刺五加叶皂苷B对过氧化氢诱导的心肌细胞凋亡的抑制作用

Inhibitory effects of Ciwujianoside B on apoptosis induced by hydrogen peroxide in neonatal rat cardiomyocytes

目的:探讨刺五加叶皂苷B(C-B)对过氧化氢(H2O2)诱导的乳鼠心肌细胞凋亡的影响,阐明其对氧化应激损伤所致的心肌细胞凋亡的抑制作用。方法:乳鼠心肌细胞行原代培养,取自主搏动良好的心肌细胞随机分为正常对照组、H2O2损伤组(200μmol.L-1H2O2诱导心肌细胞凋亡)、100mg.L-1C-B组和200mg.L-1C-B组,倒置相差显微镜观察细胞形态,MTT法检测细胞存活率,AnnexinⅤ-FITC流式细胞术检测凋亡率,分光光度法检测Caspase-3活性,免疫印迹法检测细胞Caspase-3、PARP、Bcl-2和Bax蛋白表达。结果:与正常对照组比较,200μmol.L-1H2O2组心肌细胞有损伤性改变,细胞存活率明显降低(P<0.001),凋亡率升高(P<0.001),Caspase-3活性增加(P<0.01),细胞Bcl-2蛋白表达减少,Caspase-3及Bax蛋白表达增多,PARP蛋白降解增多。与H2O2损伤组比较,100及200mg.L-1C-B组心肌细胞损伤性改变均减轻,细胞存活率增加(P<0.05或P<0.01),凋亡率降低(P<0.001),Caspase-3活性降低(P<0.05或P<0.01),细胞Bcl-2蛋白表达增加,Bax蛋白表达和Caspase-3蛋白表达减少,PARP蛋白降解减少(依据电泳条带的宽窄及明暗度判断蛋白表达程度),且200mg.L-1C-B组的作用更明显。结论:C-B对H2O2诱导的心肌细胞凋亡有一定的抑制作用,可能与其调节细胞内凋亡相关蛋白Caspase-3、Bcl-2、Bax的表达有关。

Objective To observe the effect of Ciwujianoside B （C-B） on apoptosis induced by hydrogen peroxide （H2O2 ） in cardiomyocytes and the expressions of its related regulating proteins, and elanify the inhibitory effect of C-B on apoptosis induced by injury of oxidative stress in cardiomyocytes. Methods The neonatal rat cardiomyocytes were primary cultivated and the cells which beat autonomously well were randomly divided into normal control group, H2O2 group, 100 mg · L^-1 C-B group and 200 mg· L^-1 C-B group. The cardiomyocytes were exposed to 200 t, mol · L^-1 H2O2 for 120 min to induce apoptosis. The morphological changes of cardiomyocytes were observed by microscope, the survival rate was detected by MTT. The apoptotic rate was analyzed by flow cytometry labelled with Annexin V-FITC. The activity of Caspase-3 was detected by spectrophotometric method. The expressions of Caspase-3, PARP, Bcl-2 and Bax proteins were determined by immunoblotting. Results Compared with normal control group, the injuried cardiomyocytes were seen and the survival rate was decreased significantly （P〈0. 001）, the apoptotic rate and Caspase-3 activity were increased （P〈 0. 00） or P〈0.01）; the expressions of Caspase-3, Bax and the degradation of PARP were raised, and the expression of Bcl-2 was decreased in 200 μmol · L^-1H2O2 group. Compared with 200 μmol · L^-1 H2O2 group, the injurled changes of cardiomyoeytes were reduced, the survival rates were significantly increased （P〈0.05 or P〈 0.01）, the apoptotie rates were decreased （P〈0. 001）; the activities of Caspase-3 were decreased significantly （P〈0.05 or P〈0.01）; the expressions of Caspase-3 and Bax were decreased, the degradation of PARP was attenuated, and the expressions of Bcl-2 were increased in 100 and 200 mg· L ^-1 C-B groups. Conclusion C-B can obviously inhibit the apoptosis induced by H2O2 in cardiomyoeytes, its mechanism may be related to the regulation of the expressions of the apoptosis-related proteins Caspase-3, Bax and Bcl- 2.