[关键词]
[摘要]
目的 探讨黄芪多糖通过Wnt/β-catenin信号通路对人肝癌HepG2细胞增殖及凋亡的影响。方法 四甲基偶氮唑蓝(MTT)法检测HepG2细胞增殖能力和存活率;Annexin V-FITC/PI双染和Caspase-3活性检测细胞凋亡;荧光素酶实验检测黄芪多糖(100、200 mg/L)处理后HepG2细胞Wnt/β-catenin通路活性改变;实时荧光定量PCR(qRT-PCR)、Western blotting法检测细胞内的β-catenin、c-myc和Cyclin D1表达水平。结果 与对照组比较,随着黄芪多糖质量浓度的增加和作用时间的延长,HepG2细胞存活率显著降低(P<0.05)。与对照组比较,黄芪多糖100、200 mg/L组HepG2细胞凋亡率显著升高(P<0.05);凋亡关键因子Caspase-3的相对活性显著升高(P<0.05),cleaved Caspase-3蛋白水平显著升高,Bcl-2蛋白表达水平显著降低;荧光素酶活性显著降低(P<0.05);β-catenin、c-myc和Cyclin D1 mRNA及蛋白表达水平显著降低(P<0.05、0.01)。结论 黄芪多糖通过下调Wnt/β-catenin信号通路抑制凋亡相关基因Bcl-2的表达,促进HepG2细胞凋亡。
[Key word]
[Abstract]
Objective To investigate the effects of Astragalus polysaccharides (APS) on HepG2 cell proliferation and apoptosis through Wnt/β-catenin signaling pathway. Methods Cell proliferation ability and cell viability was measured by MTT assay. Annexin V-FITC/PI stain and Caspase-3 activity assay were used to detect cell apoptosis. The changes of Wnt/β-catenin signaling pathway activity after treated by APS (100 and 200 mg/L) was measured by luciferase assay. The expression levels of β-catenin, c-myc, and Cyclin D1 were detected by qRT-PCR and Western blotting. Results As the concentration of APS increases, the cell viability of HepG2 cells decreased significantly (P < 0.05). Compared with control group, the cells apoptosis rates of in APS 100 and 200 mg/L groups were increased remarkably (P < 0.05); The relative activities of Caspase-3 in APS 100 and 200 mg/L groups increased significantly (P < 0.05); Meanwhile, the activity of luciferase in APS 100 and 200 mg/L groups decreased significantly (P < 0.05). The expression level of cleaved Caspase-3 was increased significantly. The mRNA 1evel of β-catenin, Cyclin D1, and c-myc in APS 100 and 200 mg/L groups were decreased significantly (P < 0.05), and the expression levels of Bcl-2, β-catenin, c-myc, and Cyclin D1 protein in APS 100 and 200 mg/L groups were decreased significantly (P < 0.05, 0.01). Conclusion APS promote the apoptosis of HepG2 cells through the down-regulation of Wnt/β-catenin signaling pathway, which leads to the inhibition of apoptosis-related gene Bcl-2 expression.
[中图分类号]
[基金项目]
河南省自然科学基金项目(162300410289)