脂多糖诱导巨噬细胞中核受体协同抑制因子启动子甲基化对炎症因子的调控作用

目的探讨核受体协同抑制因子(NCOR)在脂多糖(LPS)诱导巨噬细胞炎症反应中的作用及其调控机制。方法 1μg/ml的LPS分别处理小鼠巨噬细胞RAW264.7 24 h和48 h,应用Western blot和Real time-PCR检测NCOR的表达水平以及肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)mRNA水平,荧光素酶报告基因检测核因子-κB(NF-κB)的启动子活性。LPS处理细胞48 h后,应用MSP检测NCOR启动子是否发生甲基化以及Western blot检测DNMT3b的表达变化。Real time-PCR检测5'-aza和LPS联合处理细胞后NCOR mRNA的表达水平;转染DNMT3b siRNA后,分别应用Western blot和Real time-PCR检测DNMT3b的表达水平,以及DNMT3b siRNA和LPS联合作用下NCOR、TNF-α、IL-6的表达水平和NF-κB的启动子活性。结果 LPS干预细胞24、48 h后,NCOR蛋白和mRNA表达显著下调(P0.05),而TNF-α、IL-6 mRNA表达水平、DNMT3b蛋白的表达水平以及NF-κB的启动子活性显著上升(P0.05)。MSP检测说明LPS可介导NCOR的启动子甲基化。用5'-aza和LPS联合处理细胞后NCOR mRNA水平较LPS组有显著上升(P0.05)。采用DNMT3b siRNA可显著下调DNMT3b蛋白和mRNA水平,并可部分逆转LPS介导的抑制NCOR表达的效应,抑制TNF-α、IL-6的表达水平和NF-κB的启动子活性(P0.05)。结论 NCOR启动子的甲基化是LPS介导巨噬细胞炎症反应发生、发展的关键步骤,其可作为治疗ALI/ARDS的潜在靶点。

Lipopolysaccharide modulation the promoter methylation of nuclear receptor corepressor regulated inflammation mediators in macrophages

Objective To investigate the role and potential mechanism of nuclear receptor corepressor (NOCR) at lipopolysaccharide (LPS) mediation inflammation response in macrophages.Methods Western blot,realtime-PCR and luciferase assays was used to detect the expression of NCOR,interleukin-6(IL-6) and tumour necrosisfactor(TNF-α) and promoter activity of nuclear factor-κB (NF-κB) when RAW264.7 cells were treated with 1 μg/ml LPS.The promoter methylation of NCOR was analyzed by methylating-specific PCR (MSP) analysis,and the protein of DNMT3b was evaluated by western blot after cells stimulation with 1 μg/ml LPS for 48 h.Real time-PCR was also used to evaluate the expression of NCOR mRNA when RAW264.7 was treated with 1 μg/ml LPS combine with 1μM 5'-aza.After DNMT3b siRNA was transfected into RAW264.7 for 48 h,the mRNA and protein of DNMT3b was detected by western blot and real time-PCR.Additional,the expression of NCOR,TNF-α and IL-6 and NF-κB activity was analyzed after RAW264.7 was treated with 1 μg/ ml LPS combine with DNMT3b siRNA.Results The expression of NCOR mRNA and protein was significantly decreased (P＜0.05) and the expression of TNF-α and IL-6 mRNA,DNMT3b protein and activity of NF-κB was elevated (P＜0.05) after cells was treated with 1 μg/ml LPS for 24 and 48 h,respectively.MSP assay showed LPS could modulate the NCOR promoter methylation.The expression of DNMT3b mRNA and protein was decreased after cells were transfected by DNMT3b siRNA.Additionally,down-regulation of DNMT3b was partly reversed LPS modulation the inhibition of NCOR,and decreased the expression of TNF-α and IL-6 mRNA,and activity of NF-κB (P＜0.05).Conclusion NCOR promoter methylation is key step of occurrence and development of LPS mediation inflammation response.It might be a potential target for acute lung injury / acute respiratory distress syndrome (ALI/ARDS) treatment.