蚤休醇提物对H2O2损伤的ECV304细胞的细胞周期与凋亡的影响

Effects of bistortae ethanol extraction on cell cycle and apoptosis of ECV304 injured by hydrogen peroxide

目的制备中药蚤休醇提物,研究其对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,然后分为五个实验处理组:①正常对照组;②氧化损伤组;③高浓度蚤休醇提物(EFB500ng.L-1)组;④中浓度蚤休醇提物(50ng.L-1)组,⑤低浓度(5ng.L-1)组。应用流式细胞仪AnnexinV/PI染色检测该药物干预H2O2氧化损伤前后的细胞凋亡、PI染色检测细胞周期与凋亡的关系;RT-PCR检测各实验组caspase-3mRNA表达的变化。结果采用流式细胞仪PI染色检测结果对细胞周期进行分析,表明损伤作用于ECV-304细胞后,细胞在G0/G1期的数目增多,在S期的数目减少,同时流式细胞仪检测可见亚二倍体峰,而蚤休醇提物预处理以后,S期细胞数明显增多。流式细胞仪AnnexinV/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。caspase-3mRNA水平在各实验组的表达显示损伤组与内参照相比的灰度值最大,经过预处理之后其值明显下降(P<0.01)。结论蚤休醇提物可以保护H2O2造成的ECV304细胞的氧化损伤,是通过保护细胞周期正常进行、抑制凋亡蛋白caspase-3介导的凋亡信号转导通路实现的。

Aim To make ethanol from bistortae （EFB） from traditional Chinese medicine bistortae, and investigate the protection on the injury of the human umbiliar vein endothelial cell （ECV304） induced by hydrogen peroxide （H2O2）. Methods ECV304 cells were cultured and divided by five experimental groups including normal, injury model induced by H2O2 and three pretreated groups with EFB; flow cytomctry （FCM） methods Annexin V/PI staining and PI staining were used to analyze the effect of the medicine on the apoptosis and cell cycle;reverse transcription PCR （RT-PCR） was used to detect the mRNA expression level of apoptosis associated protein caspase-3 in all experiment-al groups. Results FCM Annexin V/PI stai-ning result showed early and advanced stage apoptosis rate was lower when predisposed than that of the damaged group, FCM PI staining showed there were more cells in G0/G1 stage in oxidation damage group, but less in S stage, and hypodiploid peak could be observed,RT-PCR showed that the expression of caspase- 3 was weakened when predisposed by EFB than without this disposal （ P 〈 0. 01 ）. Conclusions EFB has the protective action on oxidative damage of ECV304 induced by H2O2 ,the mechanism may relate to the protection of nomal cell cycle and the decrease of the cell signal transduction of aoootosis via casnase-3.