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目的:研究淫羊藿苷(ICA)对同型半胱氨酸(HCY)诱导的增殖血管平滑肌细胞(VSMC)葡萄糖调节蛋白78(GRP78)基因表达的影响,探讨ICA抗动脉粥样硬化的机制。方法:建立HCY诱导的兔VSMC增殖模型,与不同浓度的ICA共同培养48h后,用流式细胞仪Annexin/PI双染法检测凋亡率;RT-PCR检测GRP78mRNA的表达;目的基因克隆、鉴定并测序。结果:0.2,0.4mg.L-1的ICA可显著促进兔VSMC凋亡,且存在剂量依赖性,与半胱氨酸组相比,差异显著(P<0.01);RT-PCR结果显示:0.2,0.4mg.L-1的淫羊藿苷作用于VSMC48h后,GRP78mRNA表达水平显著升高,与半胱氨酸组相比差异显著(P<0.05);测序结果表明,来源于VSMC的全长648bp的基因片断与兔葡萄糖调节蛋白78基因高度同源。结论:ICA促进GRP78mRNA表达,诱导兔VSMC凋亡,可能是其抗动脉粥样硬化的机制之一。
Objective: To study the effects of icariin (ICA) on homocysteine (HCY)-induced proliferating vascular smooth muscle cells (VSMC) Glucose Regulatory Protein 78 (GRP78) gene expression, and to explore the mechanism of ICA anti-atherosclerosis. METHODS: The HCY-induced VSMC proliferation model was established. After cultured with different concentrations of ICA for 48 hours, the apoptosis rate was detected by flow cytometry Annexin/PI double staining method; the expression of GRP78 mRNA was detected by RT-PCR; the target gene was cloned and identified. And sequencing. RESULTS: 0.2, 0.4 mg.L-1 ICA significantly promoted the apoptosis of rabbit VSMC in a dose-dependent manner. Compared with cysteine group, the difference was significant (P<0.01); RT-PCR showed: 0.2 After treatment with icariin 0.4 mg.L-1 for 48 h, the mRNA level of GRP78 increased significantly (P<0.05) compared with the cysteine group. The sequencing results showed that the total length of VSMCs was increased. The 648 bp gene fragment is highly homologous to the rabbit glucose regulation protein 78 gene. Conclusion: ICA promotes the expression of GRP78 mRNA and induces apoptosis of VSMCs in rabbits, which may be one of the mechanisms of anti-atherosclerosis.