白藜芦醇通过抑制线粒体凋亡途径缓解热刺激诱导的小鼠心肌细胞凋亡

Alleviation of resveratrol on the mouse cardiac myocytes apoptosis induced by thermal stimulation via regulating mitochondrial-mediated signaling pathway

目的:探讨白藜芦醇(Res)对热刺激诱导的小鼠心肌细胞(MCM)凋亡的保护作用。方法:复制热刺激诱导MCM凋亡模型。首先采用实时无标记细胞分析技术检测Res对MCM存活率的影响;然后采用Annexin V-FITC/7-AAD双染法检测不同浓度Res给药组对MCM凋亡率的影响。JC-1探针法检测线粒体膜电位,初步确证MCM凋亡依赖于线粒体凋亡途径。利用全自动荧光酶标仪检测荧光探针(Ac-LEHD)2-R110标记的Caspase-9相对表达量;利用流式细胞仪检测荧光探针TF2-DEVD-FMK标记的Caspase-3/7的相对表达量;Western Blot法检测Bax、Bcl-2、COXⅣ蛋白的表达;ELISA方法检测细胞色素C(Cyt-C)含量、比色法测定Cyt-C氧化酶活性。结果:与模型组比较,Res高剂量及中剂量组细胞凋亡率显著降低(P<0.05,P<0.01),各组均可显著改善线粒体膜电位(P<0.01),Res高剂量及低剂量组Cyt-C释放量显著降低(P<0.05,P<0.01),Res各组均可显著降低Caspase-9活性(P<0.01),R es高剂量组可显著降低Ca s p a s e-3/7活性(P<0.01),B c l-2/B ax比值显著升高(P<0.01,P<0.05),Res干预后Cyt-C氧化酶活性均升高但差异无统计学意义。结论:Res通过抑制线粒体凋亡途径缓解热刺激诱导的MCM凋亡。

Objective:To explore the protective effect of resveratrol(Res)on the mouse cardiac myocytes(MCM)apoptosis induced by thermal stimulation.Methods:The model of MCM apoptosis induced by thermal stimulation was replicated.Firstly,the survival rate of Res to MCM was detected by real time cell analysis system.Then,Annexin V-FITC/7-AAD double staining method was used to detect the effects of different concentrations of Res on MCM apoptosis.JC-1 probe method was used to detect mitochondrial membrane potential,which preliminarily confirmed that MCM apoptosis induced by depended on mitochondrial apoptosis pathway.The relative expression levels of Caspase-9 labeled with fluorescent probe(Ac-LEHD)2-R110 and Caspase-3/7 labeled with fluorescent probe TF2-DEVD-FMK were detected by enzyme-linked immunosorbent assay and flow cytometry,respectively.The expression of Bax,Bcl-2 and COXⅣprotein was detected by Western Blot.Cytochrome C(Cyt-C)was detected by ELISA,and the activity of Cyt-C oxidase was determined by colorimetry.Results:Compared with the model group,the apoptosis rate in the high dose and middle dose groups of Res were significantly reduced(P<0.05,P<0.01),the mitochondrial membrane potential was significantly improved in all giving drug groups(P<0.01),and the release of Cyt-C in the high dose and low dose groups of Res were significantly reduced(P<0.05,P<0.01),the activity of Caspase-9 was significantly decreased in all groups of Res(P<0.01),the activity of Caspase-3/7 was significantly decreased in high dose group of Res(P<0.01),and the ratio of Bcl-2/Bax was significantly increased(P<0.01,P<0.05).Cyt-C oxidase activity was increased after Res intervention but the difference was not marked between Res groups and model group.Conclusion:Res can alleviate the apoptosis of MCM induced by thermal stimulation via regulating mitochondrial-mediated signaling pathway.