The Role Of Cdc42 Deletion Mediated Epithelial Barrier Dysfunction And Repair Delay In Acute Lung Injury

Acute lung injury is one of the common critical respiratory diseases.The main pathophysiological changes of acute lung injury are the decline of the barrier function of alveolar epithelium and vascular endothelial cells,resulting in pulmonary edema.Maintaining the integrity of the barrier function of alveolar capillary membrane and promoting the repair of alveolar capillary membrane after injury are of great significance for alleviating and preventing acute lung injury.However,the role of impaired airway epithelial barrier function and impaired injury repair in acute lung injury has been poorly studied.Cell division cyclin 42(Cdc42)has biological activities such as regulating cell cycle,promoting cell proliferation,migration and maintaining intercellular connections.The effect of decreased airway epithelial barrier and delayed injury repair caused by the absence of Cdc42 on acute lung injury remains unclear.The purpose of this study was to explore the role and mechanism of cdc42-mediated airway epithelial barrier decline and repair delay in acute lung injury,so as to further clarify the role of airway epithelial barrier function and injury repair in acute lung injury,and to lay a foundation for the research on the corresponding targets for the treatment of acute lung injury.Methods:Part I The effect of cdc42 knockout on barrier function and injury repair of airway epithelial cells1.In vivo experiments:1.1 A mouse model of Cdc42 gene knockout in airway epithelial cells was established by using Cre/loxp technology.1.2 Lung tissues of mice in the control group and cdc42 knockout group were collected,and the proliferation of airway epithelial cells and the expression and distribution of intercellular connective proteins were evaluated by immunohistochemical staining.2.In vitro experiments:2.1Airway epithelial cells knockdown of Cdc42 were stably constructed by transfection of siCdc42 plasmid into 16HBE(human bronchial epithelioid cell line).2.2 The proliferation capacity and the expression of intercellular connective proteins in the control group and knockout group were evaluated by immunofluorescence and WB detection.Part Ⅱ:The effects of Cdc42-mediated impaired airway epithelial barrier function and delayed repair on acute lung injury.1.In vivo experiments:Lipopolysaccharide(LPS)was directly dropped into the trachea to construct a mouse model of acute lung injury.Lung tissues were collected from mice at different time points on day 1,3 and 7 after administration,and lung tissue sections were stained with HE.Meanwhile,pathological scores were calculated to evaluate the severity of acute lung injury.2.In vitro experimentsLPS stimulation was given after the establishment of a cell model with low Cdc42 gene knockout to evaluate the effect of low Cdc42 knockout on cell repair after airway epithelial cell injury and the formation of intercellular connective protein after injury.Results:Part I:effects of cdc42 knockout on barrier function of airway epithelial cells and repair after injury1.After the establishment of the animal model,the expression of Cdc42 protein in the airway epithelial cells of mice in the knockout group was significantly lower than that in the non-knockout group.2.The function of airway epithelial barrier decreased in mice with cdc42 knockout.3.The proliferation of cdc42 knockout mouse airway epithelium decreased.4.The proliferation and migration ability of airway epithelial cells with low Cdc42 was decreased and the expression of intercellular connexin was down-regulated.Part II:The effects of cdc42 deficiency on lung injury in patients with impaired airway epithelial barrier fiunction and delayed repair.1.The scores of acute lung injury and neutrophil infiltration around the airway of mice in Cdc42 knockout group were significantly higher than those in the control group at 1,3 and 7 days after administration,and the airway repair was significantly delayed.2.After the injury of airway epithelial cells with low Cdc42,the cell migration and proliferation ability decreased,and the expression of intercellular connexin was down-regulated.3.The expression of PAK-1 in the airway of Cdc42 knockout mice was decreased after acute lung injury.Conclusions:1.A mouse model of specific knockout of Cdc42 gene in airway epithelial cells was successfully constructed for the first time.2.Knockout of Cdc42 gene reduces the migration and proliferation ability of airway epithelial cells and reduces the formation of intercellular connections,so Cdc42 is an important regulator of airway epithelial barrier function maintenance and repair.3.Lack of Cdc42 mediated airway barrier function decline and cell damage after repair delay,significantly increased in acute lung injury,prove the airway epithelial barrier function and repair ability in the occurrence of acute lung injury(ali)play an important role in the development and repair,airway epithelial cells and acute lung injury of important therapeutic targets,promote restoration of the airway epithelial barrier function recovery and injury may alleviate the acute lung injury.4.Cdc42 is one of the most important molecules that regulate the function recovery and injury repair of airway epithelial cell barrier.Intervention to enhance the activity of Cdc42,or to enhance the activity of downstream molecular targets of Cdc42,may reduce acute lung injury by strengthening the function recovery and injury repair of airway epithelial cell barrier.4.The expression change of pak-1 is consistent with the expression change direction of cdc42,which may regulate the maintenance and repair of airway epithelial barrier function through pak-1 signaling pathway,and pak-1 may be a candidate therapeutic target for acute lung injury.