基于TGF-β1/Smad信号通路探讨针刺调控巨噬细胞极化治疗类风湿性关节炎的抗炎机制研究

Macrophages play an important role in rheumatoid arthritis, and their functional polarization affects the course and outcome of the disease. Previous studies have confirmed that acupuncture could regulate the functional polarization of macrophages in target organs and up-regulate the expression of TGF-beta 1 in immune organs to exert anti-inflammatory effects. Based on the current research status of acupuncture regulating immunity and TGF-beta 1 mainly activating of Smad pathway, we supposed that acupuncture may promote differentiation of Treg cells in immune organs and secrete TGF-beta 1, and activate Smad pathway to regulate macrophage polarization so as to exert anti-inflammatory effects. In this study, using AIA rat model, treated with electro-acupuncture at bilateral Zusanli, we will access to the anti-inflammatory effect of electro-acupuncture. To clarify the effect of acupuncture on differentiation of Treg cells and macrophage polarization, flow cytometry, ELISA and quantitative PCR will be used to detect the contents of M1/M2 macrophages and Treg cells in lymph nodes and the protein and gene levels of related factors. And then via several missing/capture methods, namely TGF-β1 receptor blockage, the macrophage subsets, the anti-inflammatory effect of acupuncture and activation of Smad pathway will be observed, to determine whether TGF-beta 1/Smad signaling pathway mediates acupuncture effect and macrophage polarization, so as to potentially elucidate the mechanism of acupuncture regulating macrophage polarization and provide scientific basis for acupuncture anti-inflammatory effect.

巨噬细胞在RA中起着至关重要的作用，其功能极化影响疾病的进程和转归。前期研究证实，针刺可调控靶器官巨噬细胞功能极化并上调免疫器官TGF-β1表达以发挥抗炎效应。结合针刺调节机体免疫及TGF-β1激活Smad通路实现信号传递的研究现状，提出针刺促进免疫器官Treg细胞分化并分泌TGF-β1，激活Smad通路调控巨噬细胞极化从而发挥抗炎效应的科学假说。本课题将复制AIA大鼠模型，电针双侧足三里穴，建立稳定有效的针刺效应平台；采用流式细胞术、ELISA、定量PCR检测靶器官M1/M2型巨噬细胞和淋巴结Treg细胞含量及相关因子的蛋白和基因水平，明确针刺对Treg细胞分化、巨噬细胞极化的影响，进一步拮抗靶器官TGF-β1，观察其对巨噬细胞极化、Smad通路激活及针效的影响，以明确TGF-β1/Smad信号通路是否介导针效及巨噬细胞极化，以期阐明针刺调控巨噬细胞极化的机制，为针刺抗炎提供科学依据。

本研究通过复制佐剂性关节炎炎性痛大鼠和小鼠模型，以足肿胀和关节病理评分为效应指标，发现针刺双侧足三里穴可显著改善踝关节的炎性症状和病理损害，下调局部促炎因子的表达，表明针刺具有确切的抗炎效应。采用流式细胞术检测靶器官M1/M2型巨噬细胞及局部淋巴结Treg细胞的细胞水平，采用qRT-PCR、ELISA检测其相关因子的基因和蛋白水平，肯定针刺可下调靶器官局部M1型巨噬细胞及其相关因子的含量，从而有利于M2型巨噬细胞功能的发挥；针刺可诱导局部淋巴结Treg细胞分化并上调其相关因子TGF-β1的蛋白表达。进一步采用缺失/捕获技术调控TGF-β1的含量，予AIA模型小鼠足底注射1μmol/L、5μmol/L、20μmol/L不同剂量的TGF-βR1/ALK5拮抗剂SB431542，发现拮抗剂SB431542可加重AIA模型小鼠的足肿胀程度，采用流式细胞术、Western blot检测靶器官局部M1/M2型巨噬细胞及其相关因子、TGF-βRⅠ、TGF-βRⅡ及TGF-β1/Smad信号通路相关分子的蛋白表达，发现针刺可激活TGF-β1/Smad信号通路，上调Smad2、Smad3的磷酸化水平，低、中、高不同剂量的TGF-β1受体拮抗剂SB431542可反转针刺的抗炎效应，下调靶器官M2型巨噬细胞、Treg细胞的含量，下调p-Smad2、p-Smad3、Smad4、TGF-βRⅠ的蛋白水平，表明TGF-β1/Smad信号通路介导针刺抗炎效应及巨噬细胞极化。筛选膜联蛋白A2（annexin A2，ANXA2）并在小鼠单核巨噬细胞RAW264.7中对ANXA2进行沉默，以发现ANXA2在全基因组水平如何调控转录，为针刺抗炎及调控巨噬细胞极化提供可能的新靶标。