密蒙花方对缺氧状态下脐静脉血管内皮细胞VEGF-VEGFR信号转导通路的影响

目的从血管内皮生长因子-血管内皮生长因子受体(vascular endothelial growth factor,VEGF-VEGFR)信号转导机制方面深入探讨密蒙花方抑制缺氧状态下人血管内皮细胞增殖的作用机制。方法采用人脐静脉血管内皮细胞(human umbilical vasculal endothelial cell,HUVEC)进行体外培养,利用二氯化钴(CoCl2)模拟缺氧状态,同时分别将10g·L-1、20g·L-1、40g·L-1密蒙花方水提液作用于缺氧状态下的HUVEC24h,采用水溶性四氮唑-8(WST-8)法检测密蒙花方对缺氧状态下HUVEC增殖的影响,采用Westernblot法检测密蒙花方对缺氧状态下HUVEC内血管内皮生长因子(vascular endothelial growth factor,VEGF)、fms样酪氨酸激酶受体1(fms-like tyrosine kinase-1,Flt-1)、胎肝激酶-1/激酶插入区受体(fetal liver kinase-1/kinase insert domain-containing receptor,Flk-1/KDR)蛋白磷酸化水平的影响。采用免疫组织化学法检测HUVEC内细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)蛋白表达的情况。结果 WST-8法结果显示:缺氧组吸光度值为1.112±0.060,各密蒙花方组吸光度值分别为0.860±0.050、0.567±0.072、0.263±0.057,表明密蒙花方可有效抑制缺氧状态下HUVEC增殖(均为P<0.01)。Western blot结果显示:缺氧组HUVEC内VEGF、Flk-1/KDR蛋白磷酸化水平分别为1.922±0.048、1.341±0.054,较正常组升高,Flt-1蛋白磷酸化水平为1.265±0.065,较正常组降低,密蒙花方可使缺氧状态下HUVEC内VEGF、Flk-1/KDR蛋白磷酸化水平降低,Flt-1蛋白磷酸化水平升高,差异均有显著统计学意义(均为P<0.01)。免疫组织化学显示:正常组ICAM-1吸光度值161.416±4.905,缺氧组ICAM-1吸光度值为213.464±10.282,各密蒙花组ICAM-1吸光度值分别为186.841±9.039、171.862±8.202、165.053±6.088。密蒙花方可使缺氧状态下HUVEC内ICAM-1蛋白表达减少。结论下调细胞内VEGF、Flk-1/KDR蛋白磷酸化水平,上调细胞内Flt-1蛋白磷酸化水平,干预细胞内VEGF-VEGFR信号转导通路,是密蒙花方抑制缺氧状态下HUVEC增殖的作用机制之一。

Effect of Mimeng Flower decoction on VEGFR signal transduction pathway in human umbilical vascular endothelial cells under chronic hypoxia

Objective To study the mechanism of vascular endothelial growth factor(VEGF)-VEGF receptor(VEGFR) signal transduction,and discuss the effect of Mimeng Flower decoction on inhibiting the proliferation of human umbilical vascular endothelial cell(HUVEC) under chronic hypoxia.Methods HUVEC were cultured in vitro.CoCl 2 was used to set up the cell chemistry hypoxia model,in the same time,10 g·L-1 20 g·L-1,40 g·L-1 Mimeng Flower decoction extract were added to the HUVEC separately,the following studies were made after 24 hours incubation.The inhibitive effect of Mimeng Flower decoction on the proliferation of HUVEC under chronic hypoxia was investigated by WST-8 method.The effects of Mimeng Flower decoction on phosphorylation level of VEGF,fms-like tyrosine kinase-1(Flt-1) and fetal liver kinase-1 /kinase insert domain-containing receptor(Flk-1 /KDR) in HUVEC were detected by Western blot method.The protein expression of intercellular adhesion molecule-1(ICAM-1) in HUVEC was detected by immunohistochemistry method.Results WST-8 methods showed that the IOD in hypoxic group was 1.112 ± 0.060,which in each Mimeng Flower decoction group were 0.860 ± 0.050,0.567 ± 0.072 and 0.263 ± 0.057,indicated that the Mimeng Flower decoction could effective inhibit the proliferation of HUVEC(both P < 0.01).The results of Western blot showed that the phosphorylation levels of VEGF,Flk-1 /KDR in hypoxic group were 1.922 ± 0.048,1.341 ± 0.054,which higher than those in normal group,Flt-1 in hypoxic group was 1.265 ± 0.065,which lower than that in normal group,there were significant differences(both P < 0.01).The results of immunohistochemistry method showed that IOD of ICAM-1 in normal group and hypoxic group were 161.416 ± 4.905,213.464 ± 10.282,which in each Mimeng Flower decoction group were 186.841 ± 9.039,171.862 ± 8.202 and 165.053 ± 6.088.Mimeng Flower decoction could down-regulate the expression of ICAM-1.Conclusion Mimeng Flower decoction can markedly inhibit the HUVEC proliferation under chronic hypoxia,the effect mechanism include down-regulating the phosphorylation level of VEGF,Flk-1 /KDR,up-regulating phosphorylation level of Flt-1 and interfering the VEGF-VEGFR signal transduction pathway.