蚤休皂苷对过氧化损伤致ECV304细胞凋亡机制的研究

Effects of Pariphyllin on ECV304 apoptosis induced by overoxidation

目的研究蚤休皂苷对H2O2诱导的脐静脉内皮细胞(ECV304细胞)损伤的保护作用及其机制。方法体外培养ECV304细胞,建立氧化损伤的细胞模型,然后分为5个组:正常对照组,氧化损伤组,蚤休皂苷高浓度(1000pg/ml)组、中浓度(100pg/ml)组、低浓度(10pg/ml)组;应用流式细胞仪Annexin V/PI染色检测药物干预H2O2氧化损伤前后的细胞凋亡情况,应用激光共聚焦显微镜观察其凋亡的形态学变化。并利用激光共聚焦显微镜观察、图像分析仪分析各组细胞中游离Ca2+的表达;结果流式细胞仪Annexin V/PI检测方法显示,损伤组的早期与晚期凋亡率之和最高,预处理后细胞的早期与晚期凋亡率之和降低。在损伤组细胞核着红色,细胞膜绿色或者细胞核将游移出细胞,而蚤休皂苷药物浓度增加,表现为红色细胞核的细胞量即晚期凋亡量逐渐减少。损伤组细胞内游离Ca2+浓度明显高于正常组(P<0.01),而各蚤休皂苷预处理组均使Ca2+荧光强度下降,显著低于损伤组(P<0.01),并呈剂量依赖性效应。结论蚤休皂苷可以保护ECV304细胞因H2O2造成的氧化损伤,并与抑制钙离子介导的凋亡通路有关,达到保护内皮细胞、抗动脉粥样硬化的目的。

Objective To study the protection of Pariphyllin on human umbilical vein endothelial cell （ECV304） apoptosis induced by hydrogen peroxide（ H2O2 ）. Methods The cells were divided into normal, oxidative damage, high density Pariphyllin （ 1 000 pg/ml）, medium density Pariphyllin（ 100 pg/ml） and low density Pariphyllin（ 10 pg/ml） groups. Apoptosis rate was tested by flow cytometry （FCM） Annexin V/PI staining. The density of calciumion （ Ca^2＋） was observed by laser confocal microscopy （LCM）. Results The apoptosis rate in early and advanced stage of oxidative damage group was the highest, and it decreased after predisposed. In oxidative damage, the nucleus appeared red and cell membrane appeared green. With the increase in drug concentration, the apoptosis rate was gradually reduced. Further- more in damaged group, the density of external CaCa^2＋ was higher than that of normal group （ P 〈 0. 01 ）. But in pretreated groups, the density of external Ca^2＋ was lower than that of oxidative damage group （P 〈 0. 01 ） , and this effect was dose dependent. Conclusions By decreasing the cell apoptosis induced by Ca^2＋ mediated signal transduction, Pariphyllin has protective action on oxidative damage of HUVEC induced by H2O2, to achieve the purpose of protection endothelial cell and anti-atherosclerosis.