白术多糖对肝癌细胞干样特性、上皮间质转化和Akt/NF-κB磷酸化的影响

The effect of atractylodes macrocephalaon polysaccharide on stem cell-like properties of hepatoma cells,epithelial-mesenchymal transition and phosphorylation of Akt/NF-κB pathway

目的探讨白术多糖(AMPs)对肝癌细胞的干样特性和上皮间质转化(EMT)以及Akt/NF-κB磷酸化的影响。方法培养肝癌HepG2细胞,随机分为空白对照组,AMPs低、中、高浓度处理组(50、100、200μg/ml),显微下观察细胞成球情况,蛋白印迹(Western blotting)检测AMPs对HepG2细胞生长标记物、干样标记物及Akt/NF-κB通路的表达,实时荧光定量PCR(qPCR)和免疫荧光检测EMT标记物的表达。结果与空白对照组比较,100、200μg/ml浓度的AMPs处理后,HepG2细胞Ki-67、PCNA蛋白表达下调,p21蛋白表达上调(P<0.05);细胞成球数量减少,球体直径明显降低(P<0.05);SOX2、Oct4和Nanog蛋白表达下调(P<0.05);E-cad mRNA表达水平升高,N-cad mRNA表达水平和Vimentin阳性染色细胞数量降低(P<0.05);Akt和p-P65 NF-κB磷酸化水平降低(P<0.05)。50μg/ml AMP组上述指标与空白对照组比较,差异无统计学意义(P>0.05)。结论AMPs可抑制HepG2细胞的活性、干细胞样特性及EMT能力,抑制Akt/NF-κB磷酸化水平可能是其潜在的作用机制。

Objective To explore the effects of atractylodes macrocephalaon polysaccharide(AMPs)on cell-like properties of hepatoma cells,epithelial-mesenchymal transition(EMT)and phosphorylation of Akt/NF-κB.Methods The hepatoma cells HepG2 were cultured and randomly divided into blank control group,low-concentration,medium-concentration and high-concentration AMPs groups(50,100,200μg/ml).The cells pelletization was observed under microscope.The expression of growth markers,stem cell-like markers and Akt/NF-κB pathway in HepG2 was detected by Western blotting.The expression of EMT markers in hepatoma cells was detected by real-time fluorescent quantitative PCR and immunofluorescence.Results Compared with the blank control group,treatment with AMPs at concentrations of 100 and 200μg/ml resulted in down-regulation of Ki-67 and PCNA protein expression and up-regulation of p21 protein expression in HepG2 cells(P<0.05);The number of cell spheroids and the diameter of spheroids significantly decreased(P<0.05);The expression of SOX2,Oct4,and Nanog proteins was down-regulated(P<0.05);The expression level of E-cad mRNA increased,while the expression level of N-cad mRNA and the number of Vimentin positive stained cells decreased(P<0.05);The phosphorylation levels of Akt and p-P65 NF-κB decreased(P<0.05).There was no statistically significant difference in the above indicators between the 50μg/ml AMP group and the blank control group(P>0.05).Conclusion AMPs can inhibit the activity of HepG2,stem cell-like properties and EMT ability.The inhibition of Akt/NF-κB phosphorylation may be its potential action mechanism.