基于中性粒细胞胞外诱捕网（NETs）和中性粒细胞源的外泌体研究泄浊化瘀法对急性痛风性关节炎的作用及机制

Monosodium urate（MSU）crystals trigger the occurrence of gouty arthritis（GA）,which by inducing neutrophils to form neutrophil extracellular traps（NETs）.NETs and neutrophil exosomes（NE）may participate in the mechanisms of GA inflammation self-resolving and the information of tophi. Dispelling turbid and removing stasis method which proposed by Professor Zhu Liangchun have certain curative effect to guide clinical application. The hypothesis is proposed：dispelling turbid and removing stasis method may contributes to the mechanisms of GA inflammation self-resolving by NETs and neutrophil exosomes. In this study, these works will be done as followed：（1）Both acute and chronic GA patients and healthy volunteers will be selected. All the selected person will be taken the blood sample to isolate neutrophils and neutrophil exosomes, and cellular and molecular biology techniques will be used to explore the biological characteristics and clinical relevance of NETs and neutrophil exosomes in GA.（2）Hyperuricemia and acute gouty arthritis rat model will be made with Wistar rats. The rats will be treated with dispelling turbid and removing stasis method, then the blood and synovium samples will be taken, cellular and molecular biology techniques will be used to explore the the effects and mechanisms of dispelling turbid and removing stasis method on GA by NETs and neutrophil exosomes. Purpose to explore the effects and mechanisms of dispelling turbid and removing stasis method on acute gouty arthritis based on NETs and neutrophil exosomes, and provide a basis for clinical diagnosis and treatment and enriche the modern connotation of “Zhuoyu Bi”.

尿酸盐结晶的沉积在痛风性关节炎（GA）的发生中有重要作用，其诱导中性粒细胞形成的中性粒细胞胞外诱捕网（NETs）和中性粒细胞源的外泌体（NE）可能参与GA的自我缓解机制和痛风石的形成；朱良春教授提出的泄浊化瘀法指导临床疗效确切。故提出假说：泄浊化瘀法可能通过NETs和NE参与急性痛风性关节炎的缓解机制。基于以上开展下列研究：（1）纳入GA急性期、间歇期患者及健康志愿者，外周血分离中性粒细胞及外泌体，应用细胞学、分子生物学等技术，探讨NETs和NE在GA中的生物学特征及与临床相关性；（2）Wistar大鼠建立高尿酸血症+急性痛风性关节炎动物模型，泄浊化瘀法干预后取血及滑膜组织，研究泄浊化瘀法通过NETs和NE对痛风性关节炎的干预作用，与临床研究相呼应。目的：以NETs及NE为核心，研究泄浊化瘀法对痛风性关节炎的作用和调控机制，为临床诊疗提供应用依据，丰富“浊瘀痹”的现代内涵。

本课题以参与痛风性关节炎（GA）自我缓解机制和痛风石形成的中性粒细胞胞外诱捕网（NETs）和中性粒细胞源的外泌体（NE）作为研究切入点，从整体、组织、细胞层面，研究泄浊化瘀法对GA的作用和调控机制。以GA急性期、间歇期患者及健康志愿者为研究对象，外周血分离中性粒细胞及外泌体，初步探索NETs和中性粒细胞源的外泌体（NE）在GA中的生物学特征及与临床相关性；并构建高尿酸血症+急性痛风性关节炎动物模型，泄浊化瘀法干预后取血及滑膜组织等，研究泄浊化瘀法通过NETs和NE对痛风性关节炎的干预作用。.研究发现，痛风急性期及间歇期患者中性粒细胞诱导后均可见NETs形态，dsDNA/NETs浓度显著升高，48h内NETs对THP-1细胞的活性有促进作用，趋化值下降；而对FLSs的活性有抑制作用，趋化值增强。通过蛋白定量、粒径分析、流式细胞检测及透射电镜等方法可验证各组外泌体生物学特征。痛风患者急性期和间歇期IL-10、TGF-β、IL-37水平降低，TNF-α、IL-1β水平升高。在体实验构建高尿酸血症+急性痛风性关节炎动物模型，并进行模型评价后分别给予大中小剂量痛风汤及秋水仙碱干预，痛风汤各组降低大鼠踝关节滑膜NLRP3、IL-1β、ASC、caspase-1表达及NETs的生成，降低血清中促炎因子TNF-α、IL-6、IL-1β水平，升高抗炎细胞因子TGF-β1、IL-10、IL-37表达，并与剂量呈正相关，优于秋水仙碱组。DAPI染色后观察各组NETs形态，痛风汤大、中剂量组及秋水仙碱组显著降低血清 dsDNA/NETs 的浓度，并通过蛋白定量、粒径分析、流式细胞术及透射电镜等方法观察各组外泌体的表达及形态。提示NETs和中性粒源的外泌体参与痛风性关节炎自我缓解机制，以痛风汤为代表的泄浊化瘀法可能通过NETs和中性粒源的外泌体参与急性痛风性关节炎的缓解机制。