硫酸乙酰肝素激活NLRP3炎性小体介导细胞炎症反应：急性胰腺炎重症化的新机制

There is increasing evidence that excessive inflammatory responses initiated by heparan sulfate (HS) is mainly responsible for the aggravation of acute pancreatitis (AP), but little is known about the detailed mechanism. In previous work we have found, for the first time, that HS exhibited the potential to induce activation of the NLRP3 inflammasome, which could be blocked by either the monoclonal antibody of Toll like receptor 4 (TLR4) , or the inhibitor of MaxiK channel. Moreover, our data revealed that blocking the NLRP3 inflammasome activation contributed to a reduction in the severity of AP. Base on the findings above, a hypothesis was proposed that the HS-induced cellular inflammatory responses and resultant aggravation of AP results from its potential to activate the NLRP3 inflammasome, which could be probably regulated by TLR4 and MaxiK. In present study, the relationship between the HS-induced NLRP3 inflammasome activation and the severity of AP will be analyzed in vitro and in vivo. Furthermore, we will identify the roles of HS-triggered NLRP3 inflammasome activation in the inflammatory cascades during the progression of AP, by means of RNA interference or over-expression. In order to elucidate the molecular mechanism underlying the HS-caused activation of NLRP3 inflammasome, the roles of TLR4-mediated transcriptional or non-transcriptional pathway in the priming of NLRP3 inflammasome activation induced by HS and the MaxiK channel in the assembly of inflammasome muti-proteins complex will be revealed using immunofluorescence, immunoprecipitation, fluorescence resonance energy transfer (FRET) and other techniques. This study will bring new insight into the understanding of the progression of AP and facilitate the development of novel therapeutics that target the HS-mediated inflammatory signaling pathway.

硫酸乙酰肝素（HS）诱发过度的细胞炎症反应是导致急性胰腺炎（AP）重症化的重要原因，但确切机制尚不明确。我们首次发现HS在体外可诱导巨噬细胞NLRP3炎性小体激活，抑制TLR4或阻断钾通道MaxiK能不同程度抑制该过程；而降低NLRP3活性则能有效抑制小鼠AP重症化，据此我们推测：在TLR4和MaxiK的调控下HS通过激活NLRP3介导细胞炎症反应是AP重症化的新机制。本项目拟从不同层面分析HS激活NLRP3的水平与AP严重程度的相关性；采用干扰沉默和转染过表达技术明确NLRP3在HS诱导的细胞炎症反应及AP重症化中的作用；利用免疫荧光、共沉淀和FRET等技术研究TLR4及其调控的转录/非转录途径在HS启动NLRP3激活过程中的作用以及MaxiK对NLRP3多蛋白体组装过程的调控作用，从而揭示HS激活NLRP3炎性小体的分子机制。本项目的实施将为AP重症化及其防治策略的研究提供新的思路。

急性胰腺炎（AP）发生后，胰腺局部的炎症反应加重并向全身扩散是导致AP重症化的主要原因，但其确切机制仍有待进一步探明。课题组在前期研究中发现，硫酸乙酰肝素（HS）在体外可诱导细胞NLRP3炎性小体激活，阻断钾通道MaxiK或抑制TLR4能不同程度阻断该过程；在AP小鼠中，抑制NLRP3活性可有效阻止AP的重症化进展，据此我们推测：HS通过MaxiK和TLR4介导的信号通路激活NLRP3炎性小体，从而加重胰腺炎症反应并促进AP重症化。本项目的主要研究内容包括验证HS对NLRP3炎性小体的激活，明确NLRP3炎性小体活化对AP重症化的促进作用，在此基础上揭示HS激活NLRP3炎性小体的分子机制。项目的研究结果包括：（1）在AP动物模型中，AP大鼠血浆HS水平与胰腺炎症反应及损伤程度正相关；（2）HS可通过TLR4/NF-κB途径上调胰腺腺泡细胞中NLRP3的表达，同时通过刺激细胞K+ 外流来启动炎性小体的组装，从而介导NLRP3炎性小体激活，该过程同时受到P2X7R和MaxiK调控；（3）AP小鼠注射HS后胰腺组织炎症反应加重并释放大量促炎细胞因子入血，敲除NLRP3后胰腺炎症反应及细胞因子的释放被明显抑制；（4）AP进展过程中，血浆外泌体通过诱导NLRP3依赖的AMs焦亡引起肺损伤，促进了AP重症化。本项目揭示了AP重症化进展的新机制，为其防治策略的研究提供了新的思路。