人参多糖对K562细胞凋亡与细胞周期的影响及其机制

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。

Mechanism of ginseng polysaccharide on apoptosis and cell cycle in leukemia K562 cells

Objective To investigate the mechanism of ginseng polysaccharide(GPS) on the cell cycle and apoptosis in leukemia K562 cells.Methods Leukemia K562 cells in logarithmic phase with density of 7×108L-1 were incubated with(GPS group) and without(control group) 400 mg/L GPS,respectively.The effects of GPS on K562 cell cycle and apoptosis were determined by flow cytometry(FCM).The mRNA expression of ERK was detected by RT-PCR.The distributions and protein expressions of ERK,p-ERK,NF-κB and cyclin D1 were detected by immunohistochemical staining and Western blotting.Results Compared with the control group,the apoptosis rate of the K562 cells in the GPS group significantly increased after treatment for 24,48 and 72 h(P<0.05).The number of the K562 cells arrested in G0/G1 phase increased significantly in a time-dependent manner in the GPS group(P<0.05),and that of the K562 cells in G2+M and S phases decreased significantly(P<0.05).The mRNA level of ERK after 48 h treatment was significantly lower in the GPS group than in the control group(0.20 vs 0.50,P<0.05).The immunohistochemical staining results showed that p-ERK,NF-κB and cyclin D1 mainly located in the nucleus and cytoplasm,and the expressions of the three proteins significantly decreased in the GPS group compared with those in the control group.The Western blotting results showed that there as no significant change in total ERK(P>0.05),but the differences of p-ERK,NF-κB and cyclin D1 were statistically significant between the GPS group and the control group(P<0.05).Conclusion GPS can induce cell cycle arrest in G0/G1 phase and cell apoptosis of K562 cells in a time-dependent manner,probably through inhibiting ERK/NF-κB signaling pathway and thus reducing cyclin D1 expression.