The Mechanism Of Saikosaponin D Promoting Autophagy And Apoptosis In Lung Adenocarcinoma A549 Cells By Inhibiting The PI3K/AKT/mTOR Pathway

Objective To investigate the effect of Saikosaponin D(SSD)on autophagy and apoptosis in lung adenocarcinoma A549 cells and whether it acts roles via the PI3K/AKT/mTOR pathway.MethodsCulture of lung adenocarcinoma A549 cells in vitro.(1)Apoptosis: A549 cells were intervened with 5 μM,10 μM and 20 μM SSD for 24 h respectively,and the proliferation inhibition rate of each group of cells was detected by CCK-8 and the optimal drug concentration was selected for subsequent experiments;A549 cells were treated with different concentrations(1 μM,5 μM,10 μM)of SSD,and Western blot detected the levels of important apoptotic factors Bcl-2,Bax;A549 cells were treated with the optimal drug concentration,and qRT-PCR determined the mRNA of Bcl-2 and Bax,as well as Annexin V-FITC/PI flow cytometry determined the apoptosis rate contrasted to the control group.(2)Autophagy: A549 cells were treated with different concentrations(1 μM,5 μM,10μM)of SSD,and the formation of autophagosomes was detected by MDC staining;The expression of LC3 B,an important marker factor of autophagy,was detected using western blot;A549 cells were treated with the optimal drug concentration,and the mRNA level of the autophagy-related factors beclin-1,Atg5,Atg7 were measured by qRT-PCR compared with the control group.(3)Mechanism: A549 cells were treated with different concentrations(1μM,5μM,10μM)of SSD,and the specific inhibitor,LY294002,the protein level of p-PI3 K,p-AKT,p-mTOR,LC3 B,Bcl-2 and Bax were detected by Western blot.Results(1)Saikosaponin D promoted apoptosis by regulating the Bcl-2/Bax signaling axis in A549 cells.The proliferation inhibition rate of A549 cells were increased with the improvement in drug concentration by CCK-8.Western blot showed that when SSD concentration Improved,the expression level of Bax increased and the expression level of Bcl-2decreased.Flow cytometry showed that the early and late apoptosis rate of A549 cells treated with SSD was significantly higher than that of the control group.QRT-PCR showed that the ratio of Bcl-2 to Bax of A549 cells treated with SSD was significantly decreased compared with the control group.(2)Saikosaponin D promoted autophagy by upregulating the expression of LC3B-II in A549 cells.MDC staining showed that the number of autophagosomes increased with increasing SSD concentration.The expression level of LC3B-II was increased with improving the drug concentration by western blot.QRT-PCR showed that the mRNA expression levels of Beclin-1,Atg5 and Atg7 were significantly increased after SSD treatment in A549 cells were significantly up-regulated compared with the control group.(3)Saikosaponin D induced autophagy and apoptosis by inhibiting PI3K/AKT/mTOR pathway in A549 cells.With the increase of SSD concentration,the expression of p-PI3 K,p-AKT and p-mTOR decreased.When A549 cells were treated with SSD and LY294002,p-PI3 K,p-AKT,p-mTOR and Bcl-2 were significantly decreased,while LC3B-Ⅱ and Bax were significantly increased.ConclusionSSD promotes autophagy and apoptosis in lung adenocarcinoma A549 cells by inhibiting the PI3K/AKT/mTOR pathway.