The Study Of Saponin D Anti-pulmonary Fibrosis By Inducing Alveolar Epithelial Cell Autophagy And Its Mechanism

ObjectiveTo investigate the changes of autophagy in pulmonary fibrosis model established by type II Alveolar epithelial cells(AEC2)and the regulatory effect of saikosaponin d(SSd)on autophagy in this model.Its regulatory mechanism,the activation of autophagy-related mTOR pathway in pulmonary fibrosis cell model and the intervention effect of SSd on it,were further studied.It provides a new idea for the clinical treatment of Idiopathic pulmonary fibrosis(IPF).MethodsThe safe concentration of A549 cells treated by SSd was detected by CCK-8method,and the cell model of pulmonary fibrosis was established by transforming growth factor β1(TGF-β1)(5ng/m L)induced A549 cells for 48 h,and cells were respectively treated with SSd at low,medium and high concentrations(2.5,5,10μg/m L)and rapamycin(100ng/m L).CCK-8 assay was used to detect the effect of TGF-β1 and SSd combined with TGF-β1 treatment on the cell viability of A549 cells.The expression levels of epithelial and interstitial proteins,autophagy associated proteins and mTOR pathway key proteins were detected by Western blot.The formation of autophagosomes in cytoplasm was observed by MDC staining under fluorescence microscopy.A549 cells were transfected with m RFP-GFP-LC3 lentivirus,and m RFP-GFP-LC3 double-fluorescent aggregates were observed under confocal microscopy to detect autophagy flow.Results(1)The results of CCK-8 showed:SSd had no significant effect on the cell viability of A549 cells within the concentration range of 0-10ug/m L.Compared with the normal group,the cell viability rate after TGF-β1 treatment was decreased(P <0.05),and the cell viability after SSd combined with TGF-β1 treatment was increased,and the effect of SSd high concentration was statistically significant(P < 0.05).(2)Epithelial and interstitial associated proteins were detected by Western blot:Compared with the normal group,the expression level of epithelial characteristic protein E-cadherin in A549 cells treated with TGF-β1 was decreased(P < 0.05),and the type 1 collagen(collagen 1)、α-smooth muscle actin(α-SMA)expression level were high(P < 0.05),suggesting that the epithelial characteristics of AEC2 were weakened and interstitial phenotype was obtained.Compared with the model group,the expression of E-cadherin was increased combined with SSd intervention,while the expression of Col-1 and α-SMA protein decreased,and the effect of SSd with high concentration was the most obvious(P < 0.05).It was suggested that SSd could antagonize the fibrosis promoting effect of TGF-β1 on AEC2 in a concentration-dependent manner.(3)Autophagy-related proteins were detected by Western blot:Compared with the normal group,the expression of Beclin1 and LC3B-II/LC3B-I in A549 cells treated with TGF-β1 were decreased(P < 0.05),while the expression of p62 protein increased(P < 0.05),suggesting the formation and degradation of autophagosomes were inhibited.Compared with model group,the expression levels of Beclin1 and LC3B-II/LC3B-I increased after SSd intervention(P < 0.05),and the expression level of p62 protein decreased(P < 0.05),suggesting that SSd improved TGF-β1-induced autophagy inhibition.(4)MDC method was used to detect the formation of autophagosomes in the cytoplasm: Compared with the normal group,the fluorescent spots of MDC-labeled autophagosomes in the cytoplasm of cells in the model group were less and the fluorescence was weaker.The number of fluorescent spots in the cytoplasm increased significantly treated with SSd in high concerntration and rapamycin.These results indicated that SSd could antagonize the inhibition of TGF-β1 on autophagosome formation.(5)Autophagy flow was detected by m RFP-GFP-LC3 dual fluorescent autophagy indicator system: Compared with the normal group,there were fewer green and red fluorescent spots in the model group,and the color was relatively uniform yellow-green after co-localization.Compared with the model group,the green and red fluorescent spots in the SSd high concentration group and the rapamycin group were significantly enhanced,and the color was orange-red with obvious spots after co-localization.It was suggested that the formation and degradation of autophagosomes in the model group were inhibited,and SSd intervention could promote autophagy flow and improve the level of autophagy.(6)Western blot was used to detect mTOR pathway key proteins:In the model group,the activation levels of mTOR pathway key proteins p-mTOR/mTOR,p-AKT1/AKT1,p-S6K1/S6K1 were increased(P < 0.05),TGF-β1 activated mTOR pathway,while the activation levels of mTOR pathway key proteins were decreased after SSd high concentration and rapamycin intervention(P < 0.05).It was suggested that SSd inhibit mTOR pathway activation in pulmonary fibrosis.ConclusionSSd could induce autophagy of AEC2 cells by inhibiting the activation of mTOR pathway,and thus play an antagonistic role of TGF-β1 in promoting fibrosis for AEC2 cells.It may provide a new idea for SSd treatment of IPF.