西黄丸下调HIF-1α/VEGFA/VEGFR2信号通路抑制脑胶质瘤细胞血管生成拟态形成的作用及其机制

The effect and mechanism of Xihuang pill inhibits the formation of vasculogenic mimicry in human glioblastoma cells by downregulating HIF-1α/VEGFA/VEGFR2 signaling pathway

探究西黄丸含药血清调控低氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)/血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)/血管内皮生长因子受体2(vascular endothelial growth factor receptor 2,VEGFR2)信号通路,抑制脑胶质瘤细胞血管生成拟态(vasculogenic mimicry,VM)形成的作用及机制。采用对雄性SD大鼠连续灌胃给药7天,制备西黄丸含药血清(动物实验伦理审批号:202105A051)。采用200μmol·L^(-1)CoCl2构建U251细胞低氧模型,给予西黄丸含药血清后,CCK-8法、细胞克隆实验检测U251细胞活力和增殖情况;流式细胞术检测U251细胞凋亡及周期;细胞划痕愈合、Transwell侵袭实验检测U251细胞的迁移与侵袭能力;3D细胞培养检测U251细胞VM的形成情况;Westernblot法检测U251细胞HIF-1α、VEGFA、VEGFR2、磷酸化VEGFR2(phosphorylated-VEGFR2,p-VEGFR2)、血管内皮钙黏着蛋白(vascular endothelial-cadherin,VE-cadherin)、Eph受体酪氨酸激酶A2(Eph receptor tyrosine kinases A2,EphA2)、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)、基质金属蛋白酶14(matrix metalloproteinase 14,MMP14)和层黏连蛋白γ2(lamininγ2)等蛋白表达水平。结果显示,低氧状态下,10%西黄丸含药血清对U251细胞活力、增殖、凋亡和细胞周期影响较小。与低氧模型组相比,10%1.0、1.5和2.0 h组西黄丸含药血清均显著减慢U251细胞的迁移速率(P<0.01),显著减少侵袭的U251细胞数量(P<0.01)。10%2.0 h组西黄丸含药血清显著抑制低氧状态下U251细胞的VM管状结构形成(P<0.01)。Western blot实验显示,10%西黄丸含药血清显著下调HIF-1α、VEGFA、phospho-VEGFR2、VE-cadherin、EphA2、MMP14等蛋白表达(P<0.05)。综上所述,西黄丸可通过下调HIF-1α/VEGFA/VEGFR2信号通路,抑制脑胶质瘤U251细胞VM形成,发挥抗血管生成的作用。

Our studies were aimed to explore the effect and mechanism of the inhibition of the formation of vasculogenic mimicry(VM)in human glioblastoma cells by Xihuang pill(XHP)medicated serum through regulating the hypoxia inducible factor-1α(HIF-1α)/vascular endothelial growth factor A(VEGFA)/vascular endothelial growth factor receptor 2(VEGFR2)signaling pathway.The medicated serum of XHP was prepared by gavage for 7 days to male SD rats(approval number of animal experiment ethics:202105A051).The hypoxia model of U251 cells was established using 200μmol·L-1 of CoCl2.After treatment with XHP-medicated serum,cell viability and proliferation of U251 cells were detected by CCK-8 and cell cloning experiment.Cell apoptosis and cell cycle of U251 cells were determined by flow cytometry.Cell migration and invasion were evaluated by wound healing and Transwell invasion assay.The formation of VM was assessed by three-dimensional cell culture of U251 cells.The protein expression levels of HIF-1α,VEGFA,VEGFR2,phosphorylated-VEGFR2(p-VEGFR2),vascular endothelial-cadherin(VE-cadherin),Eph receptor tyrosine kinases A2(EphA2),matrix metalloproteinase 2(MMP2),matrix metalloproteinase 14(MMP14)and lamininγ2 in U251 cells were detected by Western blot.The results showed that 10%XHP-medicated serum had little effect on the cell viability,proliferation,apoptosis and cell cycle of U251 cells under hypoxia.Compared with the model group,10%XHP-medicated serum at 1.0,1.5 and 2.0 h significantly decreased the migration rate(P<0.01)and the number of invading U251 cells(P<0.01).10%XHP-medicated serum at 2.0 h significantly suppressed the formation of VM tubular structures in U251 cells under the condition of hypoxia(P<0.01).Western blot experiment showed that 10%XHP-medicated serum significantly down-regulated the expression of HIF-1α,VEGFA,phospho-VEGFR2,VE-cadherin,EphA2 and MMP14 proteins(P<0.05).In conclusion,XHP could inhibit the formation of VM in human glioblastoma U251 cells to suppress the angiogenesis by down-regulating the HIF-1α/VEGFA/VEGFR2 signaling pathway.