黄芪多糖抑制人肝癌细胞增殖的作用机制研究

Mechanism of Astragalus polysaccharide in inhibiting proliferation of human hepatocellular carcinoma cells

目的研究黄芪多糖(Astragalus polysaccharide,APS)对人肝癌细胞增殖的影响,并探讨其潜在的药理作用机制,为临床用药提供依据。方法采用人肝癌Hep G2细胞,分别给予0、25、50、100μg·m L-1APS处理,并用10μmol·L-1Akt通路抑制剂LY294002联合干预。噻唑蓝(MTT)法检测不同浓度APS对Hep G2细胞增殖活性的影响;免疫印迹法(Western Blot)检测LC3A、LC3B、P62、Akt、p-Akt、Bcl-2、Bax等蛋白的表达水平;流式细胞术(Flowcy tometry)检测细胞周期分布和细胞凋亡比例。结果APS处理Hep G2后,细胞形态变圆变亮;MTT实验显示:APS对Hep G2细胞生长具有抑制作用,且呈剂量依赖性;Western blot检测细胞的自噬水平发现:APS可减少LC3A、P62的表达,增加LC3B表达的水平;APS处理Hep G2细胞后,呈现G1期细胞阻滞和细胞凋亡水平显著增高,Akt、p-Akt、Bcl-2蛋白表达的水平降低,Bax蛋白表达的水平增加;与单独处理组比较,APS联合LY294002干预后,细胞凋亡的水平显著增加,Akt、p-Akt、Bcl-2蛋白表达的水平显著降低,Bax蛋白表达的水平显著增加。结论APS通过增加细胞自噬和抑制Akt通路抑制人肝癌细胞增殖,提示靶向细胞自噬和Akt可成为增强抗肝癌药物敏感性的潜在靶点。

OBJECTIVE To study effect of Astragalus polysaccharide(APS)on proliferation of human hepatocellular carcinoma cells and to explore its potential pharmacological mechanism,thus to provide scientific basis for clinical drug use.METHODS Hep G2 cells were treated with APS(0,25,50,100μg·m L-1)and that combined with Akt pathway inhibitor LY294002(10μmol·L-1).MTT assay was used to detect effect of different APS concentrations on proliferation of Hep G2 cells.Western Blot was used to detect expression levels of proteins including LC3 A,LC3 B,P62,Akt,p-Akt,Bcl-2 and Bax.Flow cytometry was used to detect cell cycle and apoptosis.RESULTS After treated with APS,Hep G2 cells became round and bright in morphology.The result of MTT assay showed that APS inhibited the growth of Hep G2 cells in a dose-dependent manner.Compared with control group,autophagic protein level of LC3 A and P62 significantly decreased in APS group,and that of LC3 B significantly increased.G1 arrest and cell apoptosis of Hep G2 significantly increased.Expression levels of Akt,p-Akt and Bcl-2 decreased,and Bax expression level increased.Compared with single treatment group,apoptosis level of APS combined LY294002 intervention significantly increased;the expression levels of Akt,p-Akt and Bcl-2 significantly decreased,and Bax expression level significantly increased.CONCLUSION APS inhibits the proliferation of human hepatocellular carcinoma cells by increasing autophagy and inhibiting Akt pathway,suggesting that targeting autophagy and Akt may be a potential approach for enhancing sensitivity of anti-hepatoma drug.