三七皂苷R1通过线粒体相关通路促进白血病细胞株HL-60凋亡

Notoginsenoside R1 enhances apoptosis of human leukemia cell line HL-60 through mitochondria-dependent pathway

目的:观察三七皂苷(notoginsenoside)R1对白血病细胞株HL-60凋亡的影响,探讨其可能的作用机制。方法:采用MTT法和流式细胞术(Annexin V-FITC双染法)分别检测三七皂苷R1(10、20、40及80μmol/L)处理HL-60细胞12、24、36及48 h后HL-60细胞的凋亡情况,Western blotting检测HL-60细胞内Bcl-2、Bax及细胞色素C(cytochrome-C,Cyt C)蛋白的表达水平,JC-1染色法观察HL-60线粒体膜电位的变化。结果:MTT和流式细胞术检测显示三七皂苷R1浓度依赖性诱导HL-60细胞的凋亡,且细胞存活率随处理时间的增加而降低;与空白对照组相比,加入三七皂苷R1后细胞中Bcl-2蛋白表达显著减少[(0.45±0.03)vs(1.00±0.00),P<0.05]、Bax蛋白表达显著增加[(1.72±0.08)vs(1.00±0.00),P<0.05];Bcl-2/Bax比值减小[(0.21±0.01)vs(1.00±0.00),P<0.05];线粒体膜电位降低[(0.56±0.09)vs(1.00±0.00),P<0.05];胞质(cyto)中Cyt-C蛋白表达水平显著下降[(0.42±0.03)vs(1.00±0.00),P<0.05]。结论:三七皂苷R1可显著诱导HL-60细胞凋亡,其作用机制可能是通过线粒体通路促进细胞的凋亡;本实验可为三七皂苷R1用于临床治疗白血病提供实验依据。

Objective： To explore the effect of Notoginsenoside R1 on the apoptosis of human leukemia cell line HL-60 and possible mechanisms. Methods：After HL-60 cells were incubated with different concentrations of Notoginsenoside R1 （10, 20, 40 and 80 μmol/L） for 12, 24, 36 h and 48 h, apoptosis of the HL-60 cells was determined by MTY and An- nexin V-FITC flow cytometry assays respectively. The expression of Bcl-2, Bax and cytochrome C （Cyt-C） in the HL-60 cells was determined by Western blotting. The change of mitochondrial membrane potential was assessed by JC-1 dyeing method. Results：The results of MTY and flow cytometry assays showed that Notoginsenoside R1 induced the apoptosis of HL-60 cells in dose-dependent manners, and survival rate of the HL-60 cells decreased with increase of incubation time ; after adding Notoginsenoside R1, expression of Bcl-2 protein in the HL-60 cells remarkably decreased （ [0.45 ± 0. 03 ] vs [ 1.00 ± 0.00 ], P 〈 0.05, compared with control group） ; expression of Bax protein significantly increased （ [ 1.72± 0. 08] vs [ 1.00 ±0.00] ,P 〈0.05, compared with control group） ; and the ratio of Bcl-2/Bax decreased （ [0.21 ±0.01 ] vs [ 1.00± 0.00 ], P 〈 0.05 ） ; the mitochondrial membrane potential decreased （ [ 0.56± 0. 09 ] vs [ 1. 00 ± 0. 00 ], P 〈 0.05 ） ; and expression level of Cyt-C protein in cytoplasm remarkably decreased （ [ 0.42 ±0.03 ] vs [ 1. 00 ± 0. 00 ], P 〈 0.05 ]. Conclusion ： Notoginsenoside R1 can remarkbly incluce apoptosis of HL-60 ceils. Mitochondria-dependent path- way may be involved inNotoginsenoside Rl-induced apoptosis of HL-60 ceils. These findings suggest that Notoginsenoside R1 may be a new strategy for treatment of leukemia.