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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 investigate the effect and mechanism of diosgenin-containing serum on cardiomyocyte apoptosis by serum pharmacology. Methods Serum pharmacological methods were used to collect serum containing drugs. Primary neonatal rat cardiomyocytes were cultured, oxidative damage model was established, and drug-containing serum was used for intervention. Cell viability was detected by MTT assay. Ho-echst33258 fluorescence staining was used to observe the morphological changes of myocardial cells. To explore the anti-apoptotic pathway of diosgenin by caspase-3 activity assay. The effect of diosgenin on the expression of Bcl-2 and Bax protein by Western blotting. Results The results of MTT assay showed that when the original serum was diluted to 0.4, 0.8 and 1.2 g (crude drug) .kg-1, the myocardial cells had a higher survival rate than the H2O2 injury group and the blank serum group Rate (P <0.01), and showed a certain dose-dependent manner. Hoechst33258 fluorescence staining showed: Compared with H2O2 group, drug-containing serum group with the drug concentration increased nuclear condensation shrinkage reduced, followed by the reduction of debris. Compared with H2O2 group, caspase-3 activity decreased with the increase of serum concentration. Compared with H2O2 group, diosgenin administration group could down-regulate Bax level and up-regulate Bcl-2 level in a dose-dependent manner. Conclusion Diosgenin can increase the survival rate of neonatal rat cardiomyocytes induced by H2O2, reduce the activity of caspase-3, reduce the nuclear condensation and aggregation, reduce the nuclear fragmentation, reduce the expression of Bax and increase the expression of Bcl-2.