Enhanced Ion Transport By Mouse Bone Marrow Mesenchymal Stem Cell Exosome In Lung Epithelium And Related Mechanisms

Objective: Acute lung injury(ALI),an injury to the lungs,is caused by a variety of diseases,which is prone to the development of respiratory distress syndrome or even pulmonary fibrosis.It is usually manifested by the accumulation of large neutrophils,the interstitial water and sodium transport disturbance,and epithelial injury.Epithelial sodium channel(ENaC)is mainly responsible for the transport of water and salt,and removal of alveolar fluid in the alveolar cavity.Dysfunction of ENaC increases the occurrence and development of ALI.Exosomes are active molecules secreted by cells to participate in various activities.Exosomes with a diameter of 30-120 nm can effectively change the biological response of recipient cells by delivering protein metabolites and nucleic acids.Bone marrow mesenchymal stem cells derived exosome(BMSC-exo)are likely to carry a variety of active substances with anti-inflammatory and repair properties,exerting similar effects of bone marrow mesenchymal stem cell.By targeting target genes,miRNAs regulate some receptor proteins and participate in the pathogenesis of lung diseases.We speculated that ENaC in alveolar epithelial Ⅱ type cells(AT Ⅱ)is regulated by miRNAs carried by BMSC-exo and plays a therapeutic role in edematous diseases.The purpose of this study was to explore the therapeutic potential of BMSC-exo and the mechanism of action between miRNAs and ENaC,so as to provide a basis for its clinical application.Methods: 1.The exosomes isolated from BMSCs conditioned medium were identified by transmission electron microscopy and western blot assay.2.CCK-8assay was used to detect the effect of BMSC-exo on AT Ⅱ cell viability.3.The TUNEL kit was used to detect the effect of BMSC-exo on LPS-induced apoptosis of AT Ⅱ.4.In ALI cell model induced by LPS,the effect of BMSC-exo on ENaC protein expression was detected by western blot and immunofluorescence assay,and the effect of BMSC-exo on ENaC mRNA level was detected by qRT-PCR assay.5.Biochemistry analysis and literature review were used to determine the differentially expressed miR-199a-3p between normal and ALI patients.The differences of miR-199a-3p among different treatment groups and the transfection efficiency of miR-199a-3p were detected by qRT-PCR.The effect of miR-199a-3p Mimic/Inhibitor on ENaC protein content was detected by Western blot.6.The expression of mammalian target of rapamycin(mTOR)was detected through western blot experiments when miR-199a-3p Mimic/Inhibitor was transfected into H441 cells.Results: 1.BMSC-exo could enhance AT Ⅱ cell viability.2.BMSC-exo could reduce LPS-induced cell pyroptosis.3.BMSC-exo could increase the expression of α-ENaC and γ-ENaC in AT Ⅱ,which might be related to the increase of transcription level ofα-ENaC and γ-ENaC.4.The results of qRT-PCR showed that BMSC-exo reversed the LPS-induced reduction of miR-199a-3p in AT cells.5.The transfection efficiency of miR-199a-3p Mimic/Inhibitor was high,and the increase of miR-199a-3p content enhanced the expression of α-and γ-ENaC protein in AT Ⅱ /H441 cells.6.MiR-199a-3p could decrease the expression of mTOR protein in H441 cells.Conclusion: BMSC-exo may affect ENaC through miR-199a-3p targeting mTOR,and thus have a certain therapeutic effect on ALI.