黄芪甲苷抑制血管紧张素Ⅱ诱导的心肌H9c2细胞凋亡

Astragaloside Ⅳ attenuates apoptosis of H9c2 cardiomyocytes induced by angiotensin Ⅱ

目的:探讨黄芪甲苷(ASⅣ)对血管紧张素Ⅱ(Ang Ⅱ)诱导的心肌H9c2细胞凋亡的作用及其机制。方法:用不同浓度Ang Ⅱ及ASⅣ处理心肌H9c2细胞,CCK-8法检测Ang Ⅱ及ASⅣ对心肌细胞活力的影响,选取Ang Ⅱ的最佳作用浓度为1μmol/L,ASⅣ浓度梯度设为25、50和100μmol/L。实验分为6组:对照组、ASⅣ组、Ang Ⅱ组、Ang Ⅱ+ASⅣ (25μmol/L)组、Ang Ⅱ+ASⅣ (50μmol/L)组和Ang Ⅱ+ASⅣ (100μmol/L)组。倒置相差显微镜下观察细胞形态并进行细胞计数检测细胞生长情况,TUNEL法检测细胞凋亡,DCFH-DA标记法检测活性氧簇(ROS)的水平, Western blot法检测Bax、Bcl-2、核因子E2相关因子2(Nrf2)及其下游因子血红素加氧酶1(HO-1)的表达情况。用negative control shRNA(NC)或Nrf2-shRNA(shRNA)质粒转染H9c2细胞,转染后实验分为8组,NC+control组、NC+AngⅡ组、NC+ASⅣ组、NC+AngⅡ+ASⅣ组、shRNA+control组、shRNA+AngⅡ组、shRNA+ASⅣ组和shRNA+AngⅡ+ASⅣ组,检测各组ROS水平及Nrf2和HO-1蛋白表达情况。结果:Ang Ⅱ呈浓度依赖性降低心肌H9c2细胞的活力,ASⅣ能逆转Ang Ⅱ对心肌H9c2细胞活力降低的作用,并呈浓度依赖性(P<0.05);与对照组相比,Ang Ⅱ组细胞凋亡率显著增加,ROS水平显著升高,Bax蛋白表达水平显著升高,Bcl-2、Nrf2和HO-1蛋白表达水平显著降低;与Ang Ⅱ组相比,ASⅣ可呈浓度依赖性逆转Ang Ⅱ诱导的心肌细胞凋亡率升高情况,降低ROS水平,下调Bax蛋白表达水平,上调Bcl-2、Nrf2和HO-1蛋白的表达水平(P<0.05)。转染shRNA后,ASⅣ降低Ang Ⅱ诱导的ROS产生及上调Nrf2和HO-1表达的作用被消除。结论:ASⅣ抑制Ang Ⅱ诱导的心肌H9c2细胞凋亡,这一保护作用与其降低ROS水平、介导Nrf2/HO-1信号通路相关。

AIM: To investigate the protective effect of astragaloside Ⅳ(ASⅣ) on angiotensin Ⅱ(Ang Ⅱ)-induced apoptosis of H9 c2 cardiomyocytes. METHODS: H9 c2 cardiomyocytes were treated with different concentrations of Ang Ⅱ and ASⅣ. The effects of Ang Ⅱ and ASⅣ on the viability of H9 c2 cells was measured by CCK-8 assay. The optimum concentration of Ang Ⅱ was 1 μmol/L and the concentrations of ASⅣ were 25, 50 and 100 μmol/L. The H9 c2 cells was divided into 6 groups: control group, ASⅣ group, Ang Ⅱ group, Ang Ⅱ+ASⅣ(25 μmol/L) group, Ang Ⅱ+ASⅣ 50(μmol/L) group and Ang Ⅱ+ASⅣ(100 μmol/L) group. The morphological changes of the H9 c2 cells were observed under inverted phase-contrast microscope. Apoptosis was detected by TUNEL assay. The generation of reactive oxygen species(ROS) was detected by DCFH-DA staining. The protein expression of Bax, Bcl-2, nuclear factor E2-related factor 2(Nrf2) and heme oxygenase-1(HO-1) was determined by Western blot. H9 c2 cardiomyocytes were transfected with negative control shRNA(NC) or Nrf2-shRNA(shRNA), and the cells were divided into 8 groups: NC+control group, NC+AngⅡgroup, NC+ASⅣ group, NC+AngⅡ+ASⅣ group, shRNA+control group, shRNA+AngⅡgroup, shRNA+ASⅣ group and shRNA+AngⅡ+ASⅣ group. ROS level was detected by ROS detection kit. The protein expression of Nrf2 and HO-1 was determined by Western blot. RESULTS: Ang Ⅱ decreased the viability of H9 c2 cells in a concentration-dependent manner(P<0.05). ASⅣ reversed the effect of Ang Ⅱ on the viability of H9 c2 cells in a concentration-dependent manner(P<0.05). Compared with control group, the apoptotic rate, the level of ROS and the protein expression of Bax in Ang Ⅱ group were increased significantly, while the protein expression of Bcl-2, Nrf2 and HO-1 was decreased significantly(P<0.05). Compared with Ang Ⅱ group, ASⅣ reversed the increase in apoptotic rate of H9 c2 cells induced by Ang Ⅱ in a concentration-dependent manner, reduced ROS level, down-regulated the protein expression of Bax and up-regulated the protein expression of Bcl-2, Nrf2 and HO-1(P<0.05). After shRNA transfection, the effects of ASⅣ decreasing ROS production induced by Ang Ⅱ and up-regulating the expression of Nrf2 and HO-1 were eliminated. CONCLUSION: ASⅣ protects H9 c2 cardiomyocytes from apoptosis induced by Ang Ⅱ, which may be related to reducing ROS generation and mediating the Nrf2/HO-1 signaling pathway.