西红花苷对3β,5α,6β-三羟胆甾烷致内皮细胞凋亡和相关基因表达的影响

Effects of Crocin on Cholestane-3β,5α,6β-triol-induced Apoptosis and Related Gene Expression of Cultured Endothelial Cells

目的:研究西红花苷(crocin)对3β,5α,6β-三羟胆甾烷(cholestane-3β,5α,6β-triol,Triol)致血管内皮细胞凋亡和相关基因表达的影响。方法:将内皮细胞分成正常对照组、模型组(25μmol/LTriol)、西红花苷(10-6mol/L,10-7mol/L,10-8mol/L)+25μmol/LTriol组。采用诱导内皮细胞凋亡的模型,用比色法测定丙二醛含量。光学显微镜、电子显微镜观察细胞形态和超微结构的变化,电泳法检测DNA梯形条带、流式细胞仪分析法检测细胞凋亡率,逆转录聚合酶链式反应(RTPCR)法检测Bcl2和BaxmRNA表达,免疫细胞化学方法检测Bcl-2、Bax和Caspase-3蛋白表达。结果:用Triol处理后,培养液中MDA含量增加(P<0.01vscontrol),细胞收缩,核浓缩,深染,线粒体肿胀空化,出现凋亡小体、DNA梯形条带和亚二倍体峰,细胞凋亡率为30.62%,BaxmRNA、Bax蛋白和Caspase3蛋白表达明显高于正常对照组(P<0.01);Bcl2mRNA和蛋白表达低于正常对照组(P<0.01);西红花苷(10-7mol/L、10-6mol/L)+Triol组,MDA含量减少,内皮细胞形态结构完整,细胞凋亡率分别为24.4%、6.3%,细胞Bax和Caspase3基因表达明显下调,Bcl2基因表达上调(P<0.05orP<0.01vsTriol)。结论:西红花苷可能是通过调节Bcl-2,Bax和Caspase-3基因表达抑制Triol诱导的内皮细胞凋亡。

AIM:To investigate the effects of crocin on cholestane-3β,5α,6β-triol-induced apoptosis and related gene expression of cultured endothelial cells (ECs).METHODS:ECs were divided as control,25 μmol/L cholestane-3β,5α,6β-triol(Triol),crocin(10^(-6) mol/L,10^(-7) mol/L,10^(-8) mol/L)+25 μmol/L Triol groups.Apoptosis model induced by Triol was established.Malondialdehyde (MDA) content was measured by colorimetric assay.The morphology and ultrastructure were observed with the light microscope and electron microscope;DNA ladder was analyzed by DNA electrophoresis;Apoptosis rate was assessed with the flow cytometer assay;Expressions of Bcl-2 mRNA and Bax mRNA were detected by RT-PCR;Expressions of Bcl-2,Bax and Caspase-3 protein were detected by immunocytochemical staining technique.RESULTS:MDA content in the media increased (P<0.01) after being treated with Triol,accompanying ECs shrinking,nuclei concentrating,mitochondria swelling as well as the appearance of apoptic bodies,DNA ladder and hypodiploid cusp.Apoptotic rate of cells was 30.62%.The expression of Bcl-2 was decreased,Bax and Caspase-3 were increased in Triol group (P<0.01 vs control).In the crocin +Triol group,MDA content decreased.Apoptotic rates of crocin (10^(-7) or 10^(-6) mol/L) + Triol groups were 24.4% and 6.3%,respectively.The expression of Bcl-2 increased while the expressions of Bax and Caspase-3 decreased (P<0.05or P<0.01 vs Triol).CONCLUSION:Crocin possibly inhibit Triol-induced apoptosis of ECs by decreasing the gene expression of Bax and Caspase-3 and increasing the gene expression of Bcl-2.