芍药苷对慢性髓系白血病耐阿霉素细胞株K562/ADR多药耐药性的逆转作用及机制  

Effect of paeoniflorin in reversing multidrug resistance of chronic myeloid leukemia K562/ADR cells and its molecular mechanism

作　　者：朱聪[1] 贾秀红[1] 刘迎雪 ZHU Cong;JIA Xiuhong;LIU Yingxue(The Affiliated Hospital of Binzhou Medical University,Binzhou 256603,China)

机构地区：[1]滨州医学院附属医院,山东滨州256603

出　　处：《山东医药》2018年第47期24-27,共4页Shandong Medical Journal

基　　金：山东省自然科学基金资助项目(ZR2014HL032);山东省医药卫生科技发展计划项目(2014WS0183)

摘　　要：目的观察芍药苷对慢性髓系白血病(chronic myelogenous leukemia,CML)耐阿霉素(adriamycin,ADR)细胞株K562/ADR多药耐药性的逆转作用,并探讨其作用机制。方法 CCK-8法检测ADR对K562/ADR细胞和K562细胞的半抑制浓度(IC50),计算K562/ADR细胞对ADR的耐药倍数; CCK-8法检测芍药苷对K562/ADR细胞的细胞毒性,选择细胞增殖抑制率<10%的芍药苷浓度用于后续实验。将K562/ADR细胞分为A(分别加入0、4、8、16、32μg/m L的ADR)、B(分别加入0、4、8、16、32μg/m L的ADR及2μmol/L的芍药苷)、C(分别加入0、4、8、16、32μg/m L的ADR及4μmol/L的芍药苷)三组,CCK-8法检测三组ADR对K562/ADR细胞的IC50,计算其耐药逆转倍数。将K562/ADR细胞分为a(加入3μg/m L的ADR)、b(加入2μmol/L的芍药苷及3μg/m L的ADR)、c(加入4μmol/L的芍药苷及3μg/m L的ADR)三组,流式细胞仪检测三组K562/ADR细胞内ADR浓度,Western blotting法检测三组K562/ADR细胞中多药耐药相关蛋白MRP1、MAPK信号通路蛋白p38和磷酸化p38 (p-p38)。结果 ADR对K562/ADR细胞、K562细胞的IC50分别为(48. 37±2. 10)、(1. 47±0. 36)μg/m L,K562/ADR细胞对ADR的耐药倍数为32. 91倍。芍药苷在K562/ADR细胞内的无毒剂量为2、4μmol/L。A、B、C组ADR对K562/ADR细胞的IC50分别为(29. 97±1. 72)、(20. 03±0. 75)、(13. 78±0. 63)μg/m L,两两相比,P均<0. 05; B、C组ADR的耐药逆转倍数分别为1. 51、2. 17倍。a、b、c组ADR荧光强度分别为320±14、676±62、883±28,两两相比,P均<0. 05。a、b、c组K562/ADR细胞中MRP1蛋白的相对表达量分别为87. 69%±1. 54%、80. 95%±4. 10%、67. 72%±5. 98%,两两相比,P均<0. 05; p-p38蛋白的相对表达量分别为72. 43%±2. 67%、63. 18%±2. 32%、52. 23%±6. 02%,两两相比,P均<0. 05。结论芍药苷能逆转K562/ADR细胞对ADR的耐药性,其逆转作用与抑制细胞P38 MAPK信号通路及下调MRP1蛋白表达有关。Objective To investigate the effect and its molecular mechanism of paeoniflorin in reversing multi-drug resistance of human chronic myeloid leukemia(CML)K562/ADR cells which are resistant to adriacin(ADR).Methods CCK-8 was used to detect the half inhibitory concentration(IC 50)of K562 cells and K562/ADR cells and we calculated the fold of resistance of paeoniflorin to K562/ADR cells.CCK-8 was used to detect the intracellular toxicity of paeoniflorin to K562/ADR cells.Paeoniflorin concentration with cell proliferation inhibition rate<10%was selected for the subsequent experiments.K562/ADR cells were divided into three groups:group A(which was added with 0,4,8,16 and 32μg/mL ADR),group B(which was added with 2μmol/L paeoniflorin and 0,4,8,16,32μg/mL ADR,respectively),group C(which was added with 4μmol/L paeoniflorin and 0,4,8,16,32μg/mL ADR,respectively).CCK-8 was used to detect the IC 50 and we calculated the fold of resistance of the above three groups.K562/ADR cells were divided into three groups:group a(3μg/mL ADR alone),group b(3μg/mL ADR and 2μmol/L paeoniflorin)and group c(3μg/mL ADR and 2μmol/L paeoniflorin).ADR concentration(expressed by fluorescence intensity)was detected by flow cytometry.Multidrug resistance-related protein MRP1,MAPK signaling pathway protein p38,and phosphorylated p38(p-p38)were detected by Western blotting.Results The IC 50 of ADR to K562/ADR cells and K562 cells was(48.37±2.10),(1.47±0.36)μg/mL and the resistant factor of K562/ADR cells to ADR was 32.91 times.The nontoxic dose of paeoniflorin in K562/ADR cells was 2 and 4μmol/L.The IC 50 of ADR in K562/ADR cells of groups A,B and C were(29.97±1.72),(20.03±0.75),(13.78±0.63)μg/mL,all P<0.05.And the reversal times of ADR in the group B and group C were 1.51 and 2.17 times.The fluorescence intensity of ADR in the groups a,b and c was 320±14,676±62 and 883±28,all P<0.05.The relative expression levels of MRP1 protein in K562/ADR cells of groups a,b and c were 87.69%±1.54%,80.95%±4.10%and 67.72%±5.98%,all P<0.05.The

关 键 词：芍药苷 多药耐药 慢性髓系白血病 阿霉素 多药耐药相关蛋白 MAPK信号通路 

分 类 号：R788.72[医药卫生—口腔医学]