5α-羟基木香酸抑制VEGF164诱导大鼠脉络膜血管内皮细胞增殖、迁移及血管形成

5α-HA inhibits VEGF164-induced proliferation,migration and angiogenesis of choroidal vascular endothelial cells

目的探讨5α-羟基木香酸(5α-hydroxycostic acid,5α-HA)体外抑制脉络膜新生血管(choroidal neovascularization,CNV)模型的作用及其机制。方法体外培养大鼠脉络膜血管内皮细胞(choroidal vascular endothelial cells,CECs)后分为(n=3):空白对照组、模型组[30 ng/mL抗血管内皮生长因子(vascular endothelial growth factor,VEGF)164]、干预组(30 ng/mL VEGF164+100μmol/L 5α-HA)、抑制剂组[30 ng/mL VEGF164+5μmol/L Semaxanib(VEGFR2特异性阻断剂)]。CCK-8实验检测各组CECs的增殖情况,细胞划痕实验、Transwell实验以及Matrigel基质胶管腔形成实验分别观察各组CECs的增殖、迁移以及管腔形成。RT-qPCR检测各组细胞渗漏相关因子如血管生成素2(Angiopoietin 2,Ang 2)、血管内皮钙黏蛋白(VE-Cadherin)、紧密连接蛋白(ZO-1)的mRNA水平的表达;Western blot检测各组细胞信号通路及渗漏相关蛋白P-VEGFR2、P-Tie2、P-FAK、VE-Cadherin、ZO-1、Occudin的表达;免疫荧光检测各组细胞中Ang 2蛋白的表达,统计分析上述各组间的指标差异。结果CCK-8实验表明:与模型组比较,干预组5α-HA作用于CECs后能抑制CECs的异常增殖(P<0.01)。细胞划痕实验、Transwell实验以及Matrigel基质胶管腔形成实验表明:干预组能扭转模型组VEGF164引起的CECs迁移及管腔形成(P<0.01)。RT-qPCR结果显示:干预组能抑制模型组中Ang 2 mRNA的上调以及VE-Cadherin和ZO-1的下调(P<0.01)。Western blot结果显示:干预组能减少模型组中P-VEGFR2、P-Tie2及P-FAK蛋白的表达(P<0.01),同时上调VE-Cadherin、ZO-1以及Occudin蛋白的表达(P<0.05)。细胞免疫荧光实验结果显示,与模型组比较,干预组能减少模型组中Ang 2蛋白的表达(P<0.01)。结论5α-HA可能通过抑制VEGFR2、Tie2双途径磷酸化以及抑制VEGFR2/FAK信号通路抑制VEGF164诱导的CECs增殖、迁移以及管腔形成。

Objective To investigate the role and underlying mechanism of 5α-hydroxycostic acid(5α-HA)in inhibiting choroidal neovascularization(CNV)model in vitro.Methods Rat choroidal vascular endothelial cells(CECs)were randomly grouped into blank control group,model group(30 ng/mL VEGF164),5α-HA treatment group(30 ng/mL VEGF164+100μmol/L 5α-HA)and inhibition group(30 ng/mL VEGF164+5μmol/L semaxanib)(n=3).CCK-8 assay was used to detect the proliferation of CECs in each group.Cell scratch assay,Transwell assay and Matrigel tube formation assay were used to observe the proliferation,migration and tube formation of the CECs.RT-qPCR was employed to detect the mRNA expression of leakage-related factors angiopoietin 2(Ang 2),VE-Cadherin and ZO-1.Western blotting was performed to measure the expression of p-VEGFR2,p-Tie2,p-FAK,VE-Cadherin,ZO-1 and occudin.Cell immunofluorescence assay was conducted to observe the expression of Ang 2 protein in the cells.Results CCK-8 assay showed that 5α-HA treatment inhibited the abnormal proliferation of CECs in the model group(P<0.01).Cell scratch assay,Transwell assay and Matrigel tube formation assay displayed that 5α-HA treatment also reversed the migration and tube formation of CECs induced by VEGF164 in the model group(P<0.01).RT-qPCR indicated that the treatment also inhibited the up-regulation of Ang 2 mRNA and the down-regulation of VE-Cadherin and ZO-1 in the model group(P<0.01).Western blotting results showed that 5α-HA significantly reduced the protein levels of p-VEGFR2,p-Tie2 and p-FAK in the model group(P<0.01),and up-regulated the levels of VE-Cadherin,ZO-1 and occudin(P<0.05).The results of cell immunofluorescence showed 5α-HA reduced the expression of Ang 2 protein in the model group(P<0.01).Conclusion 5α-HA inhibits VEGF164-induced proliferation,migration and tube formation in CECs,possibly by inhibiting the phosphorylation of VEGFR2 and Tie2 and VEGFR2/FAK signaling pathway.