葛根总黄酮联合三氧化二砷体外诱导NB4-R1细胞凋亡的实验研究

目的:探讨葛根总黄酮联合三氧化二砷(As2O3)对维甲酸耐药细胞株NB4-R1增殖及凋亡的影响。方法:采用葛根总黄酮(0、10、30、50μg/mL)联合As2O3(1μmol/L)处理NB4-R1细胞24、48、72 h,MTT法检测细胞增殖抑制率;光学显微镜及荧光显微镜观察细胞形态改变;流式细胞仪检测细胞周期变化;FITC-AnnexinV/PI双染法检测细胞凋亡率,蛋白印迹法检测凋亡相关蛋白表达。结果:(1)一定浓度葛根总黄酮对NB4-R1细胞生长有抑制效应,呈剂量和时间依赖性,相同条件下,葛根总黄酮联合1μmol/L As2O3对NB-R1细胞抗增殖作用强于单用葛根总黄酮,差异有统计学意义(P<0.05)。(2)FITC-Annexin V/PI双染流式细胞术检测细胞凋亡结果发现葛根总黄酮(0、10、30、50μg/mL)及葛根总黄酮联合1μmol/L As2O3处理NB4-R1细胞48 h后,早期凋亡率呈浓度依赖,差异有统计学意义(P<0.05)。(3)瑞氏染色,在光镜下观察到葛根总黄酮和葛根总黄酮+As2O3联用组呈现不同程度的细胞凋亡形态学特征,且联用组较单药组明显。(4)流式细胞术检测细胞周期变化发现葛根总黄酮和葛根总黄酮+As2O3联用可影响细胞进程,表现为S期细胞增多,葛根总黄酮+As2O3联用组比各单用葛根总黄酮组Sub-G1增加更为明显。(5)Western blottimg结果显示葛根总黄酮可上调NB4-R1细胞中JNK表达,下调ERK1/2表达。结论:低浓度葛根总黄酮体外对维甲酸耐药细胞株NB4-R1细胞具有增殖抑制作用,呈时间-浓度依赖性,与1μmol/L As2O3具有协同作用;其诱导凋亡作用机制可能为阻滞NB4-R1细胞周期进程,激活JNK途径,下调ERK通路。

Experimental Study of Puerariae Radix Flavones Combined with Arsenic Trioxide Inducing Apoptosis of NB4-R1 Cells in Vitro

Objective:To investigate the effect of Puerariae radix flavones combined with arsenic trioxide(As2O3)on the proliferation and apoptosis of retinoic acid resistant cell line NB4-R1.Methods:Puerariae radix flavones(0,10,30,50μg/mL)combined with As2O3(1μmol/L)were used to treat NB4-R1 cells for 24,48,and72 h.The cell proliferation inhibition rate was detected by MTT assay,cell morphology was observed by light microscope and fluorescence microscope,cell cycle was detected by flow cytometry,apoptosis rate was detected by FITC-Annexin V/PI double staining,and the expression of apoptosis related proteins was detected by Western blotting.Results:(1)A certain concentration of Puerariae radix flavones had an inhibitory effect on the growth of NB4-R1 cells in a dose and time-dependent manner.Under the same conditions,Puerariae radix flavones combined with 1μmol/L As2O3 had a stronger anti-proliferation effect on NB-R1 cells than the Puerariae radix flavones alone,the difference was statistically significant(P<0.05).(2)FITC-Annexin V/PI double staining flow cytometry to detect cell apoptosis showed that Puerariae radix flavones(0,10,30,50μg/mL)and Puerariae radix flavones combined with 1μmol/L As2O3 were treated with NB4-R1 After 48 h,the early apoptosis rate was concentration-dependent,and the difference was statistically significant(P<0.05).(3)Wright’s staining results showed that the morphological characteristics of apoptosis in different degrees in Puerariae radix flavones group,Puerariae radix flavones(0,10,30,50μg/mL)combined with As2O3(1μmol/L)group,and the combination group was more obvious than Puerariae radix flavones group.(4)Flow cytometry analysis of cell cycle showed that Puerariae radix flavones,Puerariae radix flavones(0,10,30,50μg/m L)combined with As2O3(1μmol/L)could affect the cell process,showing the increase of S phase cells.The Puerariae radix flavones combined with As2O3 increased Sub-G1 more significantly than the Puerariae radix flavones alone group.(5)Western blottimg results showed that Puerariae radix flavones can up-regulate the expression of JNK and down-regulate the expression of ERK1/2 in NB4-R1 cells.Conclusion:Low concentration of Puerariae radix flavones can inhibit the proliferation of retinoic acid resistant NB4-R1 cells in a time concentration dependent manner,and has synergistic effect with 1μmol/L As2O3.The mechanism of inducing apoptosis may be to block the cell cycle progression of NB4-R1 cells,activate JNK pathway and down-regulate ERK pathway.