The Role Of CD47 In Lung Epithelial-mesenchymal Transition

BackgroundAcute respiratory distress syndrome(ARDS)is an acute inflammatory lung disease caused by severe lung and systemic alveolar capillary membrane damage,leading to acute hypoxic respiratory failure.The mortality rate of ARDS is related to the severity of lung injury at the time of onset,ranging from 35% to 46%.Diffuse alveolar damage is the pathological hallmark of ARDS,and the pathophysiology of ARDS includes inflammation,barrier disruption,interstitial and airspace edema,cell injury,and cell death.As ARDS enters the exudative and proliferative phases,lung damage will increase,and the progression of diffuse alveolar damage in ARDS patients,with severe abnormal repair of alveolar epithelium and pulmonary capillary endothelium,can develop into a fibrotic stage.Pulmonary fibrosis is a common complication of ARDS.LPS(lipopolysaccharide)is a component of the cell wall of Gram-negative bacilli and is also the main component that causes disease.50% of ARDS cases are caused by Gram-negative bacilli.In the process of LPS-induced pulmonary fibrosis in mice,TNF-α,as an early pro-inflammatory factor,can induce a subsequent storm of inflammatory factors and promote the development of pulmonary fibrosis.Studies have shown that TNF-α can up-regulate the expression level of CD47 protein.CD47(cluster of differentiation 47),also known as integrin-related protein,is a membrane protein widely expressed in different cell types in the body,including epithelial cells.Animal studies have found that CD47-deficient mice are protected from lipopolysaccharide-induced acute lung injury.The expression level of CD47 protein is up-regulated in fibrotic diseases,and inhibiting CD47 can reduce the degree of fibrosis,but its role in pulmonary fibrotic diseases is unclear,and the role of CD47 in the EMT process of BEAS-2B cells is also lack of in-depth research.Therefore,we focused on the role of changes in CD47 in TNF-α-induced EMT progression of BEAS-2B cells,looking for new mechanisms and drug therapeutic targets for lung epithelial injury.ObjectiveThe purpose of this study is To explore the role of CD47 in promoting lung epithelial-mesenchymal transition,and to find new mechanisms and drug therapeutic targets for lung injury and fibrosis MethodIn In vivo experiment: LPS was instilled into the trachea at a dose of 10 mg/kg to construct a model of LPS-induced pulmonary fibrosis in C57BL/6 mice.The lungs were collected on the third day,and the secretion level of the inflammatory factor TNF-α in BALF was detected by enzyme-linked immunosorbent assay.On the 14 th day,H&E staining was performed to evaluate the pathological changes of lung tissue,Masson staining was used to evaluate the content of collagen fibers in lung tissue,immunohistochemistry and immunofluorescence were used to evaluate the expression level of CD47,and Western blot was used to detect markers of epithelial-mesenchymal transition(E-cadherin,α-SMA)protein expression level.In vitro experiment: Taking human lung epithelial BEAS-2B cells as the research object,the cells were stimulated with different concentrations of TNF-α(12.5ng/m L,25ng/m L,50ng/m L)for 48 hours.The expression level of CD47 protein and the protein expression level of epithelial-mesenchymal transition differentiation marker proteins(E-cadherin,Vimentin)were detected by Western blot method.Choose TNF-α 25ng/m L to stimulate BEAS-2B cells,add CD47 inhibitor JQ1 to treat cells for 48 hours.Western blot was used to detect changes in the expression level of CD47 protein,as well as changes in the expression levels of epithelial-mesenchymal transition marker proteins(E-cadherin,Vimentin).Results1.LPS induced pulmonary fibrosis in mice.The increased secretion of inflammatory factor TNF-α in the alveolar lavage fluid of the mice was detected on the first day and the third day.Fibroproliferative remodeling of lung tissue and collagen fiber deposition in lung tissue were observed on day 14,and the pulmonary fibrosis score was increased.An increase in CD47 expression on the lung tissue surface was observed on day 14.At the same time,on the 14 th day,the expression level of epithelial marker protein E-Cadherin decreased,and the expression level of mesenchymal marker protein α-SMA increased in lung tissue.2.After TNF-α stimulated BEAS-2B cells for 48 hours,the expression level of CD47 protein increased,causing BEAS-2B cells to undergo EMT,that is, epithelial cells lost the epithelial marker protein(E-Cadherin)and obtained the mesenchymal marker protein(Vimentin).3.Treating BEAS-2B cells stimulated by TNF-α with JQ1,the expression level of CD47 protein is reduced,and the progression of EMT in lung epithelial cells is alleviated,that is,the increase of epithelial marker protein(E-Cadherin)and the expression level of mesenchymal marker protein(Vimentin)reduce.Conclusion ConclusionIn the mouse lung epithelial-mesenchymal transition model established by LPS tracheal perfusion,the secretion of TNF-α increased,and the expression level of CD47 protein increased.TNF-α stimulation of EAS-2B cells can lead to an increase in the expression of CD47 protein and promote the development of EMT.CD47 inhibitor(JQ1),can inhibit the expression level of CD47 and alleviate the development of EMT.