薯蓣皂苷含药血清抗氧化损伤致心肌细胞凋亡的研究

Research on the Mechanism of Dioscin-Containing Serum Against Apoptosis of Cardiomyocytes Induced by Hydrogen Peroxide

目的采用血清药理学方法探讨薯蓣皂苷含药血清对氧化损伤心肌细胞凋亡的作用及其机制。方法以血清药理学方法采集含药血清。培养原代乳鼠心肌细胞,建立氧化损伤模型,以含药血清进行干预,MTT法检测细胞存活率。Ho-echst33258荧光染色法观察药物作用后心肌细胞形态学变化。caspase-3活性测定方法探讨薯蓣皂苷抗凋亡途径。Westernblot方法测定薯蓣皂苷对Bcl-2及Bax蛋白表达的影响。结果 MTT法作用结果显示,将原始收集血清稀释为相当于灌胃0.4、0.8、1.2 g(生药).kg-1浓度时,相对于H2O2单纯损伤组和空白血清组心肌细胞有较高的存活率(P<0.01),且呈现一定的剂量依赖性。Hoechst33258荧光染色结果显示:与H2O2组相比,含药血清组随着药物浓度升高细胞核固缩凝聚减轻,碎片依次减少。与H2O2组相比,caspase-3活性随含药血清浓度增高而降低。与H2O2组相比,薯蓣皂苷给药组能够下调Bax水平,上调Bcl-2水平,且呈现剂量依赖性。结论薯蓣皂苷能够提高H2O2诱导的原代乳鼠心肌细胞存活率,降低caspase-3活性;减轻细胞核固缩和聚集,减少细胞核碎片;且能够降低Bax表达水平,提高Bcl-2表达水平。

OBJECTIVE To discuss the mechanism of dioscin against apoptosis of cardiomyocytes induced by hydrogen peroxide through Serum Pharmacologic Method. METHODS The drug-containing serum [ 0. 6 g（ crude drug）· kg- 1] was prepared by serum pharmacologic method. Cardiomyocytes from neonatal SD rats were cultured in Dulbecco Modified Eagle Medium（DMEM）. The primary cultured eardiomyocytes were injured by hydrogen peroxide before giving the drug-containing serum with different concentrations. The cardiomyocyte viability was determined by MTT method. Morphological changes of cardiomyocytes were observed by fluorescence micro- scope after treating with the drug-containing serum at different concentrations. The anti-apoptotic effect of dioscin was indicated by de- tecting the activity of easpase-3. The protein expression of Bcl-2 and Bax were semi-quantified by Western-blot method after treating with the drug-containing serum. RESULTS The cardiomyoeyte viability was elevated （P 〈 0. 01 ） after being treated with different concen- trates of drug-containing serum （0. 4, 0. 8, 1.2 g （crude drug） · kg-1, prepared from the original drug-containing serum）. Typical apop- totic features of the cardiomyocytes such as membrane blebbing, call shrinkage and detachment, and nucleus condensation and fragmenta- tion were dose-dependently improved after being treated with drug-containing serum. The activity of easpase-3 decreased with the increas- ing concentration of drug. Western blot approach showed that the Bcl-2 level increased meanwhile the Bax decreased. CONCLUSION Dioscin could increase the viability of the primary cultured eardiomyocytes in hydrogen peroxide induced injury. It could also decrease the activity of caspase-3, improve nucleus condensation and fragmentation, increase Bel-2 level and decrease the Bax level.