甲基化转移酶SETD4调控炎症因子释放的分子机制研究

Unctrolled systemic inflammatory response resulted by a massive release of proinflammatory mediator leads to the development of sepsis and septic shock. We previously discovered a methyltransferase SETD4 which belongs to the SET Family.There is little no about the function of SETD4. We found SETD4 up-regulation or down-regulation resulted in low inflammatory cytokine releasing from macrophages following LPS treatment. The results indicated that SETD4 may plays a key role in the release of inflammatory cytokines. SETD4-deficient mice exhibited significantly lower levels of inflammatory cytokines in serum than wild-type mice during the endotoxin shock. These findings indicated that SETD4 took part in the pathology of endotoxin shock by controlling the release of inflammatory cytokines. On the basis of above findings, we will identify the target of SETD4 as well as its active site, to explain the molecular mechanism of the expressions of inflammatory cytokines modulated by SETD4, to confirm the critical role that SETD4 plays on imflammation and endotoxin shock using SETD4 knockout mice and a series of molecular biology methods such as Chromatin immunoprecipitation (ChIP), real time PCR and molecular clone. The results of this project have important theoretical significance and practical value for amplifying the machinery of histone modification, further illustrating the occurrence mechanism of imflammation and endotoxin shock, also helping to explore more rational and effective anti-inflammatory methods.

炎症介质大量释放导致失控性全身炎症反应是脓毒症、脓毒性休克发生发展最为重要的原因。我们前期在探索脓毒性休克的发生机制时发现了SETD4，该蛋白属于甲基化修饰酶SET家族蛋白中的一种，功能尚不清楚。我们发现上调或者下调巨噬细胞SETD4表达时，内毒素（LPS）刺激所致的炎性细胞因子如TNFa等的释放增加或者减少；利用SETD4敲基因小鼠复制内毒素休克模型，发现SETD4基因敲除小鼠血清中炎性细胞因子的水平明显低于野生型小鼠,提示SETD4可能通过调控炎性细胞因子的释放参与了内毒素血症的发生与发展。本项目拟在前期工作的基础上，进一步利用染色质免疫沉淀（ChIP）、蛋白质组学技术等分子生物学手段查明SETD4作用的靶分子、靶点及其发挥作用的活性位点，阐明SETD4调控细胞因子释放的分子机制。本项目的研究结果对于进一步阐明内毒素休克的发生机理，探索更合理有效的抗炎措施具有重要的理论意义和实践价值。

探讨脓毒症的确切发生机制仍是相关研究领域的重点和关键问题。已有的研究结果表明，由内毒素（LPS）等病原相关分子模式（PAMPs）诱导的大量炎症因子的产生和活化是导致脓毒症和脓毒症休克最为关键的环节。目前认为，炎性细胞因子的产生除了与PAMPs 激活Toll样受体（TLR）及其介导的细胞信号转导通路MAPK、NF-kB有关外，与组蛋白的修饰如甲基化修饰密切相关。组蛋白甲基化修饰主要由一类含有高度保守的SET结构域的SET家族蛋白完成。SETD4是本课题组前期研究小鼠内毒素休克发生机制时发现的，属于甲基化修饰酶SET家族蛋白中的一种，功能尚不清楚。本研究发现SETD4能正向调控巨噬细胞受内毒素（LPS）等刺激时所致的炎性细胞因子如TNF-等的表达，该作用主要是通过其SET结构域实现的；敲除SETD4不影响LPS刺激BMDMs介导的MAPK和NF-kB磷酸化程度；质谱鉴定和特异性抗体检测体外甲基化反应产物均发现SETD4能够使H3K4甲基化增强，该结果在BMDMs细胞实验得到进一步证实。本研究结果提示SETD4可能通过H3K4甲基化增强进而促进炎性细胞因子如TNF-a和IL-6等的转录及释放，在内毒素血症的发生及发展过程中发挥了重要作用。本项目的研究结果对于进一步阐明内毒素休克的发生机理，探索更合理有效的抗炎措施具有重要的理论意义和实践价值。