Mechanism Of PHLDA1 Inhibits Type Ⅱ Alveolar Epithelial Cell Apoptosis In COPD Emphysematous Phenotype

Objectives: Emphysematous phenotype is one of the major clinical phenotypes of chronic obstructive pulmonary disease(COPD),with high incidence and high mortality.These patients show a poor response to current pharmacologic treatments of COPD,which results in a progressive destruction of the lungs.Therefore,it is important and urgent to explore the pathological mechanisms of emphysema and search for the key targets of its repairing and development.Type II alveolar epithelial cells(AECIIs)play a critical role in wound repair and tissue regeneration,the reduced number or dysfunction of which will influences the repair of alveolar epithelial cells severely,resulting in damaged alveolus.Previous studies have revealed that the enhanced apoptosis is an essential mechanism for the formation of emphysema.PHLDA1(Pleckstrin Homology Like Domain Family A Member 1)is a member of Platelet and leukocyte C kinase substrate Pleckstrin Homology Like Domain Family,which has been reported to be either directly or indirectly involved in the regulation of apoptosis.However,there is ongoing controversy concerning its role in apoptosis.Its function and specific mechanism remain unclear in lung disease,as well.In this study,we explore the regulatory mechanisms and specific roles of PHLDA1 in the apoptotic process of ACEIIs in emphysematous phenotype of COPD,in order to provide new insights and strategies for targeted therapies of emphysema.Methods:Part Ⅰ: Cell apoptosis in type Ⅱ alveolar epithelial cells of chronic obstructive pulmonary disease emphysematous phenotype1.The eligible COPD patients were screened according to clinical manifestations and spirometry test,and all involved patients were divided into COPD non-emphysematous phenotype(COPD-NE)group and COPD emphysematous phenotype(COPD-E)group.2.Immunofluorescence and Td T-mediated d UTP Nick-End Labeling(TUNEL)assays were carried out to detect the number and apoptotic level of AECIIs in COPD-NE and COPD-E groups.3.Images analysis software and Hematoxylin-eosin(HE)staining were used to observe morphological changes in lungs of emphysema patients with preserved lung function and COPD-E groups.4.Western blot and quantitative real-time PCR were performed to quantify levels of protease-related molecules,apoptosis-related molecules and inflammatory factors in COPD-NE and COPD-E groups.Glutathione Colorimetric Detection Kits and superoxide dismutase assay kits were used to compare the levels of antioxidant substances in emphysema patients with preserved lung function and COPD-E groups.Part Ⅱ: PHLDA1 regulates the apoptosis of type Ⅱ alveolar epithelial cells to affect the development of COPD emphysematous phenotype1.GEO(Gene Expression Omnibus)database was used to perform differential gene expression and functional analysis in emphysematous phenotype of chronic obstructive pulmonary disease.2.Western blot and quantitative real-time PCR were performed to identify relative levels of PHLDA1 in COPD-NE and COPD-E groups.Immunohistochemical staining was performed to detect the protein expression levels and localization of PHLDA1.3.ACEIIs were treated by different concentration of cigarette smoke extracts to construct cigarette-treated cell models,and the Cell Counting Kit-8(CCK8)assays were used to detect cell viability of cell models.4.Downregulation and upregulation of PHLDA1 were carried out using small interfering RNA(si RNA)transfection and p EX4-c DNA plasmids transfection individually.Apoptosis levels of ACEIIs induced by cigarettes were determined using flow cytometry techniques,while the apoptosis-related proteins were detected by western blot after 24 h and 48 h treatment of cigarette smoke extracts.Part Ⅲ: PHLDA1 was bonded to 14-3-3 in the apoptosis of type Ⅱ alveolar epithelial cells1.The proteins bonded to PHLDA1 were assessed by co-immunoprecipitation experiments to determine the binding relationship between PHLDA1 and 14-3-3.2.Western blot was used to detect the protein levels of 14-3-3 epsilon and 14-3-3 theta,to explore the potemtially regulatory relationships between PHLDA1 and each subtype of 14-3-3.The expression of PHLDA1 was regulated by si RNA or p EX4-c DNA plasmids transfection to determine the regulatory function of PHLDA1 to 14-3-3 epsilon.3.Immunofluorescence was used to explore the co-localization relationship between PHLDA1 and 14-3-3 in ACEIIs and lung tissue sections of COPD patients.Results:Part Ⅰ: Cell apoptosis is enhanced in type Ⅱ alveolar epithelial cells of chronic obstructive pulmonary disease emphysematous phenotype1.The results of immunofluorescence and TUNEL suggested the number of SPC+ACEIIs in COPD-E group was significantly lower than COPD-NE group,and the apoptosis of ACEIIs was enhanced in COPD-E group.2.The results of CT images analysis and HE staining suggested that the severity and distribution of emphysema were no significant difference in emphysema patients with preserved lung function and COPD-E groups.3.The results of rt-q PCR,Western blot and oxidative stress kits suggested that,there were no significant difference observed in inflammatory factors,protease,protease inhibitor,apoptosis-related factors and antioxidant substance between emphysema patients with preserved lung function and COPD-E groups.Part Ⅱ: PHLDA1 inhibits the apoptosis of type Ⅱ alveolar epithelial cells to affect the development of COPD emphysematous phenotype1.The result of GEO datasets analysis and suggested that,PHLDA1 was a potential differently expressed gene in the comparation of lung tissues in COPD-E and COPD-NE group.2.The results of western blot,immunohistochemical staining and immunofluorescence suggested that PHLDA1 had low expression in the lung tissue of COPD-E group,with a primary expression in cytoplasm of ACEIIs,some expressions were also observed in nucleus.The results of correlation analysis suggested that emphysema index was inversely correlated with the expression of PHLDA1,and emphysema percentile density was positively correlated with it.3.The overexpression of PHLDA1 partly reversed the AECIIs apoptosis induced by 6%CSE,but the apoptostic rate cannot be recovered to its original level.Knockdown of PHLDA1 can induce the AECII apoptosis in the condition of 4% CSE exposure.The changes of cleaved-PARP and cleaved-Caspase3 level were consistent with the above results of apoptosis.Part Ⅲ: PHLDA1 was bonded to 14-3-3 in the apoptosis of type Ⅱ alveolar epithelial cells1.The results of western blot showed that the relative expression level of 14-3-3 epsilon decreased significantly,after 6% CSE treating for 24 h.2.The expression of 14-3-3 epsilon increased significantly in PHLDA1 overexpression ACEIIs,while the expression of 14-3-3 epsilon exhibited a large decrease in PHLDA1 knockdown ACEIIs.3.The results of immunofluorescence suggested that,PHLDA1 and 14-3-3 epsilon were co-expressed in intact ACEIIs,regardless of the presence of CSE.PHLDA1 and 14-3-3epsilon were colocalized in lung tissue of COPD patients,and the protein level of PHLDA1 and 14-3-3 epsilon were lower in lung tissue of COPD–E group.4.The results of co-immunoprecipitation suggesed that PHLDA1 was always bonded to14-3-3 epsilon,regardless of the existence of CSE.After CSE treated,the protein level of14-3-3 epsilon that binding to PHLDA1 decreased obviously.Conclusion: 1.The number of ACEIIs decreases in emphysematous phenotype of COPD patients,with an enhanced apoptosis level.2.Emphysema patients with preserved lung function and COPD patients with emphysematous phenotype show similar imaging manifestations,pathological results,as well as the same pathogenesis.3.The relative expression of PHLDA1 is lower and correlated with emphysema severity in emphysematous phenotype of COPD.4.PHLDA1 inhibits the apoptosis of ACEIIs induced by CSE.5.PHLDA1 affects the protein level of 14-3-3 epsilon in ACEIIs.6.The binding between PHLDA1 and 14-3-3 epsilon is weaken in the condition of CSE explosure.