术(培养48 h)考察细胞凋亡情况,采用实时荧光定量-聚合酶链式反应法检测培养24 h后细胞中凋亡相关因子(Bcl-2、Bax、Caspase-3)和心房钠尿肽(ANP)mRNA表达水平.结果:与正常对照组比较,D-gal组细胞的活性以及细胞中Bcl-2 mRNA水平、Bcl-2/Bax比值降低,细胞凋亡率和细胞中Bax、Caspase-3、ANP mRNA水平升高,差异均有统计学意义(P<0.05或P<0.01).与D-gal组比较,黄芪甲苷各浓度组细胞的活性以及细胞中Bcl-2 mRNA水平、Bcl-2/Bax比值均升高,细胞凋亡率和细胞中Bax、Caspase-3、ANP mRNA水平均降低,差异均有统计学意义(P<0.05或P<0.01),且具有一定浓度依赖性.结论:黄芪甲苷对D-gal诱导的原代心肌细胞凋亡具有一定的保护作用,其机制可能与升高细胞中Bcl-2/Bax比值和下调细胞中Cas-pase-3、ANP mRNA的表达有关.%OBJECTIVE:To study the protective effect and mechanism of astragaloside Ⅳ on D-galactose(D-gal)-induced primary cardiomyocytes apoptosis. METHODS:Wistar neonatal rat primary cardiomyocytes were isolated and cultured. The cardiomyocytes were divided into normal control group(DMEM high glucose medium without serum),D-gal group(DMEM high glucose medium without serum containing 5 g/L D-gal)and astragaloside Ⅳ low-concentration,medium-concentration and high-concentration groups(after cultured with DMEM high glucose medium without serum containing 25,50,100 μg/mL astragaloside Ⅳ for 1 h,and then replaced with DMEM high glucose medium without serum containing corresponding concentration of astragaloside Ⅳ and 5 g/L D-gal). Cardiomyocytes activity was detected by CCK-8 kit after cultured for 48 h.The apoptosis level was detected by Hoechst staining(cultured for 24 h)and flow cytometry(cultured for 48 h). The mRNA expressions of apoptosis related factors(Bcl-2,Bax,Caspase-3)and ANP in cardiomyocytes were detected by RT-PCR after cultured for 24 h. RESULTS:Compared with normal control group,the activity of cardiomyocytes,mRNA level of Bcl-2 and ratio of Bcl-2/Bax were decreased in D-gal group,while apoptosis rate and mRNA levels of Bax,Caspase-3 and ANP were increased, with statistical significance(P<0.05 or P<0.01). Compared with D-gal group,the activity of cardiomyocytes,mRNA level of Bcl-2 and ratio of Bcl-2/Bax were increased in astragaloside Ⅳ groups,while apoptosis rate and mRNA levels of Bax,Caspase-3 and ANP were decreased,with statistical significance(P<0.05 or P<0.01),and in concentration-dependent manner. CONCLUSIONS:Astragaloside Ⅳ can protect D-gal induced primary cardiomyocytes from apoptosis,the mechanism of which may be associated with the increase of Bcl-2/Bax ratio and the decrease of mRNA expressions of Caspase-3 and ANP.