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目的探讨葡萄籽提取物F2对人恶性胶质瘤细胞U-87增殖的影响及对其甲酰肽受体(FPR)功能的影响。方法采用MTT法检测F2对U-87细胞成活率的影响。光镜及AO/EB染色观察细胞形态学变化。流式细胞术考察F2对U-87细胞周期及线粒体膜电位的影响。趋化实验方法考察F2对fMLF诱导的U-87细胞趋化的影响。采用Fura-2双波长测定法考察F2对fMLF诱导的U-87细胞钙动员的影响,用westernblot方法考察F2对fMLF诱导的U-87细胞ERK1/2磷酸化的影响,并用激光共聚焦方法考察F2对U-87细胞FPR表达的影响。结果F2能显著抑制U-87细胞的增殖,其作用呈明显的时间和浓度依赖性,药物作用24h、48h、72h的IC50分别为75μg·mL-1、33.6μg·mL-1、25μg.mL-1。光镜及AO/EB观察F2作用48h能使U-87细胞形态发生明显改变,包括细胞变圆,胞浆内出现许多大大小小的空泡。F2能显著降低U-87细胞线粒体膜电位,诱导U-87细胞G2/M期阻滞并能诱导有典型paraptosis形态特征的细胞死亡。非细胞毒浓度的F2还能显著抑制FPR激动剂fMLF(10nM)诱导的U-87细胞趋化,且能显著抑制fMLF诱导的U-87细胞钙动员及ERK1/2磷酸化。而且F2还能显著降低在胶质瘤的增殖、迁移及血管生成中起重要作用的FPR的表达。结论F2不仅能显著抑制U-87细胞的增殖,还能通过抑制FPR激动剂fMLF诱导的U-87细胞的趋化而抑制肿瘤细胞迁移,能抑制其FPR的表达,是一种很有开发潜力的新型抗胶质瘤药物。
Objective To investigate the effect of grape seed extract F2 on the proliferation of human malignant glioma U-87 cells and its effect on the function of formyl peptide receptor (FPR). Methods The effect of F2 on the survival rate of U-87 cells was detected by MTT assay. Light microscopy and AO / EB staining observed cell morphological changes. The effect of F2 on the cell cycle and mitochondrial membrane potential of U-87 cells was investigated by flow cytometry. Chemotaxis assay was used to investigate the effect of F2 on fMLF-induced chemotaxis of U-87 cells. The effect of F2 on fMLF-induced calcium mobilization in U-87 cells was investigated by Fura-2 dual-wavelength assay. The effect of F2 on the phosphorylation of ERK1 / 2 induced by fMLF in UML-87 cells was investigated by western blotting and examined by laser scanning confocal microscopy F2 on FPR expression in U-87 cells. Results F2 could significantly inhibit the proliferation of U-87 cells in a time-and dose-dependent manner. The IC50 values of F2, 24h, 48h and 72h were 75μg · mL-1, 33.6μg · mL-1 and 25μg · mL -1. Observation of light microscope and AO / EB for 48 hours could change the morphology of U-87 cells significantly, including the rounding of cells and the appearance of many vacuoles in the cytoplasm. F2 can significantly reduce the mitochondrial membrane potential of U-87 cells, induce G2 / M arrest in U-87 cells and induce cell death with morphological features of typical paraptosis. Non-cytotoxic concentrations of F2 also significantly inhibited fMLF (10 nM) -induced chemotaxis of U-87 cells and significantly inhibited fMLF-induced calcium mobilization and ERK1 / 2 phosphorylation in U-87 cells. Moreover, F2 also significantly reduces the expression of FPR, which plays an important role in glioma proliferation, migration and angiogenesis. Conclusion F2 can not only significantly inhibit the proliferation of U-87 cells, but also inhibit tumor cell migration and inhibit the expression of FPR by inhibiting the chemotactic effect of FMLF-induced U-87 cells New anti-glioma drug.