黄芪多糖对肺癌A549细胞自噬的作用及机制研究

Effect and mechanism of Astragalus polysaccharides on autophagy in lung cancer A549 cells

目的探讨黄芪多糖(APS)对黄嘌呤氧化酶(XOD)诱导的肺癌A549细胞自噬模型及磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路的影响及对自噬相关调节因子表达。方法实验分为6组:正常组、模型组(20 U·L^(-1) XOD处理24 h)、低、中、高3个浓度实验组(在模型组基础上分别使用100,200和400 mg·L^(-1) APS进行处理)及3-MA抑制组(在模型组基础上使用2 mmol·L^(-1)3-甲基腺嘌呤进行处理)。以免疫荧光法检测自噬相关蛋白,以蛋白质印迹法检测PI3K/Akt/mTOR信号通路蛋白,以实时荧光定量聚合酶链反应法检测mRNA的表达水平。结果正常组、模型组、高浓度实验组及3-MA抑制组的微管相关蛋白1轻链3B(LC3B)的蛋白荧光强度分别为6368.50±17.90,2.57×10^(4)±224.03,7443.00±19.20和8356.50±18.52;抗坏死骨片1(P62)的蛋白荧光强度分别为2.86×10^(4)±194.85,3225.50±12.69,1.04×10^(4)±209.50和1.04×10^(4)±96.78。这4组的PI3K的蛋白相对表达水平分别1.11±0.05,1.93±0.06,1.30±0.04和1.40±0.08;这4组的Akt蛋白表达水平为0.64±0.06,0.27±0.06,0.50±0.09和0.66±0.03;这4组的mTOR蛋白表达水平为1.94±0.06,0.93±0.06,1.28±0.03和0.91±0.04;这4组的Beclin1蛋白表达水平为0.48±0.04,1.03±0.04,0.77±0.09和0.65±0.09。mRNA表达的趋势与蛋白一致。自噬相关调节因子(LC3B、P62、Beclin1)及PI3K、Akt、mTOR信号因子,模型组与正常组相比,或高浓度实验组与模型组相比,差异均有统计学意义(均P<0.05)。结论黄芪多糖防治肺癌的分子机制之一可能是通过调节PI3K/Akt/mTOR信号通路,抑制肺癌A549细胞自噬而发挥作用。

Objective To explore the effects of Astragalus polysaccharide(APS)on xanthine oxidase(XOD)-induced autophagy and phosphoinositide-3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway in lung cancer A549 cells and regulate the expression of autophagy-related factor.Methods The experiment was divided into 6 groups:normal group,model group(treatment with 20 U·L^(-1) XOD for 24 h),experimental-L,experimental-M,experimental-H groups(treatment with 100,200 and 400 mg·L^(-1) APS on the basis of model group)and 3-MA inhibition group(treated with 2 mmol·L^(-1)3-methyladenine on the basis of model group).Autophagy-related proteins were detected by immunofluorescence,PI3K/Akt/mTOR signaling pathway proteins were detected by Western blotting,and mRNA expression levels were detected by real-time fluorescent quantitative polymerase chain reaction.Results The protein fluorescence intensity of microtubule-associated protein 1 light chain 3B(LC3B)in normal group,model group,high concentration experimental group and 3-MA inhibition group were 6368.50±17.90,2.57×10^(4)±224.03,7443.00±19.20,8356.50±18.52,respectively.The protein fluorescence intensity of P62 was 2.86×10^(4)±194.85,3225.50±12.69,1.04×10^(4)±209.50,1.04×10^(4)±96.78,respectively.The relative expression levels of PI3K in these four groups were 1.11±0.05,1.93±0.06,1.30±0.04 and 1.40±0.08,respectively.The expression levels of Akt protein in these four groups were 0.64±0.06,0.27±0.06,0.50±0.09,0.66±0.03;the mTOR protein expression levels in these four groups were 1.94±0.06,0.93±0.06,1.28±0.03,0.91±0.04;beclin 1 protein expression levels in these four groups were 0.48±0.04,1.03±0.04,0.77±0.09,0.65±0.09.The trend of mRNA expressions in these four groups were consistent with that of proteins.Autophagy related regulatory factors(LC3B,P62,Beclin1),PI3K,Akt,mTOR signal factors,there were statistically significant differences between the model group and the normal group,and between the experimental-H group and the model group(all P<0.05).Conclusion One of the molecular mechanisms of APS against lung cancer may be through regulating PI3K/Akt/mTOR signaling pathway and inhibiting autophagy in lung cancer A549 cells.