从心理应激探讨“肝主疏泄”的中枢神经生物学机制

【作者】 李艳；

【导师】 邓中炎；

【作者基本信息】 广州中医药大学 ， 中医基础理论， 2002， 博士

【摘要】 1．理论探讨 情志与心理应激在概念、发病条件以及临床范畴方面均有着极大的可沟通之处。从心理应激的角度来研究情志、情志疾病和脏腑功能失调的本质在理论上有着坚实的基础。情志疾病多与肝有关。中医多从肝入手治疗情志疾病，疗效显著。肝的脏腑功能失调可以表现为神经-内分泌功能紊乱。心理应激病理特点是以下丘脑—垂体—肾上腺皮质轴(HPAA)被激活为基本变化的一系列神经内分泌反应的过程。可见，肝脏功能失常与心理应激的内在联系是神经内分泌网络。对于情志因素(心理应激)引起的各种变化，肝是机体调节心理应激反应的核心。因此从心理应激的角度可能在更深层次上揭示中医肝主疏泄的生理机制以及肝失疏泄的病理机制。 本实验在参考国内外造模方法以及联系中医情志致病特点的基础上，采用反复限制制动的方法建立心理应激动物模型。制造模型的原则是尽量模仿人情绪异常发生过程，尽量排除躯体应激因素。我们根据中医“肝喜调达而恶抑郁”、“肝为刚脏，体阴而用阳”等理论以调肝为主要治法进行组方用药，根据其对应激大鼠调节作用机制，从而探讨“肝主疏泄”的神经生物学机制。 2．实验研究 2．1建立反复制动心理应激动物模型 本实验连续21d动态观察了反复制动心理应激大鼠血浆皮质醇(CORT)与β-内啡肽(β-EP)水平的变化。第15d血浆COPT和血浆β-EP水平均同时达到了最高值，初步确定15d是大鼠应激反应较为强烈的时期，神经内分泌反应较为明显。故本实验初步选择15d模型大鼠作为观察对象。 而后，观察了反复制动心理应激15d模型大鼠的一般行为学(旷场行为和鼠尾悬挂测定)、血清CORT、血浆ACTH以及其他生理常数(体重、脾脏指数、胸腺指数、垂体指数、肾上腺指数等)。结果发现本模型大鼠HPAA功能亢进，并出现抑郁样行为。生理常数符合应激改变。根据对造模大鼠的一般行为学观察和一些经典反应心理应激状态的指标，本实验初步确定了本造模方法的可行性，结合对模型大鼠血浆CORT和β-EP动态观察，根据中医病因病机理论，初步确认反复束缚制动心理应激模型适合于研究中医所讲述的“怒伤肝，肝失疏泄”的病理改变。 2．2肝主疏泄的中枢神经生物学机制初步研究 2．2．1各组应激大鼠血浆、垂体、下丘脑β-EP检测 方法采用放射性免疫分析。结果发现应激大鼠血浆β-EP水平明显升高，垂体 广州中医药大学99级博士生毕业论文 李艳和下丘脑 D上P明显降低。结论：①反复制动应激 ZW Wistar模型大鼠下丘脑和垂体pEP水平明显下降，血浆卜EP水平明显升高。②反复制动应激ZW模型Wistar大鼠中医病理变化机制之一可能是肝疏泄功能失常，调肝法的本质与上调下丘脑pEP有关。2．2．二各组大鼠下丘脑CRH、血浆p－EP、血清CORT测定 方法采用放射性免疫分析法。模型组大鼠下丘脑CRll、血浆B＊P和血清CORT水平与正常对照组相比均明显升高。调肝组下丘脑CRH和血清CORT水平与模型组相比明显下降。人参总皂贰组下丘脑＊KH和血清＊＊RT水平与模型组相比明显下降，其血浆pEP水平明显高于模型组。补肾组血浆p＊P明显低于模型组。结论：①反复制动 ZW Wistar心理应激大鼠 HPAA功能明显亢进。②肝主疏泄功能与调整HPAA的功能有关。2．2．3各组大鼠下丘脑及海马＊、ASp、GIS和Tall含量变化 方法采用OPA柱前衍生反相高效液相色谱法。结果发现：模型组海马G山和Tau水平明显下降。调肝组下丘脑GABA、Tau水平和下丘脑指数均明显下降。补肾组海马＊IU、ASp、＊＊＊A、Ta S、海马指数和下丘脑＊Aj IA均明显升高，补肾组下丘脑GABA＊平明显下降。健脾组下丘脑ASp、GABA矛Tall水平明显降低，健脾组海马m、ASP、GABA、Tall、海马EA／IA和海马指数均明显升高。人参总皂贰组下丘脑Gill、ASp、GABA和 Tau水平明显下降，人参总皂贰组海马0、ASp、GABA和 Tau海马指数水平均明显升高。结论：①反复制动应激ZW后 WISt盯模型大鼠下丘脑神经元可能具有抑制倾向，而海马神经元可能具有兴奋倾向。②调肝法本质可能在于下调下丘脑氨基酸的含量；健脾法本质可能与其降低下丘脑Asp、GABA、Tyr、Tau，升高海马各氨基酸水平，尤其是GABA有关；补肾法本质可能与其下调下丘脑＊＊＊A水平，上调海马各氨基酸水平（尤其是mU与ASp）有关。③疏泄法与调补法的内在机制在调节海马氨基酸水平方面是有差别的。2．2．4各组大鼠下丘脑酪氨酸羟化酶（TH）和酪氨酸（Tyr）的变化 前者方法采用免疫组织化学观察ABC法。后者方法采用OPA柱前衍生反相高效液相色谱法。模型组弓状核与下丘脑腹内侧核TH阳性细胞数量以及阳性纤维均明显增多。调肝方药对两核TH阳性细胞数明显增加。肾气丸对两核TH阳性细胞数量无明显的影响。四君子汤和人参总皂贰明显上调两核TH阳性细胞数量。 结论：①本实验反复制动心理应激大鼠下丘脑弓状核和下丘脑腹内侧核的TH细胞功能明显增强。②调肝、健脾和人参总皂贰能增强TH阳性细胞的功能，进而增强机体应对应激的能力。2．2．5各组大鼠血清及下丘更多还原

【Abstract】 1. Research on basic theory of emotional reactions in TCM andpsychological stress in Western MedicineThere are many similarities between emotional reactions and psychological stress considering the concepts, pathogens and the related diseases. There are reliable proofs to study the mechanisms of emotions and emotional diseases from the view of the psychological stress. Not only most of the emotional diseases are related to the Liver (Traditional Chinese Medicine, TCM), but also the methods adopted to treat them according to the theory about liver (TCM) showed the great efficiency. The pathological changes of liver’s irregulatory represent the neuroendocrinological disturbance. The traits of psychological stress are series of neuroendocrinological reactions characterized by the activation of hypothalamus-pitutary-adrenal gland axis (HPAA). Therefore, the common basis of the liver irregulatory and the psychological stress is the neuroendocrinological network. The liver plays very important role in regulating psychological stress reactions. It will be feasible to study deeply the modern physiological and pathological mechanisms of Ganzhushuxie (TCM) with the psychological stress theory.The model rats with repeated restraint psychological stress had been established on the base of characteristics of emotional diseases and the methods adopted by the researchers outside or inside. The rules to make the model according with the natural procedure which is similar to the way of emotional diseases will happen then try to avoid the stimulus of physical body factors. The formula of the herbs was made up according to the Liver theory (TCM). We made efforts to probe the neurobiological mechanism of Ganzhushuxie (TCM) by the effects of the formula on the model rats.2. The experimental study2.1 Establishing the model rats with repeated psychological stressThe consecutive dynamic observations of plasma CORT and 3 -EP changes, lasted for 21d , had been carried out. The results of them in the 15th day showed the highestlevel among all of the groups which suggested the intense reaction of psychological stress. That was the reason why the model rats in 15th day was chosen for the further research.Then, the behaviors, serum CORT, plasma ACTH and other physiologic constants (weight, indexes of spleen, thymus, pituitary, and adrenal glands) were observed. The results proved that the model rats characterized by the overreactions of HPAA and showed the signs of depression. The results provided us the further proofs of the feasibility of adopting the model rats of the 15" day to study the psychological stress with the changes of classical indexes. Meanwhile, analyzing all of the results mentioned above including the dynamic changes of plasma CORT and p -EP combined with TCM theory, we made a conclusion that the model is suitable for studying the mechanism of Ganzhushuxie (TCM) in the light of the psychological stress.2.2 The pilot study of nerve center neurobiological mechanism on the Ganzhushuxie (TCM)2.2.1. The detections of plasma, pituitaiy and hypothalamus 3 -EP in each group. Detecting Methods: Radio-immunity analysis.The content of plasma P -EP increased and those of pituitary and hypothalamus decreased significantly. Conclusions:The P-EP in the pituitary and hypothalamus decreased but that of the plasma increased significantly in model group. According to TCM, the pathological mechanism of model rats may be irregnlatory of Liver, and one of the effects of Regulating Liver Therapy was related to enhancing P-EPin hypothalamus.2.2.2. The detections of hypothalamus CRH, plasma P -EP and serum CORT in each group. Detecting Methods: Radio-immunity analysis.Compared with control group, the levels of hypothalamus CRH, plasma P -EP and serum CORT in model rats were all higher. But the levels of CRH in hypothalamus and serum in both Adjusting Liver Group and the Ginsenoside group were lower than those in model group. The plasma P -EP in the Ginsenoside group was higher than更多还原