基于Cyr61/CCN1在痛风炎性微环境中作用探讨清热利湿通络法调控机制

Based on the clinical experience of the XiaShi surgery treatment of gout by “Clearing heat and removing dampness and dredging collaterals method”, and our previous studies demonstrated “Clearing heat and removing dampness and dredging collaterals method”can inhibi neutrophil activation and chemotaxis to sites of inflammation、aggregation, and reduce the release of inflammatory mediators,combines the reserch progress of the relationship of Cyr61 and synovial inflammatory microenvironment, our research group hypothesized that:“Clearing heat and removing dampness and dredging collaterals method”antagonize gout’s key link has a very close relationship with Cyr61/CCN1 regulats inflammatory synovial microenvironment. This project uses the in vivo and in vitro experiments combining, respectively using sodium urate suspension induced rat model of gout、normal rats and their FLS for the study, using shRNA Lentiviral Particles and the expression method of Anti-Cyr61 antibody interference in Cyr61 to studied on the function of the Cyr61 in the gout inflammatory microenvironment and investigating “Clearing heat and removing dampness and dredging collaterals method” ’s cellular and molecular biological mechanisms as well as its internal regular pattern,to provide important theoretical basis and experimental basis for the Cyr61 protein as a new target for the treatment of gout by “Clearing heat and removing dampness and dredging collaterals method”.

基于夏氏外科“清热利湿通络法”治疗痛风的临床经验，在前期研究证实“清热利湿通络法”通过抑制中性粒细胞活化和向炎症部位趋化、聚集，同时减少炎症介质释放来拮抗痛风的基础上，结合Cyr61与免疫性疾病炎性微环境研究进展，课题组提出假说：“清热利湿通络法”拮抗痛风的关键环节与Cyr61/CCN1调控痛风炎性微环境密切相关。项目组采用在体和离体实验相结合，分别以尿酸钠悬浊液诱导大鼠痛风模型和正常大鼠及其FLS为研究对象，采用shRNA慢病毒颗粒、与Anti-Cyr61抗体共同孵育方法，明确Cyr61/CCN1作为一种新的炎症因子在痛风炎性微环境的形成及发病过程中的作用，探讨“清热利湿通络法”治疗痛风的细胞分子生物学机制及其内在规律，为将Cyr61蛋白作为“清热利湿通络法”治疗痛风的新靶点提供重要的理论依据和实验基础。

基于夏氏外科“清热利湿通络法”治疗痛风的临床经验，项目组前期研究已证实“清热利湿通络法”通过抑制中性粒细胞活化和向炎症部位趋化，同时减少炎症介质释放来拮抗急性痛风，近期研究发现Cyr61是多种自身免疫疾病炎性微环境的重要启动和维持者。在此基础上，我们进一步从Cyr61调控痛风炎性微环境探讨清热利湿通络法治疗急性痛风的细胞分子生物学机制。体内实验部分，我们运用MSU诱导痛风大鼠模型同时使用shRNA慢病毒注入大鼠病变关节腔造成Cyr61表达沉默，采用Realtime-PCR、Western blot、ELISA、免疫组化等技术观察该蛋白在尿酸钠大鼠痛风模型炎症过程中的功能，研究“清热利湿通络”中药的调控机制。体外实验部分，以大鼠成纤维样滑膜细胞（FLS）为研究对象，将FLS与Cyr61-shRNA共同孵育，阻断Cyr61/CCN1表达，以及使用中药含药血清干预，运用荧光显微镜、流式细胞仪观察细胞生物学行为变化，并运用Realtime-PCR、Western blot等技术，深入研究Cyr61/CCN1这一关键基因功能，阐释“清热利湿通络”中药对滑膜细胞生长及IL-1β、IL-6、TNF-α、PGE2的影响。实验结果表明，Cyr61蛋白作为一种重要的促炎症因子参与GA的炎症过程，能促进IL-1β、IL-6、TNF-α、PGE2的表达；清热利湿通络法可通过下调Cyr61蛋白表达水平抑制IL-1β、IL-6、TNF-α、PGE2的分泌，从而减轻局部炎症。本研究得出结论：Cyr61作为一种重要的促炎症因子参与GA的炎症过程，能促进IL-1β、IL-6、TNF-α、PGE2的分泌；清热利湿通络法可通过下调Cyr61水平抑制炎症因子的分泌，从而拮抗急性痛风炎症。该成果丰富和完善目前指导痛风临床诊疗的理论，为将Cyr61蛋白作为“清热利湿通络法”治疗痛风的新靶点提供理论依据和实验基础。