白藜芦醇抑制叔丁基过氧化物诱导的外周血内皮祖细胞损伤

摘要

目的:观察白藜芦醇对叔丁基过氧化物诱导的人外周血内皮祖细胞(EPC)凋亡的影响,探讨其可能机制.方法:密度梯度离心法获取人外周血单个核细胞,培养4 d后,收集贴壁细胞.实验分为对照组、叔丁基过氧化物诱导组和白藜芦醇+叔丁基过氧化物组.采用二苯基四氮唑溴盐(MTT)比色法和5-溴脱氧尿嘧啶核苷(BrdU)ELISA法检测EPC增殖能力;采用改良Boyden小室法检测EPC增殖及迁移能力;Annexin V-FITC/PI染色及流式细胞术检测EPC凋亡率;比色法检测caspase-3活性;荧光探针H2DCF-DA法检测细胞内活性氧簇(ROS)水平;Western blot检测cleaved caspase-3、Bax及Bcl-2蛋白的表达.结果:白藜芦醇能抑制叔丁基过氧化物诱导的EPC凋亡,并增加EPC增殖及迁移能力;白藜芦醇降低EPC细胞内ROS水平,抑制caspase-3的活性及cleaved caspase-3的蛋白水平,同时能促进Bcl-2及抑制Bax蛋白表达.结论:白藜芦醇对叔丁基过氧化物诱导的EPC凋亡有抑制作用,其机制可能与抗氧化应激有关.%AIM:To investigate the effect of resveratrol on the apoptosis of endothelial progenitor cells (EPC) induced by tert-butyl hydroperoxide (tBHP) and the underlying mechanisms.METHODS:Total mononuclear cells were isolated from peripheral blood by density gradient centrifugation, and the cells were cultured on fibronectin-coated culture dishes.After 4 d of culture, attached cells were divided into control group, tBHP group, and resveratrol plus tBHP group (pretreated with resveratrol for 24 h and then cultured with 100 μmol/L tBHP for 6 h).The proliferation and migration abilities of the EPC were assessed by MTT assay, BrdU incorporation assay and modified Boyden chamber assay.The proportion of apoptotic EPC was determined by flow cytometry after staining with fluorescein isothiocyanate-conjugated Annexin V and propodium iodide.The level of reactive oxygen species (ROS) was determined by H2DCF-DA method.Caspase-3 activity was assay using a caspase-3 colorimetric assay kit.The protein levels of cleaved caspase-3, Bcl-2 and Bax were determined by Western blot.RESULTS:Resveratrol decreased EPC apoptosis induced by tBHP in a dose-dependent manner.Moreover, resveratrol increased the proliferation and migration abilities of the EPC.Resveratrol decreased intracellular ROS level, caspase-3 activity and the protein level of cleaved caspase-3.Resveratrol also decreased protein expression of Bax and increased protein expression of Bcl-2.CONCLUSION:Resveratrol attenuates EPC apoptosis induced by oxidative stress, and its mechanisms may be related to protecting the mitochondrial function.