青藤碱靶向胆碱能抗炎通路中α7nAChR治疗RA的机制研究

Sinomenine (SIN) is a major effective component of the Chinese materia medica Sinomenium acutum for treatment of rheumatoid arthritis (RA). Our team had elucidated some anti-inflammatory mechanism of SIN before, but the exact binding sites or proteins on target cells remain unclear. We found recently the anti-inflammation effects and the inhibition of activation of NF-κB of SIN were mediated by nicotinic acetylcholine receptor alpha 7 (α7nAChR). α7nAChR is the key receptor in cholinergic anti-inflammatory pathway (CAP), and is involved in cell activation and proliferation. So we forward hypothesis that SIN inhibition effects on inflammation and synovial hyperplasia depend on α7nAChR, which is the major target of SIN in treatment of RA. The anti-inflammation mechanism of SIN targeting α7nAChR will be revealed by analysis SIN effect on macrophages and α7-deficient mice. The correlation between α7nAChR and SIN effect on inflammation and proliferation of fibroblast-like synoviocytes cells will be detected in vitro. The therapeutic effects of SIN on RA rat model by acting on α7nAChR will be investigated. From the angle of neuro-immune to study the mechanism of sinomenine acting on α7nAChR of cholinergic anti-inflammatory pathway will provide theoretical basis on treatment of RA, and be helpful to explain Qu-Feng-Tong-Luo function of Sinomenium acutum. The study will also provide a new target and a new strategy to study treatment of RA.

青藤碱（SIN）是中药青风藤的主要有效成分，是治疗类风湿性关节炎（RA）的常用药。但其作用于靶细胞时确切结合或作用的靶分子尚未明确。我们最新研究发现SIN的抗炎作用和抑制NF-κB活化依赖于烟碱型乙酰胆碱受体α7（α7nAChR，简称α7）。α7是机体胆碱能抗炎通路（CAP）中的关键受体，并参与细胞活化、增殖。据此我们提出SIN通过作用于α7抑制炎症及滑膜组织的异常增生、α7是SIN治疗RA的关键靶点的假设。本项目应用体外巨噬细胞和α7基因敲除小鼠进一步明确SIN靶向α7的抗炎作用和胞内信号机制，体外实验观察SIN抑制成纤维样滑膜细胞的炎症、增殖与靶向α7的关系，采用RA动物模型观察SIN的治疗作用与靶向α7的关系。本研究从神经-免疫、CAP角度探讨SIN治疗RA的作用机制，为阐释SIN治疗RA、青风藤祛风通络功能的现代药理学内涵提供依据，也为靶向α7抗关节炎的新治疗策略提供实验依据。

本项目围绕青藤碱（SIN）靶向作用于胆碱能抗炎通路中烟碱型乙酰胆碱受体α7（α7nAChR），从巨噬细胞、滑膜成纤维细胞（FLS）、RA动物模型三个方面研究SIN靶向α7nAChR的抗炎及治疗RA的作用机制。研究表明：① SIN对体外LPS刺激的巨噬细胞炎症模型、对小鼠内毒素血症模型的抗炎作用，以及对佐剂性关节炎模型（AIA）大鼠的抗关节炎作用、对FLS的抗炎抗增殖作用能被α7nAChR siRNA及α7nAChR选择性拮抗剂阻断；② 活化的巨噬细胞、淋巴细胞、滑膜成纤维细胞及内毒素血症小鼠、AIA大鼠中α7nAChR表达异常升高，α7nAChR表达升高与关节炎发病进程、与关节炎指标之间呈正相关，SIN下调异常升高的α7nAChR，而对照药物小檗碱、甲氨蝶呤和吲哚美辛对α7nAChR表达无明显影响；③ SIN通过α7 nAChR/ERK/Egr-1通路抑制巨噬细胞炎症反应，并反馈下调巨噬细胞中异常高表达的α7；采用shRNA长效沉默巨噬细胞α7nAChR，发现M1极化表型分子表达下调，M2表型分子表达升高，表明高表达的α7 nAChR参与巨噬细胞M1极化，青藤碱下调α7 nAChR表达能抑制巨噬细胞M1极化；④ FLS体外实验中，siRNA沉默实验表明α7 nAChR表达参与FLS增殖，SIN通过抑制ERK/Egr-1通路下调α7 nAChR表达；⑤ 腺苷受体A2A表达水平与大鼠AIA模型发病进程呈负相关，SIN能上调肝脏、脾脏、滑膜中异常降低的A2A的表达； FLS增殖和炎症水平升高时，A2A表达降低，SIN通过作用于α7nAChR抑制FLS增殖、抑制炎症因子释放、上调A2A表达。.研究中探讨了α7nAChR的表达与炎症、滑膜增生、RA发展的关系，明确RA疾病进展中存在α7nAChR表达的异常升高，分析了青藤碱靶向作用于α7nAChR并调节α7nAChR表达水平在细胞增殖、活化中的作用机制，明确了SIN与其他抗关节炎药物不同的作用机制，研究结果为阐释青藤碱及青风藤治疗RA、青风藤祛风通络功能的现代药理学内涵提供理论依据，也为阐明α7nAChR作为治疗RA的重要靶点提供实验依据。目前共发表基金第一标注SCI收录文章3篇及国内核心期刊文章4篇，预期再发表3篇SCI文章，2篇国内核心期刊文章。