由α7nAChR介导的胆碱能抗炎通路对RA滑膜单核/巨噬细胞浸润的调控作用及机制研究

Our previous studies have shown that the cholinergic anti-inflammatory pathway(CAP) mediated by α7nAChR exerts a protective effect on rheumatoid arthritis(RA). Applicants recently perceived that nicotine is capable of reducing the cell number of CD11b-positive macrophages in the synovium and decreasing the serum level of MIP-1α and MCP-1. These discoveries indicate that CAP might participate in the regulation of macrophage infiltration 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 employed the whole-body imaging technique to monitor the macrophage migration of CIA mice by nicotine or vagotomy. And the polarization of migrated macrophages was tested by immunofluorescence histochemical double staining technique. Then it use CIA mice and peripheral blood monocyte of normal and RA patients, and explore the mechanism in-depth on how CAP regulate and control the macrophage infiltration from the following aspects, adhesion, chemotaxis and polarization of macrophages. The carried out study is expected to elaborating the protection of CAP on RA and to reveal its cellular molecular mechanism from another angle, thus providing experimental basis for further outline of the network diagram about how CAP regulates inflammatory and immune reaction in RA.

我们既往研究已证实由α7nAChR介导的胆碱能通路（CAP）对RA具有保护作用，申请者新近发现烟碱可减少胶原性关节炎（CIA）小鼠关节CD11b+细胞数量， 降低血清MIP-1、MCP-1水平。这提示CAP可能参与调控RA单核/巨噬细胞浸润。但CAP能否调控以及如何调控这一过程，目前尚未见报道。本研究首先通过活体荧光检测系统动态观察激活及阻断CAP对CIA小鼠单核巨噬细胞迁移轨迹的影响，进一步通过组织免疫荧光法检测迁移至关节的巨噬细胞的极化状态。随后以CIA为动物模型、以分离的正常人及RA患者外周血单核细胞为细胞模型，从影响单核巨噬细胞的粘附、趋化以及极化过程深入探讨胆碱能抗炎通路调控RA单核巨噬细胞浸润的机制。开展本项目研究，可望从另一个角度揭示胆碱能抗炎通路对RA关节炎症的抑制作用及其细胞分子机制，从而为进一步勾勒胆碱能抗炎通路调控RA炎症及免疫反应的网络图谱提供实验依据。

在国家自然科学基金项目资助基础上，针对胆碱能抗炎通路对类风湿关节炎滑膜单核巨噬细胞浸润的调控作用及机制进行了相关实验研究。分别阐述了两种胆碱能抗炎通路的激活剂烟碱及GTS-21对单核巨噬细胞浸润的影响及潜在的作用机制。共获得如下发现：（1）乙酰胆碱受体激动剂烟碱显著减轻 CIA小鼠关节炎症，这与烟碱可以减低滑膜组织CD11b阳性细胞的分布，降低血清中MIP-1及MCP-1水平有关；同时乙酰胆碱受体激动剂烟碱可减少滑膜组织趋化因子受体CCR2的表达以及滑膜组织粘附分子ICAM-1的表达。（2）IL-6可呈时间依赖性的促进CCR2及CD11b的表达。烟碱通过α7nACh受体抑制CCR2及CD11b的表达；烟碱可以抑制IL-6刺激所致巨噬细胞的伪足形成，抑制F-actin的表达；而MLA可逆转烟碱对巨噬细胞伪足形成的抑制作用。（3）α7nACh受体激动GTS-21可以抑制CIA小鼠的关节炎症，且GTS-21(8mg/kg)组在达峰前对关节炎症的抑制作用优于GTS-21(4mg/kg)组。活体成像技术显示GTS-21（8mg/kg）可以抑制外周血单核细胞向关节局部迁移。GTS-21可减低CIA小鼠滑膜组织F4/80巨噬细胞的分布，且GTS-21(8mg/kg)组优于4mg/kg组，这可能与其影响趋化因子CCL2及CCL5的表达有关。