人参总皂苷TSPG联合EPO对白血病细胞KG1-α增殖的影响

Effect of total saponins of panax ginseng combined with erythropoietin on proliferation in leukemia KG1-α cells

目的探讨人参总皂苷TSPG单独或联合人促红细胞生成素(EPO)对人白血病细胞KG1-α增殖的影响及机制探讨。方法体外培养KG1-α细胞,选取处于对数生长期的细胞用于实验。空白对照组予以常规培养;人参总皂苷TSPG组分别加入25、50、100、200、400 mg/L TSPG;人参总皂苷+EPO组加入上述等量TSPG,同时加入0.5 U/L EPO,并设置EPO对照组。培养3、7、14 d后用CCK-8法检测TSPG单独或联合EPO对KG1-α细胞增殖的影响;运用Real-time PCR技术检测EPOR、STAT5、Jak2、AKT等EPO/EPOR通路相关基因的表达。用Western blot检测Bcl-2、Bax、Cleaved Caspase-3、P53、P16等蛋白的表达,EPOR蛋白的表达及其活性。结果人参总皂苷TSPG在体外能明显抑制KG1-α细胞的增殖,100μmol/L为最适作用浓度,7 d为最适作用时间,如果在EPO存在的条件下,其最适浓度则降低到75 mg/L;与对照组相比,在EPO存在的条件下人参总皂苷TSPG诱导可明显使细胞阻滞于G0/G1期,若不使用EPO则TSPG对细胞周期无影响;Real-time PCR检测结果显示TSPG诱导组KG1-α细胞内EPOR、Jak2、STAT5等由EPOR介导的JAK-STAT通路相关基因的表达均随TSPG浓度增加而降低,其中高浓度具有明显差异(P<0.05);Western blot检测经单独使用TSPG处理了7 d的KG1-α细胞,与对照组相比,其EPOR及p-EPOR表达明显下调,Jak2、STAT5、p-STAT5、Bcl-2蛋白表达下调,而Bax、Cleaved Caspase-3表达增高,P53及P16无变化;然而添加了EPO处理的各组p-EPOR、Jak2、p-STAT5、P53及P16的表达明显增加,Bcl-2、Bax、Cleaved Caspase-3的表达没有变化。结论 TSPG能下调细胞内由EPOR介导的Jak-STAT通路相关蛋白的表达,并降低EPOR的活性,进而下调Bcl-2、上调Bax和Cleaved Caspase-3来促进细胞凋亡;而添加了EPO后这种作用被拮抗,抑制细胞增殖的作用是通过上调P53及P16促进细胞衰老而产生的。

Objective To determine the effect of total saponins of panax ginseng（ TSPG） separately or jointly with erythropoietin（ EPO） on the proliferation of human leukemia cell line KG1-α and investigate the possible underlying mechanism. Methods The KG1-α cells were respectively treated by 25,50,100,200 and 400 mg / L TSPG in presence or absence of 0. 5 U / L EPO. The cells cultured in normal condition served as control. CCK-8 assay was utilized to detect the proliferation of KG1-α cells in 3,7 and 14 d after treatment.Real-time PCR was employed to detect the mRNA expression of EPO / EPO receptor（ EPOR） pathway related genes,such as EPOR,STAT5,Jak2,and AKT. Western blot analysis was used to measure the expression of apoptosis related proteins, Bcl-2, Bax, Cleaved Caspase-3, P53, P16 and EPOR. Results TSPG significantly inhibited the proliferation in KG1-α cells,with 100 μmol / L as optimal concentration and 7 d as best treatment period. In the presence of 0. 5 U / L EPO,its optimal concentration was decreased to 75 mg / L.Compared with the control,TSPG significantly arrested the cell cycle at G0/ G1 phase in the presence of EPO,but without the EPO,TSPG had no effect on cell cycle. Real-time PCR analysis showed that the expression of the genes related to EPOR-mediated Jak-STAT signaling pathway,such as EPOR,Jak2 and STAT5 genes were in a negatively dose-dependent manner with TSPG in KG1-α cells,especially in the cells treated by high dose TSPG（ P〈0. 05）. Western blotting indicated that the expression of EPOR,p-EPOR,Jak2,STAT5,p-STAT5 and Bcl-2 was down-regulated,while that of Bax and Cleaved Caspase-3 proteins was increased,and that of P53 and P16 had no change in the KG1-α cells treated by TSPG in absence of EPO. However in the presence of EPO,the expression of p-EPOR,Jak2,p-STAT5,P53 and P16 was significantly increased,while Bcl-2,Bax and Cleaved Caspase-3 had no change in expression. Conclusion TSPG down-regulates the expression of EPOR and related proteins in Jak-STAT pathway mediated by EPOR,decreases the activity of EPOR,then reduces the expression of Bcl-2 protein,and increases the expression of Bax and Cleaved Caspase-3 proteins to promote the cell apoptosis in the KG1-α cells. While,when combining EPO,these effects are antagonized. The inhibition of cell proliferation is induced by increasing the expression of P53 and P16 and promoting cell senescence.