消癌平注射液抗血管生成作用及其机制研究

Effects and mechanisms of Xiao-Ai-Ping injection on angiogenesis

研究消癌平注射液对血管生成的影响,并探索其作用机制与血管内皮生长因子(VEGF)信号通路的相关性。采用CCK8实验和Brd U掺入细胞免疫荧光实验检测消癌平注射液对人脐静脉内皮细胞(HUVECs)的增殖影响;采用细胞划痕愈合和小室迁移实验考察消癌平注射液对HUVECs的迁移能力影响;采用HUVECs管腔形成实验、大鼠动脉环出芽实验以及鸡胚尿囊膜(CAM)实验等考察消癌平注射液的抗血管生成作用;分别采用ELISA酶联免疫吸附实验和Western blot检测HUVECs中VEGF的分泌情况以及血管内皮生长因子受体2(VEGFR2)等蛋白的表达变化。结果显示,消癌平注射液可剂量和时间依赖性地抑制HUVECs增殖,在24、48和72 h各时间点对HUVECs的IC50值(mg·m L-1)分别为48.7±7.14,29.1±2.25和22.0±4.53。消癌平注射液可抑制HUVECs的DNA合成以及迁移作用;还可抑制HUVECs管腔样结构形成,并且一定浓度下可抑制大鼠动脉环切口处微血管样结构萌发以及鸡胚尿囊膜血管生长;此外,消癌平注射液还可抑制HUVECs中VEGF分泌并下调p-VEGFR2和磷酸化蛋白激酶B(p-AKT)蛋白表达。本研究结果表明,消癌平注射液可在体外和离体水平抑制血管生成,其作用机制可能与抑制VEGF分泌、VEGF引起的VEGFR2及其下游AKT信号通路活化有关。

This study was designed to investigate the effect of Xiao-Ai-Ping injection on cancer angiogenesis. CCK8 assay and Brd U incorporation immunofluorescence assay were used to detect the effect of Xiao-Ai-Ping injection on HUVECs proliferation; wound healing assay and transwell assay were employed to test the effect of Xiao-Ai-Ping injection on HUVECs migration. The anti-angiogenic effect of Xiao-Ai-Ping injection was examined by tube formation assay, rat aortic ring assay and chicken chorioallantoic membrane（CAM） assay. ELISA assay was used to measure the secretion of vascular endothelial growth factor（VEGF）; and the activation of vascular endothelial growth factor receptor 2（VEGFR2） protein and its downstream signaling pathways were examined by Western blot. Our data demonstrated that Xiao-Ai-Ping injection inhibited HUVECs proliferation in a time- and dose-dependent manner, and the IC50（mg·m L-1） values for 24, 48 and 72 h were 48.7 ± 7.14, 29.1 ±2.25 and 22.0 ± 4.53, individually. Xiao-Ai-Ping injection inhibited HUVECs DNA synthesis and migration. Xiao-Ai-Ping injection suppressed HUVECs tube formation, and reduced microvessel sprouting from rat aortic rings and vessel growth in CAMs. Furthermore, Xiao-Ai-Ping injection attenuated the secretion of VEGF, and inhibited the expression of p-VEGFR2 and phosphorylation of protein kinase B（p-AKT）. We conclude that Xiao-Ai-Ping injection inhibits angiogenesis by down-regulation of VEGF signaling and AKT pathway.