The Effects Of Exosomes Derived From Macrophages Stimulated By Lipopolysaccharide On TGF-β1-induced Epithelial-Mesenchymal Transtition In Pulmonary Epithelial Cells

Pulmonary fibrosis is a fatal chronic disease caused by abnormal long-term damage and repair of lung tissue and the final pathological manifestation of various interstitial lung diseases.Epithelial mesenchymal transformation is a biological process in which epithelial cells transform into cells with mesenchymal phenotype.The study found that inflammation of the lung macrophages produce inhibition is the main cause of lung tissue injury and fibrosis,between different cells in the lung tissue,especially the interaction of alveolar epithelial cells and macrophages in steady-state and maintain lung tissue fibrosis pathological process,Exosomes are secretory vesicles containing many various biological macromolecules,which play an important role in the process of communication between cells.The effect of macrophage-derived exosomes on epithelial mesenchymal transformation in inflammatory environment has not been reported.To investigate the effect of macrophage-derived exosomes on the interstitial transformation of pulmonary epithelial cells in inflammatory environment and its related mechanisms.In this study,an epithelial mesenchymal transformation model of pulmonary epithelial cells was induced by TGF-β1,and the lipopolysaccharide(LPS)was used for induce inflammatory response to cells.Exosomes from LPS-stimulated macrophages were isolated and identified.A549 epithelial cells treated with exosomes and/or TGF-β1 were used for analyzing the effect of macrophage derived exosomes on the characteristics of epithelial mesenchymal transformation(EMT)and its mechanism in an inflammatory environment.The experimental results are as follows:1.The epithelial mesenchymal transformation model was successfully generated using A549 cells by exposing cells to TGF-β1.After induction of A549 cells with TGF-β1,the expression of E-cadherin protein was down-regulated(P<0.01),while the expressions of Vimentin,α-SMA protein and mRNA were up-regulated(P<0.01).In addition,A549 cells were observed to gradually became polygonal and spindle-shaped,suggesting the successful generation of an epithelial-mesenchymal transformation model.It laid a foundation for further exploring the effect of macrophage-derived exosomes on TGF-β1 induced interstitial transformation of A549 cells.2.Successfully isolated and identified macrophage-derived exosomes,and confirmed that macrophage-derived exosomes could enter epithelial cells.The macrophage-derived exosomes were round or oval under transmission electron microscope,and their diameters were distributed between 30-150 nm.They expressed TSG101,ALIX,CD81,CD9 and other exosome marker proteins.Exosomes are able to enter epithelial cells and accumulated around the nuclear membrane.It laid an experimental foundation for further stufy on the effect of macrophage-derived exosomes on epithelial-mesenchymal transformation.3.Macrophage-derived exosomes could inhibit the proliferation of epithelial cells and reverse the proliferation of epithelial cells induced by TGF-β(P<0.01).Macrophages exosomes could promote epithelial cell migration and EMT property,by reducing the expression of E-cadherin,increasing expression of Vimentin,α-SMA,Collagen-1 in A549 cells(P<0.01).The effect of exosomes derived from macrophages treated with LPS showed more robust effect than that from naive macrophages,and exosomes could play a synergistic effect with TGF-β1 in the EMT process.4.Exosomes derived from macrophags stimulated with LPS were able to to up-regulate the protein level of p-Smad2/3 in epithelial cells and activate the TGF-β1/Smad2/3 signaling pathway(P<0.01),along with its downstream transcription factors ZEB1 and Snail(P<0.01).They could further promote the mesenchymal transformation of epithelial cells induced by TGF-β1.These results suggested that LPS could stimulate macrophag-derived exosomes to promote the epithelial cell mesenchymal transformation induced by TGF-β1/Smad2/3 signaling pathway.In summary,macrophage-derived exosomes are involved in epithelial mesenchymal transformation in inflammatory environments,By a mechanism of upregulating the expression level of p-Smad2/3,the activation of TGF-β1/Smad2/3 signal transduction pathway subsequentially resulted in the high expression of downstream transcription factors,thus promoted the EMT of epithelial cells.