Research On The Mechanism Of PBEF Regulating Sodium Water Transport System Inalveolar Epithelial Cells

Objective:The sodium water transport system plays an important role in the clearance of fluid in the alveoli.The pulmonary edema caused by extracorporeal circulation is mainly due to the increased capillary permeability of the alveoli and the reduction of fluid clearance in the alveoli.Our previous studies have demonstrated that pre-B cell cloning enhancer(PBEF)can increase alveolar capillary permeability and inhibit the sodium water transport system,and the specific mechanism by which PBEF inhibits the sodium water transport system is unclear.Therefore,this experiment explores the specific mechanism by which PBEF regulates the sodium-water transport system in alveolar epithelial cells.Methods:1.The hypoxic reoxygenation model was constructed using HPAEpiC(alveolar type II epithelial cells)to simulate the extracorporeal circulation microenvironment.2.Different concentrations of PBEF(10ng/ml,50ng/ml,100ng/ml,200ng/ml,500ng/ml)were used to stimulate the expression of apoptosis and proliferation of alveolar type II epithelial cells by flow cytometry and CCK8 reagent.3.Three concentrations of PBEF(10ng/ml,50ng/ml,100ng/ml)were selected to stimulate the expression of ENaC,NKA and AQP1 in the alveolar type II epithelial cells,and ERK,P38 and AKT were detected by Western blot.4.Instigate alveolar type II epithelial cells with 50 ng/ml PBEF,and add specific inhibitors of three signaling pathways,respectively,to verify the role of signaling pathways in sodium water transport system.Results:1.The measured concentration of PBEF is essentially non-toxic to alveolar epithelial cells.2.PBEF inhibits the expression of ENaC,NKA and AQP1 and activates the ERK,P38 and AKT signaling pathways.3.ERK pathway inhibitors can slow down PBEF inhibition of ENaC protein and gene expression,alleviate PBEF-inhibited NKA gene expression,increase NKA activity,and alleviate PBEF inhibition of AQP1 gene expression.P38 pathway inhibitors only attenuated PBEF-inhibited NKA gene expression.AKT pathway inhibitors potentiated the inhibitory effect of PBEF,decreased expression of ENaC protein and gene,decreased NKA activity and gene expression,and decreased AQP1 protein expression.Conclusions:1.PBEF inhibits the sodium water transport system.2.PBEF activates the ERK signaling pathway and inhibits the AKT signaling pathway to inhibit the sodium water transport system.