芪玉三龙汤调节mTOR/Beclin1/LC3信号轴相关分子的表达诱导肺癌A549细胞自噬

Effect of Qiyu Sanlong Decoction in Inducing Autophagy of A549 Cells by Regulating Molecular Expression Related to Signal Axis of mTOR-Beclin1-LC3

目的:从细胞水平探讨芪玉三龙汤干预肺癌A549细胞对哺乳动物雷帕霉素靶蛋白(mTOR)/自噬关键分子酵母Atg6同系物(Beclin1)/微管相关蛋白1轻链3(LC3)信号轴关键分子表达的影响。方法:选择肺癌人A549细胞作为研究对象,采用细胞增殖检测试剂盒(CCK-8)法检测芪玉三龙汤含药血清对A549细胞活性的影响;原位末端标记法(TUNEL),透射电镜(TEM)及实时荧光定量聚合酶链式反应(Real-time PCR),蛋白免疫印迹法(WB)分别检测芪玉三龙汤对A549细胞凋亡、自噬体形成及自噬标记分子表达的影响。结果:芪玉三龙汤血清能以浓度依赖性的方式抑制细胞活性;与空白血清组比较,芪玉三龙汤血清组A549细胞的凋亡数量显著增多(P<0.01),自噬体形成增加;与空白血清组比较,芪玉三龙汤血清组A549细胞的mTOR mRNA和蛋白表达显著降低(P<0.01),Beclin1,自噬相关基因5(Atg5),自噬相关基因13(Atg13)mRNA和蛋白表达水平均显著升高(P<0.01)。结论:芪玉三龙汤能够诱导肺癌A549细胞发生自噬,其具体作用机制可能与其下调mTOR的表达,上调Beclin1,Atg5,Atg13,LC3的表达,促进LC3Ⅰ转化为LC3Ⅱ有关。

Objective:To observe the effect of Qiyu Sanlong decoction(QYSL)on the expressions of key molecules in signal axis of mammalian rapamycin target protein(mTOR)/yeast Atg6 homologous(Beclin1)/microtubule-associated protein1 light chain3(LC3)in A549 cells. Method:With A549 cells as the research object,the effect of QYSL medicated serum on cell viability of A549 cells were detected by cell counting kit-8(CCK-8)method. The effect of QYSL decoction on A549 cell apoptosis, autophagosome formation and the expression of autophagy markers were detected by Terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)method,transmission electron microscope(TEM),Real-time polymerase chain reaction(Real-time PCR)and Western blot. Result: QYSL medicated serum could inhibit the viability of A549 cells in a concentration-dependent manner. Compared with the blank serum group,the number of apoptotic A549 cells in the QYSL medicated serum group was significantly increased(P<0.01),and the formation of autophagosome was significantly increased. Compared with the blank serum group,the mRNA and protein expressions of mTOR in A549 cells in the QYSL serum group were significantly decreased(P<0.01),while mRNA and protein expressions of Beclin-1,autophagy related genes 5(ATG5),autophagy related genes 13(ATG13)were significantly increased(P<0.01). Conclusion: QYSL decoction can induce autophagy in A549 cells,and its specific mechanism may be related to the down-regulation of mTOR expression,the up-regulation of Beclin1,ATG5,ATG13 and LC3 expression,and the promotion of LC3Ⅰ conversion to LC3Ⅱ.