人参皂苷Rh_(2)逆转HepG2细胞对5-氟尿嘧啶耐药性作用研究  

Reversal of 5-fluorouracil resistance in HepG2 cells by ginsenoside Rh_(2)

作　　者：许洪亮 苗红 李军 刘艳萍 孙漫原 XU Hongliang;MIAO Hong;LI Jun;LIU Yanping;SUN Manyuan(Department of pharmacy,Jinan City People's Hospital,Jinan 271199,China;Department of traditional Chinese medicine,Fuping Second People's Hospital,Weinan 711799,China)

机构地区：[1]济南市人民医院药剂科,山东济南271199 [2]富平县第二人民医院中医内科,陕西渭南711799

出　　处：《药物评价研究》2021年第9期1876-1881,共6页Drug Evaluation Research

基　　金：陕西省中医药管理局中医药科研课题(LCMS016)。

摘　　要：目的研究人参皂苷Rh_(2)逆转HepG2细胞对5-氟尿嘧啶(5-Flu)耐药性的作用。方法构建对5-Flu耐药的HepG2细胞系(HepG2-5-Flu),MTT法分别测定人参皂苷Rh_(2)和5-Flu(二者质量浓度均为15.63、31.25、62.50、125.00、250.00、500.00、1000.00、2000.00μg/mL)、人参皂苷Rh_(2)和5-Flu不同比例(5∶1、3∶1、1∶1、1∶3、1∶5)的混合药物(总浓度同单给药)对HepG2-5-Flu细胞存活率的影响,利用协同作用系数(CI)获得人参皂苷Rh_(2)和5-Flu的最佳协同作用浓度;通过Annexin V/PI双染色流式细胞术检测5-Flu(375μg/mL)、人参皂苷Rh_(2)+5-Flu(人参皂苷Rh_(2)∶5-Flu=1∶3,浓度为125、375μg/mL)对细胞凋亡影响;实时荧光定量PCR(qRT-PCR)法检测细胞P-糖蛋白(P-gp)和多药耐药相关蛋白(MRP)的mRNA表达水平。结果人参皂苷Rh_(2)对HepG2-5-Flu细胞的半数抑制浓度(IC_(50))为223.3μg/mL,而5-Flu的IC_(50)为8.34μg/mL。人参皂苷Rh_(2)与5-Flu浓度比为5∶1、3∶1、1∶1、1∶3、1∶5时,混合药物对细胞的IC_(50)分别为324.7、224.0、219.0、135.6、105.9μg/mL。人参皂苷Rh_(2)和5-Flu比例为5∶1时,二者没有协同作用(CI>1);当人参皂苷Rh_(2)和5-Flu比例为3∶1和1∶1时,药物浓度大于62.5μg/mL时才有较小的协同作用(0.6<CI<1);当人参皂苷Rh_(2)和5-Flu比例为1∶3和1∶5时,大多数药物浓度下都有较好的协同作用(0<CI<0.5)。与5-Flu组比较,人参皂苷Rh_(2)+5-Flu组细胞坏死、早期凋亡、晚期凋亡的比例均显著升高,活细胞比例显著下降(P<0.05、0.01、0.001),MDR1和P-gp 2个耐药相关蛋白的mRNA表达显著下调(P<0.01)。结论人参皂苷Rh_(2)能够逆转HepG2细胞对5-Flu的耐药性,机制可能与下调MDR1和P-gp表达相关。Objective To study anti-liver cancer efficacy and overcome the drug resistance of 5-fluorouracil(5-Flu)that synergistically enhanced by ginsenoside Rh_(2).Methods HepG2 cell line(HepG2-5-Flu)resistant to 5-Flu was constructed,Ginsenoside Rh_(2) and 5-Flu(15.63,31.25,62.50,125.00,250.00,500.00,1000.00,2000.00μg/mL)were determined by MTT method,different proportions(5∶1,3∶1,1∶1,1∶3,1∶5)of ginsenoside Rh_(2) and 5-Flu(the total concentration was the same as single administration)on the survival rate of HepG2-5-Flu cells,and the optimal synergistic concentration of Ginsenoside Rh_(2) and 5-FLu was obtained by using the synergistic coefficient.The effects of 5-Flu(375μg/mL)and ginsenoside Rh_(2)+5-Flu(Ginsenoside Rh_(2)∶5-Flu=1∶3,125,375μg/mL)on apoptosis were detected by Annexin V/PI double staining flow cytometry.The mRNA expression levels of P-gp and MRP were detected by real-time quantitative PCR(qRT-PCR).Results The 50%inhibitory concentration(IC_(50))of Ginsenoside Rh_(2) against HepG2-5-Flu cells was 223.3μg/mL,and that of 5-Flu was 8.34μg/mL.The IC_(50) of ginsenoside Rh_(2) and 5-Flu were 324.7,224.0,219.0,135.6 and 105.9μg/mL when the concentration ratio of ginsenoside Rh_(2) and 5-flu was 5∶1,3∶1,1∶1,1∶3 and 1∶5,respectively.When the ratio of ginsenosides Rh_(2) and 5-Flu was 5∶1,there was no synergistic effect(CI>1).When the ratio of ginsenoside Rh_(2) and 5-Flu was 3∶1 and 1∶1,the concentration of ginsenoside Rh_(2)and 5-Flu was greater than 62.5μg/mL(0.6<CI<1).When the ratio of ginsenoside Rh_(2) and 5-Flu was 1∶3 and 1∶5,most drugs had a good synergistic effect(0<CI<0.5).Compared with the 5-Flu group,the percentages of cell necrosis,early apoptosis and late apoptosis in ginsenoside Rh_(2)+5-Flu group were significantly increased,and the percentages of living cells were significantly decreased(P<0.05,0.01,0.001),and the mRNA expressions of MDR1 and P-GP were significantly down-regulated(P<0.01).Conclusion Ginsenoside Rh_(2) can reverse the resistance of HepG2 cell

关 键 词：人参皂苷 5-氟尿嘧啶 肝癌 药物协同 逆转耐药性 

分 类 号：R285.5[医药卫生—中药学]