“内湿致痹”发病观指导下痛风宁方干预高尿酸血症防治痛风的机制研究

The high level of serum uric acid and urate deposition caused tissue damage and inflammation are the key link in the pathogenesis of gout. Research has confirmed that urate transporter in tubules of the kidney play an important role in reabsorption and secretion of uric acid, which regulate the renal excretion of uric acid and play a decisive role in the onset of hyperuricemia. Meanwhile, series of studies find that NALP3 inflammasome activation is a core of acute gout attacks. Thus, the research of drug for gout prevention targeted at urate transporter and NALP3 inflammasome has become a hotspot in recent years. TongFengNing is an effective prescription summarized from the pathogenesis of'Bi syndrome caused by internal dampness accumulation' in gout, which holds the function of relieving pain and detumescence, expelling turbid and eliminating dampness. Preliminary series of studies have confirmed its exact effect of improving acute inflammation and reducing serum uric acid level in gout. Hypothesis of this project is that TongFengNing Decoction works by intervening urate transporter and NALP3 inflammasome. Our workgroup designed a multi-level, multi-perspective research scheme，which targeted to clarify the effect of TongFengNing Decoction on intervention urate transporter and NALP3 inflammasome in protein and gene expression level, to further clarify the mechanism of TongFengNing Decoction treatment of gout, to determine experimental relationships between the time-effect and does-effect,to intensify 'Bi syndrome caused by internal dampness accumulation' recognition of the pathogenesis of gout in this program and to lay the foundation for clinical application.

血尿酸升高及尿酸盐沉积引起的组织炎症损伤是痛风发病的关键环节。研究已明确尿酸盐转运体在肾小管重吸收和分泌尿酸过程中至关重要，能调节尿酸在肾脏的排泄量，对高尿酸血症的发生起决定性作用；同时，研究发现NALP3炎性体活化是急性痛风发作的核心节点。故近年来以尿酸盐转运体与NALP3炎性体为靶标的痛风防治药物研究成为热点。痛风宁方是针对痛风"内湿致痹"发病观总结的效验方，有利湿排浊、消肿止痛功效，前期系列研究证实其降尿酸及改善痛风急性炎症的疗效确切。本项目的假说：对尿酸盐转运体及NALP3炎性体的干预是痛风宁方利湿排浊、消肿止痛功效的主要发挥途径。为验证假说，本项目设计了多角度、多层面的研究方案，旨在从蛋白及基因表达等多途径阐明痛风宁方对尿酸盐转运体及NALP3炎性体的干预效应，进一步明确该方降尿酸防治痛风的机制，并确定其量效与时效关系，以深化痛风"内湿致痹"发病观的认识，为临床推广应用奠定基础。

本研究采用分子生物学相关技术与方法，分别从蛋白及基因表达的层面阐明了痛风宁对尿酸盐转运体及NALP3炎性体的干预效应，进一步明确该方降尿酸防治痛风的机制，并确定其量效与时效关系，为深化痛风“内湿致痹”发病观的认识及该方的临床推广应用奠定了扎实的理论和实验基础。. 在痛风宁对尿酸盐转运体的干预效应实验中，首先在大鼠整体层面，通过两种方法分别建立尿酸生成增多及尿酸排泄障碍的高尿酸血症大鼠模型，再采用不同剂量的痛风宁药液进行干预，在不同时间点采集大鼠标本并检测相关指标；其次在细胞层面，通过尿酸分别刺激小肠上皮细胞（FHs）及肾近曲小管上皮细胞（HK-2）建立细胞模型，再采用不同剂量的痛风宁含药血清进行干预，在不同时间点收集细胞及上清液并检测相关指标。结果提示：痛风宁干预一定时间后能显著降低高尿酸血症模型大鼠的血尿酸水平，并一定程度改善大鼠损伤的肾功能；可抑制XOD活性及表达而减少尿酸生成；可调控肾脏组织及HK-2尿酸盐转运体表达及促进小肠组织及FHs ABCG2表达而促进尿酸排泄，降低血尿酸。. 在痛风宁对NALP3炎性体的干预效应实验中，首先在大鼠整体层面，通过关节腔局部注射尿酸盐混悬液建立急性痛风性关节炎（AGA）大鼠模型，再采用不同剂量的痛风宁药液进行干预，在不同时间点采集大鼠标本并检测相关指标；其次在细胞层面，通过尿酸盐刺激THP-1建立细胞炎症模型，采用不同剂量的痛风宁含药血清进行干预，在不同时间点收集THP-1细胞及上清液进行相关指标检测。结果提示：痛风宁可明显改善AGA模型大鼠关节皮温、肿胀度、步态，对AGA有明显的消肿止痛效果，中、高剂量消肿止痛效果最佳，在48 h时痛风宁的作用最明显；痛风宁含药血清可抑制THP-1中 NALP3炎性体相关蛋白及mRNA表达，减少IL-1β、PGE2产生，以中剂量痛风宁含药血清干预24h的效果相对最好。