ZFP91参与调控经典和非经典炎症小体活化及侧金盏花E干预机制研究

The significant findings based on the preliminary experimental results of Thousands of Foreign Experts Plan (WQ20122200065): ZFP91 is functioned as a factor promoting inflammatory cytokine IL-1β release, as well as induces the expression of NLRP3, ASC, caspase-1, caspase-8 protein and mRNA. The IL-1β secretion level of mice with ZFP91 gene knockout is lower compared to the wild type. These results make us hypothesize that ZFP91 is involved in the regulation of canonical and non-canonical inflammasome activation and induced IL-1β secretion. However, the exact mechanism remains unclear. In this proposal, we will perform experiments at both molecular (over-expression, dominant negative mutation and RNA interference) and cellular (function) levels, as well as animal models (ZFP91 gene knockout mice), to figure out the role of ZFP91 in the regulation of canonical NLRP3-ASC-caspase-1 and noncanonical NLRP3-ASC-caspase-8 inflammasome activation. Finally, targeting the ZFP91/inflammasome using Calenduloside E as a therapeutic approach will be studied.

基于前期外专千人计划（WQ20122200065）项目研究中发现：ZFP91促进炎症因子IL-1β分泌，同时能够诱导NLRP3、ASC、caspase-1、caspase-8蛋白和mRNA的表达；ZFP91基因敲除小鼠IL-1β分泌水平低于野生型，因此认为ZFP91参与调控经典NLRP3-ASC-caspase-1和非经典NLRP3-ASC-caspase-8炎症小体活化从而诱导了IL-1β的分泌，然而其确切机理尚不明确。研究拟从分子（过表达、显性失活突变和RNA干扰）、细胞（功能）和整体（ZFP91基因敲除模型）不同层面，通过免疫共沉淀、免疫荧光和流式细胞等技术，以炎症小体活化为切入点，探索ZFP91对经典NLRP3-ASC-caspase-1和非经典NLRP3-ASC-caspase-8炎症小体调节机制，同时探讨侧金盏花E干预作用，以期为ZFP91/炎症小体为靶点的炎症诊治提供新策略。

白介素-1β（IL-1β）是重要的炎症介质之一，在急慢性炎症的致病过程中发挥重要作用。据报道锌指蛋白91（ZFP91）参与各种生物学过程，但对于ZFP91调节IL-1β的表达机制尚未明确，该研究揭示ZFP91可促进pro-IL-1β的产生和IL-1β的分泌，Convallatoxin是我们课题组从长白山药用植物侧金盏花的干燥根部分离提取的一种活性单体化合物，尚未见国内外有解析该种化合物在91介导的炎性小体中作用机理的研究报道。本研究利用蛋白免疫印记、RT-PCR、免疫共沉淀。免疫荧光等明确观察到（1）ZFP91上调pro-IL-1β的产生及活化；（2）ZFP91促进LPS-刺激的THP-1细胞和BMDM细胞中非经典caspase-8炎性小体组分的表达；（3）ZFP91通过非经典caspase-8炎性小体路径促进LPS刺激的THP-1细胞中IL-1β的表达；（4 ZFP91促进LPS刺激的THP-1细胞中非典型caspase-8炎性复合体集聚；（5）ZFP91促进LPS刺激的THP-1细胞和BMDM细胞中MAPKs的活化；（6）ZFP91缺失改善了DSS诱导的结肠炎小鼠的症状和结肠炎症；（7）ZFP91缺失改善明矾诱导的腹膜炎；（8）Convallatoxin通过下调ZFP91抑制IL-1β的释放；（9）Convallatoxin显著降低ZFP91调节的K63连接Pro-IL-1β的泛素化，从而限制过度的炎性体活性。（10）Convallatoxin通过降低ZFP91抑制非经典Caspase-8炎症小体复合物的组装和MAPK信号通路的激活。（12）Convallatoxin通过下调ZFP91改善D-GalN/LPS诱导的小鼠肝损伤、DSS诱导的小鼠结肠炎和明矾诱导的小鼠腹膜炎。本研究为开发Convallatoxin作为靶向ZFP91的新型抗炎药提供了新视角。