新藤黄酸通过PTEN-PI3K/AKT/VEGF/eNOS信号通路干预人脐静脉内皮细胞血管生成的研究
Gambogenic Acid Intervenes Against Angiogenesis in Human Umbilical Vein Endothelial Cells via the PTEN-PI3K/AKT/VEGF/eNOS Signaling Pathway
  
DOI:
中文关键词:  新藤黄酸  人脐静脉内皮细胞  血管生成  分子机制
英文关键词:Gambogenic acid  Human umbilical vein endothelial cell  Angiogenesis  Molecular mechanism
基金项目:国家自然科学基金项目(81673650,81903859)
作者单位
高 俊,程 卉,李庆林 1.安徽中医药大学药学院安徽 合肥 2300122.安徽中医药大学新安医学教育部重点实验室安徽 合肥 230038 
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中文摘要:
      目的 探究PTEN-PI3K/Akt/VEGF/eNOS信号通路在新藤黄酸(gambogenic acid,GNA)抑制人脐静脉内皮细胞(human umbilical vein endothelial, HUVECs)迁移中的作用。方法 倒置显微镜下观察HUVECs形态变化;Boyden chamber实验以及细胞划痕实验观察GNA对HUVECs迁移能力的影响;硝酸还原酶法检测NO水平;Western blot法检测GNA及LY29400预处理对HUVECs内磷脂酰肌醇3激酶(phosphatidylinositol 3 kinase,PI3K)、p-PI3K、蛋白激酶B(protein kinase B,AKT)、p-AKT表达水平的影响;HUVECs转染PTEN-siRNA后,RT-PCR实验检测VEGF mRNA的表达水平。结果 GNA能有效抑制HUVECs迁移,其抑制效果与剂量相关(P<0.05)。GNA和抑制剂LY294002以及L-NAME均能有效抑制NO的产生,且GNA+LY294002组、GNA+L-NAME组抑制效果更好(P<0.05);Western blot检测结果显示,GNA上调PTEN蛋白表达水平,下调eNOS、p-PI3K、p-AKT蛋白表达水平(P<0.05)。RT-PCR检测结果表明,PTEN-siRNA转染后,GNA能上调VEGF mRNA基因的表达水平(P<0.05)。结论 GNA通过PTEN-PI3K/Akt/VEGF/eNOS信号传导通路,抑制HUVECs迁移,阻止HUVECs血管生成。
英文摘要:
      Objective To investigate the role of the PTEN-PI3K/Akt/VEGF/eNOS signaling pathway in the inhibition of the migration of human umbilical vein endothelial cells (HUVECs) by gambogenic acid (GNA). Methods An inverted microscope was used to observe the morphological changes of HUVECs; Boyden chamber experiment and wound-healing assay were used to observe the effect of GNA on the migration ability of HUVECs; the nitrate reductase method was used to measure the level of nitric oxide (NO); Western blot was used to measure the effect of GNA and LY29400 pretreatment on the expression levels of phosphoinositide 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (AKT), phosphorylated AKT (p-AKT) in HUVECs; after HUVECs were transfected with PTEN-siRNA, RT-PCR was used to measure the mRNA expression of vascular endothelial growth factor (VEGF). Results GNA effectively inhibited the migration of HUVECs in a dose-dependent manner (P<0.05). GNA and the inhibitors LY294002 and L-NAME effectively inhibited the production of NO, and the GNA+LY294002 group and the GNA+L-NAME group had a significantly better inhibitory effect (P<0.05). Western blot showed that GNA upregulated the protein expression of PTEN and downregulated the protein expression of endothelial nitric oxide synthase, p-PI3K, and p-AKT (P<0.05). RT-PCR showed that after PTEN-siRNA transfection, there was a significant increase in the mRNA expression of VEGF (P<0.05). Conclusion GNA inhibits the migration of HUVECs and prevents angiogenesis in HUVECs via the PTEN-PI3K/Akt/VEGF/eNOS signaling pathway.
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