蜕皮甾酮对同型半胱氨酸诱导人血管内皮细胞凋亡的影响

摘要

Objective To investigate the effect of ecdysterone ( EDS) against homocysteine( Hcy)-induced apopto-sis of human umbilical vein endothelial cells(CRL-1730). Methods CRL-1730 cells were cultured in vitro routine-ly with different concentrations of EDS. The viability of CRL-1730 cells was detected by cell counting Kit-8 assay. Hcy treatment induced apoptosis in CRL-1730 cells, CRL-1730 cells were divided into 5 groups: control group, high dose of EDS ( Hcy+1. 5 μmol/L EDS ) , middle dose of EDS group ( Hcy+1. 0 μmol/L EDS ) , low dose of EDS group(Hcy+0. 5 μmol/L EDS), and Hcy group. The apoptosis of CRL-1730 cells was determined by flow cytometry assay and was observed by fluorescent staining. Real-time quantitative reverse transcriptase polymerase chain reaction was used to detect Bax, Bcl-2 and Caspase-3 mRNA expressions in CRL-1730 cells. Western blot was used to detect Bax, Bcl-2, cleaved-Caspase-3 protein expressions in CRL-1730 cells. Results EDS in selec-tive concentration had no effect on the viability of CRL-1730 cells. The apoptosis rates of CRL-1730 cells were sig-nificantly increased with the treatment of Hcy, but Hcy-induced apoptosis in CRL-1730 cells was inhibited by EDS treatment in a dose-dependent manner. The protein expressions of Bax and cleaved-Caspase-3 in CRL-1730 cells in the Hcy group showed significant elevation in comparison with the control group. The expression of Bcl-2 protein declined in the Hcy group compared with the control group, but high, middle and low doses of EDS treatments down-regulated the protein expressions of Bax, cleaved Caspase-3 and up-regulated the expression of Bcl-2 in Hcy-treated CRL-1730 cells. The mRNA expressions of Bax and Caspase-3 of CRL-1730 cells in the Hcy group were el-evated significantly in comparison with the control group. The expression of Bcl-2 mRNA was reduced in the Hcy group compared with the control group, but high,middle and low doses of EDS treatments down-regulated the mR-NA expressions of Bax, Caspase-3 and up-regulated the mRNA expression of Bcl-2 in Hcy-treated CRL-1730 cells. Conclusion EDS has a protective effect on Hcy-induced apoptosis of human umbilical vein endothelial cells. This study provides evidence for the efficacy of EDS in the treatment of cardiovascular disease.%目的 探讨蜕皮甾酮对同型半胱氨酸(Hcy)诱导人脐静脉内皮细胞株CRL-1730凋亡的影响.方法 体外常规培养人血管内皮细胞CRL-1730,细胞计数试剂盒法检测蜕皮甾酮对CRL-1730细胞增殖活力的影响;Hcy处理人血管内皮细胞CRL-1730建立损伤模型,分组为空白对照组、蜕皮甾酮高剂量组(Hcy+1.5μmol/L蜕皮甾酮)、蜕皮甾酮中剂量组(Hcy+1.0μmol/L蜕皮甾酮)、蜕皮甾酮低剂量组(Hcy+0.5μmol/L蜕皮甾酮)、Hcy组;流式细胞术检测CRL-1730细胞凋亡率,并利用荧光染色进行细胞凋亡形态学观察;RT-PCR法检测CRL-1730细胞内Bax、Bcl-2及Caspase-3基因的表达;采用Western blot法检测CRL-1730细胞内Bax、Bcl-2、cleaved-Caspase-3蛋白表达.结果 所选浓度蜕皮甾酮对CRL-1730细胞增殖活性无明显影响;流式细胞术结果显示经Hcy处理后的CRL-1730细胞凋亡率明显增高,但经过不同浓度的蜕皮甾酮处理后,Hcy诱导的CRL-1730细胞凋亡率明显下降;与空白对照组比较,Hcy组CRL-1730细胞Bax和cleaved-Caspase-3的表达明显增高,Bcl-2的表达明显降低,差异有统计学意义.与Hcy组比较,经过低、中、高剂量蜕皮甾酮处理后,Hcy诱导的CRL-1730细胞Bcl-2表达上调,Bax和cleaved-Caspase-3表达下调;与空白对照组比较,Hcy组CRL-1730细胞Bax mRNA和Caspase-3 mRNA的表达明显增高,Bcl-2 mRNA的表达明显降低,差异有统计学意义.与Hcy比较,经过低、中、高剂量蜕皮甾酮处理后,CRL-1730细胞Bcl-2 mRNA表达上调,Bax mRNA和Caspase-3 mRNA表达下调.结论 蜕皮甾酮对Hcy诱导人脐静脉内皮细胞CRL-1730凋亡具有保护作用,这为其治疗心血管疾病提供了依据.