Act1与IL-17/IL-17R串话在口腔扁平苔藓发生发展中作用的研究

Oral lichen planus (OLP) is a T cell-mediated immune-related disease. The etiology and pathogenesis of OLP have not been well elucidated. Recently, IL-17R, the target of cytokine IL-17 secreted by Th17 subset, has been playing an important role in the pathogenesis of several epithelial immune-related diseases. However，role of IL-17R in etiology and pathogenesis of OLP have not been reported yet. Recently, Act1 was proved to specifically bind to IL-17R, and further mediate the consequent activation of several signaling pathways in several tumor cell lines. However, the significance of IL-17R signaling pathways in OLP is still unknown. According to the results of previous studies and recent advances of our research with this field: ①Act1 and IL-17R showed a significantly higher expression level in OLP local lesions; ②Act1 and IL-17R were expressed in several oral mucosa cell lines; and further investigation showed that human recombinant IL-17 can activate the Act1 phosphorylation, while interference with IL-17R (IL-17R-shRNA) attenuate the Act1 phosphorylation. Then, we made a hypothesis that Act1 might have participated in the pathogenesis of OLP by mediating IL-17R associated signaling pathways. Therefore, the following studies are designed to prove the hypothesis: ①Expression and distribution of IL-17R and Act1 with its related cytokines in the clinical samples of OLP and healthy controls local lesions; ②The crosstalk between Act1 and IL-17/IL-17R on the proliferation, apoptosis, migration, differentiation and inflammation of oral mucosa cell lines and the possible signaling pathways. ③A lentivirus vector-expressing human Act1 and lentivirus-mediated shRNA interference will be constructed to evaluate biologic effects. The result of this research will shed a new light on the mechanism of crosstalk between Act1 and IL-17/IL-17R in mediating signaling pathways of oral lichen planus, thus facilitate the development of new therapeutic strategies to treat OLP disease.

口腔扁平苔藓(OLP)是T细胞介导的慢性免疫相关性疾病。Act1、IL-17R介导的信号通路与黏膜免疫疾病相关性日益受到关注，但在OLP中的表达及作用不明。申请人前期研究发现：①OLP组织中IL-17、IL-17R较正常组织表达上调；②IL-17R表达于多种口腔角质细胞株，人重组IL-17能够激活胞内Act1磷酸化；沉默IL-17R表达后IL-17激活Act1磷酸化减弱。据前期成果，本课题拟研究：通过干扰口腔角质细胞IL-17R表达，考察Act1在介导IL-17R信号通路中的作用；过表达和沉默Act1基因，探索口腔角质细胞中Act1调控的相关炎性介质在IL-17/IL-17R信号通路持续激活中的反馈作用；了解Act1与IL-17/IL-17R在OLP角质形成细胞中的复杂交互作用机制（简称“串话”），阐明Act1与IL-17/IL-17R串话在OLP发生发展中的作用，并探讨其临床防治价值。

本课题按照原定计划开展了如下实验，顺利完成了预定目标。（1）研究发现了细胞因子IL-17家族(A、C、E、F各亚型)阳性细胞显著高表达于固有层。OLP外周血中存在高表达的IL-17+Th17细胞，主要类型为IL-17A+Th17，其作用要强于IL-17F+Th17，糜烂型OLP中IL-17+IL-9+ Th17细胞也显著升高。（2）研究发现了OLP组织中IL-17RA、IL-23、Act1 mRNA显著升高。对口腔角质细胞IL-17RA受体采用ShRNA干扰，可导致IL-17诱导的Act1磷酸化表达降低。干扰IL-17R表达后通过PCR芯片筛选下游目标炎性因子，得到高度相关因子TNF-、CCL20和CXCL8。干扰胞内Act1表达后，IL-17A对细胞分泌CCL20、CXCL8和TNF-诱导作用显著降低。体外分别干扰IL-17R、Act1、TRGF6相关信号通路分子，能显著降低IL-17A/F诱导IL-6、CXCL8分泌，其中TRAF6与Act1干扰对炎性介质分泌的影响作用更强。TRAF6作为Act1下游的重要信号分子，对于承接Act1介导的IL-17R信号通路传导发挥重要作用。上述研究结果提示TRAF6在OLP角质细胞的IL-17/Act1信号通路中可能扮演中枢作用，与OLP疾病发病机制密切相关。（3）发现IL-17A能够显著激活胞内信号通路NF-kB及ERK，同时IL-17F也能够发挥类似激活作用；而在干扰IL-17RA的表达后IL-17A、IL-17F对下游信号通路的激活作用显著减弱。IL-23能显著提高OLP中IL-17A+ Th17及IL-17F+ Th17细胞的比例。在OLP外周血淋巴细胞中，IL-23不能显著提高IL-17A+IL-17F+ Th17细胞分化的比例。提示IL-23差异性调控Th17细胞分化，能够更加促进其向IL-17A+Th17分化。综上所述，本课题主要发现IL-17+Th17、IL-17R、Act1在OLP中表达升高，TRAF6可通过IL-17R/Act1介导的NF-kB、MAPKs信号通路诱导炎性介质TNF-、IL-6、CXCL8、CCL20上调，IL-23、IL-6等细胞因子在诱导趋化Th17细胞正反馈扮演促进作用。TRAF6在OLP角质细胞IL-17/Act1信号通路中可能扮演重要中枢性作用，与OLP疾病发病机制密切相关。