氯吡格雷通过上调miR-145表达抑制VSMC增殖的机制研究

摘要

目的 本研究旨在探究miR-145是否对血管平滑肌细胞(VSMC)的增殖起调控作用,以及氯吡格雷如何通过调控CD40来发挥其消炎作用,以期为氯吡格雷的药用作用发挥提供新的理论依据.方法 实验分组为①DMSO组,即溶剂对照组;②TNF-α组;③miR-145抑制剂对照组;④氯吡格雷组;⑤miR-145抑制剂+氯吡格雷组.并通过EdU标记检测细胞增殖;qRT-PCR用于检测miR-145,CD40和VSMC中Calponin mRNA的表达;Western blot检测CD40的蛋白表达水平;通过ELISA检测细胞培养物上清液中IL-6的水平.结果 与氯吡格雷组相比,miR-145抑制剂+氯吡格雷组的Calponin mRNA水平降低(P <0.01);与DMSO组相比,TNF-α组的miR-145 mRNA水平下降(P<0.01);与TNF-α组相比,氯吡格雷组的miR-145 mRNA水平上升(P<0.01);与氯吡格雷组相比,miR-145抑制剂+氯吡格雷组的miR-145 mRNA水平下降(P<0.01).miR-145抑制剂对照组不影响CD40的水平;与DMSO组相比,TNF-α组的CD40 mRNA水平上升(P<0.01);与TNF-α组相比,氯吡格雷组的CD40 mRNA水平下降(P<0.01);与氯吡格雷组相比,miR-145 抑制剂+氯吡格雷组的CD40 mRNA水平上升(P<0.01).TNF-α组上清液中IL-6的水平高于DMSO组(P<0.01);氯吡格雷组中IL-6水平低于TNF-α组(P<0.01);miR-145抑制剂+氯吡格雷组IL-6的水平高于氯吡格雷组(P<0.01).结论 氯吡格雷通过抑制CD40的表达,诱导miR-145抑制VSMC细胞增殖并发挥消炎作用.%Objective To investigate whether miR-145 is involved in the regulation of vascular smooth muscle cell (VSMC) proliferation and to determine the anti-inflammatory effects of clopidogrel via its regulation of CD40 for providing a new theoretical basis for the pharmacological effect of clopidogrel.Methods The TNF-α induced proliferation models of VSMC were divided into different groups with or without clopidogrel.RT-PCR was used to detect mRNA expression of miR-145, CD40 and Calponin (a VSMC differentiation marker gene).Western blot was used to detect CD40 protein expression;ELISA was used to determine the concentration of inflammatory cytokine IL-6 in the supernatant of cells.Results Compared with the clopidogrel group, the Calponin and mRNA levels in the miR-145 inhibitor + clopidogrel group decreased significantly (P<0.01).Compared with DMSO group, TNF-group of miR-145 alpha mRNA decreased significantly (P<0.01);compared with the TNF-Alpha group, clopidogrel group had significantly increased miR-145 levels (P<0.01);compared with clopidogrel group, clopidogrel/miR-145 group had reduced the mRNA level of miR-145 (P<0.01).In control group, miR-145 inhibitors did not affect CD40 level;compared with DMSO group, in TNF-group, CD40 mRNA alpha level increased (P<0.01);compared with the TNF-Alpha Group, in clopidogrel group CD40 mRNA decreased (P<0.01);compared with clopidogrel group, in miR-145 inhibitor + clopidogrel group, the mRNA level of CD40 increased (P<0.01).The concentration of IL-6 in supernatant of TNF-group was higher than that in DMSO group (P<0.01).The IL-6 concentration in the clopidogrel group was lower than that in the TNF group (P<0.01).The concentration of IL-6 in the +miR-145 inhibitor + clopidogrel group was higher than that in the clopidogrel group (P<0.01).Conclusion miR-145 is involved in the anti-proliferative and anti-inflammatory effects of clopidogrel on VSMCs by inhibiting the expression of CD40.