摘要
目的:探讨茶黄素调节磷脂酰肌醇 3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对A549 细胞增殖、凋亡及自噬的影响.方法:测定 0、2.5、5、10、20、40、80、160μmoL/L茶黄素干预A549 细胞后增殖率,筛选出合适的作用浓度.将 A549 细胞分为 control 组、低-茶黄素组(20μmoL/L茶黄素)、中-茶黄素组(40μmoL/L茶黄素)、高-茶黄素组(80μmoL/L茶黄素)、高-茶黄素+740Y-P 组(80μmoL/L茶黄素+30μmoL/L的PI3K/Akt/mTOR信号通路激活剂 740Y-P).CCK-8 法、平板集落形成实验测定A549 细胞增殖;流式细胞术、MDC 法分别测定 A549 细胞凋亡及自噬空泡生成;Western blot 测定 A549 细胞 PI3K/Akt/mTOR 信号通路蛋白及自噬蛋白表达.结果:与0μmoL/L茶黄素对比,2.5、5、10、20、40、80、160μmoL/L的茶黄素均可抑制A549 细胞增殖,经计算茶黄素对A549 细胞的IC50 值为36.72μmoL/L.与control组比较,低-茶黄素组、中-茶黄素组、高-茶黄素组A549 细胞增殖率、集落形成率、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR均降低,凋亡率、自噬空泡相对含量、LC3II/LC3I、Beclin-1 蛋白表达均升高,且均呈茶黄素剂量依赖性变化(P<0.05).与高-茶黄素组对比,高-茶黄素+740Y-P 组 A549 细胞增殖率、集落形成率、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR均升高,凋亡率、自噬空泡相对含量、LC3II/LC3I、Beclin-1 蛋白表达均降低(P<0.05).结论:茶黄素可通过抑制PI3K/Akt/mTOR信号通路激活,诱导保护性自噬,进而促进肺癌细胞凋亡,抑制其增殖.
Abstract
Objective:To investigate the effects of theaflavin on A549 cell proliferation,apoptosis,and autophagy by regulating the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian tar-get of rapamycin(mTOR)signaling pathway.Methods:The proliferation rate of A549 cells was measured af-ter intervention with 0,2.5,5,10,20,40,80,and 160 μmol/L theaflavin,and the appropriate concentra-tion of action was screened.A549 cells were separated into control group,low theaflavin group(20 μmol/L theaflavin),medium theaflavin group(40 μmol/L theaflavin),high theaflavin group(80 μmol/L theafla-vin),and high theaflavin+740Y-P group(80 μmol/L theaflavin+30 μmol/L PI3K/Akt/mTOR signaling pathway activator 740Y-P).CCK-8 method and plate colony formation experiment were applied to determine the proliferation of A549 cells.Flow cytometry and MDC methods were applied to determine apoptosis and au-tophagic vacuole formation in A549 cells,respectively.Western blot was applied to determine the expression of PI3K/Akt/mTOR signaling pathway proteins and autophagy proteins in A549 cells.Results:Compared with 0 μmol/L of theaflavin,2.5,5,10,20,40,80,and 160 μmol/L of theaflavin can inhibit the prolifera-tion of A549 cells.The IC50 value of theaflavin on A549 cells was calculated to be 36.72 μmol/L.Compared with the control group,the proliferation rate,colony formation rate,p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR of A549 cells in the low theaflavin group,medium theaflavin group,and high theaflavin group all reduced,the apoptosis rate,relative content of autophagic vacuoles,LC3II/LC3I,and Beclin-1 protein expression all increased,and all showed dose-dependent changes in theaflavin(P<0.05).Compared with the high theaflavin group,the proliferation rate,colony formation rate,p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR of A549 cells in the high theaflavin+740Y-P group all increased,the apoptosis rate,relative content of autophagic vacuoles,LC3II/LC3I,and Beclin-1 protein expression all reduced(P<0.05).Con-clusion:Theaflavin can induce protective autophagy by inhibiting the activation of the PI3K/Akt/mTOR sig-naling pathway,thereby promoting apoptosis and inhibiting the proliferation of lung cancer cells.
基金项目
陕西省汉中市人民医院项目计划(sy-2023-02)
国家科技期刊平台
NSTL
万方数据