人参皂甙对人白血病-60细胞凋亡的影响

Effect of ginsenoside on apoptosis of human leukemia-60 cells

摘要:

目的：探讨人参皂甙对白血病细胞的致凋亡作用及其相关作用机制。方法：采用MTT细胞毒实验检测不同浓度（100、50、25、12.5、6.25、3.125、1.5625 μmol/L）人参皂甙对人白血病-60（HL-60）细胞的生长抑制活性；流式细胞术Annexin V/PI双染法检测不同浓度（0、5、10、20 μmol/L）人参皂甙诱导HL-60细胞凋亡情况；提取各种浓度人参皂甙处理的细胞总蛋白，Western blot检测caspase-8、caspase-9和caspase-3裂解情况；Western blot检测10 μmol/L人参皂甙处理HL-60细胞后caspase-8和caspase-9 抑制剂对caspase-3 裂解情况的影响。结果：人参皂甙对白血病HL-60细胞具有高效的细胞毒作用，IC50值为7.3±1.2 μmol/L；0、5、10、20 μmol/L 人参皂甙处理HL-60细胞48 h后，流式细胞仪检测细胞的凋亡率呈剂量依赖性，各处理组之间差异有统计学意义（F=12.67，P<0.01）；Western blot检测细胞中caspase-9和caspase-3出现裂解带，而caspase-8未出现裂解带；10 μmol/L人参皂甙诱导caspase-3裂解可以被caspase-9的特异性抑制剂Z-LEHD-FMK所抑制，而caspase-8的特异性抑制剂Z-IETD-FMK却没有这一作用。结论：人参皂甙对人白血病HL-60细胞具有高效的致凋亡作用，激活线粒体凋亡途径可能是其致凋亡的主要作用机制。

Abstract:

OBJECTIVE: To study the effect of ginsenoside on apoptosis of human leukemia-60 (HL-60) cells and its mechanism. METHODS: MTT cytotoxicity assay was used to determine the growth inhibition activity of ginsenoside (100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μmol/L) on HL-60 cells. The apoptosis of HL-60 cells after treatment with ginsenoside (0,5,10 and 20 μmol/L) was determined by Annexin V-FITC/PI staining and flow cytometry. The cleavage of total proteins by caspase-8, caspase-9 and caspase-3 was evaluated by Western blot. The cleavage of caspase-3 protein was detected by Western blot after treatment with 10 μmol/L ginsenoside and caspase-8 and 9 inhibitors. RESULTS: Ginsenoside had potent cytotoxicity on HL-60 cells, with an IC50 value of 7.3±1.2 μmol/L. After treatment with ginsenoside (0, 5, 10 and 20 μmol/L) for 48 hours, the apoptotic rate displayed a dose dependency, as shown by flow cytometry, with significant differences between the groups (F=12.67, P<0.01). Western blot showed that there were caspase-9 and caspase-3 cleavage bands, but without caspase-8 cleavage band. The specific inhibitor of caspase-9 Z-LEHD-FMK could block the caspase-3 cleavage induced by 10 μmol/L ginsenoside, but the specific inhibitor of caspase-8 Z-IETD-FMK did not have this effect. CONCLUSIONS: Ginsenoside can induce apoptosis of HL-60 cells, which may be related to a mitochondria-dependent pathway.