胆碱能抗炎通路对类风湿关节炎树突状细胞分化和成熟的调控作用及其机制研究

Our previous studies have shown that the cholinergic anti-inflammatory pathway (CAP) exerts a protective effect on rheumatoid arthritis (RA). While the specific mechanism has not been fully elucidated, especially the immune regulation mechanism of CAP on of RA is still far from clear. Applicants recently perceived that nicotine is capable of reducing differentiated-marker expression and cytokine production from RA monocyte-derived dendritic cells (DCs). These discoveries indicate that CAP might participate in the regulation of DCs differentiation maturation of RA. However, there has no report on whether CAP can control this process and how to control it. In the first place, the study observes the distribution and function of DCs in CIA mice by cholinergic agonists or vagotomy. Then it induces DC differentiation and maturation from CIA bone marrow and RA monocytes ex vivo, and explores the mechanism in-depth on how CAP regulate and control the differentiation and maturation of RA DCs. The carried out study is expected to elaborating the immune regulation of CAP on RA and to reveal its cellular molecular mechanism from a new angle, thus providing theoretical basis for the immunotherapy of RA based on the CAP.

我们既往研究已证实胆碱能抗炎通路（CAP）对RA具有保护作用，但其具体机制尚未完全阐明，尤其是CAP对RA的免疫调控机制还远未明晰。申请者新近发现胆碱能激动剂可减少RA单核细胞来源树突状细胞（DCs）分化标志物表达及细胞因子产生，这提示CAP可能参与调控RA DCs分化及成熟。但CAP能否调控以及如何调控这一过程，目前尚未见报道。本课题在前期研究基础上，以胶原诱导性关节炎（CIA）小鼠为模型，从整体观察胆碱能激动剂及迷走神经切断术对DC分布及功能的影响；并进一步体外诱导CIA小鼠骨髓细胞及RA患者外周血单核细胞向DCs分化成熟，从细胞水平观察激活及阻断CAP对DC分化及成熟的影响，深入探讨CAP及其受体在DC分化及成熟中的作用及机制。开展本项目，可为发展基于CAP信号的免疫治疗提供新的理论依据。

胆碱能抗炎通路对胶原诱导的关节炎小鼠（CIA）起保护作用，但其免疫调节机制尚不明确。本项目旨在探究胆碱能抗炎通路对于CIA DCs分化成熟的影响。①整体观察GTS-21对CIA小鼠的影响：40只DBA/1小鼠随机分为5组，即对照组：假手术+每天腹腔注射PBS一次；模型组：假手术+每天腹腔注射PBS一次+CIA模型复制；迷走神经切断组：迷走神经切断术+每天腹腔注射PBS一次+CIA模型复制；GTS-21 4mg预处理组：假手术+每天腹腔注射GTS-21一次（4mg/kg）+CIA模型复制；GTS-21 8mg预处理组：假手术+每天腹腔注射GTS-21一次（8mg/kg）+CIA模型复制。于初次免疫的第21天起，每3天对小鼠四肢关节进行关节炎指数评分。于观察终点，行HE、TRAP染色评估关节组织病理学改变。流式细胞术检测小鼠脾脏中CD11c+DCs MHC-Ⅱ、CD80的表达。免疫组化检测小鼠滑膜中CD11c+DCs 表达情况。Luminex多因子检测技术检测小鼠血清中IL-6、TNFα、IL-10的表达情况。研究发现：GTS-21 4mg组、GTS-21 8mg组可显著减轻CIA小鼠的关节炎症,可降低脾脏CD11c+DCs CD80、MHC-II的表达，减少滑膜组织CD11c+DCs浸润，降低血清中 IL-6、TNFα水平。②观察GTS-21对小鼠骨髓来源DCs分化成熟的影响：分离C57小鼠骨髓细胞，予GM-CSF+IL-4体外诱导小鼠骨髓细胞向 DCs 分化，予 GTS-21 或和α7nAChR 拮抗剂MLA干预 DCs 分化过程:流式细胞术检测CD11c+DCs CD80、MHC-Ⅱ的表达。体外诱导小鼠骨髓细胞向DCs分化后予LPS诱导DCs成熟，并予GTS-21干预DCs成熟过程，流式细胞术检测CD11c+DCs CD80、MHC-Ⅱ的表达情况。研究发现：GTS-21 10umol/l可显著抑制骨髓细胞分化为未成熟DCs过程中CD11c+DCs CD80、MHC-II的表达。而MLA可逆转此过程。GTS-21对DCs成熟无明显抑制作用。本项目深入阐明胆碱能抗炎通路对RA DCs 的免疫调节作用，有望为RA的治疗提供新的靶点。