Study On The Mechanism Of Bone Marrow Mesenchymal Stem Cells Alleviating The Inhibitory Effect Of LPS On Epithelial Sodium Channel By MiR-130b

Objective: Acute lung injury(ALI)is a disease syndrome with high morbidity and mortality,and one of the characteristics is severe pulmonary edema.The epithelial sodium channel(ENaC)located on the apical membrane side of alveolar type ?epithelial cells(AT?)is essential for the completion of sodium-water transport and edema clearance in the alveolar space.Mesenchymal stem cells(MSCs)have the ability to regulate immune responses to tissue damage and promote repair in vivo.They have been suggested to act on a variety of lung diseases(including ALI).MSCs can release a variety of cytokines,growth factors,exosomes,and microRNAs(miRNAs),etc.MiRNAs play important regulatory roles in biological processes.In addition,they are involved in the pathogenesis of some lung diseases.We hypothesized that mouse bone marrow mesenchymal stem cells(BMSCs)may affect the function of ENaC in AT?through miRNAs and thus play a role in ALI.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 possible molecular mechanisms.Methods: 1.The effect of BMSCs on the viability of H441 cells was tested by CCK-8cell survival rate test.2.Western blot technique was used to examine the effect of BMSCs on ENaC and phosphatase and tensin homolog deleted on chromosome ten(PTEN)protein levels in AT? cells.The effects of BMSCs on ENaC mRNA levels in AT? cells were examined by qRT-PCR.3.The effect of BMSCs on short-circuit current of H441 monolayer cells was analyzed using Ussing chamber assay.4.qRT-PCR was used to test the co-culture of BMSCs with AT? cells,the changes of miR-130 b in AT?cells,and the transfection efficiency of miR-130 b mimics and inhibitors.5.The effect of miR-130 b and BMSCs transfected with miR-130 b mimics on ENaC and PTEN protein levels in AT? cells was determined by Western blot.6.Small interfering RNA was used to specifically knock down PTEN in AT? cells,and Western blot experiments were used to detect whether miR-130 b affected ENaC protein expression level by targeting PTEN.Results: 1.BMSCs could improve the viability of H441 cells.2.BMSCs increased the expression level of ?-,?-ENaC and decreased the expression level of PTEN in AT?cells at the protein level,and could increase the expression level of ENaC at the transcriptional level.3.BMSCs increased the amiloride-sensitive current of H441 cells,which indicated that BMSCs could enhance the activity of ENaC.4.qRT-PCR results showed that co-culture of BMSCs with AT? cells could increase the expression of miR-130 b in AT? cells.5.MiR-130 b mimics and their inhibitors had high transfection efficiency.6.Mi R-130 b and BMSCs transfected with miR-130 b mimics could increase the expression of ?-,?-ENaC protein and decrease the expression of PTEN protein in AT? cells.7.After knockdown of PTEN,the protein expression of ?-ENaC and ?-ENaC in AT? cells was enhanced.Conclusions: BMSCs may affect ENaC by targeting PTEN through miR-130 b,which has a certain therapeutic effect on ALI.