南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (3): 321-329.doi: 10.12122/j.issn.1673-4254.2022.03.02

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p300诱导的乙酰化修饰参与脂多糖诱导的炎症介质合成

胡 柯，曹湘玉，李玉娴，刘灵丽，陈月富，陈立军，黄民江，谭碧峰，尹辉明   

  1. 湖南医药学院医学院，第一附属医院心血管内科，第一附属医院呼吸与危重症医学科，湖南 怀化 418000
  • 出版日期:2022-03-20 发布日期:2022-04-11

Coactivator p300-induced H3K27 acetylation mediates lipopolysaccharide-induced inflammatory mediator synthesis

HU Ke, CAO Xiangyu, LI Yuxian, LIU Lingli, CHEN Yuefu, CHEN Lijun, HUANG Minjiang, TAN Bifeng, YIN Huiming   

  1. Medical College of Hunan University of Medicine, Department of Cardiology, First Affiliated Hospital, Hunan University of Medicine, Department of Respiratory and Critical Care Medicine, First Affiliated Hospital, Hunan University of Medicine, Huaihua 418000, China
  • Online:2022-03-20 Published:2022-04-11

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摘要： 目的 探讨辅助激活因子p300诱导的乙酰化修饰介入脂多糖（LPS）诱导的炎症介质合成过程及其作用机制。方法 Agilent Sureprint G3 Mouse Gene Expression V2 微阵列芯片以及蛋白免疫印迹（WB）技术联合于小鼠巨噬细胞（RAW246.7）中筛选表达水平与LPS刺激强度相关的分子；凝胶电泳迁移实验（EMSA）以及染色质免疫共沉淀（chip-qpcr）方法验证相关分子与炎症基因IL-6以及TNF-α启动子部位的结合现象；相关分子过表达或干扰质粒转染RAW246.7后，WB法检测转染质粒的作用效果，ELISA方法检测IL-6以及TNF-α合成水平，染色质免疫沉淀-测序技术（chip-seq）分析炎症基因启动子部位相关分子的结合以及H3K27的乙酰化修饰水平。结果 微阵列芯片以及WB技术共同证实p300的表达与LPS刺激强度存在较好关联。免疫共沉淀证实了p300与c-myb存在结合现象。EMSA证实c-myb可与炎症基因启动子序列结合，而p300不能直接结合；chipqPCR表明当c-myb被干扰时，p300不能与炎症基因启动子序列结合。WB检测显示过表达或干扰质粒均能够干预相关分子的表达，且p65、p300以及c-myb 3者在表达上无相互影响。ELISA联合chip-seq显示与对照组比较，p300过表达以及LPS刺激均可导致炎症基因启动子部位结合的p300和H3K27乙酰化修饰水平增加，从而促进p65与启动子的结合和炎症基因转录（P<0.05）；而c-myb表达被干扰时，p300过表达以及LPS刺激诱导的p300与p65在启动子的结合、H3K27乙酰化修饰和炎症介质合成均被遏制（P<0.05）。在p65干扰相关组别中，p65与启动子的结合以及炎症基因转录均被抑制（P<0.05），但未对p300的结合以及H3K27乙酰化水平造成影响。结论 LPS刺激可诱导p300合成，后者在c-myb介导下结合于炎症基因启动子区域，并通过乙酰化H3K27易化启动子与p65结合，从而促进炎症基因表达。

关键词: p300；乙酰化；脂多糖；炎症介质；合成

Abstract: Objective To investigate the role of acetylated modification induced by coactivator p300 in lipopolysaccharide (LPS)-induced inflammatory mediator synthesis and its molecular mechanism. Methods Agilent SurePrint G3 Mouse Gene Expression V2 microarray chip and Western blotting were used to screen the molecules whose expression levels in mouse macrophages (RAW246.7) were correlated with the stimulation intensity of LPS. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (chip-qPCR) were used to verify the binding of the molecules to the promoters of IL-6 and TNF-α genes. The effects of transfection of RAW246.7 cells with overexpression or interfering plasmids on IL-6 and TNF-α synthesis were evaluated with ELISA, and the binding level of the target molecules and acetylation level of H3K27 in the promoter region of IL-6 and TNF-α genes were analyzed by chromatin immunoprecipitation sequencing technique (chip-seq). Results Gene microarray chip data and Western blotting both confirmed a strong correlation of p300 expression with the stimulation intensity of LPS. Immunocoprecipitation confirmed the binding between p300 and c-myb. The results of EMSA demonstrated that c-myb (P<0.05), but not p300, could directly bind to the promoter region of IL-6 and TNF-α genes; p300 could bind to the promoters only in the presence of c-myb (P<0.05). The expressions of p65, p300 and c-myb did not show interactions. Both p300 overexpression and LPS stimulation could increase the level of promoter-binding p300 and H3K27 acetylation level, thus promoting p65 binding and inflammatory gene transcription; such effects were obviously suppressed by interference of c-myb expression (P<0.05). Interference of p65 resulted in inhibition of p65 binding to the promoters and gene transcription (P<0.05) without affecting p300 binding or H3K27 acetylation level. Conclusion LPS can stimulate the synthesis of p300, whose binding to the promoter region of inflammatory genes via c-myb facilitates the cohesion of p65 by inducing H3K27 acetylation, thus promoting the expression of the inflammatory genes.

Key words: p300; acetylation; lipopolysaccharide; inflammatory mediator; synthesis