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目的:观察狼毒大戟根部提取物12-去氧佛波醇-13-棕榈酸酯(12-deoxyphorbol 13-palmitate,DP)对MCF-7细胞血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响,及其是否通过Von Hippel-Lindau蛋白(VHL)/低氧诱导因子(HIF-1α)信号通路进行调节。方法:实验分为常氧空白组、常氧实验组(10,20,40μmol·L-1DP)、乏氧空白组和乏氧实验组(10,20,40μmol·L-1DP),其中乏氧空白组和乏氧实验组用150μmol·L-1Co Cl2预处理细胞。当细胞密度达70%~80%时进行药物处理;MTT法检测DP对MCF-7细胞在6,12,24 h细胞存活率的影响;酶联免疫吸附测定(ELISA)检测DP对MCF-7细胞分泌VEGF的影响;实时荧光定量PCR法(RT-q PCR)检测DP对VEGF mRNA表达的影响;Western blot检测DP对MCF-7细胞中VEGF,HIF-1α和VHL蛋白表达;细胞免疫荧光法(IF)检测DP对HIF-1α表达及其在细胞中分布的影响。结果:MTT法结果显示,乏氧状态下细胞存活率在12,24 h且DP浓度为20,40μmol·L-1时与对照组相比有显著性差异(P<0.05);乏氧条件下,VEGF和HIF-1α的表达较常氧条件下升高,与乏氧组相比,DP能降低VEGF(P<0.05)和HIF-1α(P<0.05)的表达;乏氧条件下VHL表达较常氧条件下降低,当药物处理后,相较于乏氧组,VHL浓度明显上升(P<0.05)。结论:DP可能通过调节VHL/HIF-1α信号通路从而下调MCF-7细胞VEGF表达。
Objective: To observe the effect of 12-deoxyphorbol 13-palmitate (DP) on the growth of vascular endothelial growth factor (VEGF) in MCF-7 cells. Expression and whether it is regulated by the Von Hippel-Lindau protein (VHL) / hypoxia inducible factor (HIF-1α) signaling pathway. Methods: The experiment was divided into three groups: normoxia group, normoxia group (10,20,40μmol·L-1DP), hypoxia blank group and hypoxia experimental group (10,20,40μmol·L-1DP) The blank group and the hypoxia experimental group pretreated the cells with 150μmol·L-1Co Cl2. The effect of DP on the survival rate of MCF-7 cells at 6, 12 and 24 h was detected by MTT assay and the effect of DP on the survival rate of MCF-7 cells by enzyme-linked immunosorbent assay (ELISA) The effect of DP on the expression of VEGF mRNA was detected by real-time fluorescence quantitative PCR (RT-qPCR). The expressions of VEGF, HIF-1α and VHL protein in MCF-7 cells were detected by Western blot. (IF) was used to detect the effect of DP on the expression of HIF-1α and its distribution in cells. Results: The results of MTT assay showed that the survival rate of cells under hypoxic condition was significantly lower than that of control group (P <0.05) at 12 and 24 h and at DP of 20 and 40 μmol·L-1, respectively. Under hypoxia , While the expression of VEGF and HIF-1α was higher than that of normoxia. Compared with hypoxia group, DP could decrease the expression of VEGF (P <0.05) and HIF-1α (P <0.05) Compared with hypoxia group, the concentration of VHL increased significantly (P <0.05). Conclusion: DP may down-regulate the expression of VEGF in MCF-7 cells by regulating the VHL / HIF-1α signaling pathway.