桔梗皂苷D对人肝癌细胞株放射敏感性的影响及其相关机制

Effect of platycodin D on radiosensitivity of human hepatoma cell line and related mechanisms of action

目的研究桔梗皂昔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5ug／ml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（DO），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）-κB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）、t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）ug／ml；SMMC-7721细胞24h的IC50为（23．8±0．57）ug／ml，48h的IC50为（8．63±0．86）μg／ml；桔梗皂苷D联合放射处理后，Dq（P=0．002）、DO（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，pIκBα（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-KB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 fraction （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 Western 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 （pit, Bet）. 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 ±0.86μg/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 of pIκBα （P = 0.003）. Conclusion Platycodin D can increase the radiosensitivity 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.