Regulatory Effect Of NCOR Promoter Methylation On Macrophage Inflammatory

Background:Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are a serious threaten to people health.Severe infection,such as sepsis,is one of the major causes of ALI / ARDS.Although the pathogenesis of ALI/ARDS was study deeply,the effective drugs and therapy are absent and the mortality of ALI/ARDS still remains high.Therefore,it is very important and urgent to explore the ALI/ARDS inflammatory response mechanism in order to find new treatments methods for ALI/ARDS.Previously studies have shown that alveolar macrophages play an important role in the progress of acute lung injury.Explor the role of macrophages in this process in-depth is expected to provide a new therapeutic strategy for ALI/ARDS.Nuclear receptor corepressor(NCOR)is a nuclear transcriptional inhibitory factor.Previous studies have shown that NCOR regulated the inflammatory response through TLR4 signal transduction pathway.Other study showed that NCoR was involved in inflammation by inhibiting the activation of NF-?B and AP-1.However,the potential regulation mechanism of NCOR was unclear.DNA methylation is the main epigenetic modification of the higher eukaryotic genome,which is involved in gene regulation under the catalysis of DNA methyltransferase(DNMTs),leading to the development of diseases.In our study,we used lipopolysaccharide(LPS)-induced macrophage inflammation model to investigate the effect of NCOR on inflammation and the regulation mechanism.Objectives:1.Explore the role of NCo R in LPS induced macrophages inflammation response.2.Investigate the mechanism of NCOR promoter methylation and its effect in inflammatory response.Methods:1.Effects on the expression of NCOR and inflammatory afer RAW264.7 cells treated with LPS.After RAW264.7 macrophages were treated with 1ug/ml LPS for 24 h and 48 h.Western blot was used to detect the expression of NCOR protein,the expression of NCOR,TNF-? and IL-6 mRNA were detected by real time-PCR.The luciferase reporter assay detected NF-?B promoter activity.2.Effects of LPS treatment to RAW264.7 cells on methylation of NCOR gene promoter.After be stimulated by LPS for 48 h,MSP was used to detect the methylation of NCOR promoter and the expression of DNMT3b was detected by Western blot.Real time-PCR was used to detect the expression of NCOR mRNA after 5'-aza and LPS combined treatment.3.Effects of down-regulation of DNMT3b on the expression of NCOR and inflammatoryAfter Macrophages were transfected with DNMT3b siRNA.Western blot and Real time-PCR detecte the expression levels of DNMT3b,NCOR?TNF-? and IL-6 and the activation of NF-?B treated by DNMT3b si RNA combined with LPS.Results:1.The expression level of NCOR was decreased after RAW264.7 cells stimulated by LPS for 24?48h(p <0.05),The expression of TNF-? and IL-6 mRNA and the activity of NF-?B were increased(p <0.05).2.LPS could induce methylation of NCOR promoter,and the expression level of DNMT3b protein was increased.The level of NCOR m RNA was increased after treatment with 5'-aza and LPS(p <0.05).3.DNMT3b si RNA can downregulated the levels of DNMT3b protein and m RNA,increasing the level of NCOR expression,inhibiting TNF-?,IL-6 expression and NF-?B promoter activity(p <0.05).Conclusions:1.LPS stimulates macrophages suppress the expression of NCOR,result in promoting inflammation.2.LPS mediated the methylation of NCOR promoter by DNMT3b,which upregulated macrophage inflammatory response.3.Silence DNMT3b could suppress macrophage inflammatory response by demethylation of NCOR promoter.