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目的探讨人参多糖(ginseng polysaccharide,GPS)诱导白血病细胞K562凋亡和周期阻滞的机制。方法取对数生长期的K562细胞,调整密度为7×108/L,空白对照组予以常规培养;GPS组加入400 mg/L GPS。流式细胞仪测定细胞的凋亡率及细胞周期分布变化;RT-PCR检测细胞ERK表达的变化。免疫组化的方法检测细胞中p-ERK、NF-κB、Cyclin D1蛋白定位及表达量的变化。Western blot检测细胞中ERK、p-ERK、NF-κB、Cyclin D1蛋白的变化。结果与对照组比较,K562细胞在400 mg/L GPS的作用下,体外培养24、48、72 h后,GPS组细胞凋亡率显著增高(P<0.05),周期分布检测结果显示,与对照组相比,GPS组K562细胞G0/G1期细胞数量呈时间依赖性增多(P<0.05),G2+M、S期细胞数量则明显减少(P<0.05)。RT-PCR检测结果显示,400 mg/L GPS处理48 h组ERK mRNA的表达水平明显低于对照组(0.20vs 0.50,P<0.05)。免疫组化显示p-ERK、NF-κB、Cyclin D1主要分布在细胞核和细胞质,与对照组相比,GPS组K562细胞内p-ERK、NF-κB、Cyclin D1的表达明显减弱。Western blot检测结果显示,ERK总蛋白无明显变化(P>0.05),而p-ERK、NF-κB、Cyclin D1随时间变化有减少趋势,且在48 h减少明显,差异有统计学意义(P<0.05)。结论人参多糖GPS可促使K562细胞周期阻滞在G0/G1期,并诱导K562细胞凋亡,且均呈时间依赖性。GPS促进K562细胞凋亡、导致其细胞周期阻滞的机制可能是通过抑制ERK/NF-κB信号通路的激活,进而下调Cyclin D1来实现的。
Objective To investigate the mechanism of ginseng polysaccharide (GPS) -induced apoptosis and cell cycle arrest in leukemia K562 cells. Methods The logarithmic growth phase K562 cells were cultured in a density of 7 × 108 / L, and the control group was routinely cultured. The GPS group was given 400 mg / L GPS. Flow cytometry was used to determine the changes of cell apoptosis and cell cycle distribution. The changes of ERK expression were detected by RT-PCR. The expression of p-ERK, NF-κB and Cyclin D1 in the cells was detected by immunohistochemistry. Western blot was used to detect the changes of ERK, p-ERK, NF-κB and Cyclin D1 in the cells. Results Compared with the control group, the apoptotic rate of K562 cells in GPS group was significantly increased (P <0.05) after being cultured with 400 mg / L GPS for 24, 48 and 72 h. The results of cycle distribution showed that compared with the control Compared with the control group, the number of cells in G0 / G1 phase of K562 cells in GPS group increased in a time-dependent manner (P <0.05), and the number of cells in G2 + M and S phase decreased significantly (P <0.05). The results of RT-PCR showed that ERK mRNA expression in 48 h treated with 400 mg / L GPS was significantly lower than that in control (0.20 vs 0.50, P <0.05). Immunohistochemistry showed that p-ERK, NF-κB and Cyclin D1 mainly distributed in the nucleus and cytoplasm. Compared with the control group, the expression of p-ERK, NF-κB and Cyclin D1 in K562 cells of GPS group was significantly decreased. The result of Western blot showed that there was no significant change in the total protein of ERK (P> 0.05), while the expression of p-ERK, NF-κB and Cyclin D1 decreased with time and decreased significantly at 48 h (P <0.05). Conclusion Ginseng polysaccharide GPS can promote the arrest of K562 cell cycle in G0 / G1 phase and induce the apoptosis of K562 cell in a time-dependent manner. The mechanism by which GPS can promote the apoptosis of K562 cells and lead to cell cycle arrest may be through inhibiting the activation of ERK / NF-κB signaling pathway and then down-regulating Cyclin D1.