王晓霞 刘天龙 刘晶 刘小玲 张勇 肖云峰 刘小雷

中图分类号 R285 文献标志码 A 文章编号 1001-0408（2018）09-1189-05

DOI 10.6039/j.issn.1001-0408.2018.09.09

摘 要 目的：研究黄芪甲苷对D-半乳糖（D-gal）誘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、100 ?g/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Caspase-3、ANP mRNA的表达有关。

关键词 黄芪甲苷；D-半乳糖；原代心肌细胞；凋亡；保护作用

ABSTRACT 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 ?g/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.

KEYWORDS Astragaloside Ⅳ； D-galactose； Primary cardiomyocytes； Apoptosis； Protective effect

黄芪（Astragalus membranaceus）为豆科植物蒙古黄芪或膜荚黄芪的干燥根，是中蒙医临床处方中较重要的药材，而黄芪甲苷（Astragaloside Ⅳ）是黄芪的主要有效成分之一[1]。黄芪皂苷在心血管疾病的治疗中具有较大的应用潜力，有研究显示，其能够通过提高心肌细胞内Ca2+水平、抑制环磷腺苷水解、抗氧化应激及调节血压和脂质代谢等方式对多种原因引起的心肌损伤、心力衰竭及生理性衰老等具有一定保护作用[2]。但上述研究多使用的是黄芪总皂苷，而对于黄芪皂苷单体的生物学活性研究报道较少。……