细梗香草总皂苷联合5-氟尿嘧啶抗胆管癌作用及其机制研究

The Anti-Cholangiocarcinoma Effect and Its Mechanism of Lysimachia Capilliposide in Combination with 5-Fluorouracil

[目的]研究细梗香草总皂苷(Lysimachia capilliposide,LC)单独使用及与5-氟尿嘧啶(5-fluorouracil,5-Fu)联用对胆管癌细胞QBC939增殖和凋亡的影响,并初步探究其机制。[方法]分别以0～32μg·mL-1的LC和0～1 000μg·mL-1的5-Fu培养QBC939细胞48h,以3-(4,5-二甲基吡啶-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑内盐[3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-etrazolium inner salt,MTS]法检测细胞增殖抑制率,计算半数抑制浓度(half maximal inhibitory concentration,IC50)。根据所得IC50,在QBC939细胞中同时加入不同浓度5-Fu和LC,计算联合作用指数(combined index,CI),确定两药联用浓度,并将细胞分为空白对照组、5-Fu单药组、LC单药组和联合用药组。AnnexinⅤ/PI法检测各组细胞凋亡比例和周期分布。Western blot检测天冬氨酸特异性的半胱氨酸蛋白水解酶-3 (cysteine aspartic acid specific protease-3,caspase-3)、聚腺苷二磷酸-核糖聚合酶(poly-ADP-ribose polymerase, PARP)的蛋白表达。[结果]与空白对照组比较,5-Fu和LC均抑制QBC939细胞增殖,并且两药联用有协同作用。5-Fu和LC可显著增加QBC939细胞凋亡比例(P<0.05),且联合用药组凋亡细胞比例高于LC或5-Fu单药组(P<0.01)。5-Fu增加G1期细胞比例,降低G2期细胞比例(P<0.05),而LC对细胞周期无明显影响。5-Fu、LC及两药联用可明显增加caspase-3表达,降低PARP表达(P<0.05,P<0.01)。[结论]LC可抑制胆管癌细胞增殖,与5-Fu有协同作用。LC诱导胆管癌细胞凋亡,但不影响细胞周期,其机制可能与上调caspase-3表达、下调PARP表达有关。

[Objective]To investigate the effect of Lysimachia capilliposide(LC) alone and combined with 5-fluorouracil(5-Fu) on proliferation and apoptosis of human cholangiocarcinoma cell line QBC939 and its mechanism.[Methods]Cholangiocarcinoma cells QBC939 were cultured with different concentrations of LC or 5-Fu respectively for 48 hours.The inhibition rate was measured with 3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-etrazolium inner salt(MTS) assay.Half maximal inhibitory concentration(IC50) was calculated.According to IC50,different concentrations of 5-Fu and LC were combined and used for culture of cholangiocarcinoma cells.Combined index(CI) was calculated and the combination concentration was confirmed.QBC939 cells were divided into blank control group,5-Fu group,LC group and combination group.The apoptotic cells and cell cycle were detected by flow cytometry with Annexin Ⅴ/PI assay.The expression of apoptotic proteins cysteine aspartic acid specific protease-3(caspase-3) and poly-ADP-ribose polymerase(PARP) was detected by Western blot.[Results]Compared with blank control group,both 5-Fu and LC could inhibit proliferation of cholangiocarcinoma cells.Combination of the two medicines had synergistic effect.Apoptotic cells significantly increased under the effect of 5-Fu and LC(P <0.05).Combination of the two medicines increased apoptotic cells significantly when compared with 5-Fu or LC alone(P<0.01).With the effect of 5-Fu,cells proportion of G1 phase increased and cells proportion of G2 phase decreased.However,LC didn’t result in significant difference in cells of each phase.5-Fu,LC and combination of the two medicines obviously upregulated the expression of caspase-3 and downregulated the expression of PARP,the difference was statistically significant(P <0.05,P <0.01).[Conclusion]LC can inhibit proliferation of cholangiocarcinoma cells and have synergistic effect if combined with 5-Fu.LC induces apoptosis and doesn ’t change cell cycle of cholangiocarcinoma cells,whose mechanism may be related to upregulation of caspase-3 and inhibition of PARP.