针刺调控巨噬细胞表型极化以抗炎的神经免疫学机制研究

Inflammation plays an important role in the development of various diseases. Lines of clinical studies show that acupuncture displays definite anti-inflammatory effects in its indications, but the mechanism is still unclear. Our previous studies have confirmed that acupuncture could promote M2 macrophage polarization and up-regulate Treg cell (Tregs) population in surrounding lymph nodes. In view of acupuncture can modulate the activity of autonomic nerve system and differentiation of T cells, we supposed that acupuncture may increase the expression of Foxp3, promote the proliferation of Tregs via neuro-transmitters secreted from innervated autonomic nerve terminals, and Tregs promote M2 macrophage differentiation by secreting anti-inflammatory factors. In this study, using CFA mice models, treated with electro-acupuncture at bilateral Zusanli, we will access to the anti-inflammatory effect of electro-acupuncture. To investigate which T cell subsets and the dominating type of autonomic nerves are regulated by electro-acupuncture, T cell sub-populations, neurotransmitters including catecholamines and acetylcholine in the spleen, lymph nodes, blood and inflamed organs will be detected by qPCR, ELISA, flow cytometry and LC-MS method. And then via several missing/capture methods, namely Tregs-associated factor blockage, Foxp3 knock out mice and autonomic nerve blockage, the anti-inflammatory effect and the macrophage subsets will be observed, to clarify the neuro-immunological mechanism by which acupuncture modulate the polarization of macrophage. In this study, we aim to systematically disclose the potential mechanism of anti-inflammatory effects of acupuncture.

炎症在多种疾病发生发展中发挥重要作用，临床研究表明针刺在多种适应症中起到抗炎效应，但机制未明。申请者前期研究证实，针刺可调控巨噬细胞表型极化并上调淋巴结Treg细胞以抗炎。结合针刺可调控自主神经的既往研究，提出针刺通过自主神经上调Foxp3的表达，促进Treg细胞增殖，分泌抗炎因子促进巨噬细胞表型向M2型极化，发挥抗炎作用的科学假说。本课题将复制类风湿性关节炎小鼠模型，电针双侧足三里，采用qPCR、ELISA、流式细胞术及液质联用法检测脾脏、淋巴结、血液和靶器官的Treg等T细胞亚群和自主神经（交感和迷走神经）递质——儿茶酚胺和乙酰胆碱含量，以明确针刺影响T细胞和自主神经的类型和作用靶点。进一步通过Foxp3基因敲除、Treg相关因子阻断及自主神经调控，观察对针刺抗炎效应和巨噬细胞极化的影响，以阐明针刺调控巨噬细胞表型极化的神经免疫机制，为针刺抗炎的共同机制提供科学依据。

炎症是多种疾病发生、发展过程的共通环节，改善炎症性疾病及疾病的炎性状态是针刺疗法的重要适应病症之一，但机制有待于进一步阐明。申请者前期研究证实，针刺可调控巨噬细胞表型极化并上调淋巴结Treg细胞以抗炎。结合针刺可调控自主神经的既往研究，提出针刺通过自主神经上调Foxp3的表达，促进Treg细胞增殖，分泌抗炎因子促进巨噬细胞表型向M2型极化，发挥抗炎作用的科学假说。本课题采用针刺治疗类风湿性关节炎小鼠（AIA）模型，针刺双侧足三里穴，以行为学、关节形态学等为效应指标，明确了针刺足三里可抑制AIA小鼠关节和足爪部位的M1型巨噬细胞群，促进巨噬细胞由促炎的M1型优势向抗炎的M2型优势转化。同时运用高通量检测技术，发现针刺足三里可促进踝关节局部细胞因子及趋化因子恢复炎性平衡。运用RNA测序技术和数据分析，筛查出针刺改善关节炎症和促进组织修复的信号通路。在关节炎受累腘窝淋巴结中，发现针刺可下调Th1、Th17细胞比例，上调Th2细胞比例，促进Treg及Th2细胞优势，可调节多种T细胞亚群相关的转录因子及细胞因子的基因和蛋白水平，同时，针刺可抑制腘窝淋巴结副皮质区T细胞增殖。应用炎性淋巴结RNA测序和生物信息学分析，筛查出针刺调节免疫细胞极化及增殖的关键信号通路，并验证了关键分子的基因变化。运用选择性反应/多反应检测技术、免疫荧光染色等技术，发现针刺足三里可抑制关节炎模型腘窝淋巴结中升高的神经兴奋性、多种交感和感觉神经递质含量。交感神经化学阻断剂6羟基多巴胺和受体α1拮抗剂可模拟针刺调控淋巴结和炎性关节炎症，提示交感神经可能参与了针刺调节腘窝淋巴结CD4+T细胞、关节巨噬细胞的极化平衡。本课题系统阐明了“针刺——自主神经——T细胞极化/巨噬细胞极化——抗炎”的神经免疫学机制，整体把握，系统研究，阐明了针刺通过自主神经调控免疫细胞增殖、极化，以发挥抗炎作用的机制，可促进针刺治疗炎性疾病的应用和推广。