基于腹腔巨噬细胞微环境调控研究艾灸治疗溃疡性结肠炎的效应与作用机制

Moxibustion can improve some clinical symptoms and pathological changes in patients with ulcerative colitis. However, the undefined action mechanism has restricted its application and popularization. There is always a controversy on the action mechanism of moxibustion between the moxibustion heat and moxibustion smoke. Yet there have no articles reported about the regulation of the moxibustion heat and smoke on the peritoneal macrophages' microenvironment..Our research is based on the hypothesis that the irregulation of macrophages' microenvironment should be a crucial factor leading to the happening and development of ulcerative colitis, and it will be an effective way to analyze the action machanism of moxibustion by observing the influence of the heat and smoke produced on the peritoneal macrophages' microenvironment.By detecting the macrophages' microenvironment,and adopting pathological techniques and flow cytometry and microarray techniques to analyse the cytokines specially related with ulcerative colitis in peritoneal fluids and their probable network of signal transduction, we aimed to testify the relationship between the imbalance of macrophages' microenvironment and the pathogenesis of ulcerative coloitis, to prove that the regulation of macrophages' microenvironment is an important immunological mechanism of moxibustion in treating ulcerative colitis, and to make clear the differences between the heat and smoke in affecting the microenvironment of macrophages in peritoneum and their ineractions, for providing evidences for the study on the action mechanism of moxibustion.

艾灸能显著改善溃疡性结肠炎患者临床症状与结肠病理变化，但因起效机制不明限制了艾灸应用与有效推广。有关艾灸起效机制研究，素有灸热、灸烟之争，尚未有灸烟、灸热调控腹腔巨噬细胞微环境的相关文献报道。.本项目以"腹腔巨噬细胞微环境失调是溃疡性结肠炎发生发展的重要影响因素，观察灸热、灸烟对腹腔巨噬细胞微环境的影响是分析艾灸起效机制的有效途径"为工作假说。从腹腔巨噬细胞微环境（腹膜组织、巨噬细胞、相关细胞因子）角度，应用多种生物学研究技术，分析腹腔液中可能与溃疡性结肠炎高度相关的细胞因子及可能信号转导通路网络，证实腹膜巨噬细胞微环境失调与溃疡性结肠炎发病相关性，揭示腹膜巨噬细胞微环境调控是艾灸干预治疗溃疡性结肠炎的重要免疫学机制，明确艾灸起效两因素灸热、灸烟对腹腔巨噬细胞微环境的调控及其对溃疡性结肠炎病变改善的效应差异，明确灸热、灸烟两者是否存在交互效应，为艾灸起效机制研究提供一定科学依据。

背景 溃疡性结肠炎（Ulcerative colitis, UC）以结肠慢性非特异性炎症为主要病理变化。巨噬细胞（Macrophage, Mφ）异常激活和趋化是造成结肠炎性损伤的重要原因。以天枢、气海为主穴的艾灸对UC具有良好临床疗效，是目前公认临床治疗UC的替代疗法。关于艾灸作用机制尚无明确定论，热效应与艾烟是重要的两大影响因素。目的 观察艾灸对UC大鼠Mφ重要表型蛋白的表达及其活性的影响，证实艾灸对Mφ分化的调节效应；并基于对Mφ分化关键细胞因子的观察，研究艾灸调节Mφ分化的作用机制；分析艾灸两因素（温度和烟）在免疫调控效应中的交互作用，探索艾灸治疗UC的起效机制。方法 SD大鼠随机分为正常组、模型组、高温艾灸组、低温艾灸组、高温无烟灸组、低温无烟灸组、西药组，除正常组外，制备UC模型，艾灸组、无烟灸组大鼠双侧天枢穴分别给予艾灸和无烟灸治疗，治疗持续10分钟，每日1次，共治疗8次。高温组、低温组大鼠灸温分别控制在41~43℃和37~38.5℃范围。治疗结束后，观察组织结构变化，采用FCM法观察腹腔内Mφ功能表型及吞噬活性，运用PCR法检测腹腔Mφ中iNOs/ArgⅠmRNA的表达，采用ELISA法测定腹腔、肺中多种细胞因子含量，运用IHC法观察结肠中MIF蛋白的表达，采用WB技术检测肺组织中Mφ重要功能表型蛋白CD86、CD163的表达。结果 UC大鼠结肠存在炎症反应与损伤，大体评分和组织学评分升高，体重增加缓慢，腹腔内CD86+/CD163+ Mφ比例上调伴吞噬活性增强，Mφ内iNOs/ArgⅠmRNA比例上调（均P<0.05），该组大鼠腹腔冲洗液中IFN-γ、TNF-α含量增加，IL-4、IL-13含量降低（均P<0.05），该组大鼠结肠中MIF蛋白表达降低（均P<0.05）。与正常组比较，模型组大鼠肺组织中IFN-γ、TNF-α含量增加，IL-4、IL-13含量降低，CD86/CD163蛋白表达比例升高（均P<0.05）。与模型组比较，艾灸治疗组大鼠结肠损伤有所改善，表现为溃疡修复、炎症减轻，该组大鼠结肠大体及组织学评分降低（均P<0.05），艾灸能减少腹腔内TNF-α、IFN-γ含量和促进IL-4、IL-13表达（均P<0.05），降低大鼠腹腔内CD86+/CD163+Mφ比值，抑制吞噬能力，下调Mφ内iNOs/ArgⅠmRNA比例（均P<0.05），该