摘要
目的 探讨姜黄素对糖皮质激素耐药的急性淋巴细胞白血病(ALL)Jurkat细胞株生物学特性和NF-κB通路相关蛋白表达的影响.方法 选择ALL耐药细胞株Jurkat,以1μmol/L地塞米松作为Jurkat细胞耐药最佳浓度,传代培养细胞.将细胞分为10、25、50μmol/L姜黄素组,以及50 μmol/L吡咯烷二硫基甲酸酯(PDTC)组、对照组(加入等体积不含药物的培养液).各组细胞培养72 h,倒置显微镜下观察细胞形态.采用CCK-8法检测Jurkat细胞增殖能力,流式细胞术检测Jurkat细胞凋亡能力和细胞周期,实时荧光定量聚合酶链反应(qRT-PCR)检测NF-κB p65、NF-κB p50、IκBα、A20 mRNA的相对表达情况,蛋白质印迹法检测 NF-κB p65、NF-κB p50、IKBα、caspase-8、caspase-3、bcl-2、A20蛋白的表达.结果 10、25、50 μmol/L姜黄素及50 μmol/L PDTC分别处理Jurkat细胞72 h,对照组细胞膜基本完整,大小一致,圆形透明,细胞核荧光均匀;不同浓度姜黄素组和50 μmol/L PDTC组均有大量变形的细胞及细胞碎片,表现出不同程度的细胞核浓缩、破碎,凋亡现象明显.不同浓度姜黄素及50 μmol/L PDTC分别处理Jurkat细胞24、48、72 h后,不同浓度姜黄素组和PDTC组细胞增殖抑制率均较对照组高(均 P＜0.01).对照组、10 μmol/L姜黄素组、25 μmol/L姜黄素组、50 μmol/L姜黄素组、50 μmol/L PDTC组 72 h 时细胞凋亡率分别为(4.9±0.1)％、(99.2±0.1)％、(99.9±0)％、(100.0±0)％、(100.0±0)％,差异有统计学意义(F=2 876 604.40,P＜0.001);不同浓度姜黄素组和50 μmol/L PDTC组的细胞凋亡率均高于对照组,差异均有统计学意义(均P＜0.01).不同浓度姜黄素组及50 μmol/L PDTC组72 h时S期、G2期细胞比例均低于对照组,G1期细胞比例均高于对照组,差异均有统计学意义(均P＜0.01).与对照组比较,不同浓度姜黄素组及50 μmol/L PDTC组细胞NF-κB p65、NF-κB p50蛋白和mRNA表达均低(均 P＜0.01),IκBα、caspase-8、caspase-3 蛋白表达均高(均 P＜0.01),bcl-2 蛋白表达低(P＜0.01),A20蛋白和mRNA表达均高(均P＜0.01).结论 姜黄素可有效逆转Jurkat细胞糖皮质激素耐药,促进其凋亡,这可能与姜黄素影响NF-κB通路相关蛋白有关.
Abstract
Objective To explore the effects of curcumin on the biological characteristics and expressions of NF-κB pathway-related proteins in glucocorticoid-resistant acute lymphoblastic leukemia(ALL)cell line Jurkat.Methods The drug-resistant ALL cell line Jurkat was selected,and 1 μmol/L dexamethasone was used as the optimal concentration for drug resistance of Jurkat cells,and the cells were passaged and cultured.The cells were divided into 10,25 and 50 μmol/L curcumin groups,as well as 50 μmol/L pyrrolidinedithiocarbamate(PDTC)group,and control group(equal volume of culture medium without drug was added).The cells in each group were cultured for 72 h,and the cell morphology was observed under an inverted microscope.The CCK-8 method was used to detect the proliferation ability of Jurkat cells,flow cytometry was used to detect the apoptosis ability and cell cycle of Jurkat cells,real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative expressions of NF-κB p65,NF-κB p50,IκBα,and A20 mRNA,and Western blotting was used to detect the expressions of NF-κB p65,NF-κB p50,IκBα,caspase-8,caspase-3,bcl-2,and A20 proteins.Results Jurkat cells were treated with 10,25,50 μmol/L curcumin and 50 μmol/L PDTC for 72 h.In the control group,the cell membranes were basically intact,the size was uniform,the cell was round and transparent,and the cell nucleus had uniform fluorescence;a large number of deformed cells and cell fragments were observed in curcumin groups with different concentrations and 50 μmol/L PDTC group,with concentrated and fragmented nuclei and obvious apoptosis.After treating Jurkat cells with different concentrations of curcumin and 50 μmol/L PDTC for 24,48 and 72 h,respectively,the cell proliferation inhibition rates in curcumin groups with different concentrations and PDTC group were higher than those in the control group(all P＜0.01).The apoptosis rates at 72 h in the control group,10 μmol/L curcumin group,25 μmol/L curcumin group,50 μmol/L curcumin group,and 50 μmol/L PDTC group were(4.9±0.1)％,(99.2±0.1)％,(99.9±0)％,(100.0±0)％,and(100.0±0)％,respectively,and the difference was statistically significant(F=2 876 604.40,P＜0.001);compared with the control group,the apoptosis rates in curcumin groups with different concentrations and 50 μmol/L PDTC group were higher,and the differences were statistically significant(all P＜0.01).Compared with the control group,the proportions of S-phase and G2-phase cells were lower and the proportion of G1-phase cells was higher in curcumin groups with different concentrations and 50 μmol/L PDTC group at 72 h,and the differences were statistically significant(all P＜0.01).Compared with the control group,the protein and mRNA expressions of NF-κB p65 and NF-κB p50 in curcumin groups with different concentrations and 50 μmol/L PDTC group were lower(all P＜0.01),while the protein expressions of IκBα,caspase-8 and caspase-3 were higher(all P＜0.01),the protein expression of bcl-2 was lower(P＜0.01),and the protein and mRNA expressions of A20 were higher(both P＜0.01).Conclusions Curcumin can effectively reverse glucocorticoid resistance and promote apoptosis in Jurkat cells,which may be related to the influence of curcumin on NF-κB pathway-related proteins.
基金项目
贵州省科技计划(黔科合基础-ZK[2022]一般269)
贵州省卫生健康委科学技术基金(gzwkj2021-153)
NETL
NSTL
万方数据