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目的:探讨大蒜素(allicin)抑制胰岛素(insulin)诱导的血管平滑肌细胞(VSMCs)增殖和迁移及其作用机制。方法:采用组织贴块法培养大鼠的VSMCs,并进行平滑肌α-肌动蛋白(α-SMA)免疫荧光鉴定,取3~5代细胞进行实验。建立胰岛素刺激的VSMCs模型。实验分5组:对照组、胰岛素组、大蒜素组、ERK抑制剂PD98059组、大蒜素+PD98059组。噻唑蓝(CCK8)比色法检测VSMCs的增殖;细胞计数法检测VSMCs的迁移;免疫印迹实验(Western blotting)测定total ERK,PhosphoERK(p-ERK),PCNA蛋白表达的情况。结果:原代培养的VSMCs生长良好,光镜下呈长梭形及“峰与谷”样生长。α-SMA免疫荧光显示培养的细胞具有典型的VSMCs特征。胰岛素能刺激VSMCs的增殖和迁移,且以100 nmol·L-1浓度时效果最明显。大蒜素预处理能显著抑制胰岛素刺激的VSMCs的增殖和迁移,且呈剂量依赖性。PD98059、大蒜素+PD98059预处理亦能显著抑制胰岛素刺激的VSMCs的增殖和迁移。胰岛素可明显促进VSMCs表达p-ERK,PCNA蛋白。大蒜素能显著抑制VSMCs表达p-ERK,PCNA蛋白,且呈剂量依赖性。结论:大蒜素能显著抑制胰岛素诱导的VSMCs的增殖与迁移,其作用机制可能是抑制ERK信号通路的激活。
Objective: To investigate the effect of allicin on the proliferation and migration of vascular smooth muscle cells (VSMCs) induced by insulin. Methods: Rat VSMCs were cultured by tissue patch method, and the smooth muscle α-actin (α-SMA) immunofluorescence was used to identify the cells. Establish insulin stimulated VSMCs model. The experiment was divided into 5 groups: control group, insulin group, allicin group, ERK inhibitor PD98059 group, allicin + PD98059 group. The proliferation of VSMCs was detected by CCK8 colorimetric assay. The migration of VSMCs was detected by cytometry. The expression of total ERK, phospho ERK and PCNA protein was detected by Western blotting. RESULTS: Primary cultured VSMCs grew well, with long spindle shape and “peak and valley” growth under light microscope. α-SMA immunofluorescence showed that the cultured cells have typical characteristics of VSMCs. Insulin stimulated the proliferation and migration of VSMCs, and the effect was the most obvious when the concentration was 100 nmol·L-1. Allicin pretreatment significantly inhibited the proliferation and migration of insulin-stimulated VSMCs in a dose-dependent manner. PD98059, allicin + PD98059 pretreatment also significantly inhibited insulin-stimulated VSMCs proliferation and migration. Insulin can significantly promote VSMCs expression of p-ERK, PCNA protein. Allicin can significantly inhibit VSMCs expression of p-ERK, PCNA protein, and in a dose-dependent manner. CONCLUSION: Allicin can significantly inhibit the proliferation and migration of insulin-induced VSMCs, and its mechanism may be that it inhibits the activation of ERK signaling pathway.