基于TLR/MyD88与Dectin-1/Syk信号通路及其介导的NLRP3炎症小体研究白头翁汤正丁醇提取物治疗外阴阴道念珠菌病（VVC）作用机制

The hyphae of Candida albicans (Ca) collaborate with TLRs/MyD88 and Dectin-1/Syk pathways to produce IL-1β and IL-18 by stimulating NLRP3 inflammasome of macrophage, playing an important role in VVC. The mechanism of BaiTouWeng decoction in the treatment of VVC is unclear. Our team found that butyl alcohol extract of BaiTouWeng decoction (BAEB) inhibited IL-1β and IL-18 produced by Ca hyphae stimulated macrophage and the critical proteins of the two pathways. Therefore, we speculate that VVC treatment by BAEB might be associated with the regulation of the critical proteins of the two pathways and/or NLRP3 inflammasome to inhibit the releases of IL-1β and IL-18. VVC mice and macrophage are used to investigate the effects of BAEB on two pathways and NLRP3 inflammasome. Two pathways are blocked separately to observe the effect on the other pathway. Further, protein agonists and plasmid transfection are used to upregulate the expression of critical protein for studying the mechanism of BAEB against VVC and potential target. The completion of this program will provide new ideas and experimental proofs for VVC treatment by BAEB.

白念珠菌（Ca）菌丝通过TLRs/MyD88与Dectin-1/Syk通路协同激活巨噬细胞NLRP3炎症小体产生IL-1β与IL-18，在外阴阴道念珠菌病（VVC）中发挥重要作用。白头翁汤临床上治疗VVC疗效显著但机制不详。课题组发现该复方正丁醇提取物（BAEB）能显著抑制VVC小鼠及Ca菌丝刺激下巨噬细胞产生IL-1β与IL-18，以及两条通路中关键蛋白。因此推测BAEB干预VVC可能是通过调节两条通路关键蛋白或/和NLRP3炎症小体表达进而抑制IL-1β与IL-18释放。故本课题以VVC小鼠和巨噬细胞为对象，研究BAEB含药血清对两条通路及NLRP3炎症小体调控作用，并分别阻断一条通路观察对另一条通路影响，进一步采用两条通路中重要蛋白激活剂及运用质粒转染技术上调关键蛋白表达，在分子、细胞及整体水平上研究BAEB抗VVC作用机制和效应靶标，为该药治疗VVC提供新的思路和实验依据。

外阴阴道念珠菌病（vulvovaginal candidiasis，VVC）是最常见的妇科真菌感染性疾病，目前治疗仍以唑类药为主，但耐药菌株分离率逐年升高。课题组研究发现，中药复方白头翁汤正丁醇提取物（BAEB）能通过调控宿主固有免疫重要组分NLRP3炎症小体而发挥其治疗VVC的作用，但NLRP3炎症小体受到多重因素的调控，因此，进一步从对NLRP3炎症小体上游调控的角度揭示BAEB治疗VVC作用机制具有重要意义。本项目从BAEB对VVC小鼠TLR/MyD88与Dectin-1 /Syk受体信号通路关键蛋白以及NLRP3炎症小体的调控作用、BAEB对白念珠菌丝刺激巨噬细胞TLR/MyD88与Dectin-1/Syk两条受体信号通路关键蛋白以及NLRP3 炎症小体的调控作用、阻断TLR/MyD88通路后BAEB主要组分对白念珠菌丝刺激巨噬细胞Dectin-1/Syk受体信号通路关键蛋白和NLRP3 炎症小体的调控作用；阻断Dectin-1/Syk通路后BAEB主要组分对白念珠菌丝刺激巨噬细胞TLR/MyD88受体信号通路关键蛋白和NLRP3 炎症小体的调控作用等方面研究BAEB对VVC的作用机制。本研究经体内外实验证实，BAEB通过调节TLR/ MyD88与Dectin-1/ Syk受体信号通路关键蛋白表达以抑制IL-1β与IL-18前体的产生及通过调节NLRP3炎症小体抑制caspase-1活化进而阻断IL-1β与IL-18的激活，发挥抗VVC作用。同时还发现，BAEB对VVC时损伤的阴道黏膜上皮具有修复作用，BAEB主要组分小檗碱能抑制白念珠菌对阴道上皮细胞的黏附以及抑制“无能”中性粒细胞向阴道腔的募集。BAEB这些作用的发现，不仅阐释了复方中药通过整体调控作用对相关疾病治疗的机制，同时也为最终将BAEB开发为从宿主免疫调控角度治疗VVC的药物奠定坚实基础。