从TLR4炎性信号通路调控失衡研究肝郁化火焦虑症的生物学机制以及丹栀逍遥散干预的整合机理

Anxiety Disorder has become a pronounced public heath problem for seriously affecting people's physical and mental heath. PFSL is a main pathogenesis and common syndrome of Anxiety Disorder. Persistent inflammatory reaction is one of the ways how Anxiety Disorder causes psychosomatic diseases, while the TLR4 signaling pathway is a key inflammatory circuit. In this program which was based on the preclinical evidence that inflammatory reaction exists in the internal body of the patient with Anxiety Disorder resulting from PFSL, the patient was tested for expression levels of the TLR4 signaling pathway-related factors in the blood and the regular patterns of the pathway before and after Danzhi Xiaoyao Powder intervention. Meanwhile, the psychological stress rat model was dynamically tested for the changing patterns of the TLR4 signaling pathway-related factors and the related indicators of the neuroendocrine in the serum and brain tissue, so as to investigate how the activation of TLR4 signaling pathway was related to the neuroendocrine unbalance and how the Traditional Chinese medicines intervened.The effects of the neurotransmitters and hormones upon the TLR4 signaling pathway of neurogliocyte and neurons were studied in vitro to reveal how the TLR4 signaling pathway was activated. It was expected that the study at three levels-experiments on human body and animal and cell culture would show that the functional imbalance of TLR4 signaling pathway was the biological mechanism of Anxiety Disorder resulting from PFSL, how Danzhi Xiaoyao Powder intervened , and what exactly happened to Anxiety Disorder resulting from PFSL, thus to provide clinic prevention and treatment with rationales and effective prescriptions.

焦虑症已是当今社会突出的公共卫生问题。肝郁化火是焦虑症的主要病机和常见证型。炎性反应持续存在是焦虑症导致心身疾病的机制之一，TLR4信号通路是重要的炎性通路。本项目在前期临床证实肝郁化火焦虑症患者体内呈现出炎性反应的基础上，以肝郁化火焦虑症患者为对象，检测血TLR4信号通路相关因子表达及丹栀逍遥散干预前后规律。同时通过心理应激大鼠模型为对象，动态检测血清和脑组织TLR4信号通路相关因子及神经内分泌相关指标变化规律，探讨TLR4信号通路激活与神经内分泌失衡的相关性及中药干预机制。在体外研究神经递质、激素对神经胶质细胞及神经元细胞TLR4信号通路的影响，揭示TLR4通路激活机制。通过人体、动物实验和细胞培养三个层面的研究期待证明：TLR4信号通路调控失衡是肝郁化火焦虑症的生物学机制，诠释肝郁化火焦虑症病因病机的科学内涵，阐明丹栀逍遥散干预效应作用环节和机制，为临床防治提供理论依据和有效方药。

焦虑症已成为突出的公共卫生问题。肝郁化火是焦虑症的主要病机和常见证型。炎性反应持续存在是焦虑症导致心身疾病的机制之一。前期临床揭示肝郁化火焦虑症患者体内呈现炎性反应，而TLR4信号通路是重要的炎性通路，该通路调控失衡是否为肝郁化火焦虑症的生物学机制及丹栀逍遥散效应机制仍有待阐明。. 本项目以广泛性焦虑症肝郁化火证患者为对象，与肝气郁结患者、正常人对比，血清TNF-α、IL-1、IL-6、IL-10升高，单核细胞TAK1和JNK蛋白及TLR4mRNA表达增加，表明肝郁化火焦虑症患者TLR4信号通路激活；丹栀逍遥散治疗4周后中医证候和HAMA积分及皮质醇降低，抗焦虑和调节炎症。. 采用单笼饲养和不确定性空瓶饮水应激，设立正常组、模型组、地西泮组和丹栀逍遥散低/中/高剂量组,建模同步用药，14天后与正常组比较，行为学提示模型组开放臂中运动距离和停留时间百分比、中央区停留时间、进入明箱次数及停留时间百分比均减少，表明成功构建焦虑大鼠模型；杏仁核TLR4-IRAK-TAK1-p38-AP1-p65升高，血清和杏仁核TNF-α、IL-1β、IL-6、MIP-1α均升高，IL-10降低，炎症因子mRNA表达结果类似，TLR4信号通路被激活；血清NE、5-HT和CORT和杏仁核Glu均升高，血清和杏仁核中GABA降低，其中CORT、Glu、GABA造模第7天就出现差异，14天更明显，即神经内分泌失衡早于TLR4信号通路激活。丹栀逍遥散通过TLR4-IRAK-TAK1-p38-AP1-p65改善焦虑，高剂量与地西泮相当，某些方面优于后者。. 用皮质酮、肾上腺素等分别孵育神经细胞（PC12）及小胶质细胞（BV-2）2-24h后检测TLR4表达水平。CORT对两种细胞、Glu对BV-2细胞有作用，12h达到峰值；5-HT对PC12细胞有作用，8h和16h存在双峰。皮质酮孵育PC12细胞，设立空白组、空白含药血清组、模型组、地西泮组、丹栀逍遥散低/中/高剂量组，与空白含药血清组比较，模型组TLR4-IRAK-TAK1-P65-P38-AP1升高,表明TLR4信号通路被激活；与模型组比较，高剂量组TLR4-IRAK-TAK1-P65下降，P38、JNK和AP1无变化，通过调节TLR4-IRAK-TAK1-P65-NF-kB通路起改善作用；而地西泮无作用。