蛋白激酶MK2调控巨噬细胞miRNA表达在急性炎症反应中的作用研究

Sepsis causes millions of deaths each year worldwide, however the treatment for sepsis is not satisfying so far. To advance the understanding of the cellular and molecular networks that regulate acute inflammation (such as sepsis) will provide more chances to develop effective treatment of acute inflammatory diseases. Mitogen activated protein kinase activated protein kinase 2 (MAPKAPK-2 or MK2), a substrate of p38 MAPK, is known to influence the activation of macrophages in response to the Toll-like receptor (TLR)-4 ligand bacterial lipopolysaccharide (LPS). Recent evidence also reveals a critical role for a group of noncoding RNAs, i.e. microRNAs (miRNAs), in regulating the inflammatory process. Despite the importance of both MK2 and miRNAs in macrophage activation and acute inflammation, it still remains unclear whether miRNAs contribute to MK2 targeted cytokine production in the condition of acute inflammation and tissue injure. Our previous study showed that the deletion of MK2 gene can significantly decrease the expression of several cytokines such as TNF-α, IL-6, and MIP-2(CXCL2). The MK2 knockout mice also demonstrate a divergent expression map of miRNA in macrophages in comparison with wild type. Using the MK2 knock-out mice and miRNA microarray analysis, the present study will examine the role of MK2 in the regulation of microRNAs expression in LPS-stimulated macrophages. By administration of miRNA mimics and inhibitors into mice macrophages, we will also test whether miRNA regulated by MK2, such as let-7e, miR155 and miR21, contribute to the acute inflammation induced lung injury and inflammatory cytokines production in LPS and CLP mouse models. Furthermore, we will determine the targets of these miRNAs and itsthe regulation molecular mechanism. The findings will shed a new light on molecular networks that regulate inflammation and provide potential drug target for therapy.

败血症是感染导致临床病人死亡的重要原因，一直缺乏有效治疗手段。MK2激酶和microRNA（miRNA）在炎症反应中发挥重要作用，但这两者在急性炎症诱导组织损伤中的相互关联和作用机制尚无专门的研究报道。我们的前期研究显示，MK2基因敲除可引起急性炎症条件下的细胞因子表达下调和miRNA表达变化。本研究将利用MK2基因敲除小鼠和miRNA芯片分析，构建细菌内毒素LPS和小鼠盲肠结扎穿孔诱导的败血症和脓毒症模型，在前期结果基础上，从整体动物和细胞水平两个层面进一步阐明MK2如何通过调控巨噬细胞miRNA表达，如miR155，miR21，let-7e等，参与调节急性炎症诱导肺损伤和炎症因子释放，并解析MK2参与调节LPS介导的TNF-α、IL-6、MIP-2（CXCL2）、MyD88等细胞因子和信号分子表达的具体分子机制，为MK2在巨噬细胞活化和急性炎症反应中的作用提供新的视野和潜在药物靶点。

败血症伴随急性炎症，是导致临床病人死亡的重要原因。作为一个潜在的抗炎药物靶点，有很多报道指出MK2蛋白激酶在炎症反应中发挥着极为重要的作用。MK2激酶是p38 MAPK的直接底物之一，在TLR4配体细菌内毒素LPS引起的炎症反应和巨噬细胞活化中起重要作用。近年来，一类非编码的小RNA在调控炎症进程中的作用也引起了相当的关注。但这两者在急性炎症诱导组织损伤中的相互关联和作用机制尚无专门的研究报道。我们的前期研究显示，MK2基因敲除可引起急性炎症条件下的细胞因子表达下调和miRNA表达变化。本研究在前期结果的基础上，利用MK2基因敲除小鼠，构建细菌内毒素LPS和小鼠盲肠结扎穿孔（CLP）诱导的败血症和脓毒症模型。结果显示MK2敲除小鼠中CLP和LPS诱导的小鼠致死率有明显降低。与野生型小鼠相比，MK2敲除小鼠在LPS诱导的炎症反应均有症状减轻的现象，特别体现在体内和体外实验中炎症因子（TNF-α、IL-6、MIP-2）的表达量显著降低。接下来的miRNA分析显示，在MK2敲除的骨髓巨噬细胞BMDM中，LPS诱导的let-7e miRNA表达相比野生型BMDM中有明显减少。体外转染let-7e miRNA相似片段和抑制片段，表明let-7e miRNA可抑制BMDM中LPS诱导的炎症因子（TNF-α、IL-6、MIP-2）表达。在进一步的机制研究中发现，MK2信号通路（p38/MK2/CREB）在巨噬细胞中通过Lin28蛋白调控miRNA let-7e的表达，继而在LPS刺激的炎症反应中调节炎症因子（TNF-α、IL-6、MIP-2）的产生和释放。本项目还首次利用MK2（髓系细胞）特异性敲除小鼠阐明巨噬细胞中MK2激酶的表达在细菌诱导的炎症反应中起着关键性的作用。我们的结果为MK2在巨噬细胞活化和急性炎症反应中的作用提供新的潜在药物靶点。