葛根素抑制HCY所致血管内皮细胞凋亡及其相关机制的研究

Puerarin results in down-regulation of apoptosis in cultured human aortic endothelial cells induced by homocysteinemia

目的:差异筛选葡萄糖调节蛋白78(glucose regulated protein78,GRP78)基因,观察其在动脉粥样硬化(athemsclero-sis,AS)病灶中的表达水平,探讨葛根素抗AS的可能机制。方法:建立同型半胱氨酸(homocysteinemia,HCY)诱导的人主动脉血管内皮细胞损伤模型,与不同浓度葛根素(10-7mol/L、10-6mol/L、10-5mol/L)共同培养48h,Annexin V-FITC检测血管内皮细胞凋亡率,RT-PCR筛选差异表达的GRP78基因片段,制备地高辛标记探针,高脂饲料复制兔AS模型,主动脉组织原位杂交检测GRP78mRNA表达水平。结果:经葛根素处理48h后,各组血管内皮细胞凋亡率均低于HCY组(P<0.05、P<0.01),且随葛根素剂量增加而减少;筛选出全长648bp的基因片断与兔葡萄糖调节蛋白78基因高度同源,组织原位杂交显示该基因在AS病灶血管内皮细胞胞质中高表达。结论:GRP78基因参与AS病变形成;葛根素拮抗HCY诱导的血管内皮细胞凋亡,下调GRP78基因表达水平可能是其作用机制之一。

AIM： To select differentially the expressed gene,glucose regulated protein 78（GRP78）,to observe the expression of GRP78 in atherosclerosis tissue and to elucidate the mechanism for anti-atherosclerosis of puerarin.METHODS： The model was established by using homocysteinemia in vitro.The vascular endothelial cells were incubated with various concentrations of puerarin（10 -7 mol/L,10 -6 mol/L,10 -5 mol/L） for 48 h.The apoptosis rate was detected by flow cytometry using Annexin/PI-staining.GRP78 gene was amplificated by PCR.These differentially expressed fragments were cloned,selected,confirmed,and sequenced.Digoxigenin-labeled DNA Probe and cholesterol-rich rabit model were prepared,GRP78 mRNA was determined by in situ hybridization in atherosclerotic lesions and normal tissue.RESULTS： Three concentrtions of puerarin significantly inhibited the apoptosis of endothelial cells.The early apoptosis rate was decreased magnificently in a dose dependent manner compared with that of HCY group （P 0.05,P 0.01）.Furthermore,the fragment,648bp,was similar to the gene of glucose regulated protein GRP78 from endothelial cells and the level of expression was significantly higher in high cholesterol group than that in the control group.CONCLUSION： The GRP78 plays a key role in inducing atherosclerotic lesions.The mechanism of anti-atherosclerosis of puerarin might be partly due to down-regulation of apoptosis and decrease in the GRP78mRNA expression of vascular endothelial cells.