HUCMSCs-derived Exosomes Alleviate Pulmonary Fibrosis In Mice By Inhibiting Epithelium-interstitial Transition

BackgroundIdiopathic pulmonary fibrosis(IPF)is a chronic,progressive and interstitial pulmonary disease and its etiology is complex,which can cause extensive pulmonary structure lesions.However,corresponding clinical treatment is not ideally effective.Recent literatures indicate that the process of epithelial interstitial transition(EMT)activated by TGF-β1/Smad2/3 plays an important role in IPF.Mesenchymal stem cell derived exosomes(MSCs)have been shown to relieve pulmonary fibrosis.Furthermore,MSCs-derived exosomes have become a new cell-free therapeutic method due to their high stability and non-tumor-inducing properties.ObjectiveTo investigate the effect of hUCMSC-EXOs on pulmonary fibrosis in mice and the potential mechanism and provide experimental basis for clinical treatment of IPF.Methods1.hUCMSC-EXOs were extracted from the culture supernatants of hUCMSCs and identified by exosome particle size analysis,western blotting and electron microscopy.2.In vitro,the effect of hUCMSC-EXOs on the cytotoxicity of A549 cells was detected by CCK8 assay.hUCMSC-EXOs were co-cultured with A549 cells at the concentration of μg/ml、20 μg/ml、40 μg/ml、80 μg/ml.The expression of the EMT process markers(E-cadherin and vimentin)and the phosphorylated of the downstream signal protein Smad2/3 were detected by western blotting in three groups in A549 cells:control group,model group(TGF-β1,2ng/ml)and treatment group(TGF-β1,2ng/ml;hUCMSC-EXOs,40μg/ml).3.In vivo,twenty-four micewere divided into four groups on average:control group,model group(bleomycin was given by intratracheal in 3mg/kg),EXOsl group(hUCMSC-EXOs were given on the second day after modeling,100μg/250μl,inflammatory phase),EXOs2 group(hUCMSC-EXOs were given on the eleventh day after modeling,100μg/250μl,fibrosis phase).The mice were sacrificed on the twentyfirst day after modeling,and the lung tissues of both sides of the mice were taken to measure the pulmonary index.The lung tissue pathology and collagen deposition were examined by HE staining and Masson staining,respectively.The expression level of TGF-β1 in serum was detected by ELISA.Vimentin,E-cadherin and phosphorylated Smad2/3(p-Smad2/3)were observed by immunohistochemical staining.Results1.The expression of CD9,CD63,and CD81 of hUCMSC-EXOs were positive;2.Compared to the model group,hUCMSC-EXOs could significantly reduce the pulmonary index,collagen deposition and lung tissue pathological changes in lung tissue slices,inhibit the expression of TGF-β1 in serum(P<0.05),and downregulate vimentin and p-Smad2/3 in mice with pulmonary fibrosis while upregulating Ecadherin.And the inhibition effect was more obvious when administered on the second day than on the eleventh day;3.CCK8 assay results showed that hUCMSC-EXOs had no toxic effects on A549 cells proliferation in those dosage.Stimulated by TGF-β1,the EMT process in A549 cells featured the characteristics including Smad2/3 phosphorylation,E-cadherin downregulation and vimentin up-regulation,while hUCMSC-EXOs could reverse these changes.Conclusion1.hUCMSC-EXOs could inhibit the EMT process in A549 cells activated by TGF-β1;2.hUCMSC-EXOs could alleviate the development of pulmonary fibrosis in mice,and the inhibition effect was more obvious when administered in the inflammatory stage;3.hUCMSC-EXOs could alleviate pulmonary fibrosis in mice by inhibiting epitheliuminterstitial transition activated by TGF-β1/Smad2/3 signaling pathway.