从NF-κB炎症通路和NLRP3炎性体轴协同激活IL-1β角度探讨土茯苓总黄酮防治痛风性关节炎的机制

At present, the morbidity rate of gout in China is 2.2%, which is similar to the western developed countries. Latest research data showed that TLR/MyD88/NF-κB signaling pathway and NLRP3/ASC/caspase-1 axis could active IL-lβ synergistically, which play a vital role in monosodium urate crystals (MSU) induced gouty arthritis. These data bring new opportunity for the development of drugs to treat gout. Our previous results showed that total flavonoids from Smilax glabra (TFSG) has obvious anti-gout effects, and could significantly inhibit the IL-1β secretion and NLRP3 inflammasome mRNA and protein expression in mouse bone marrow-derived macrophages (BMDMs) stimulated by MSU. This project aimed to study the regulatory mechanisms of TFSG to some key proteins in TLR/MyD88/NF-κB signaling pathway and NLRP3/ASC/caspase-1 axis by using modern molecular biotechnology techniques, associate with flow cytometry, confocal laser microscope, transmission electron microscope, and immunohistochemistry. Moreover, in order to study the new mechanism of TFSG to treat gouty arthritis, we use NF-κB agonist, NLRP3 activator, ROS inducer or plasmid transfection technique, to up-regulate the key proteins expression in above-mentioned signaling pathway and axis. This project could offer the innovative ideas and new targets to study the nosogenesis of gout and anti-gout drug screening.

目前我国痛风患病率高达2.2%，接近西方发达国家水平。最新资料显示，TLR/MyD88/NF-κB通路与NLRP3/ASC/caspase-1轴协同激活IL-1β，在尿酸钠晶体（MSU）诱导的痛风性关节炎（GA）中发挥关键作用，这为痛风治疗药物的研制带来了新的契机。我们前期研究发现土茯苓总黄酮（TFSG）具有良好的抗大鼠GA功效，并能显著抑制MSU刺激的小鼠骨髓来源巨噬细胞（BMDMs）中IL-1β产生及NLRP3炎性体表达。本项目拟以GA大鼠和单核巨噬细胞（THP-1）为对象，借助现代分子生物学技术，结合流式细胞术、激光共聚焦等手段，研究TFSG对TLR/MyD88/NF-κB通路与NLRP3/ASC/caspase-1轴关键蛋白的调控作用；并分别用激动（活）剂、诱变剂或质粒转染技术上调上述通路与轴关键蛋白的表达，研究TFSG抗GA新机制；为痛风发病机制研究和药物筛选提供新思路和新靶点。

近年来，痛风患病率呈现快速上升的趋势。土茯苓是治疗痛风的传统中药，具有一定的临床和应用基础，但缺乏现代药理学的系统研究。经过四年探索，课题组已完成了项目计划书规定的任务，基本达到了预期目标。主要研究内容和成果：（1）建立MSU诱导的急性痛风THP-1细胞模型，阐明TLR/MyD88/NF-κB通路与NLRP3/ASC/caspase-1轴关键蛋白表达的特点、规律及土茯苓总黄酮（TFSG）和以土茯苓为君药组成的中药方剂加味三妙丸（MSMP）的调控作用。（2）建立急性痛风性关节炎C57BL/6小鼠模型，通过测定关节肿胀度及滑膜细胞因子水平，观察关节组织病理学改变，分析关节滑膜NF-κB通路与NLRP3炎症小体轴关键蛋白表达的特点、规律，揭示了TLR/MyD88/NF-κB通路与NLRP3/ASC/caspase-1轴能协同激活IL-lβ，在MSU诱导的痛风性关节炎中发挥关键作用，并阐明了TFSG和MSMP调控作用。本研究为痛风的病理机制研究与抗痛风中药新药研制奠定了坚实基础。（3）本课题已经完成SCI收录论文3篇，投稿SCI论文1篇。发表中文一级期刊论文3篇，核心期刊论文1篇。参加国内学术会议10次并作专题报告5次，参加国际学术会议3次。获得长三角地区研究生学术论坛优秀论文一等奖1项。授权实用新型专利1项。入选浙江中医药大学“5151远志人才工程”1人。已培养硕士研究生3名，正在培养博士研究生和硕士研究生各1名。