20（S）-人参皂苷Rg3对U266细胞增殖及其自分泌血管内皮生长因子的影响

Effect of 20（S）-ginsenoside Rg3 on the proliferation inhibition and secretion of vascular endothelial growth factor of multiple myeloma cell line U266

目的 探讨20（S）-人参皂苷Rg3[简称20（S）-Rg3]体外对多发性骨髓瘤（MM）细胞增殖及其自分泌血管内皮生长因子（VEGF）的影响.方法 以MM细胞系U266细胞为研究对象,利用MTT法检测不同浓度20（S）-Rg3对细胞增殖的影响;流式细胞术检测细胞周期及凋亡率;ELISA法检测细胞培养上清VEGF水平;Westem blot法检测凋亡相关蛋白caspase-3、8和9的表达.结果 20（S）-Rg3在一定浓度范围内可抑制U266细胞增殖,并呈浓度依赖性（P＜0.05）,作用24、48 h的IC50值分别为（71.07±2.63）μmol/L和（44.06±3.98）μmol/L;ELISA法检测结果显示20（S）-Rg3处理24 h细胞培养上清中VEGF的含量呈剂量依赖性减少（P＜0.05）,未加药组和加近IC50浓度（80 μmol/L）组的细胞培养上清中VEGF的含量分别为（419.93±36.76）和（314.82±27.05）pg/106细胞.流式细胞术检测显示0、20、40、80 μmol/L 20（S）-Rg3处理后U266细胞凋亡率分别为（0.51±0.05）％、（8.32±0.83）％、（10.72±1.29）％和（15.27±2.26）％,呈剂量依赖性升高（P＞0.05）;G0/G1期细胞比率分别为（49.11±1.71）％、（52.72±7.75）％、（60.29±5.76）％和（61.81±3.46）％,呈剂量依赖性增多（P＜ 0.05）;Western blot检测显示随20（S）-Rg3浓度的增加,procaspase-3、8和9表达略下降,cleaved caspase-3、8和9显著表达上升（P＜0.05）.结论 20（S）-Rg3可抑制U266细胞增殖,其机制可能是通过诱导细胞凋亡,上调cleaved cas-pase-3、8、9的表达及阻滞细胞周期进程实现的;20（S）-Rg3可抑制U266细胞分泌VEGF,潜在治疗MM的新制剂.

Objective To explore the effect of 20 （S）-ginsenoside Rg3 [20 （S）-Rg3] on the proliferation inhibition and secretion of vascular endothelial growth factor （VEGF） of multiple myeloma （MM） cell line U266.Methods The proliferation inhibition rate of U266 cells after treatment with different doses of 20 （S）-Rg3 was detected by MTT method,the cell cycle and apoptosis by flow cytometry,the expression of apoptosis related proteins of caspase-3,8 and 9 by Western blot,VEGF concentration in the culture supernatant by ELISA.Results It showed that 20 （S）-Rg3 could inhibit the proliferation of U266 in a dose-dependent manner （P〈0.05） with IC50 of （71.07±2.63） μmol/L and （44.06± 3.98） μmol/L at 24 h and 48 h,respectively.VEGF concentration in the culture supernatant showed a dose-dependent reduction （P〈0.05）,decreased from （419.93 ± 36.76） pg/106cells in the control group to （314.82±27.05） pg/106cells in 80 μmol/L 20 （S）-Rg3 treated group by ELISA assay.Flow cytometry with Annexin-V/PI double staining revealed that 20 （S）-Rg3 may induce U266 cells apoptosis in a concentration-dependent manner from （0.51±0.05）% at control group to （8.32±0.83）%,（10.72±1.29）% and （15.27±2.26）% at 20,40 and 80μmol/L treatment groups,respectively （P〈0.05）.Flow cytometry with PI staining showed that the ratio of cells in G0/G1 phase increased from （49.11±1.71）% to （52.72±7.75）%,（60.29± 5.76） % and （61.81±3.46） %,respectively （P〈0.05）.Western blot analysis indicated that the expression of caspase-3,8 and 9 declined,and that ofcleaved-caspase-3,8 and 9 significantly increased （P〈0.05） with 20 （S）-Rg3 concentration increased.Conclusion 20（S）-Rg3 can inhibit the proliferation ofU266 cells by cell cycle arrest in Gi phase and induce cell apoptosis by increasing the expressions of cleaved-caspase-3,-8 and-9.It can also inhibit VEGF secretion of U266 cells,which makes it a potential agent for multiple myeloma therapy.