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[目的]研究姜黄素诱导人肺癌A549细胞自噬现象及与哺乳动物雷帕霉素靶蛋白(mTOR)的关系。[方法]采用CCK-8法检测姜黄素对A549细胞增殖的抑制作用;采用吖啶橙染色(AO)及转染GFP-LC3质粒荧光显微镜下观察姜黄素处理后A549细胞的自噬现象;采用Western blot法检测微管相关蛋白轻链Ⅱ(LC3Ⅱ)、微管相关蛋白轻链Ⅰ(LC3Ⅰ)、mTOR表达的变化。[结果]姜黄素对人肺癌A549细胞增殖有明显抑制作用,且成明显的剂量—时间依赖关系。AO染色结果显示,与对照组相比,姜黄素40μmol/L组及Rapa组细胞内酸性滤泡染成亮红色荧光比例增多;与姜黄素40μmol/L组相比,姜黄素40μmol/L+自噬抑制剂3-甲基腺嘌呤(3-MA)组及3-MA组亮红色荧光比例下降。细胞转染GFP-LC3后姜黄素40μmol/L组和Rapa组胞浆内点状的绿色荧光斑点明显;与姜黄素40μmol/L组相比,姜黄素40μmol/L+3-MA组及3-MA组胞浆内点状的绿色荧光斑点明显比例有所下降。Western blot法检测结果显示,与对照组相比,姜黄素40μmol/L组作用24h后,LC3-Ⅱ/LC3-Ⅰ表达显著升高(P<0.05),姜黄素40μmol/L+3-MA组LC3Ⅱ/LC3-Ⅰ表达低于姜黄素40μmol/L组(P<0.05)。姜黄素40μmol/L组mTOR表达显著下降(P<0.01),姜黄素40μmol/L+3-MA组mTOR表达高于姜黄素40μmol/L组(P<0.05)。[结论]姜黄素能够明显抑制A549细胞增殖并诱导A549细胞发生自噬,同时还能够明显抑制mTOR蛋白的表达水平,其机理可能与mTOR的信号通路有关。
[Objective] To study the relationship between curcumin-induced autophagy in human lung cancer A549 cells and mammalian target of rapamycin (mTOR). [Method] The inhibitory effect of curcumin on the proliferation of A549 cells was detected by CCK-8 method. Autophagy was observed in A549 cells treated with curcumin by acridine orange staining (AO) and GFP-LC3 transfected with plasmids. Western blot was used to detect the expression of microtubule-associated protein light chain Ⅱ (LC3Ⅱ), microtubule-associated protein light chain Ⅰ (LC3Ⅰ) and mTOR. [Result] Curcumin significantly inhibited the proliferation of human lung cancer A549 cells in a dose-time dependent manner. Compared with the control group, AO staining showed that the percentage of bright red fluorescence in the follicles of curcumin 40μmol / L group and Rapa group was significantly higher than that of the control group. Curcumin 40μmol / L + autophagy The ratio of bright red fluorescence of 3-methyladenine (3-MA) group and 3-MA group decreased. Compared with 40μmol / L curcumin group, 40μmol / L + 3-MA curcumin group and 3-MA curcumin group showed significantly more spots in the cytoplasm of curcumin transfected with GFP- The percentage of spot-shaped green fluorescent spots in the cytoplasm of MA decreased significantly. Western blot results showed that compared with the control group, the expression of LC3-Ⅱ / LC3-Ⅰ was significantly increased after treated with 40μmol / L curcumin for 24 hours (P <0.05), while the curcumin 40μmol / L + LC3Ⅱ / LC3-Ⅰ expression was lower than curcumin 40μmol / L group (P <0.05). Curcumin 40μmol / L group mTOR expression was significantly decreased (P <0.01), curcumin 40μmol / L + 3-MA group mTOR expression was higher than curcumin 40μmol / L group (P lt; [Conclusion] Curcumin can significantly inhibit the proliferation of A549 cells and induce the autophagy in A549 cells. At the same time, curcumin also significantly inhibits the expression of mTOR protein, which may be related to the mTOR signaling pathway.