丁苯酞通过激活VEGF/VEGFR2-Notch1/Dll4信号促进HUVECs形成血管

NBP promotes angiogenesis of HUVECs by activating VEGF/VEGFR2-Notch1/Dll4 signal pathway

目的:探索缺血缺氧(H/I)条件下丁苯酞(NBP)通过激活血管内皮生长因子(VEGF)/VEGF受体2(VEGFR2)-Notch1/Delta样配体4(Dll4)信号促进人脐静脉内皮细胞(HUVECs)血管形成的机制。方法:利用无血清培养基和缺氧罐模拟H/I条件,HUVECs传代培养后设置正常对照(control)组、H/I组、NBP高剂量(H/I+NBP_(high))组和NBP低剂量(H/I+NBP_(low))组,其中control组为常规培养的HUVECs,H/I组为H/I条件下培养的HUVECs,H/I+NBP_(high)组为在H/I环境下使用20μmol/L丁苯酞干预的HUVECs,H/I+NBP_(low)组为在H/I环境下使用5μmol/L丁苯酞干预的HUVECs。CCK-8法检测各组细胞的细胞活力,细胞划痕实验检测各组细胞的迁移能力,体外血管形成实验检测各组细胞成管能力,Western blot法检测VEGFR2、Notch1和Dll4的表达,ELISA法检测培养基中VEGF的表达,qPCR检测VEGF、VEGFR2、Notch1和Dll4的mRNA表达水平。结果:丁苯酞可以提高H/I条件下HUVECs的存活率,促进细胞的迁移和体外血管的形成能力,且H/I+NBP_(high)组比H/I+NBP_(low)组更加显著。丁苯酞可以提高VEGF、VEGFR2、Notch1和Dll4的mRNA及蛋白表达。结论:丁苯酞可以在H/I条件下促进HUVECs形成血管,其机制可能与VEGF/VEGFR2-Notch1/Dll4信号的激活有关。

AIM： To investigate the effects of DL-3-n-butylphthalidle（ NBP） on angiogenesis of human umbilical vein endothelial cells（ HUVECs） and the role of vascular endothelial growth factor（ VEGF）/VEGF receptor 2（ VEGFR2）-Notch1/Delta-like ligand 4（ Dll4） signaling pathway in this process. METHODS： The serum-free medium and anoxic tank were used to simulate the conditions of hypoxia and ischemia（ H/I）. HUVECs were divided into control group,H/I group,H/I ＋ NBP（high）group and H/I ＋ NBP（low）group. The HUVECs in control group were conventionally cultured,and those in H/I group were cultured under H/I intervention. The HUVECs in H/I ＋ NBP（high）group were treated with NBP at 20 μmol/L under H/I intervention. The HUVECs in H/I ＋ NBP（low）group were treated with NBP at 5 μmol/L under H/I intervention. The cell viability of each group was measured by CCK-8 assay. The migration ability of the HUVECs in each group was detected by cell scratch test. The vessel formation ability of the HUVECs was examined by in vitro angiogenesis assay. The expression of VEGFR2,Notch1 and Dll4 at mRNA and protein levels was determined by qPCR and Western blot,and the expression of VEGF was determined by qPCR and ELISA. RESULTS： NBP increased the viability of HUVECs,and promoted the migration ability and the formation of blood vessels in vitro under H/I intervention. These effects of NBP at high dose were more significant than those at low dose. NBP increased the expression of VEGF,VEGFR2,Notch1 and Dll4 at mRNA and protein levels（ P〈0. 05）. CONCLUSION： NBP promotes HUVECs to form blood vessels under H/I intervention. The mechanism may be related to the activation of VEGF/VEGFR2-Notch1/Dll4 signaling pathway.