PEBP4 Deficiency Aggravates LPS-induced Acute Lung Injury By Impairing Alveolar Fluid Clearance

Background:Acute lung injury(ALI)is a non-cardiogenic pulmonary edema and severe hypoxemia resulted from alveolar epithelial cell and microvascular endothelial cell injury caused by a variety of factors,which greatly endangers people’s health and is also an important cause of death in critically ill patients.Pulmonary edema is one of the most significant pathophysiological changes in ALI,and timely and effective alveolar fluid clearance(AFC)is essential for the prognosis of ALI.It has been found that the damage of sodium and water activetransport system is the fundamental cause of impaired AFC,fuethermore epithelial sodium channel(ENa C)and Na,K-ATPase play important roles in AFC.Phosphatidylethano-lamine binding protein 4(PEBP4)belongs to the phosphatidylethano-lamine binding protein 4(PEBP)family,which is widely expressed in a variety of mammalian tissues,and has a unique structural domain that allows it to perform multiple functions.It has been reported that PEBP4 has an anti-apoptotic function and is involved in the development and progression of cancer.PEBP4 also has anti-inflammatory effects,whereas the onset and resolution of inflammation is critical for development of ALI.It is worth mentioning that it has reported recently that PEBP4 is identified as a novel marker of alveolar type II epithelial cells.In addition,serum PEBP4 content is negatively correlated with Na~+concentration,and active transportation of Na~+across the cell membrane is the motive power of AFC.All of the above suggest that PEBP4 may regulate AFC and play a key role in the development of ALI.The aim of this study was to investigate the effect of PEBP4 on AFC and ALI,in addition its mechanism.Objective:To investigate the effect of PEBP4 on acute lung injury and its mechanism.Methods:1.PEBP4 KO(knock out,KO)mice were constructed and bred,identified and phenotyped.2.Detect the degree of acute lung damage.H&E staining and micro-CT of small animals were used to observe histopathological changes in lung tissue;kits were used to detect MPO activity,serum secretion levels of TNF-α,IL-1β,IL-6 and Western Blot to detect COX-2 expression levels in lung tissue to assess the degree of inflammatory response;the wet weight/dry weight ratio of lung tissue and total protein concentration in bronchoalveolar lavage fluid(BALF)were used to assess the degree of pulmonary edema and damage to the alveolar epithelial-endothelial barrier.3.Detect alveolar fluid clearance abilities and PI3K/AKT signaling pathway activation levels.The protein expression levels of ENa Cα,βandγsubunits as well as Na,K-ATPaseα1 andβ1 subunits in lung tissue homogenates were detected using Western blot.The levels of PI3K and AKT protein expression were also detected.Results:1.Western blot results showed that PEBP4 protein expression levels in lung tissues of WT mice were significantly decreased after intratracheal instillation of LPS,suggesting that PEBP4 is associated with acute lung injury.Agarose gel electrophoresis and Western blot results showed that PEBP4 gene and protein were not expressed in the mouse tail and lung of PEBP4 KO mice,indicating that PEBP4KO mice were successfully constructed.H&E staining and micro-CT of small animals showed that knockout of PEBP4 had no significant effect on the phenotype of lung tissue.2.Knockout of PEBP4 aggravated alveolar structural disruption,fusion,hemorrhage,inflammatory cell infiltration,pulmonary solidification and pulmonary edema;increased total BALF protein concentration and MPO activity;promoted inflammatory cytokine IL-1β,IL-6 and TNF-αsecretion and COX-2 expression.3.Knockout of PEBP4 inhibited PI3K/AKT pathway activation,and the expression levels of p-PI3K and p-AKT were significantly decreased,while the expression levels of ENa C-αandγand Na,K-ATPaseα1 andβ1 were inhibited.4.Both PI3K-specific activator 740YP and AKT-specific activator SC79partially reversed the effects produced by knockout of PEBP4.These include reduction the degree of lung injury,promotion alveolar fluid clearance,increase the expression level of the sodium-water transport system,and activation the PI3K/AKT pathway.Conclusion:PEBP4 deficiency aggravates LPS-induced acute lung injury by impaired alveolar fluid clearance,which may be mediated by inhibition of the PI3K/AKT pathway.