Fty-720 Mediates The Autophagic Pathway To Inhibit Bleomycin-induced Pulmonary Fibrosis In Mice

Objective To explore the mechanism of FTY-720(Fingolimod)in regulating pulmonary fibrosis through autophagy signaling pathway.Methods In vivo experiment,80 clean-grade male Balb/c rates were divided into four groups at random: saline control group(Control),bleomycin model group(BLM),FTY-720 control group(FTY-720),the bleomycin+FTY-720 treatment group(BLM+FTY-720)were sacrificed on the 7th and 28 th days after bleomycin administration,respectively.H&E staining and Masson staining were used to observe the pathological changes of lung tissues in rats.The expression levels of IL-1β,TGF-β1 and TNF-α cytokines in the bronchoalveolar lavage fluid of rate were detected by ELISA.The protein expression levels of EMT-related markers E-cadherin,N-cadherin,α-SMA,COL1A1,Snail and autophagy related markers Beclin-1,LC-3BII /LC-3BI and P62 in lung tissues of rates were detected by Western Blot.The expression levels of EMT-related markers E-cadherin and α-SMA were detected by immunofluorescence method.The expression levels of autophagy related markers Beclin-1,LC-3B and P62 were detected by immunohistochemistry.In vitro experiment,MLE-12 cells were cultured and set as control group(untreated group),BLM group,FTY-720 group,BLM+FTY-720 group,and BLM+FTY-720+3-MA group.The effect of bleomycin on alveolar epithelial cell viability(MLE-12)was detected by MTT assay.The effect of FTY-720 on bleomycin-induced apoptosis of MLE-12 cells was detected by flow cytometry.Western Blot was used to detect the protein expression levels of EMT-related markers E-cadherin,N-cadherin,α-SMA,COL1A1,Snail and autophagy related markers Beclin-1,LC-3BII/LC-3BI and P62 in MLE-12 cells.Results FTY-720 significantly reduced ECM deposition in bleomycin-induced lung tissue of rates,alleviated the pathological features of bleomycin-induced fibrosis and reduced the content of inflammatory factors IL-1β,TGF-β1 and TNF-α in the bronchoalveolar lavage fluid of PF rates.The EMT process in PF was inhibited,and the expression of E-cadherin was increased,while the expression of N-cadherin,α-SMA,Snail and COL1A1 was decreased.FTY-720 up-regulated the autophagy process in PF,increased the expression of Beclin-1 and LC-3BII/LC-3BI,and decreased the level of P62 protein.FTY-720 significantly reduced BLM-induced apoptosis of MLE-12 cells,inhibited EMT process in alveolar epithelial cells,increased the expression of E-cadherin,and decreased the expression of N-cadherin,α-SMA,Snail and COL1A1.The autophagy process in alveolar epithelial cells was up-regulated,the expressions of Beclin-1 and LC-3BII/LC-3BI were increased,and the expression level of P62 protein was decreased.Conclusion 1.FTY-720 attenuates the progression of pulmonary fibrosis in mice by reducing the occurrence of ECM deposition and EMT in alveolar epithelial cells.2.FTY-720 attenuates the progression of pulmonary fibrosis in mice by upregulating alveolar epithelial cell autophagy.