温阳活血解毒复方对人脐静脉内皮细胞VEGFs/VEGFRs通路的影响

Effect of Wenyang Huoxue Jiedu Formula on The VEGFs/VEGFRs Pathways in Human Umbilical Vein Endothelial Cells

目的研究温阳活血解毒复方(WHJF)对血管内皮生长因子(VEGFs/VEGFRs)信号通路的影响,从分子层面探讨其治疗动脉粥样硬化(AS)的机制。方法将SD大鼠随机分为空白对照组(C组)、温阳活血解毒复方低、中、高剂量组(L、M、H组,剂量分别为10.35、31.05、93.15 g·kg^(-1)),于第4天首次灌胃2 h后采血制备含药血清,采用免疫印迹法(Western Blot)及逆转录-聚合酶链式反应(q RT-PCR)法分别观察含药血清对人脐静脉内皮细胞(HUVECs)的VEGFs/VEGFRs信号通路中各信号分子及其基因表达的影响。结果温阳活血解毒复方含药血清对VEGFs/VEGFRs信号通路有抑制作用。在蛋白表达方面,与空白对照组比较,高剂量组中血管内皮生长因子受体2(VEGFR-2)和磷酸化的细胞外调节蛋白激酶(pERK)的表达量明显减少,差异有统计学意义(P<0.05);中剂量组中pERK的表达量明显减少,差异有统计学意义(P<0.05);通路中其他信号分子的表达量无明显减少,差异无统计学意义(P>0.05)。在基因表达方面,与空白对照组比较,温阳活血解毒复方组血管内皮生长因子亚型A、B(VEGFA、VEGFB)及血管内皮生长因子受体1、2(VEGFR-1、VEGFR-2)的mRNA表达量无明显减少,差异无统计学意义(P>0.05)。结论温阳活血解毒复方含药血清可能通过下调VEGFR-2及下游分子pERK的表达,对VEGFs/VEGFRs信号通路发挥抑制作用,从而直接影响细胞DNA合成功能,减少血管新生,起到稳定动脉粥样硬化斑块的作用。

Objective To investigate the effect of drug-containing serum of Wenyang Huoxue Jiedu Formula(WHJF) on the vascular endothelial growth factors and their receptors(VEGFs/VEGFRs) pathways;and toexplore its anti-atherosclerosis(AS) mechanism at molecular level. Methods The SD rats were randomly dividedinto four groups,namely blank control group(C),low-dose(L),middle-dose(M) and high-dose(H) WHJFgroups,which were treated with normal saline,WHJF in the dosages of 10.35,31.05,93.15 g·kg^-1 respectively,twice a day. At 2 h after first administration of the fourth day,the drug-containing serum was taken from the rats.The expression of signaling molecules and their mRNAs of VEGFs/VEGFRs pathways in the human umbilical veinendothelial cells(HUVECs) were assayed by Western Blot and quantitative real-time polymerase chain reaction(qRT-PCR) respectively. Results The drug-containing serum of WHJF exerted an inhibitory effect on the VEGFs/VEGFRs pathways. The H group showed a reduction of the expression of vascular endothelial growth factor receptor 2(VEGFR-2) and extracellular regulated protein kinases(pERK) when compared with C group(P < 0.05),whileM group disturbed the expression of pERK(P < 0.05). The levels of other proteins showed no obviously change(P > 0.05). There was no difference in the mRNA levels of VEGFA,VEGFB,VEGFR-1 and VEGFR-2(P >0.05). Conclusion The drug-containing serum of WHJF inhibits the VEGFs/VEGFRs pathways of HUVECs viadown-regulating the mediate molecules(VEGFR-2 and pERK), which results in the obstruction of DNAsynthesis, the decrease of angiogenesis and the stabilization of atherosclerosis.