主动吸烟激活Nrf2信号通路调控NLRP3炎症小体对口腔黏膜局部免疫的影响及其机制研究

Active smoking plays an important role in occurrence of oral leukoplakia, while the mechanism is still unknown. In the previous studies, our results indicated that active smoking could promote the infection of Candida albicans in oral mucosa. These results suggested active smoking could play a substaintial part in the pathogenesis of oral leukoplakia by resulting in a direct damage to oral mucosal epithelial cells, suppressing local immunity of oral mucosa and promoting the infection of pathogenic microbe, such as Candida. Recent studies indicated that active smoking could activate Nrf2 signal pathway and NLRP3 inflammasome could play a crucial role in the sense and defense of the host to many kinds of pathogenic microorganisms, including Candida. Therefore, we presume that synergism of active smoking and Candida infection could be an important new mechanism of the occurrence of oral leukoplakia by which active smoking could negatively regulate NLRP3 inflammasome by activating Nrf2 signal pathway, inhibit the production of IL-1βand IL-18 and aggravate the invasion of pathogenic microorganism into oral mucosa, such as Candida. We plan to set up an animal model of active smoking and an in vitro cell culture model exposed to cigarette smoke in combination with the investigations on clinical specimens. By using RNA interference, colocalized flourescence, co-immunoprecipitation and other experimental methods, we will confirm our hypothesis in the ways of clinical individuals, animal model, cellular localization and molecular mechanism. We will explore whether NOD1 agonist can reverse cigarette smoke-induced suppression of local immunity in oral mucosa. This study provides some new ideas and new target to improve the prevention, treatment and prognosis of oral leukoplakia and oral candidiasis.

主动吸烟在口腔白斑的发病过程中起重要作用，但机制尚不详。本课题组前期结果表明，主动吸烟可促进口腔黏膜白色念珠菌的感染，提示主动吸烟除对口腔黏膜上皮细胞造成直接损伤外，还可削弱口腔黏膜局部免疫，促进微生物侵袭而参与口腔白斑的发生发展过程。最近研究表明，主动吸烟可激活Nrf2信号通路，而NLRP3炎症小体在宿主对多种微生物的识别和防御中起关键作用。因此，我们推测“主动吸烟激活Nrf2通路可负向调控NLRP3炎症小体，进而抑制其下游IL-1β和IL-18等防御分子的产生，导致念珠菌等微生物对上皮组织的侵袭加剧”是主动吸烟与念珠菌感染协同损伤口腔黏膜上皮细胞、诱发口腔白斑的重要新机制。我们拟构建主动吸烟动物模型和体外吸烟细胞模型，结合临床标本研究，应用基因敲除、免疫共沉淀等技术，从临床个体-动物模型-细胞定位-分子机制多层次证明我们的假说。此研究将为口腔白斑、口腔念珠菌病的防治提供新思路和新靶点。

香烟烟雾暴露和白色念珠菌感染是口腔白斑病（oral leukoplakia, OLK）的流行病学风险因子，也是其癌变的重要危险因素。NLRP3是模式识别受体的重要成员，能够形成炎症小体复合物，在固有免疫应答的调控中起关键作用，其下游的IL-1β、IL-18具有抗真菌活性，参与机体对白色念珠菌的防御反应。而香烟烟雾中含有大量的氧化剂和亲电子物质，易造成上皮的氧化异常，导致口腔黏膜损伤。Nrf2参与的抗氧化反应被认为是维持细胞内氧化还原稳态的重要途径之一，有效对抗外源性毒性物质的损伤。近年来，大量研究已证实吸烟可导致Nrf2信号通路的激活。本研究通过构建主动吸烟动物模型、体外吸烟细胞培养模型，采用免疫荧光、免疫组化、荧光定量PCR、Western blot和ELISA等研究方法，发现白色念珠菌引起 NLRP3炎症小体激活和促进细胞因子的产生；香烟烟雾削弱了口腔黏膜上皮细胞的免疫防御能力，促进白色念珠菌芽管和菌落形成；NLRP3炎症小体参与机体抵御白色念珠菌的过程；吸烟抑制NLRP3的激活和促炎因子的分泌，从而抑制口腔黏膜抗真菌免疫；Nrf2信号通路参与吸烟引起的口腔黏膜氧化损伤的过程；通过慢病毒干扰，证实Nrf2具有抗炎活性，可抑制NLRP3炎症小体的活化，减少IL-1β的分泌，从而表明Nrf2对NLRP3的负向调控作用参与吸烟影响口腔黏膜抗感染免疫。通过免疫组化和免疫印迹法检测非吸烟与吸烟组的正常口腔黏膜、口腔白斑病组织，发现吸烟抑制正常口腔黏膜上皮细胞内NLRP3、IL-1β、IL-18的表达，而在白斑病组织中增强Nrf2 的表达。本研究提出了吸烟和白色念珠菌协同作用损伤口腔黏膜防御，诱发口腔白斑病的新机制，为提高口腔白斑病等黏膜病的防治效果、阻断病损发展、改善预后提供了一定的实验基础和理论支持。