甲基化修饰酶EZH2及JMJD3对脓毒症炎症反应的调控机制

Abnormal systemic inflammation is the key factor of death in sepsis patients. However, there has been no agent tested in clinical trials show consistent benefit to date. Epigenetic modification is important for multiple biological pathways and is involved in inflammatory cytokine production. Our preliminary data showed that inhibition of JMJD3, which is a H3K27me3 demethylase, down-regulated the production of multiple inflammatory cytokines in human monocytes. EZH2, which is an H3K27me3 methylase, gene knock out protected mice from sepsis induced death and decreased production of multiple key inflammatory cytokines in LPS-induced sepsis mouse model. This inhibition effect of EZH2 knock out on cytokines is contrary to the classical role of EZH2 which represses gene expression through H3K27me3. Further study showed that LPS stimulated human monocytes induce down-regulation of H3K27me3 whereas up-regulation of EZH2, which is associated with the activation of cytokines by NF-KB, indicating EZH2 acts as an upstream regulator of NF-KB to modulate cytokine production. The aim of this study is to discover the mechanism of EZH2 and JMJD3 in regulating inflammatory cytokine production, assess the protective role of targeting EZH2 and JMJD3 in sepsis treatment and address how EZH2 and JMJD3 is involved in abnormal cytokine production in clinical sepsis patients. Through this study, we will unravel new mechanisms regulating inflammation in sepsis and provide important information for the development of novel treatment approach for sepsis

异常的全身炎症反应是脓毒症致死的关键因素，但目前尚无有效的干预手段。表观遗传修饰作为调控机体反应的重要方式参与多种炎症因子基因转录和表达。我们前期研究发现：H3K27me3去甲基化酶JMJD3抑制剂可抑制单核细胞产生多种炎症因子；H3K27me3甲基转移酶EZH2基因敲除保护小鼠免受脓毒症致死，并降低多种炎症因子水平，这与EZH2通过经典H3K27me3通路抑制下游基因的功能不一致；深入研究发现LPS诱导H3K27me3下调，但增强EZH2表达，后者与NF-κB活化的炎症因子相关，提示EZH2可能是NF-κB调控炎症反应的重要调控分子。本项目将在此基础上研究EZH2和JMJD3调控炎症因子的分子机制，评估靶向调节EZH2和JMJD3对脓毒症的保护作用，阐明临床脓毒症患者EZH2和JMJD3调控炎症因子异常表达可能的途径。项目研究成果将揭示炎症调控的新机制，为研发脓毒症治疗新策略提供重要依据

脓毒症是宿主对感染的异常反应引起的危及生命的器官功能障碍，极大威胁着儿童健康。我们通过研究发现（1）a. JMJD3通过调控单核细胞、巨噬细胞、人CD4+T细胞和人CD8+T细胞H3K27me3的去甲基化造成细胞因子的释放，GSK-J4作为调控JMJD3的特异性药物，能够有效抑制单核细胞和巨噬细胞分泌细胞因子TNF-α、IL-1b和IL-6，抑制人CD4+T细胞分泌IL-17，IL-4和IFN-γ，抑制人CD8+T细胞释放IFN-γ，可能作为我们通过调控重要免疫细胞的组蛋白甲基化状态从而调控脓毒症炎症水平的有力措施; b.通过检测收集的临床脓毒症患者入院时外周血样本发现：①患者体内单核细胞HLA-DR表达显著降低；②患者单核细胞H3K27me3表达平均水平显著高于健康对照；③患者单核细胞DOT1L催化产物H3K79me2水平显著高于健康对照；④单核细胞中H3K27me3/H3K79me2高表达与炎性因子TNF-a/IL-1b/IL-6表达成正相关；患者H3K27me3和H3K79me2表达水平是否对预判患者预后有意义尚在进一步分析中。本项目已建立的检测平台有助于临床脓毒症状态下树突状细胞和单核细胞表观修饰状态的快速检测，有助于通过区分临床脓毒症的不同表观表型而采用不同的对策。（2）急性肾损伤（AKI）是脓毒症常伴发的病理状态，我们研究发现两个较新的生物标志物用来预警AKI，血清FGF23水平与eGFR（预计肾小球滤过率）呈现负相关，尿IGFBP-7是预测危重症患儿发生AKI（急性肾损伤）的独立危险因素。（3）KPT-330，一种新型靶向染色体维持蛋白1（CRM1）的特异性小分子抑制剂具有较好的抗炎活性，对脓毒症具有保护作用。动物在体实验结果表明，KPT-330 处理能够改善脓毒症时肺组织损伤，降低炎症因子TNF-α, IL-6 和HMGB1 水平，从而对脓毒症表现出保护作用。进一步体外细胞实验发现，KPT-330 处理通过干预CRM1 的分布能够下调LPS诱导的TNF-α、IL-6 的mRNA水平以及HMGB1 的胞质转运。此外，Western blot结果显示KPT330 通过抑制下游NF-κB和MAPK p38信号的活化，从而发挥抗炎活性，因此，KPT-330具有较好的抗炎活性，对脓毒症可能具有保护作用.