草苁蓉多糖对氧化应激所致血管内皮细胞凋亡的抑制作用

Inhibition of Boschniakia rossica Polysaccharides on Oxidative Stress-Induced Apoptosis in Vascular Endothelial Cells

目的:研究草苁蓉多糖(Boschniakia rossica polysaccharides,BRPS)对氧化应激所致血管内皮细胞凋亡的抑制作用。方法:利用叔丁基过氧化氢(tert-butyl hydroperoxide,t BHP)损伤人脐静脉血管内皮细胞制备细胞氧化应激损伤模型,采用四甲基偶氮唑盐(methylthiazolyldiphenyl-tetrazolium bromide,MTT)比色法检测细胞存活率,分光光度法检测细胞内丙二醛(malondialdelyde,MDA)水平、超氧化物歧化酶(superoxide dismutase,SOD)活力和还原型谷胱甘肽(reduced glutathione,GSH)水平。采用二氯荧光乙酰乙酸盐(dichlorofluorescein diacetate,DCFH-DA)法检测细胞内活性氧(reactive oxygen species,ROS)水平,采用JC-1荧光染色法观察细胞线粒体跨膜电位水平,采用Hoechst染色和原位末端标记(Td T-mediated d UTP nick end labeling,TUNEL)法观察细胞凋亡情况。Western blot法检测细胞Bax、Bcl-2、细胞色素c(cytochrome c,Cyt c)、Bid片段(truncated Bid,t Bid)、Caspase-3、Caspase-8和Caspase-9蛋白的表达情况。结果:BRPS提高t BHP损伤的内皮细胞存活率,降低细胞ROS水平,减少MDA生成,提高SOD活性与GSH水平,升高ΔΨm水平,抑制细胞凋亡。Western blot结果显示,BRPS降低胞浆Cyt c、线粒体t Bid水平,减小细胞Bax/Bcl-2比值,抑制Caspase-3、Caspase-8和Caspase-9的活化。结论:BRPS对氧化应激所致血管内皮细胞凋亡具有抑制作用,机制可能与线粒体凋亡途径和死亡受体途径均有关。

Objective： To study the inhibitory effect of Boschniakia rossica polysaccharides （BRPS） on oxidative stressinduced apoptosis in vascular endothelial cells in vitro. Methods： A cellular model was established by treating human umbilical vein endothelial cells with tert-butyl hydroperoxide （tBHP）. Cell viability was tested by methylthiazolyldiphenyltetrazolium bromide （MTT） assay, and the levels of malondialdehyde （MDA）, superoxide dismutase （SOD） and reduced glutathione （GSH） were measured spectrophotometrically. The reactive oxygen species （ROS） was observed using the fluorescent probe dichlorofluorescein diacetate （DCFH-DA）, the mitochondrial membrane potential （ΔΨm） was evaluated by JC-1 staining, and the apoptotic changes were detected by Hoechst staining and TdT-mediated dUTP nick end labeling （TUNEL） assay. The protein expression of Bax, Bcl-2, cytochrome c （Cyt c）, truncated Bid （tBid）, caspase-3, caspase-8 and caspase-9 were determined by Western blotting. Results： Pretreatment with BRPS significantly increased cell viability,reduced ROS level, inhibited MDA formation, increased SOD activity and GSH level, elevated ΔΨm, and suppressed tBHP induced apoptosis in vascular endothelial cells. Western blotting results showed that BRPS reduced mitochondrial tBid, reduced cytoplasmic Cyt c and Bax/Bcl-2 ratio, and suppressed the activation of caspase-3, caspase-8 and caspase-9 in vascular endothelial cells. Conclusion： BRPS has an inhibitory effect on oxidative stress-induced apoptosis in vascular endothelial cells in vitro, likely through inhibiting mitochondrial apoptosis and death receptor pathways.