| Objective:Bronchopulmonary dysplasia(BPD)is a common multifactorial chronic lung disease in preterm infants.With the continuous improvement of a series of interventional alternative treatments such as mechanical ventilation and oxygen therapy,the survival rate of preterm infants has increased significantly.However,due to the imperfect development of lung tissue in preterm infants,inflammatory lung injury due to hyperoxia ventilation and causing abnormal lung tissue repair throughout the neonatal period can still occur in the face of various ventilation measures,ultimately leading to the development of BPD.Therefore,exploring the molecular mechanisms underlying the development of bronchopulmonary dysplasia is of great medical importance to reduce the incidence of BPD,optimize treatment methods,and improve prognosis.Recent studies have revealed that cellular autophagy has an important role in lung development.Cellular autophagy is maintained at low levels during embryonic development and is upregulated in various tissues after birth to adapt to unfavorable environments.Intervening in the level of alveolar epithelial cell autophagy can interfere with the formation of terminal alveoli and the development of the pulmonary vascular network,creating pathological changes similar to BPD.In addition,hyperoxia treatment was able to promote autophagy in lung tissues and accelerate the development of BPD,and these results suggest that cellular autophagy plays an important role in the early onset of BPD and in the course of the disease process.Intrinsic immunity completes the immune response process mainly through the synergy of immune cells and immune molecule-mediated signaling pathways.As the main cellular component of intrinsic immunity in lung tissue,macrophages in the lung not only have the function of antigen presentation,but also can phagocytose and secrete a variety of inflammatory factors and participate in the inflammatory response.Related studies have confirmed that the activation phenotype of macrophages in the lung microenvironment can be divided into pro-inflammatory(M1)and anti-inflammatory(M2 type),and that these two polarization states can be interconverted,thus affecting hyperoxia-induced pulmonary vascular remodeling and neutrophil infiltration and participating in the development of BPD.In our previous work,we analyzed the gene expression profile of macrophages in lungs before and after hyperoxia treatment using whole transcriptome sequencing,and found that differential genes were enriched in functional subsets related to immune processes,cell death and molecular linkage by GO analysis,and KEGG analysis revealed that differential genes were enriched in pathways related to the polarization process of macrophages to M1 type,from which we screened for the related differential gene interferon-inducible protein ISG54,also known as interferon-inducible protein 2(IFIT2)with a tetratricopeptide repeat,is a member of the interferon-stimulated gene family that is involved in a variety of biological processes including host innate immunity,antiviral immune response,virus-induced double-stranded RNA signaling,and PAMP recognition.The current understanding of ISG54 is limited to antiviral immune response and innate immunity,but the role of ISG54 in macrophages,especially whether it can influence the development of BPD by regulating the relationship between the phenotypic transformation of macrophages in the lung and their function under hyperoxia stimulation,has not been reported in the literature.In this study,we analyzed the expression of ISG54 in pulmonary macrophages under hyperoxia,and analyzed its effects on autophagy and phenotypic transformation of pulmonary macrophages,in an attempt to find the key factors affecting the impaired cytoprotective mechanisms in BPD caused by hyperoxic lung injury.theoretical significance and application value.Methods:1.A model of pulmonary macrophages co-cultured with alveolar epithelial cells was established using Transwell chambers,and full-length transcriptome sequencing was used to find the changes in the expression profile of induced macrophages in the lung microenvironment under hyperoxia stimulation.2.To analyze ISG54 expression and cellular autophagy changes in macrophages in mouse lungs under hyperoxia conditions using Western blot and real-time PCR experiments.3.To down-regulate the expression of ISG54 in pulmonary macrophages using gene recombination and gene intervention techniques,and to observe the phenotypic transformation of pulmonary macrophages under hyperoxic conditions by Western blot and real-time PCR experiments.4.To down-regulate the expression of ISG54 in pulmonary macrophages by gene recombination and gene intervention techniques,and to observe the alteration of macrophage autophagy under hyperoxic conditions by Western blot.5.To establish macrophage cell lines stably overexpressing ISG54 by lentiviral transfection technique,and to find the signaling molecules related to the alteration of macrophage function in lung by ISG54 under hyperoxia stimulation by transcriptome sequencing technique.Results:1.Mouse lung epithelial cells MLE-12 were co-cultured with mouse macrophages RAW264.7 cells under hyperoxia conditions,and full-length transcriptome sequencing was performed to obtain all differential genes before and after hyperoxia treatment.GO functional enrichment analysis revealed that the differential genes were enriched in a subset of functions related to immune process,cell death and molecular linkage;KEGG enrichment analysis revealed that the differential genes were enriched in pathways related to M1-type polarization process of macrophages.Cross-tabulation analysis further screened to obtain the differential gene-ISG54.2.With the extension of hyperoxia treatment time,the expression of ISG54 in pulmonary macrophages showed a trend of increasing and then decreasing,with a significant increase at 8-12 h after hyperoxia treatment.The results indicated that at the early stage of hyperoxia-induced BPD,with the gradual aggravation of lung injury,the altered expression of ISG54 appeared in macrophages,suggesting that ISG54 may have an impact on the development of BPD.3.With the prolongation of hyperoxia treatment,the expression of M1-type marker molecules in pulmonary macrophages was upregulated,while the expression of M2 signature molecules was decreased,suggesting that hyperoxia can induce the conversion of pulmonary macrophages to the pro-inflammatory M1 phenotype and cause inflammatory response in lung tissue.After down-regulation of ISG54 expression,the expression of M1-type signature molecules in macrophages significantly increased under hyperoxia conditions and was accompanied by a decrease in the expression of M2-type signature molecules.The above results suggest that ISG54 may not be a direct molecule that regulates the conversion of pulmonary macrophages to the M1 phenotype,but may indirectly affect the change of macrophage phenotype by regulating the autophagic function of pulmonary macrophages.4.With the prolongation of hyperoxia treatment,pulmonary macrophages underwent upregulation of autophagy level,suggesting that pulmonary macrophages resist the cellular damage caused by hyperoxia by activating their own autophagic process.After downregulation of ISG54 expression,the expression of autophagy-related proteins in pulmonary macrophages decreased.The results suggest that ISG54 in pulmonary macrophages may be a positive regulator of autophagy and can maintain the high autophagic state of cells in the early stage of lung injury.5.Overexpression of ISG54 in pulmonary macrophages followed by transcriptome sequencing revealed significant enrichment of differential genes in Toll-like receptor signaling pathway and NOD-like signaling pathway,which are pathways related to cellular autophagy and macrophage differentiation.Cross-tabulation of differential genes contained within the functional modules of cellular autophagy and cellular senescence and apoptosis associated with autophagic processes yielded MAPK3.It is hypothesized that pulmonary macrophages maintain the level of autophagy in alveolar macrophages by upregulating ISG54 expression to inhibit MAPK3-related pathways during the early stages of hyperoxia-induced BPD,while indirectly exerting an inhibitory effect on the transformation of the M1 phenotype of macrophages.Conclusion:Macrophages located in lung tissue undergo intracellular oxidative stress after hyperoxia stimulation,and the M1 polarized phenotype of pulmonary macrophages is formed while ISG54 expression level is upregulated,and the upregulated ISG54 inhibits macrophages to M1 phenotype and promotes conversion to M2 phenotype while maintaining cellular autophagy,which ultimately affects the functional remodeling of pulmonary macrophages during hyperoxic lung injury.The results of this study provide The results of this study provide new ideas for the prevention and treatment of BPD. |
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