黄芪多糖通过Wnt/β-catenin信号通路促进肝癌细胞凋亡研究

Astragalus polysaccharides downregulates apoptosis in HepG2 cell through Wnt/β-catenin signaling pathway

目的探讨黄芪多糖通过Wnt/β-catenin信号通路对人肝癌Hep G2细胞增殖及凋亡的影响。方法四甲基偶氮唑蓝(MTT)法检测Hep G2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Hep G2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CyclinD1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,Hep G2细胞存活率显著降低(P<0.05)。与对照组比较,黄芪多糖100、200mg/L组Hep G2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Hep G2细胞凋亡。

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 1 evel 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.