金丝桃苷对叠氮钠诱导PC12细胞凋亡的保护作用

摘要

AIM:To investigate the neuroprotective effects of hyperoside (Hyp) on PC 12 cells apoptosis induced by sodium azide and the mechanism of action.METHODS:To induce apoptosis,cultured PC12 cells was incubated with sodium azide 20 mmol.L-1 for 3-24 h.Pretreatment with Hyp (0.01,0.1 or 1μmol.L-1) for 4 h before sodium azide treatment.Cell viability was measured by MTT assay.The chromatin-specific dye Hoechst 33342 was used to observe nuclear changes.Caspase-3 activity was detected with a protease assay using Ac-DEVD-AMC as a substrate.Reactive oxygen species (ROS) was estimated by CM-H2DCFDA assay.Bcl-2 and Bax expression was determined by Western blotting.RESULTS:Pretreatment with Hyp (0.01,0.1 or 1 μmol.L-1) for 4 h attenuated sodium azide-mediated apoptosis,and the anti-apoptotic action of Hyp was partially dependent on suppressing the production of ROS,inhibition of caspase-3 activity and associated with increasing the expression of the anti-apoptotic protein Bcl-2,decreasing the expression of pro-apoptotic protein Bax.In addition,the caspase-3 inhibitor could reduce ROS formation in a time-dependent manner.CONCLUSION:The results suggest that Hyp has the neuroprotective capacity to attenuate sodium azide-induced apoptosis in PC12 cells.Our data also indicate that the generation of ROS is associated with the activity of caspase-3 during apoptosis.%目的:研究金丝桃苷对叠氮钠诱导的PC12细胞凋亡的保护作用.方法:将PC12细胞与不同浓度金丝桃苷共同孵育4h后,加入20 mmol.L-1的叠氮钠继续孵育3～24 h,观察金丝桃苷的保护作用.采用MTT、Hoechst 33342分别检测细胞存活率和细胞形态变化.以Ac-DEVD-AMC为底物检测caspase-3活性,CM-H2DCFDA法用于测定ROS水平.Western blotting用于分析Bcl-2和Bax表达水平.结果:金丝桃苷在0.01,0.1及lμmol.L-1浓度时,可显著提高叠氮钠作用后PC12细胞的存活率,抑制细胞凋亡,减少细胞内活性氧自由基生成和caspase-3活性,提高抗凋亡蛋白Bcl-2表达,降低促凋亡因子Bax表达.同时结果显示caspase-3抑制剂呈时间依赖性地减少细胞内活性氧自由基含量.结论:金丝桃苷对叠氮钠诱导的PC12细胞凋亡具有保护作用,活性氧自由基生成与凋亡进程中caspase-3活性密切相关.