志贺毒素(ST)虽是引起溶血性尿毒综合征（HUS)的主要因素，但宿主炎症反应可增强ST毒性效应及促进HUS发生。我们前期工作发现NLRP3炎性体参与O157:H7诱导的IL-1b释放。鉴于ST能诱导巨噬细胞分泌IL-1b已被研究证实，ST如何激活NLRP3炎性体及其在HUS中的作用尚无报道，本项目以探索ST激活NLRP3炎性体的分子机制及评价NLRP3炎性体激活对ST-HUS的影响为研究目标。表达纯化ST2，体外实验研究ST2如何影响pro-IL-1b合成和激活NLRP3炎性体的信号通路，影响pro-IL-1b合成涉及TLR/MyD88、MAPK和NF-κB通路，激活NLRP3炎性体的信号通路包括钾离子外流，有机氧产生和组织蛋白酶B释放。同时利用NLRP3缺失小鼠建立ST-HUS模型，验证NLRP3对诱导宿主炎症反应以及发生HUS的影响。研究结果将为更好地防治HUS提供理论依据。

Infection with Shiga toxin (ST)-producing Escherichia coli can induce hemolytic uremic syndrome (HUS), which draw more attention worldwide due to the outbreaks caused by O157:H7 E.coli and O104:H4 E.coli occurring in United State and German respectively. Although ST2 is the major virulence factor associated with the development of HUS, the host inflammatory response can also contribute to the pathogenesis of ST-induced vascular lesion. Our previous work has shown that NLRP3 inflammasome is involved in the production of IL-1b induced by O157:H7 E. coli. Given the fact that ST can induce macrophage to produce IL-1b and there is no information available on how ST2 activates NLRP3 inflammasome and whether NLRP3 plays a great role in the development of HUS, we propose this project to address these questions. The aim of the project is to study the molecular multi-signal mechanisms of NLRP3 activation by ST in vitro and evaluate the effect of NLRP3 activation on pathogenesis of HUS in vivo using NLRP3 deficient mouse model of ST-HUS. We will firstly express and purify the ST2 for whole project. To study the effect of ST on the protein synthesis of pro-IL-1b, we will focus on the pathway of TLR/MyD88/MAPK/NF-κB using antibodies or inhibitors to block signaling or measuring phosphorylation of key proteins in this pathway. In order to know how ST2 affects activation of NLRP3 pathway, we will explore at least three distinct NLRP3 activation pathways including efflux of intracellular potassium ions (K+), release of cathepsin B and generation of reactive oxygen species (ROS). To clarify the role of NLRP3 activation in pathogenesis of ST-HUS in vivo, we will establish ST-HUS model to compare the difference in inflammatory response and pathology sign of HUS development between NLRP3 deficient and wild type mouse. The results of this project would provide more information about complex molecular mechanism of NLRP3 inflammation activation by ST, which will shed light on the role of host factors in progressing HUS and eventually benefit the better control of HUS in the future.