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目的:观察岩大戟内酯B(jolkinolide B,JB)刺激MCF-7条件培养基对人脐静脉血管内皮细胞的影响,并分析岩大戟内酯B的作用机制。方法:分别用25,55,85 mg·L~(-1)岩大戟内酯B刺激MCF-7人乳腺癌细胞为JB刺激组,正常培养的MCF-7细胞为MCF-7组。获得25,55,85 mg·L~(-1)JB刺激的MCF-7细胞上清液为相应浓度的条件培养基。利用各组条件培养基分别培养人脐静脉血管内皮细胞(HUVEC)为25,55,85 mg·L~(-1)JB组,正常培养的HUVEC为非条件培养基组。分别用噻唑蓝(MTT)比色法,Annexin V-FITC细胞凋亡实验,细胞划痕实验和transwell细胞小室迁移实验观察25,55,85 mg·L~(-1)JB组HUVEC增殖、凋亡、迁移的变化;并利用蛋白免疫印迹法(Western blot)检测JB对MCF-7细胞的作用机制,酶联免疫吸附试验(ELISA)分析各组条件培养基中血管内皮细胞生长因子(VEGF)的含量变化。结果:与正常组比较,25,55,85 mg·L~(-1)JB组HUVEC细胞凋亡数目逐渐升高(P<0.01),HUVEC增殖显著降低(P<0.01);25,55,85 mg·L~(-1)JB组HUVEC细胞划痕面积闭合率和细胞迁移率较非条件培养基组均显著降低(P<0.01)。与MCF-7比较,25,55,85 mg·L~(-1)JB均能够明显抑制MSC-7细胞蛋白激酶B/信号传导及转录激活因子3/哺乳动物雷帕霉素靶蛋白(Akt/STAT3/m TOR)信号通路表达,下调MCF-7细胞表达的VEGF因子。结论:岩大戟内酯B通过抑制Akt/STAT3/m TOR信号通路下调MCF-7细胞旁分泌VEGF,抑制血管内皮细胞的增殖或迁移活性。
OBJECTIVE: To observe the effects of jolkinolide B (JB) on the proliferation of human umbilical vein endothelial cells (ECs) stimulated by MCF-7 conditioned medium, and to analyze the mechanism of action of ingeolide B. Methods: MCF-7 human breast cancer cells were stimulated with 25, 55 and 85 mg · L -1 halogerope B respectively. MCF-7 cells were cultured in MCF-7 group. The supernatant of MCF-7 cells stimulated with 25, 55 and 85 mg · L -1 of JB was the conditioned medium of the corresponding concentration. Human umbilical vein endothelial cells (HUVECs) were cultured in 25, 55 and 85 mg · L -1 JB groups, respectively. The normal cultured HUVECs were cultured in a non-conditioned medium. The proliferation of HUVECs in 25, 55 and 85 mg · L -1 JB groups were observed by MTT assay, Annexin V-FITC apoptosis assay, cell scratch assay and transwell cell migration assay respectively The migration mechanism of JB was detected by Western blot. The expression of vascular endothelial growth factor (VEGF) was detected by enzyme-linked immunosorbent assay (ELISA) Changes in the content. Results: Compared with the normal group, the apoptosis of HUVECs in 25, 55 and 85 mg · L -1 JB groups were significantly increased (P <0.01), while the proliferation of HUVEC was significantly decreased (P <0.01) The closure rate and the cell migration rate of HUVEC in 85 mg · L -1 JB group were significantly lower than those in the non-conditioned medium group (P <0.01). Compared with MCF-7, 25, 55 and 85 mg · L -1 JB can obviously inhibit the protein kinase B / signal transducers and activators of transcription 3 / mammalian target of rapamycin (Akt / STAT3 / m TOR) signaling pathway and downregulate the VEGF factor expressed in MCF-7 cells. Conclusions: Inhibirolide B inhibits the proliferation or migration of vascular endothelial cells by downregulating the secretion of VEGF by inhibiting Akt / STAT3 / m TOR signaling pathway in MCF-7 cells.