健脾益气祛痰法对肥胖鼠肠黏膜TLRs的良性调控机制研究

The intensive relationships of obesity and intestine mucosus immunology have been established, yet few works have been published on the regulation mechanisms of Chinese herbs. Based on our former studies, we proposal in the present paper to explore the regulation rules upon intestine TLR network by Jian-Pi-Yi-Qi-Qu-Tan, a typical principle of TCM against obesity. 90 obesity rats will be made by high-lipid and high-energy food, and be eaqually divided into three groups, e.g., acupuncture group, sham group and obesity model. Five points, i.e. Sanyinjiao, Zusanli, Tianshu, Zhongwan and Guanyuan, of the rats of acupuncture group will be electroacupuncture at every other day. After acupuncture for 1, 2 and 4 weeks,ten rats will be selected at the random rule and the following analysis will be executed: (1).obesity index such as weight,hormones,serum immunlogy and gut flora.(2).the distribution pattern of TLRs on different cells within the peryer's patch; (3).The gene expression levels of TLRs within the peryer's patch; (4). The expression status of IRF3 and MyD88, downstream moleculars of TLR pathways. The proposal will explore the dynamic pattern of intestine mucosusimmunology along with the progress of acupuncture therapy, construct a novel platform for the researches of anti-obesity by the approach of acupuncture, and set a theory of benefic regulation of mucosus immunology by acupuncture.

肥胖发生发展与肠黏膜免疫功能紊乱密切相关，但缺乏中医药调控机制的深入研究。在前期自然基金课题研究基础之上，本课题基于肠黏膜TLRs免疫调控分子网络，探索针刺健脾益气祛痰穴位对肥胖鼠肠黏膜免疫的调控机理。拟复制肥胖大鼠90只，随机平分至针刺组、假针刺组、模型组。针刺组隔日针刺三阴交、足三里、天枢、中脘、关元等穴位。针刺治疗第1、2、4周等时点，各组随机选取大鼠10只，检测肥胖体征、激素、血清免疫分子、肠道菌群构成、TLRs在肠壁细胞分布规律。从结肠中段随机采集10个派氏小体，定量PCR法测定TLR2等5个TLR基因表达水平、ELISA阵列法测定其蛋白含量。引入Native Page、ELISA阵列等敏感方法，测定TLRs下游信号通路效应分子IRF3、MyD88等含量与活性。本课题将揭示针刺减肥的肠黏膜免疫动态调控机理，搭建中医药肠黏膜免疫研究新平台，建立针刺良性调控肠黏膜免疫新学说.

本课题基于食源性脾胃虚弱型肥胖小鼠模型，研究高脂饮食肥胖造模及电针减肥进程对肠粘膜关键免疫分子网络---TLRs系统的动态调控规律。基于经典的高脂饮食法复制肥胖模型小鼠75只，采用健脾益气祛痰法隔日针刺天枢、关元、足三里、三阴交等穴位。于电针治疗第7、14、21、28天，运用免疫组化、ELISA、QRT-PCR等方法，观察了肥胖进程及电针减肥治疗过程肠中段派氏小体之中TLR1、TLR2、TLR3、TLR4、TLR7、TLR9蛋白分布密度的变化规律，以及相关mRNA的拷贝数变化特征，提出了健脾益气祛痰法电针对TLRs分子网络的动态调控模式；观察了肥胖进程及电针减肥治疗过程中，肠道粘膜及血清sIgA、LyM、β-D1、IL-2等代表性免疫分子的变化规律。采用16S rRNA测序与宏基因组等方法，平行检测了肥胖进程及电针减肥治疗过程中，肠道菌群组成、丰度、功能的变化模式。尤为重要的是，通过对肠道派氏小体转录组学分析，全面观察了电针对肥胖小鼠中枢与肠神经系统、肠道胶质细胞及其微环境、肠道（局部）粘膜免疫及（系统性）血清免疫、脑及肠道激素水平等维度的整体性调控模式，初步揭示了电针减肥的主要调控靶标与调控节点。本研究不仅搭建了针刺良性调控以TLRs分子网络为核心的肠黏膜免疫新学说，同时为进一步探讨针刺减肥的“脑-肠-菌”轴整体性调控模式，提供全新的科学依据。