H3K27me3去甲基化介导的PR3高表达在脓毒症早期全身炎症反应中的作用研究

Systemic inflammatory response that induced by enormous amounts of activated proinflammatory cytokines is the leading cause of multiple organ failure or even death. Researches have shown that blocking the function of IL-1β and IL-18 simultaneously could remarkablely increase the survival rate of early sepsis model mice，thus finding a single target that could block the activation of various kinds of proinflammatory cytokine at early stage of sepsis would make sense. Monocytes are the main source of proinflammatory cytokines, caspase-1 and PR3 are the key enzymes that can cleave these cytokines precursor into active form. We and researchers from abroad both found that caspase-1 was obviously inhibited at early stage of sepsis, while PR3 was over expressed, but the exact mechanism is still unkown. Studies have shown that over expression of PR3 in neutrophils was much related to JMJD3 mediated demethylation of H3K27me3; and LPS could induce over expression of JMJD3. Interestingly, we found that mRNA expression of JMJD3 was obviously elevated in monocytes of early sepsis, so we hypothesize that JMJD3 was over induced by LPS in monocytes of early sepsis, then mediated demethylation of H3K27me3 and subsequent massive expression of PR3, eventually lead to the activation of enormous amounts of proinflammatory cytokines. We next will carry out series experiments to verify our hypothesis, and to explore the feasibility of remitting systemic inflammatory response and improving outcomes of sepsis by the early blockage of JMJD3 and PR3 respectively, and finally provide a new therapy philosophy and target to the treatment of early sepsis.

脓毒症早期大量促炎因子活化引起的全身炎症反应是导致患者多脏器衰竭甚至死亡的重要原因。研究发现同时阻断IL-1β和IL-18显著改善脓毒症小鼠预后，因而寻找单一靶点早期阻断多种促炎因子的活化具有临床意义。促炎因子主要来自单核细胞，由caspase-1和PR3激活。我们和国外研究发现脓毒症早期单核细胞caspase-1明显受抑而PR3却过度表达，但具体机制尚不明确。研究发现中性粒细胞PR3的高表达与JMJD3介导的H3K27me3去甲基化有关；且JMJD3可被LPS诱导表达。我们发现脓毒症早期单核细胞JMJD3表达明显增多，故提出假设：脓毒症早期LPS诱导单核细胞JMJD3高表达，导致H3K27me3去甲基化并引起PR3表达增多，最终引起促炎因子大量活化。我们拟进一步通过相关实验验证假说，并探究早期分别阻断JMJD3和PR3对脓毒症炎症反应和预后的影响，为脓毒症早期干预提供新的治疗思路和靶点。

背景：脓毒症早期大量促炎因子活化引起的过度炎症反应是导致脓毒症早期患者死亡的主要原因。研究发现同时阻断IL-1β和IL-18能显著改善脓毒症小鼠预后，因此寻找靶点早期阻断多种促炎因子活化具有重要意义。中性粒细胞膜蛋白酶3（mPR3）对多种促炎因子具有正向调节作用。基因表达的表观遗传调控是调节脓毒症患者髓系细胞功能的重要机制。但中性粒细胞mPR3在脓毒症中的调控机制未有研究报道。本研究旨在阐明脓毒症早期中性粒细胞mPR3的表达及其与炎症因子的关系，探讨mPR3的调控机制。方法：纳入符合脓毒症诊断的患者，确诊24小时内采集外周血并以健康志愿者为对照组。获得中性粒细胞和血浆，检测中性粒细胞mPR3和去甲基化组蛋白酶3（JMJD3）表达，血浆IL-1β、TNF-α水平。LPS体外刺激正常人中性粒细胞，实验分为4组：对照组，LPS刺激组，LPS+GSK-J4组（GSK-J4为JMJD3的抑制剂）及GSK-J4组。以上四组中性粒细胞分别与单核细胞共培养，检测单独中性粒细胞培养组mPR3的水平，中性粒细胞组蛋白（H3K27me3）表达。检测各单独培养中性粒细胞组和共培养细胞组上清IL-1β水平。结果：与健康志愿者相比，脓毒症患者中性粒细胞mPR3和JMJD3显著升高；与mPR3低表达组相比，mPR3高表达组脓毒症患者血浆TNF-α和IL-1β水平显著升高。体外实验表明，在单独培养中性粒细胞条件下：与空白对照组相比，LPS刺激组mPR3和IL-1β均显著升高，H3K27me3表达明显下降。与LPS组相比，LPS+GSK-J4组mPR3和IL-1β均显著下降，H3K27me3水平显著升高。在共培养条件下：LPS刺激后培养上清中IL-1β的水平显著高于空白对照组；GSK-J4+LPS共培养细胞上清中IL-1β的水平显著低于LPS单独刺激共培养细胞体系；LPS刺激共培养细胞上清中IL-1β的水平显著高于各相应单独培养细胞组。结论：脓毒症早期患者外周血中性粒细胞mPR3和JMJD3表达升高，mPR3的表达升高与血浆炎症因子增高相关。中性粒细胞mPR3在脓毒症早期可能增加炎症反应中促炎因子IL-1β的释放，对炎症反应具有放大作用。JMJD3调控中性粒细胞mPR3的表达，进而可能调控IL-1β水平。JMJD3可能通过修饰H3K27me3调节中性粒细胞mPR3的表达。