| Objectives:Acute lung injury(ALI)is a respiratory syndrome characterized by uncontrolled diffuse lung injury caused by a variety of causative factors,and is often clinically manifested as acute respiratory distress syndrome(ARDS),characterized by high morbidity and mortality.The pathological changes of COVID-19 infection or pneumonia that threaten human life in the past three years are also acute lung injury.Macrophages play a key role in the development of ALI and can be recruited and involved in inflammatory injury through complex mechanisms.The quantity of proinflammatory macrophages is positively correlated with the severity of ALI,and upregulation of pro-inflammatory macrophage apoptosis can reduce macrophage count and ultimately improve ALI.KDM6B(Lysine-specific demethylase 6B)is a histone demethylase,while trimethylation at lysine 27 of histone H3(H3K27me3)is an important epigenetic marker for gene silencing,and KDM6 B can regulate H3K27me3 through site-specific demethylation through antagonizing this silence effect.When macrophages are stimulated by Lipopolysaccharide,KDM6 B is reported to mediate the vast majority(70%)of their downstream gene expression.Previous studies have shown that KDM6 B is involved in pro-inflammatory macrophage responses in abdominal aortic aneurysms and autoimmune diseases.However,the role and specific mechanism of KDM6 B and H3K27me3 in acute lung injury are still unclear.Adenosine receptor A2a(ADORA2A)is an adenosine receptor found in immune cells and somatic cells.In inflammatory relative diseases,the role of ADORA2 A in the inflammatory response of macrophages is controversial: increased adenosine receptor A2 a in the macrophage-mononuclear phagocyte system has an anti-inflammatory effect when stimulated by hyperoxia in lung,but a pro-inflammatory effect in bronchial asthma.Although adenosine receptor A2 a has been reported to inhibit apoptosis in chondrocytes and cardiomyocytes,it has not been accurately reported in macrophage apoptosis in acute lung injury.CCAAT enhancer-binding protein β beta(C/EBPβ)belongs to the family of basic leucine zipper transcription factors,and previous studies have shown that C/EBPβ plays an important role in innate immunity and macrophage inflammation.In hematologic diseases,C/EBPβ has been reported to be involved in the regulation of myeloid cells as a transcript of KDM6B-H3K27me3.However,the mechanism in acute lung injury is not clear.Firstly,this study explored the levels of histone demethylases KDM6 B and H3K27me3 in lipopolysaccharide-induced acute lung injury and clarified their effect on apoptosis of macrophages.Secondly,the mechanism by which KDM6B-H3K27me3 inhibits apoptosis of macrophages by upregulating adenosine receptor A2 a was elucidated.Finally,the specific mechanism of KDM6B-H3K27me3 promoting the expression of adenosine receptor A2 a by upregulating C/EBPβ was further explored.The purpose of this study is to provide a new target and theoretical basis for the treatment of acute lung injury.Methods:1.The first part of this study describes the role of KDM6 B in exacerbating acute lung injury by inhibiting apoptosis of macrophages.First,an animal and cellular model of lipopolysaccharide-induced acute lung injury was established.A mouse animal model of acute lung injury was established by LPS intra-tracheal administration,KDM6B-specific inhibitor GSK-J4 injected intraperitoneally simultaneously +LPS and control group were established.The severity of ALI was evaluated by lung tissue hematoxylin-eosin(H&E)staining,acute lung injury score,and lung tissue wet/dry ratio.The degree of ALI-related inflammatory response was jointly evaluated by enzyme-linked immunosorbent assay(ELISA)to detect the expression levels of inflammatory factors(IL-1β,IL-6 and TNF-α)in serum and alveolar lavage fluid.The cell model was established by LPS pretreatment of RAW264.7 macrophage line,and the LPS stimulation group,GSK-J4+LPS stimulation group and control group were established.The expression level of inflammatory factors(IL-1β,IL-6 and TNF-α)in the cell supernatant was detected by ELISA method to evaluate the degree of cellular inflammatory response.Secondly,in vivo experiments used immunofluorescence and Western blot to detect the expression of KDM6 B and H3K27me3 lung tissue proteins in each group of mice,and cell localization and semi-quantitative analysis were carried out.In vitro experiments,Western blot and real-time quantitative polymerase chain reaction(rt-q PCR)were used to detect the protein and m RNA expression levels of KDM6 B and H3K27me3 in mouse macrophage lines.Third,apoptosis of macrophages in each group was detected in vivo and in vitro experiments.The expression level of apoptosis-related protein cleaved-caspase 3 was detected in lung tissue and macrophages of mice with acute lung injury in each group,and the apoptosis of each group was detected by TUNEL(Td T-mediated d UTP Nick-End Labeling)staining to jointly evaluate the apoptosis level and evaluate the effect of KDM6 B on it.2.The second part of this study elaborates the specific mechanism by which KDM6 B upregulates macrophage adenosine receptor A2 a to participate in apoptosis.Firstly,the GEO(Gene Expression Omnibus database)database was used to analyze the Chromatin Immunoprecipitation Sequencing data to screen downstream genes related to KDM6 B and H2K37me3 regulation of macrophage inflammation.Immunofluorescence,Western blot and rt-q PCR were used to detect the protein and m RNA expression levels of adenosine receptor A2 a in mice and macrophages with acute lung injury.Secondly,by knocking down Adora2 a in macrophages,the expression of the apoptosis-related protein cleaved-caspase 3,the proportion of TUNEL-stained apoptotic cells and the sorting of apoptotic cells by flow cytometry were detected to elucidate the role of adenosine receptor A2 a in macrophage apoptosis.Thirdly,the enrichment of H3K27me3 in the transcriptional initiation region of Adora2 a was detected by Chromatin Immunoprecipitation and polymerase chain reaction(Chip-PCR),and the transcriptional regulatory relationship between the two was elucidated.3.The third part of this study elaborates the mechanism by which KDM6 B promotes the expression of adenosine receptor A2 a through upregulation of C/EBPβ in acute lung injury.First,the possible regulatory relationships between H2K37me3-C/EBPβ and C/EBPβ-adenosine receptor A2 a were analyzed based on Chip-seq.Secondly,immunofluorescence,Western blot and rt-q PCR were used to detect the protein and m RNA expression levels of C/EBPβ in mice and macrophages with acute lung injury,respectively.Thirdly,ChipPCR was used to detect the enrichment of H3K27me3 in the C/EBPβ transcription start site and C/EBPβ in the Adora2 a transcription start site,respectively,to clarify the transcriptional regulation relationship between the two.Results: 1.KDM6 B inhibits apoptosis of macrophages by downregulating H3K27me3,thereby exacerbating the pathological changes and inflammatory response of acute lung injury.The expression of KDM6 B was increased in macrophage of mouse lung tissue in acute lung injury,and the expression level of H3K27me3 was decreased,which was co-localized with macrophages.The expression level of H3K27me3 rebounded after GSK-J4 inhibition of KDM6 B.The protein and m RNA levels of KDM6 B were increased in mouse macrophage cell lines,and the protein and m RNA levels of H3K27me3 were decreased.The protein and m RNA levels of H3K27me3 increased after GSK-J4 inhibited KDM6 B.In the ALI mouse model,after the application of GSKJ4 to inhibit KDM6 B,the expression level of apoptosis-related protein cleaved-caspase3 and the proportion of TUNEL staining apoptotic cells in acute lung injury mice were significantly higher than those in LPS-induced acute lung injury.Lung injury group.2.KDM6 B participates in macrophage apoptosis by upregulating the transcription of adenosine receptor A2 a.The Chip-seq data of KDM6 B and H2K37me3 chips were screened for downstream genes involved in epigenetic regulation of macrophage inflammation,and the results showed that KDM6 B and H2K37me3 were co-enriched in the transcription initiation region of 47 genes,and the function of Adora2 a gene was related to the biological process of GO analysis of acute lung injury disease.The protein expression of adenosine receptor A2 a in mice and macrophages in acute lung injury group was higher than that in GSK-J4 inhibitor group and control group.The levels of Adora2 a in acute lung injury mice and macrophages detected by rt-q PCR were higher than those in GSK-J4 inhibitor group and control group.Adora2 a was knocked down within macrophages,and the expression level of the apoptosis-related protein cleavedcaspase 3,the proportion of TUNEL-stained apoptotic cells,and the flow cytometry were used for cell sorting to detect the level of apoptosis,and the apoptosis after application of GSK-J4 inhibitors was significantly higher than that of the LPSstimulated macrophage group.The results of Ch IP-PCR showed that H3K27me3 was enriched in the transcription start site of Adora2 a,and the enrichment decreased after LPS stimulation.3.The results of Ch IP-PCR showed that H2K37me3 was enriched in the transcription start site of C/ebpβ,and C/EBPβ was enriched in the transcription start site of Adora2 a.The protein expression level of C/EBPβ in mice and macrophages in acute lung injury group was higher than that in GSK-J4+LPS group and control group.The expression levels of C/ebpβ in acute lung injury mice and macrophages detected by rt-q PCR were higher than those in GSK-J4+LPS group and control group.The results of Ch IP-PCR showed that H2K37me3 was enriched in the transcription initiation region of C/ebpβ,and the enrichment decreased after LPS stimulation.At the same time,C/EBPβ was enriched in the transcription start site of Adora2 a,and increased after LPS stimulation,indicating that C/EBPβ upregulated the transcription of adenosine receptor A2 a.The results showed that KDM6 B upregulated the transcription of C/EBPβ through H3K27me3,while C/EBPβ as a transcription factor upregulated the transcription of adenosine receptor A2 a by H3K27me3.Conclusions:1.Increased expression of KDM6 B in macrophages of lung tissue of mice with acute lung injury accompanied by decreased expression of H3K27me3,KDM6 B aggravates pathological changes and inflammatory responses related to acute lung injury by inhibiting apoptosis of macrophages.2.In acute lung injury,KDM6 B inhibits macrophage apoptosis by demethylation and upregulates the transcription of adenosine receptor A2 a.GSK-J4 alleviates acute lung injury and reduces inflammation by downregulating KDM6 B and adenosine receptor A2 a levels.3.In acute lung injury,C/EBPβ is used as a transcript product of KDM6 B,and can be used as a transcription factor for adenosine receptor A2 a,that is,KDM6 B can upregulate the expression of adenosine receptor A2 a through the KDM6B-H3K27me3-C/EBPβ-adenosine receptor A2 a pathway.GSK-J4 can inhibit the KDM6B-C/EBPβ-adenosine receptor A2 a pathway,further promote apoptosis of macrophages,thereby alleviating the pathological changes and inflammatory response of acute lung injury. |
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