氯吡格雷通过上调miR-145表达抑制VSMC增殖的机制研究

Study on the Mechanism of Inhibiting VSMC Proliferation by up Regulating miR-145 Expressionby Clopidogrel

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(1)DMSO组,即溶剂对照组;(2)TNF-α组;(3)miR-145抑制剂对照组;(4)氯吡格雷组;(5)miR-145抑制剂+氯吡格雷组。并通过Ed U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.