摘要：
目的 研究桔梗皂苷D对人肝癌细胞株HepG2与SMMC-7721细胞放射敏感性的作用,并探讨其相关作用机制. 方法 四甲基偶氮唑盐法检测不同浓度不同处理时间桔梗皂苷D对细胞存活率的影响;预先给予细胞5μg/m1桔梗皂苷D处理24h,经过不同照射剂量的X射线照射,细胞克隆形成实验检测桔梗皂苷D对细胞的辐射增敏作用,计算准域剂量(Dq),平均致死剂量(D0),外推数(N),放射增敏比SER及不同照射剂量作用下的存活分数(SF),并依据多靶单击型模型公式SF=1-(1-e-D/D0)N拟合细胞存活曲线;流式细胞仪检测细胞周期的分布;Western blot检测细胞中磷酸化磷脂酰肌醇激酶(pPI3k)、磷酸化蛋白激酶(pAkt)、核因子(NF)-κ B与磷酸化核因子抑制蛋白(pIκBα)的蛋白表达变化.据资料不同分别单因素方差分析(ANOVA)、f检验或LSD检验进行统计学分析. 结果 桔梗皂苷D降低两种细胞存活率,且该抑制作用呈时间与剂量依赖性,HepG2细胞24h的IC50为(24.2±0.61) μg/ml,48h的IC50为(7.68±0.46)μg/ml;SMMC-7721细胞24h的IC50为(23.8±0.57)μg/ml,48h的IC50为(8.63±0.86)μg/ml;桔梗皂昔D联合放射处理后,Dq (P=0.002)、D0(P=0.002)和N值(P=0.003)均显著降低,细胞存活曲线明显左移,放射增敏比SER分别为1.347±0.04 (HepG2)与1.418±0.05(SMMC-7721);此外,桔梗皂苷D显著降低放射引起的两种细胞周期G2/M期比例的上升,以及pPI3k (P=0.002)、pAkt(P=0.003)与NF-κB(P=0.002)蛋白表达的增加,pIκ B α(P=0.003)蛋白的表达降低. 结论 桔梗皂苷D增加人肝癌细胞株HepG2与SMMC-7721细胞的放射敏感性,其机制可能与桔梗皂苷D增强发射线对细胞生长抑制作用、抑制PI3k/Akt及NF-κB通路的激活有关.%Objective To investigate the effect of platycodin D on the radiosensitivity of human hepatoma cell lines HepG2 and SMMC-7721 and related mechanisms of action.Methods MTT assay was used to analyze the effect of different concentrations of platycodin D with different treatment times on cell viability.The cells were pretreated with 5 μg/ml platycodin D for 24 hours followed by X-ray irradiation at different radiation doses.Colony-forming assay was used to measure the radiosensitizing effect of platycodin D on cells.The quasi-threshold dose (Dq),mean lethal dose (Do),extrapolation number (N),sensitizer enhancement ratio (SER),and survival f raction (SF) at different radiation doses were calculated,and the multi-target single-hit model was used to fit the cell survival curve according to the formula SF =1-(1-eD/D0)N.Flow cytometry was used to investigate the distribution of cell cycle,and Westem blotting was used to measure the changes in the protein expression of phosphorylated phosphatidylinositol 3’-kinase (pPI3K),phosphorylated protein kinase (pAkt),nuclear factor-κB (NF-κB),and phosphorylated nuclear factor inhibiting protein (pIκ Bα).A one-way analysis of variance,the t-test,or the least significant difference test was used for statistical analysis based on the type of data.Results Platycodin D reduced the viability of HepG2 and SMMC-7721 cells in a dose-dependent manner;the IC50 value for HepG2 cells was 24.2 ± 0.61 μg/ml at 24 hours and 7.68 ± 0.46 μg/ml at 48 hours,and that for SMMC-7721 cells was 23.8 ± 0.57 μg/ml at 24 hours and 8.63 ± 0.86 μg/mL at 48 hours.After the combined treatment with platycodin D and irradiation,there were significant reductions in Dq (P =0.002),Do (P =0.002),and N value (P =0.003),the survival curve markedly shifted to the left,and SER was 1.347 ± 0.04 in HepG2 cells and 1.418 ± 0.05 in SMMC-7721 cells.In addition,platycodin D significantly inhibited the increase in the proportion of cells in G2/M phase,the increases in the protein expression ofpPI3k (P =0.002),pAkt (P =0.003),and NF-κB (P =0.002),and the reduction in the protein expression ofpIκBα (P =0.003).Conclusion Platycodin D can increase the mdiosensitivity of HepG2 or SMMC-7721 cells,possibly by enhancing the growth inhibition effect of irradiation and inhibiting the activation of the PI3k/Akt and NF-κB pathways.
