Conditioned Medium Of Bone Marrow Mesenchymal Stem Cells Relieves Acute Lung Injury In Mice By Regulating Epithelial Sodium Channels Via MiR-34c/miR-124

Objective:One of the characteristics of acute lung injury is severe pulmonary edema,which is closely related to alveolar fluid clearance(AFC).Enhance of AFC contributes to the relief of pulmonary edema.The epithelial sodium channels(ENaC)is the rate-limiting step in the alveolar fluid clearance process,and is an important mechanism for the completion of sodium-water transport and edema clearance in the alveolar cavity in the lung.Therefore,regulation of ENaC is essential for the removal of pulmonary edema in patients with acute lung injury.MicroRNA(miRNAs)participate in a series of important processes in the life process and play various regulatory roles in the metabolic process of the body.In addition,they are also associated with the pathogenesis of some lung diseases.Studies have shown that miRNAs are particularly important in the homeostasis and development of the lungs,and have confirmed that miRNAs are involved in many lung diseases such as lung cancer,chronic obstructive pulmonary disease,acute respiratory distress syndrome,acute lung injury,idiopathic pulmonary fibers,and so on.We hypothesized that mouse bone marrow mesenchymal stem cells(BMSC)may secrete miRNAs through conditioned medium(BMSC-CM)to alter the function of ENaC in alveolar epithelial cells,thereby contributing to alveolar fluid clearance in acute lung injury.This study was designed to investigate the relationship between miRNAs and ENaC,to determine whether it affects the transmembrane transport of Na~+and explore its molecular mechanism.Methods:1.Flow cytometry was used to detect the purity of BMSC.2.Identification of alveolar type II epithelial cells(ATII)by immunofluorescence.3.The concentration of bovine serum albumin(BSA)was detected by microplate reader to verify the effect of BMSC-CM on AFC in mice.4.BMSC and ATII were transwelled to detect the effect of BMSC and BMSC-CM on the viability of ATII cells by CCK-8 survival assay.5.Real-time polymerase chain reaction(RT-PCR)and Western blot method were used respectively to detect the effects of BMSC-CM on mRNA levels and the protein expression of ENaC subunits.6.BMSC-CM was applied to ATII cells,and the changes of miRNAs were detected by RT-PCR.7.Verification of miRNAs transfection efficiency by RT-PCR.8.The impact of miRNAs on short-circuit current(Isc)of H441 cell monolayers was detected by Ussing chamber assay.9.The effects of miRNAs on protein levels of ATII cells ENaC were determined by Western blot.10.Dual-Luciferase Reporter Assay detects whether miRNAs act directly on ENaC.Results:1.BMSC-CM can improve the survival rate of ATII cells.2.BMSC-CM can simultaneously increase the expression levels of?-,?-ENaC at the protein and transcriptional levels.3.BMSC-CM can enhance the AFC in mice.4.BMSC-CM acts on ATII cells,and the expression of miR-34c and miR-124 has changed significantly.5.The miR-34c mimic and miR-124 mimic have high transfection efficiency.6.MiR-34c enhanced the amiloride-sensitive current of H441 cells,suggesting that miR-34c enhances ENaC activity.MiR-124 reduced the amiloride-sensitive current of H441 cells,indicating that miR-124 can reduce the activity of ENaC.7.MiR-34c is capable of enhancing the protein expression of?-ENaC,while miR-124 can reduce the protein expression of?-ENaC.8.MiR-124 can act directly on?-ENaC,thereby inhibiting its expression.Conclusions:BMSC-CM may improve the transcription and translation of?-ENaC by miR-34c/miR-124 to repair LPS-induced acute lung injury.