调肝治法方药抗心理应激损伤的部分神经免疫学机制研究
|
摘要
一、文献综述与理论探讨
心理应激是机体通过认识、评价而察觉到应激原的威胁时引起的心理、生理机能改变的过程。随着社会发展和各方面竞争压力的增加，心理应激已成为一个不可忽视的因素威胁着人类健康。明确作为“观念性”的心理社会刺激以及由此产生的心理活动是如何转变为“物质的”生理反应的机制是研究应激机制与应对方式的关键内容。
从病理生理学的角度来看，应激是指机体在受到各种强烈因素刺激时所出现的以交感神经兴奋和垂体-肾上腺皮质分泌为主的一系列神经内分泌反应，进而引起各种机能和代谢的进一步改变。机体应激系统可分为位于中枢的神经指挥系统和外周的效应器官与功能，前者以下丘脑、海马部位为主，后者则是下丘脑-垂体-肾上腺皮质(HPA)轴、传出交感／肾上腺體质系统和副交感系统两大系统的作用下完成。其中，HPA轴是应激作用的主要途径，在心理应激反应中起关键性作用。而不同的性别与年龄直接影响着心理应激反应的方式与强度。
免疫系统是HPA轴外周作用的主要靶点，研究应激及其应付方式对免疫功能的影响是心理神经免疫学研究的热点之一。在多种应激条件下人体免疫功能发生变化，主要以免疫功能抑制为主。T淋巴细胞是机体免疫系统内功能最重要的免疫细胞，正常情况下，各T淋巴细胞亚群相互作用，维持着机体正常的免疫功能，当不同淋巴细胞亚群的数量和功能发生异常时，就可导致机体细胞免疫功能紊乱。研究表明，HPA轴是应激过程中免疫应答调节的主要途径，除糖皮质激素外，CRH和ACTH也均参与免疫调节。而在中枢，HPA轴应激反应的高位调节中枢是海马。大量研究表明，正常海马可抑制HPA轴的活性，参与了应激过程HPA轴的抑制性调节，促进应激状态下亢进的HPA轴恢复到基础水平，同时，海马不仅是HPA轴应激反应的高位调节中枢，而且是应激损伤的敏感区。应激性海马损伤与HPA轴功能亢进是神经内分泌系统应激反应的恶性循环结果。
中医学虽没有“心理应激”这一概念，但中医脏象及七情学说很早就认识到不良的环境或精神刺激与躯体疾病的发生发展有着密切的关系。应激是机体对意外刺激做出的非特异性全身反应，涉及各组织器官，因而中医药抗应激的研究也需涉及多个脏腑。目前中药复方抗心理应激损伤的研究多以情志立论，从肝、肾、心、脾入手，检测指标多为抗应激能力行为学指标和神经—内分泌—免疫系统等指标为主。但从理论基础、临床应用、实验研究三方面来看，情志调节核心在于五脏，而肝脏在其中起着主要作用。肝郁证候存在心理应激的障碍，病理机制涉及神经内分泌免疫等调节网络，但现代分子生物学的研究还不能充分地解释中医整体水平上的肝郁证候的理论。从心理应激反应来研究肝脏象的中医证候是近几年研究的热点。
本研究团队以往一系列研究亦表明：中医肝主疏泄调畅情志的功能在实现对下丘脑-垂体-肾上腺皮质系统的整体性调节上，还可能存在着一定的具体的中枢神经机制；在作用机理上体现出局部(中枢神经机制)与整体(NIM网络)的有机统一。并且以加味四逆散(JWSNS)为调肝治法方药之代表方，采用“方-证-效-脏腑功能本质”的研究思路，对慢性应激损伤及调肝方药的作用机制进行了一系列的研究，探讨肝主疏泄调畅情志功能的中枢神经生物学机制，其具体机制有待一步探讨。
心理应激动物模型的建立是开展心理应激机制研究的基础，但某些重要环节的偏差与设计仍影响着中医药抗心理应激损伤机理的阐明。例如，心理应激模型中常用的应激源包括束缚和制动、条件反射性电击足等或多或少均含有一定的躯体性应激成份，某种程度上影响着单纯心理应激作用机制的阐明。Willner等受CUS模型启发于1987年提出了“慢性轻度不可预知的应激模型”主要是将动物长时间的暴露在多种不可预知的应激源下，这些应激源包括不定期的禁水、禁食、震动、电击、游泳等，由此引起动物对奖励刺激的缺乏，是抑郁的重要表现，更真实的模拟了人们在现实生活中遭遇的“困难”，可以认为此应激模型是一种有效的抑郁性应激模型。
鉴于HPA轴及其海马中枢调控在NIM网络调节机制中的重要作用，有必要进一步探讨与阐明HPA轴及其海马中枢调控作用在肝主疏泄调畅情志功能的具体机制。在动物模型不断改进的基础上，不断接近肝主疏泄调畅情志的HPA调控作用机制的实质。在上述理论基础支持下，本论文旨在进一步研究不同性别、年龄情况下慢性心理应激反应损伤的HPA轴等神经内分泌免疫学作用机制，以及中药复方对其的干预作用，为最终从整体上阐明心理应激作用机制及中医调肝治法方药抗心理应激损伤的作用机理奠定基础。
二、研究目标与内容
研究目标：开展调肝方药——加味四逆散(JWSNS)抗心理应激损伤HPA轴作用方式及其神经内分泌免疫变化的实验研究，尝试阐明调肝治法抗不良心理应激损伤的神经内分泌作用机制，探求安全有效的中医药抗心理应激方法。进而，从细胞及分子水平进一步探讨调肝方药一加味四逆散(JWSNS)在心理应激损伤状态下多层次、多靶点调控NIM网络的作用机制，更加深入揭示加味四逆散抗心理应激损伤的神经免疫学的物质基础及其作用方式。
研究内容：建立慢性轻度不可预计应激损伤大鼠模型，检测各试验组分中免疫功能的变化，及其神经内分泌调控过程中外周与中枢各类激素等主要生化指标与病理改变，观察调肝方药JWSNS的调控作用，以HPA轴为重点，探讨其作用可能的机制与途径，研讨调畅情志疏肝之法在抗心理应激损伤中的作用机理。
药物组成与立法：JWSNS组成：柴胡5g、白芍15g、枳壳6g、枸杞子15g、山栀5g、干地黄18g、石决明30g。药材由本校第一附属医院药房提供，经药剂科鉴定均为纯正药材。制备后置4℃冰箱内保存备用。各治疗组分别于应激造模前1h给予加味四逆散2ml灌胃。
动物模型制作与分组：经慢性轻度不可预计应激动物模型改制而成，去除以往采用模型中电击应激源的设置，减轻应激源的造模作用强度，以减少躯体因素的因素。接受应激处理的组别大鼠单独放在一间独立的房间，在21天接受不同应激源的刺激，应激源包括有限制空间1h，45度斜笼7h、湿笼17h(湿笼是200g铺料倒100ml水，使垫料湿透)、新入侵者(两笼并笼)23h、持续光照36h、空瓶1h、禁食禁水23h。
(一)慢性轻度不可预计应激损伤大鼠模型的建立
本实验目的在于建立慢性轻度不可预计应激模型，观察不同年龄、性别大鼠模型的特征。用表面效度、结构效度和预测效度三个效度对其进行综合评价。大鼠基本状况观察结果表明，随着造模工作的进行，模型组大鼠整体状况不断明显下降，焦虑，烦躁不安，排便增多，部分过程拒绝，呼吸急促，毛色发黯，发黄，存在修饰行为，但不断减少，直到基本消失。治疗组整体状态强于模型组，但略弱于正常组，表现为较安静，毛色虽杂乱，但略有光泽，修饰行为减少，抵抗行为减少，表明经中药复方JWSNS组干预，基本状况有好转，但在应激源未解除之前，仍较疲倦。糖水消耗偏爱度是判断大鼠抑郁应激程度的重要指标。数据表明，模型大鼠糖水消耗偏爱度减少，提示模型成功，而中药复方JWSNS干预，糖水消耗偏爱度显著提高，与正常组无显著差异，提示中药复方JWSNS可能干预慢性心理应激损伤，此结果与以往一致。其中，幼年鼠正常组因处于生长期，糖水消耗偏爱度随体重呈增长趋势，但模型组三周后糖水消耗偏爱度显著减少(p＜0.01)，表明慢性应激对其产生较为严重的损伤，导致其不升反降；成年鼠正常组糖水消耗偏爱度未显示随体重而增长趋势，模型组三周后糖水消耗显著减少(p＜0.01)，经中药治疗有显著改善(p＜0.01)，表现出一定的稳定性。体重方面，各组正常大鼠体重增长平稳，雄性组略快，而经造模后体重增长显著变缓(p＜0.01)，提示长时间应激导致机体的综合状况显著下降，经治疗后有改善，但与正常组相比仍有显著差异(p＜0.01)，无法复原，表明中药复方可以有效缓解其应激状态，但无法改变其只要应激源持续存在就必然所造成的损伤与压力。
性别是影响应激作用的重要因素。结果表明，就心理应激而言，雌性和雄性动物之间存在显著的性别差异，两种动物在行为表现、内分泌和病理生理等多个方面也存在显著差别。雌雄间比较，雄性对糖水消耗偏爱度更为稳定一些，总体趋势呈下降；雌性大鼠则在第一周即出现显著下降，随后又有所恢复，进而第三周又显著下降，提示雌性可能对急性应激更为强烈，进而转为慢性应激状态。
(二)加味四逆散对慢性心理应激大鼠下丘脑—垂体—靶腺轴的调节
本实验旨在观察慢性心理应激状态下大鼠肾上腺、甲状腺、性腺轴的外周分泌激素的改变及其主要作用途径。本研究结果显示，成年组雄激素有显著改变，雌性激素有改变趋势，而T3、T4含量无明显改变。表现为，雄性造模组雄激素显著升高(p＜0.01)，经JWSNS治疗后下降与正常组无显著差异，提示心理应激对成年雄性大鼠HPG外周功能造成了影响，经中药干预后显著下降，很好地起到调控应激损伤的作用；HPA轴是慢性心理应激反应的主要途径。本实验结果显示，慢性应激作用下HPA轴发生了显著变化，而雌雄间显示一定的差异。
从HPA轴整体分泌上来看，本实验充分显示出CRH、ACTH、CORT三者间的相互作用确实存在，同时也可解释本实验当中，雌雄组间不同的变化趋势及多组数据仅有趋势而无显著差异的结果。例如，本实验总的趋势是HPA轴的激活与亢进状态，但雌性组却CORT仅显示出趋势，其机制有待进一步阐明，但结合ACTH的显著下降，是否可以看出ACTH对其的调控作用，即正是由于模型组ACTH的减少，从而使CORT的升高变缓。雄性组方面，ACTH的变化仅有升高趋势，其同样存在CORT显著升高后，对其的反馈性调节，从而导致其增高的显著性变化减弱。而下丘脑CRH显著高于对照组，可能是对下丘脑对血浆ACTH、CORT改变的反馈性调控的结果。
本次实验结果显示，与正常组相比，模型组肾上腺皮质其形态学发生改变，提示慢性心理应激大鼠肾上腺内分泌功能处于亢奋状态，表现为皮质增生明显，细胞密度增高等，而中药治疗组明显好转，表明中药复方对肾上腺内分泌激素的分泌有很好的调控作用，可抑制其皮质增生，进而减缓糖皮质激素的分泌。结合其免疫功能显著抑制与降低，表明二者间的一定的因果关系。
(三)加味四逆散对慢性心理应激大鼠免疫功能的调节
本实验旨在观察慢性应激状态下大鼠免疫功能的变化，探讨其与神经内分泌改变的关系，尝试明确其HPA轴主要作用途径。结果表明，模型各组大鼠胸腺指数显著降低，经中药复方干预后，胸腺指数显著回升，表明中医复方JWSNS可以有效提高应激反应后的免疫功能改变。模型各组胸腺形态学发生严重改变，反映出慢性应激反应后大鼠免疫抑制显著，经中医复方JWSNS治疗后器官形态有显著改善，提示可以有效提高应激反应后的免疫功能改变。T淋巴细胞是机体免疫系统内功能最重要的免疫细胞，对人体免疫功能有重要的调节作用，本次实验结果表明，雌性大鼠模型组CD3+、CD4+、CDS+均呈下降，CD3+下降有显著差异(p＜0.05)，经中药复方治疗后T淋巴细胞亚群CD3+、CD4+显著回升，CDS+呈上升趋势，并高于正常组，也显示出雌性对应激干预手段与药物的敏感性；而雄性组方面，模型组T淋巴细胞亚群(CD3+、CD4+、CD8+)均有所下降，经中药复方JWSNS治疗后，有所回升，但均未达到统计学意义。模型各组CD4+与CDS+比值呈下降趋势，中药复方各组有所提高，但未有统计学意义，可能与实验大鼠例数相对较少有关，尚需进一步观察与探讨。雌雄间比较显示，雄性应激免疫抑制趋势更加明显一些，与以往报道一致。
(四)加味四逆散对慢性心理应激大鼠海马神经元的保护作用
观察慢性应激状态下大鼠中枢海马神经元的变化，探讨其对外周神经内分泌改变的调控，尝试明确其对HPA轴主要作用途径。海马不仅是应激反应的高级调节中枢也是应激损伤的敏感区。本次研究结果表明，与正常组相比，雌雄模型组细胞凋亡率均明显增加，海马光密度值显著低于正常组，海马组织损伤明显高于正常组；与模型组相比，雌雄JWSNS组海马神经元凋亡率明显下降，JWSNS能明显升高应激大鼠海马光密度值，减轻组织损伤，提示不可预计应激模型大鼠已引起海马神经元的形态发生变化，出现海马损伤及神经元细胞的凋亡。而经加味四逆散治疗干预后，海马组织损伤明显降低，表明其可能很好的缓解海马损伤与神经元细胞的调亡，具有较好的海马神经元保护作用。
三、研究结论
1．本实验对慢性不可预计心理应激大鼠模型和复制与改制较为成功，从表面效度、结构效度和预测效度三个效度对其进行综合评价，表明该模型大鼠行为学表现与人类相应的情绪表现具有一定的相似性；其反映出一定的情绪障碍的行为学和生理学的基本特征，以及共同的神经生物学机制——HPA轴的调控等。而中药复方JWSNS可有效调控慢性心理应激状态下机体的NIM网络改变。
2．慢性心理应激反应的神经内分泌通路主要是HPA系统，此系统的激活是应激性外周躯体反应、免疫功能紊乱等的生理学基础，而中药复方JWSNS可有效调控HPA轴，提示其抗应激损伤作用机制与HPA轴密切相关。
3．慢性心理应激反应在不同性别中存在一定差异。心理应激可影响到下丘脑—垂体—性腺(HPG)轴的功能，JWSNS显示出对HPG一定的调控作用，但由于不同性别性腺激素分泌的复杂性，JWSNS作用机制与方式有待进一研究。
3．慢性应激状态下免疫功能显著抑制，中药复方JWSNS可有效改善其免疫功能。
4．研究结果表明，慢性应激反应大鼠海马神经元凋亡率显著升高，提示海马神经元死亡增加，从而影响到对应激及HPA轴的调控。中药复方JWSNS可有效降低海马神经元凋亡，提示其抗应激损伤的中枢作用机制与海马密切相关。
四、存在问题与研究展望
因受时间、经费的限制，大鼠模型的复制与评价，及其神经内分泌免疫整体调节功能的阐明仍需进一步完善，尤其是其他调节途径的明确。同时，慢性心理应激反应损伤海马神经元的机制非常复杂，单纯的细胞凋亡的研究仅仅是开始。其作用通路与信号传导机制仍需进一步研讨。研究初步表明，JWSNS可以有效抗应激损伤，其作用途径与HPA轴及海马中枢关系密切，但作为复方，其真正起效的有效部分是什么，相应活性成分的主要作用途径是什么，将是我们今后研究的重点。
Part one: Literature summary and Theory approch
Psychological stress is the course of psychological and physiological change resulted by organism that perceive and surge the threats of stress causing psychology through knowing appraise. With the development of society and the increase of pressure from every facet, psychological stress threaten the human heath, as an important fact which can not be ignored, How the psychological and socialological stimulation and the psychological action convert into physiological change, ie from concept to substance, is the key of the psychological stress mechanism.
From the perspective of pathological physiology, psychological stress is a series of neuro-endocrine change which caused by intense stimulation. Sympathesis nerve excitation and pituitary-adrenal cortex secretion are the main mechanism. The stress system contain central and external parts. The former mainly include hypothalamic and hippocampus, however, the latter are consist of hypothalamic- pituitary-adrenal cortex axis(HPA) and sympathesis/adrenal system and sub sympathesis system. HPA is the main channel which play a key role in the psychological stress mechanism.
Immune system is the out-receptor of HPA, so the infuence on it become more popular. Immunologycal inhibition is the main response. T cell cluster's disorder in quantity and function leads to the disorder in Immune system. Research indicats that HPA is the main channel which play a key role in the psychological stress mechanism. As complement, CRH and ACTH took part in immunoregulation except glucocorticoid. On the other hand, hippocampus becomes the central immunoregulational one. Studies found that normal hippocampus can constrain the excessive response of HPA and lower it to normal level. Furthermore, hippocampus is a sensitive and easy be hurt area in stress. In a word, hippocampus's hurt in stress and HPA's excessive response are the results of infernal circle.
The traditional Chinese medicine does not have the concept called "psychological stress", but the bad circumstances and mental stimulation have close relationship with the body disease in state of viscera and seven sentiments theories. Stress is nonspecificity systemic reaction, thus the study of the Chinese medicine for anti-stress must involve many organs. At present, many researches are base on the emotion theory, involving the liver, the kidney, the heart, and the spleen, especially the liver. The detective indexes primarily involve praxiology and neuro-endocrine-immune system. But the conclusion does not sufficient in the holistic level.
The studies of our working team indicats that there are some concrete central nerve mechanism in adtion to the whole regulation of the "liver"function (liver controlling dispersion, adjusting emotion)in TCM on hypothalamic-pituitary-adrenal cortex system. Meanwhile there are unity between part and whole in mechanism of action. We take JiaWeiSiNiSan (JWSNS) as a representative regulation liver formula to regulate the psychological stress reaction and have done some relative researches which have tentatively confirmed our investing hypothesis.
To make animal model in psychological stress is the foundation, but there are still some vital segments which can't be resolved. For example, the somatic stress factor in the course of previous model-making can't be avoid, which infuenced the experimental results in some extent. Willner p. who established the chronic mild unpredictable stress model (CMUS) solve the problem.
Given to the importance in the NIM net, the HPAA and hippocampus become more significant in the regulation of the 'liver'. And with the improvement of animal model, we will get close to the essence of the regulation of the 'liver'. Base on that, we will study further the mchanism of HPAA in chronic psychological stress and the action of Chinese compouned medcine. In order to interpret the mchanism of psychological stress and Chinese compouned medcine in holistic level.
Part two: Study target and content
Target: according to study the mechanism of action of jiaweisinisan (JWSNS) against psychological stress on HPAA and the chang on neuro-endocrine-immune system, we will try to interpret the the mchanism of regulation "liver" theory against psychological stress on neuro-endocrine-immune level, and finally develop a way against psychological stress with TCM. Furthermore, we can reveal the substance foundation and the mechanism of regulation NIM net on multistrata and multitarget from cell and even molecule level.
Content: to establish the CUMS animal model and detect the chang of neuro-endoc rine-immune system, so as to observe the regulation action of TCM and discuss the mechanism.
Composition and dosage of JWSNS: Bupleurum root 15 g, White peony root 15g, Bitter orange 6g, Wolfberry fruit 15g, Capejasmine fruit 5g, Rehmannia root 18g, Concha haliotidis 30g. The formula are bought from the first affiliated hospital pharmacy of our school which concidered as pure crude herbs by relative department. Finally, we preserved it in refrigerator at 4 centigrade after being prepared. We should provide 2 ml per animal each treatment group for intragastric administration before building mode.
Model building and grouping: we take the modified CUMS animal model which to be cancel lightning stroke installation and to be lighten intensity. Model rats were placed in one independent room and accepted different stress stimulus within 21 days. The stressors consist of restraint for 1h, 45°cage tilted for 7h, cage moistened (padding: water=200g:100ml), new intruder added (two cages combined ) for 23 days, persistent illumination for 36h, bottle free lh, fasting diet and water 23h.
Section one: The establish of CMUS model
The target of this experiment is to observe the characters of different ages and sex according to establish of CMUS, and then evaluate it through three facet of face, structure and predictive validity, we saw that, in the course of building model, the model group decline in whole level. Show that, anxiety, dysphoria, increased defecation, reject in some course, shortness of breath, color pattern get darker and even become yellow, the modified behavior decrease even disappear. As compared, the treatment group was between model and normal one, normal group shows best. As to sacchar-concume, the result are similar, although there are transient increase because of baby rats' grouth. But three weeks later it come down as others. Results also similar on weight. Hence, we can come to conclude that JWSNS can relieve stress state effectively, but it can' t make them recovered as long as the stressor exist. The sex is also an important factor. In our experiment, the differences comes from behavior, neuro-endocrine-immune index ect. Compared with male rats, the female one's sacchar-concume present fluctuation state. Say it specificially, that is, decline on first week and then increase in some extent, finally low down again on third week. Base on that, we deduce female' s reaction are more intensity on acute stress and then change into chronic stress state.
Section two: JWSNS regulate CUMS on neuro-endocrine-immune system
In this section, we will observe the change and the way to act of adrenal hormone, thyroid hormone and hebin. This finding show that, female adult rats' T3, T4 of every group don't have obvious chang except the hebin and androgen of adult group. Say it specificially, in male model group, androgen increase obviously(p＜0.01), and then decrease after be given JWSNS. Compared with normal group, there isn't obvious difference. Cue that psychological stress can infuence the periphery sex gland of adult male rats, and can be lower down by JWSNS effectively. However, the female group' androgen didn't lower dow later, which need study further. We have known about the HPAA is the main chanel for chronic psychological stress, the result displayed the certain change of it, and even in different sexes.
From the whole level, the result thoroughly displayed the interaction among CRH、ACTH and CORT. At the same time, it also explain the unobvious but changeable result. For example, the general tendency is the activation and accentuation on HPAA, but the situation on female group only showed the tendency without obvious sense. Taking the degression of ATCH as reference, we can deduce the regulation action of it on female' s CORT, ie the elevated tendency get gently because of the decrease of ATCH. The same situation happened to the male group. However, the CRH in hypothalamus are higher than control group obviously, maybe it' s caused by the feedback action for ATCH and CORT in blood plasma.
Section three: the regulation of JWSNS on immune function of CMUS model
In this section we will approach the mutual relationship between immune function and neuroendocrine, and try to identify the action chanel of HPAA. Results show that each model group obviously decrease on thymus gland index, after being given JWSNS both of them rise with the former obviously. It displayed that JWSNS could effectively recover the chang on immune function. From the shape change of thymus gland to improve, we can also found the obvious effect of JWSNS on immunodepression. T cell is the most important immunocell, so we take it for example. Resuts show that, in female model group, CD3+, CD4+ and CD8+ all decrease, especially CD3+ (p＜0.05), and then increase after be given JWSNS, especially CD3+ and CD4+, the three are higher than normal one. We can deduce that the female are more sensitive for stress and medicine; In male group, the three indexes have the same change but without statistical significance.. CD4+ and CDS+ decrease in model group and increase in medicine group both without statistical significance, maybe have relative with the number, which need study further. Compared with male group, the female one's expresion are better which is equal to previous report.
The change of adrenal cortex shape, compared with normal group, displayed the excited state of endocrine function of adrenal gland. After be given JWSNS, corticohyp erplassia were restrained and glucocorticosteroid secreted become slow. Combined with the obvious degrade on immune function, we can get the causal relation between the two.
Section four: the protection of JWSNS on hippocampus neurone of CMUS model
In this section we will approach the mutual relationship between the change of hippocampus neurone and the regulation of periphery neuroendocrine, and try to identify the action chanel of HPAA. Hippocampus is not only the senior centers of accommodation but also the area of sensitivity coping with stress hurt. In this experiment, compared with normal group, apoptosis rate of model group both male and female increase obviously, hippocampus optical density value obviously lower than normal group; Compared with model group, hippocampus neurone apoptosis rate of medicine group both male and female decrease obviously, hippocampus optical density value obviously increase. Show that JWSNS can protect hippocampus neurone of CMUS model.
Part three: Conclusion
First, the duplicate and modify of CMUS model are successful, and then evaluate it through three facets of face, structure and predictive validity, we can say there are some similarity between praxiology appearance of rat and emotional expression of human; It is reflected that there are some essential character of emotional disturbance on praxiology and physiology, and collective neurobiology mechanism——the regulation of HPAA etc. Moreover, JWSNS works effectively.
Second, the main active path of neuroendocrine against chronic psychological stress is HPA axis. The activation of it is physiological foundation of periphery body reaction and immune function disorder. However, JWSNS can regulate HPA axis effectively, so we could think the mechanism of it have close relationship with HPA axis.
Third, chronic psychological stress displayed the obvious change in different sexes. the hypothalamus-pituitarium-thyroid gland axis and hypothalamus-pituitarium-sex gland axis were involved, but it need to be study further.
Fourth, immune function were restrained obviously in state of chronic psychological stress, and JWSNS can improve it effectively.
Last, hippocampus neurone apoptosis rate of chronic psychological stress rats increase obviously, we can think the regulation of HPAA were influenced by the increase death of hippocampus neurone, and JWSNS can improve it effectively, the mechanism of it have close relationship with hippocampus.
Part Four Deficiency and Prospect
Because of the limitation of time and fund, the model's duplication and evaluation as well as the whole regulation mechanism on neuro-endocrine-immune system need to be consummated, especially the relative pathes. Another, hippocampus neurone apoptosis is only the start of hippocampus hurt. In the future, we will focus on the validation section of JWSNS against chronic psychological stress.
心理应激是机体通过认识、评价而察觉到应激原的威胁时引起的心理、生理机能改变的过程。随着社会发展和各方面竞争压力的增加，心理应激已成为一个不可忽视的因素威胁着人类健康。明确作为“观念性”的心理社会刺激以及由此产生的心理活动是如何转变为“物质的”生理反应的机制是研究应激机制与应对方式的关键内容。
从病理生理学的角度来看，应激是指机体在受到各种强烈因素刺激时所出现的以交感神经兴奋和垂体-肾上腺皮质分泌为主的一系列神经内分泌反应，进而引起各种机能和代谢的进一步改变。机体应激系统可分为位于中枢的神经指挥系统和外周的效应器官与功能，前者以下丘脑、海马部位为主，后者则是下丘脑-垂体-肾上腺皮质(HPA)轴、传出交感／肾上腺體质系统和副交感系统两大系统的作用下完成。其中，HPA轴是应激作用的主要途径，在心理应激反应中起关键性作用。而不同的性别与年龄直接影响着心理应激反应的方式与强度。
免疫系统是HPA轴外周作用的主要靶点，研究应激及其应付方式对免疫功能的影响是心理神经免疫学研究的热点之一。在多种应激条件下人体免疫功能发生变化，主要以免疫功能抑制为主。T淋巴细胞是机体免疫系统内功能最重要的免疫细胞，正常情况下，各T淋巴细胞亚群相互作用，维持着机体正常的免疫功能，当不同淋巴细胞亚群的数量和功能发生异常时，就可导致机体细胞免疫功能紊乱。研究表明，HPA轴是应激过程中免疫应答调节的主要途径，除糖皮质激素外，CRH和ACTH也均参与免疫调节。而在中枢，HPA轴应激反应的高位调节中枢是海马。大量研究表明，正常海马可抑制HPA轴的活性，参与了应激过程HPA轴的抑制性调节，促进应激状态下亢进的HPA轴恢复到基础水平，同时，海马不仅是HPA轴应激反应的高位调节中枢，而且是应激损伤的敏感区。应激性海马损伤与HPA轴功能亢进是神经内分泌系统应激反应的恶性循环结果。
中医学虽没有“心理应激”这一概念，但中医脏象及七情学说很早就认识到不良的环境或精神刺激与躯体疾病的发生发展有着密切的关系。应激是机体对意外刺激做出的非特异性全身反应，涉及各组织器官，因而中医药抗应激的研究也需涉及多个脏腑。目前中药复方抗心理应激损伤的研究多以情志立论，从肝、肾、心、脾入手，检测指标多为抗应激能力行为学指标和神经—内分泌—免疫系统等指标为主。但从理论基础、临床应用、实验研究三方面来看，情志调节核心在于五脏，而肝脏在其中起着主要作用。肝郁证候存在心理应激的障碍，病理机制涉及神经内分泌免疫等调节网络，但现代分子生物学的研究还不能充分地解释中医整体水平上的肝郁证候的理论。从心理应激反应来研究肝脏象的中医证候是近几年研究的热点。
本研究团队以往一系列研究亦表明：中医肝主疏泄调畅情志的功能在实现对下丘脑-垂体-肾上腺皮质系统的整体性调节上，还可能存在着一定的具体的中枢神经机制；在作用机理上体现出局部(中枢神经机制)与整体(NIM网络)的有机统一。并且以加味四逆散(JWSNS)为调肝治法方药之代表方，采用“方-证-效-脏腑功能本质”的研究思路，对慢性应激损伤及调肝方药的作用机制进行了一系列的研究，探讨肝主疏泄调畅情志功能的中枢神经生物学机制，其具体机制有待一步探讨。
心理应激动物模型的建立是开展心理应激机制研究的基础，但某些重要环节的偏差与设计仍影响着中医药抗心理应激损伤机理的阐明。例如，心理应激模型中常用的应激源包括束缚和制动、条件反射性电击足等或多或少均含有一定的躯体性应激成份，某种程度上影响着单纯心理应激作用机制的阐明。Willner等受CUS模型启发于1987年提出了“慢性轻度不可预知的应激模型”主要是将动物长时间的暴露在多种不可预知的应激源下，这些应激源包括不定期的禁水、禁食、震动、电击、游泳等，由此引起动物对奖励刺激的缺乏，是抑郁的重要表现，更真实的模拟了人们在现实生活中遭遇的“困难”，可以认为此应激模型是一种有效的抑郁性应激模型。
鉴于HPA轴及其海马中枢调控在NIM网络调节机制中的重要作用，有必要进一步探讨与阐明HPA轴及其海马中枢调控作用在肝主疏泄调畅情志功能的具体机制。在动物模型不断改进的基础上，不断接近肝主疏泄调畅情志的HPA调控作用机制的实质。在上述理论基础支持下，本论文旨在进一步研究不同性别、年龄情况下慢性心理应激反应损伤的HPA轴等神经内分泌免疫学作用机制，以及中药复方对其的干预作用，为最终从整体上阐明心理应激作用机制及中医调肝治法方药抗心理应激损伤的作用机理奠定基础。
二、研究目标与内容
研究目标：开展调肝方药——加味四逆散(JWSNS)抗心理应激损伤HPA轴作用方式及其神经内分泌免疫变化的实验研究，尝试阐明调肝治法抗不良心理应激损伤的神经内分泌作用机制，探求安全有效的中医药抗心理应激方法。进而，从细胞及分子水平进一步探讨调肝方药一加味四逆散(JWSNS)在心理应激损伤状态下多层次、多靶点调控NIM网络的作用机制，更加深入揭示加味四逆散抗心理应激损伤的神经免疫学的物质基础及其作用方式。
研究内容：建立慢性轻度不可预计应激损伤大鼠模型，检测各试验组分中免疫功能的变化，及其神经内分泌调控过程中外周与中枢各类激素等主要生化指标与病理改变，观察调肝方药JWSNS的调控作用，以HPA轴为重点，探讨其作用可能的机制与途径，研讨调畅情志疏肝之法在抗心理应激损伤中的作用机理。
药物组成与立法：JWSNS组成：柴胡5g、白芍15g、枳壳6g、枸杞子15g、山栀5g、干地黄18g、石决明30g。药材由本校第一附属医院药房提供，经药剂科鉴定均为纯正药材。制备后置4℃冰箱内保存备用。各治疗组分别于应激造模前1h给予加味四逆散2ml灌胃。
动物模型制作与分组：经慢性轻度不可预计应激动物模型改制而成，去除以往采用模型中电击应激源的设置，减轻应激源的造模作用强度，以减少躯体因素的因素。接受应激处理的组别大鼠单独放在一间独立的房间，在21天接受不同应激源的刺激，应激源包括有限制空间1h，45度斜笼7h、湿笼17h(湿笼是200g铺料倒100ml水，使垫料湿透)、新入侵者(两笼并笼)23h、持续光照36h、空瓶1h、禁食禁水23h。
(一)慢性轻度不可预计应激损伤大鼠模型的建立
本实验目的在于建立慢性轻度不可预计应激模型，观察不同年龄、性别大鼠模型的特征。用表面效度、结构效度和预测效度三个效度对其进行综合评价。大鼠基本状况观察结果表明，随着造模工作的进行，模型组大鼠整体状况不断明显下降，焦虑，烦躁不安，排便增多，部分过程拒绝，呼吸急促，毛色发黯，发黄，存在修饰行为，但不断减少，直到基本消失。治疗组整体状态强于模型组，但略弱于正常组，表现为较安静，毛色虽杂乱，但略有光泽，修饰行为减少，抵抗行为减少，表明经中药复方JWSNS组干预，基本状况有好转，但在应激源未解除之前，仍较疲倦。糖水消耗偏爱度是判断大鼠抑郁应激程度的重要指标。数据表明，模型大鼠糖水消耗偏爱度减少，提示模型成功，而中药复方JWSNS干预，糖水消耗偏爱度显著提高，与正常组无显著差异，提示中药复方JWSNS可能干预慢性心理应激损伤，此结果与以往一致。其中，幼年鼠正常组因处于生长期，糖水消耗偏爱度随体重呈增长趋势，但模型组三周后糖水消耗偏爱度显著减少(p＜0.01)，表明慢性应激对其产生较为严重的损伤，导致其不升反降；成年鼠正常组糖水消耗偏爱度未显示随体重而增长趋势，模型组三周后糖水消耗显著减少(p＜0.01)，经中药治疗有显著改善(p＜0.01)，表现出一定的稳定性。体重方面，各组正常大鼠体重增长平稳，雄性组略快，而经造模后体重增长显著变缓(p＜0.01)，提示长时间应激导致机体的综合状况显著下降，经治疗后有改善，但与正常组相比仍有显著差异(p＜0.01)，无法复原，表明中药复方可以有效缓解其应激状态，但无法改变其只要应激源持续存在就必然所造成的损伤与压力。
性别是影响应激作用的重要因素。结果表明，就心理应激而言，雌性和雄性动物之间存在显著的性别差异，两种动物在行为表现、内分泌和病理生理等多个方面也存在显著差别。雌雄间比较，雄性对糖水消耗偏爱度更为稳定一些，总体趋势呈下降；雌性大鼠则在第一周即出现显著下降，随后又有所恢复，进而第三周又显著下降，提示雌性可能对急性应激更为强烈，进而转为慢性应激状态。
(二)加味四逆散对慢性心理应激大鼠下丘脑—垂体—靶腺轴的调节
本实验旨在观察慢性心理应激状态下大鼠肾上腺、甲状腺、性腺轴的外周分泌激素的改变及其主要作用途径。本研究结果显示，成年组雄激素有显著改变，雌性激素有改变趋势，而T3、T4含量无明显改变。表现为，雄性造模组雄激素显著升高(p＜0.01)，经JWSNS治疗后下降与正常组无显著差异，提示心理应激对成年雄性大鼠HPG外周功能造成了影响，经中药干预后显著下降，很好地起到调控应激损伤的作用；HPA轴是慢性心理应激反应的主要途径。本实验结果显示，慢性应激作用下HPA轴发生了显著变化，而雌雄间显示一定的差异。
从HPA轴整体分泌上来看，本实验充分显示出CRH、ACTH、CORT三者间的相互作用确实存在，同时也可解释本实验当中，雌雄组间不同的变化趋势及多组数据仅有趋势而无显著差异的结果。例如，本实验总的趋势是HPA轴的激活与亢进状态，但雌性组却CORT仅显示出趋势，其机制有待进一步阐明，但结合ACTH的显著下降，是否可以看出ACTH对其的调控作用，即正是由于模型组ACTH的减少，从而使CORT的升高变缓。雄性组方面，ACTH的变化仅有升高趋势，其同样存在CORT显著升高后，对其的反馈性调节，从而导致其增高的显著性变化减弱。而下丘脑CRH显著高于对照组，可能是对下丘脑对血浆ACTH、CORT改变的反馈性调控的结果。
本次实验结果显示，与正常组相比，模型组肾上腺皮质其形态学发生改变，提示慢性心理应激大鼠肾上腺内分泌功能处于亢奋状态，表现为皮质增生明显，细胞密度增高等，而中药治疗组明显好转，表明中药复方对肾上腺内分泌激素的分泌有很好的调控作用，可抑制其皮质增生，进而减缓糖皮质激素的分泌。结合其免疫功能显著抑制与降低，表明二者间的一定的因果关系。
(三)加味四逆散对慢性心理应激大鼠免疫功能的调节
本实验旨在观察慢性应激状态下大鼠免疫功能的变化，探讨其与神经内分泌改变的关系，尝试明确其HPA轴主要作用途径。结果表明，模型各组大鼠胸腺指数显著降低，经中药复方干预后，胸腺指数显著回升，表明中医复方JWSNS可以有效提高应激反应后的免疫功能改变。模型各组胸腺形态学发生严重改变，反映出慢性应激反应后大鼠免疫抑制显著，经中医复方JWSNS治疗后器官形态有显著改善，提示可以有效提高应激反应后的免疫功能改变。T淋巴细胞是机体免疫系统内功能最重要的免疫细胞，对人体免疫功能有重要的调节作用，本次实验结果表明，雌性大鼠模型组CD3+、CD4+、CDS+均呈下降，CD3+下降有显著差异(p＜0.05)，经中药复方治疗后T淋巴细胞亚群CD3+、CD4+显著回升，CDS+呈上升趋势，并高于正常组，也显示出雌性对应激干预手段与药物的敏感性；而雄性组方面，模型组T淋巴细胞亚群(CD3+、CD4+、CD8+)均有所下降，经中药复方JWSNS治疗后，有所回升，但均未达到统计学意义。模型各组CD4+与CDS+比值呈下降趋势，中药复方各组有所提高，但未有统计学意义，可能与实验大鼠例数相对较少有关，尚需进一步观察与探讨。雌雄间比较显示，雄性应激免疫抑制趋势更加明显一些，与以往报道一致。
(四)加味四逆散对慢性心理应激大鼠海马神经元的保护作用
观察慢性应激状态下大鼠中枢海马神经元的变化，探讨其对外周神经内分泌改变的调控，尝试明确其对HPA轴主要作用途径。海马不仅是应激反应的高级调节中枢也是应激损伤的敏感区。本次研究结果表明，与正常组相比，雌雄模型组细胞凋亡率均明显增加，海马光密度值显著低于正常组，海马组织损伤明显高于正常组；与模型组相比，雌雄JWSNS组海马神经元凋亡率明显下降，JWSNS能明显升高应激大鼠海马光密度值，减轻组织损伤，提示不可预计应激模型大鼠已引起海马神经元的形态发生变化，出现海马损伤及神经元细胞的凋亡。而经加味四逆散治疗干预后，海马组织损伤明显降低，表明其可能很好的缓解海马损伤与神经元细胞的调亡，具有较好的海马神经元保护作用。
三、研究结论
1．本实验对慢性不可预计心理应激大鼠模型和复制与改制较为成功，从表面效度、结构效度和预测效度三个效度对其进行综合评价，表明该模型大鼠行为学表现与人类相应的情绪表现具有一定的相似性；其反映出一定的情绪障碍的行为学和生理学的基本特征，以及共同的神经生物学机制——HPA轴的调控等。而中药复方JWSNS可有效调控慢性心理应激状态下机体的NIM网络改变。
2．慢性心理应激反应的神经内分泌通路主要是HPA系统，此系统的激活是应激性外周躯体反应、免疫功能紊乱等的生理学基础，而中药复方JWSNS可有效调控HPA轴，提示其抗应激损伤作用机制与HPA轴密切相关。
3．慢性心理应激反应在不同性别中存在一定差异。心理应激可影响到下丘脑—垂体—性腺(HPG)轴的功能，JWSNS显示出对HPG一定的调控作用，但由于不同性别性腺激素分泌的复杂性，JWSNS作用机制与方式有待进一研究。
3．慢性应激状态下免疫功能显著抑制，中药复方JWSNS可有效改善其免疫功能。
4．研究结果表明，慢性应激反应大鼠海马神经元凋亡率显著升高，提示海马神经元死亡增加，从而影响到对应激及HPA轴的调控。中药复方JWSNS可有效降低海马神经元凋亡，提示其抗应激损伤的中枢作用机制与海马密切相关。
四、存在问题与研究展望
因受时间、经费的限制，大鼠模型的复制与评价，及其神经内分泌免疫整体调节功能的阐明仍需进一步完善，尤其是其他调节途径的明确。同时，慢性心理应激反应损伤海马神经元的机制非常复杂，单纯的细胞凋亡的研究仅仅是开始。其作用通路与信号传导机制仍需进一步研讨。研究初步表明，JWSNS可以有效抗应激损伤，其作用途径与HPA轴及海马中枢关系密切，但作为复方，其真正起效的有效部分是什么，相应活性成分的主要作用途径是什么，将是我们今后研究的重点。
Part one: Literature summary and Theory approch
Psychological stress is the course of psychological and physiological change resulted by organism that perceive and surge the threats of stress causing psychology through knowing appraise. With the development of society and the increase of pressure from every facet, psychological stress threaten the human heath, as an important fact which can not be ignored, How the psychological and socialological stimulation and the psychological action convert into physiological change, ie from concept to substance, is the key of the psychological stress mechanism.
From the perspective of pathological physiology, psychological stress is a series of neuro-endocrine change which caused by intense stimulation. Sympathesis nerve excitation and pituitary-adrenal cortex secretion are the main mechanism. The stress system contain central and external parts. The former mainly include hypothalamic and hippocampus, however, the latter are consist of hypothalamic- pituitary-adrenal cortex axis(HPA) and sympathesis/adrenal system and sub sympathesis system. HPA is the main channel which play a key role in the psychological stress mechanism.
Immune system is the out-receptor of HPA, so the infuence on it become more popular. Immunologycal inhibition is the main response. T cell cluster's disorder in quantity and function leads to the disorder in Immune system. Research indicats that HPA is the main channel which play a key role in the psychological stress mechanism. As complement, CRH and ACTH took part in immunoregulation except glucocorticoid. On the other hand, hippocampus becomes the central immunoregulational one. Studies found that normal hippocampus can constrain the excessive response of HPA and lower it to normal level. Furthermore, hippocampus is a sensitive and easy be hurt area in stress. In a word, hippocampus's hurt in stress and HPA's excessive response are the results of infernal circle.
The traditional Chinese medicine does not have the concept called "psychological stress", but the bad circumstances and mental stimulation have close relationship with the body disease in state of viscera and seven sentiments theories. Stress is nonspecificity systemic reaction, thus the study of the Chinese medicine for anti-stress must involve many organs. At present, many researches are base on the emotion theory, involving the liver, the kidney, the heart, and the spleen, especially the liver. The detective indexes primarily involve praxiology and neuro-endocrine-immune system. But the conclusion does not sufficient in the holistic level.
The studies of our working team indicats that there are some concrete central nerve mechanism in adtion to the whole regulation of the "liver"function (liver controlling dispersion, adjusting emotion)in TCM on hypothalamic-pituitary-adrenal cortex system. Meanwhile there are unity between part and whole in mechanism of action. We take JiaWeiSiNiSan (JWSNS) as a representative regulation liver formula to regulate the psychological stress reaction and have done some relative researches which have tentatively confirmed our investing hypothesis.
To make animal model in psychological stress is the foundation, but there are still some vital segments which can't be resolved. For example, the somatic stress factor in the course of previous model-making can't be avoid, which infuenced the experimental results in some extent. Willner p. who established the chronic mild unpredictable stress model (CMUS) solve the problem.
Given to the importance in the NIM net, the HPAA and hippocampus become more significant in the regulation of the 'liver'. And with the improvement of animal model, we will get close to the essence of the regulation of the 'liver'. Base on that, we will study further the mchanism of HPAA in chronic psychological stress and the action of Chinese compouned medcine. In order to interpret the mchanism of psychological stress and Chinese compouned medcine in holistic level.
Part two: Study target and content
Target: according to study the mechanism of action of jiaweisinisan (JWSNS) against psychological stress on HPAA and the chang on neuro-endocrine-immune system, we will try to interpret the the mchanism of regulation "liver" theory against psychological stress on neuro-endocrine-immune level, and finally develop a way against psychological stress with TCM. Furthermore, we can reveal the substance foundation and the mechanism of regulation NIM net on multistrata and multitarget from cell and even molecule level.
Content: to establish the CUMS animal model and detect the chang of neuro-endoc rine-immune system, so as to observe the regulation action of TCM and discuss the mechanism.
Composition and dosage of JWSNS: Bupleurum root 15 g, White peony root 15g, Bitter orange 6g, Wolfberry fruit 15g, Capejasmine fruit 5g, Rehmannia root 18g, Concha haliotidis 30g. The formula are bought from the first affiliated hospital pharmacy of our school which concidered as pure crude herbs by relative department. Finally, we preserved it in refrigerator at 4 centigrade after being prepared. We should provide 2 ml per animal each treatment group for intragastric administration before building mode.
Model building and grouping: we take the modified CUMS animal model which to be cancel lightning stroke installation and to be lighten intensity. Model rats were placed in one independent room and accepted different stress stimulus within 21 days. The stressors consist of restraint for 1h, 45°cage tilted for 7h, cage moistened (padding: water=200g:100ml), new intruder added (two cages combined ) for 23 days, persistent illumination for 36h, bottle free lh, fasting diet and water 23h.
Section one: The establish of CMUS model
The target of this experiment is to observe the characters of different ages and sex according to establish of CMUS, and then evaluate it through three facet of face, structure and predictive validity, we saw that, in the course of building model, the model group decline in whole level. Show that, anxiety, dysphoria, increased defecation, reject in some course, shortness of breath, color pattern get darker and even become yellow, the modified behavior decrease even disappear. As compared, the treatment group was between model and normal one, normal group shows best. As to sacchar-concume, the result are similar, although there are transient increase because of baby rats' grouth. But three weeks later it come down as others. Results also similar on weight. Hence, we can come to conclude that JWSNS can relieve stress state effectively, but it can' t make them recovered as long as the stressor exist. The sex is also an important factor. In our experiment, the differences comes from behavior, neuro-endocrine-immune index ect. Compared with male rats, the female one's sacchar-concume present fluctuation state. Say it specificially, that is, decline on first week and then increase in some extent, finally low down again on third week. Base on that, we deduce female' s reaction are more intensity on acute stress and then change into chronic stress state.
Section two: JWSNS regulate CUMS on neuro-endocrine-immune system
In this section, we will observe the change and the way to act of adrenal hormone, thyroid hormone and hebin. This finding show that, female adult rats' T3, T4 of every group don't have obvious chang except the hebin and androgen of adult group. Say it specificially, in male model group, androgen increase obviously(p＜0.01), and then decrease after be given JWSNS. Compared with normal group, there isn't obvious difference. Cue that psychological stress can infuence the periphery sex gland of adult male rats, and can be lower down by JWSNS effectively. However, the female group' androgen didn't lower dow later, which need study further. We have known about the HPAA is the main chanel for chronic psychological stress, the result displayed the certain change of it, and even in different sexes.
From the whole level, the result thoroughly displayed the interaction among CRH、ACTH and CORT. At the same time, it also explain the unobvious but changeable result. For example, the general tendency is the activation and accentuation on HPAA, but the situation on female group only showed the tendency without obvious sense. Taking the degression of ATCH as reference, we can deduce the regulation action of it on female' s CORT, ie the elevated tendency get gently because of the decrease of ATCH. The same situation happened to the male group. However, the CRH in hypothalamus are higher than control group obviously, maybe it' s caused by the feedback action for ATCH and CORT in blood plasma.
Section three: the regulation of JWSNS on immune function of CMUS model
In this section we will approach the mutual relationship between immune function and neuroendocrine, and try to identify the action chanel of HPAA. Results show that each model group obviously decrease on thymus gland index, after being given JWSNS both of them rise with the former obviously. It displayed that JWSNS could effectively recover the chang on immune function. From the shape change of thymus gland to improve, we can also found the obvious effect of JWSNS on immunodepression. T cell is the most important immunocell, so we take it for example. Resuts show that, in female model group, CD3+, CD4+ and CD8+ all decrease, especially CD3+ (p＜0.05), and then increase after be given JWSNS, especially CD3+ and CD4+, the three are higher than normal one. We can deduce that the female are more sensitive for stress and medicine; In male group, the three indexes have the same change but without statistical significance.. CD4+ and CDS+ decrease in model group and increase in medicine group both without statistical significance, maybe have relative with the number, which need study further. Compared with male group, the female one's expresion are better which is equal to previous report.
The change of adrenal cortex shape, compared with normal group, displayed the excited state of endocrine function of adrenal gland. After be given JWSNS, corticohyp erplassia were restrained and glucocorticosteroid secreted become slow. Combined with the obvious degrade on immune function, we can get the causal relation between the two.
Section four: the protection of JWSNS on hippocampus neurone of CMUS model
In this section we will approach the mutual relationship between the change of hippocampus neurone and the regulation of periphery neuroendocrine, and try to identify the action chanel of HPAA. Hippocampus is not only the senior centers of accommodation but also the area of sensitivity coping with stress hurt. In this experiment, compared with normal group, apoptosis rate of model group both male and female increase obviously, hippocampus optical density value obviously lower than normal group; Compared with model group, hippocampus neurone apoptosis rate of medicine group both male and female decrease obviously, hippocampus optical density value obviously increase. Show that JWSNS can protect hippocampus neurone of CMUS model.
Part three: Conclusion
First, the duplicate and modify of CMUS model are successful, and then evaluate it through three facets of face, structure and predictive validity, we can say there are some similarity between praxiology appearance of rat and emotional expression of human; It is reflected that there are some essential character of emotional disturbance on praxiology and physiology, and collective neurobiology mechanism——the regulation of HPAA etc. Moreover, JWSNS works effectively.
Second, the main active path of neuroendocrine against chronic psychological stress is HPA axis. The activation of it is physiological foundation of periphery body reaction and immune function disorder. However, JWSNS can regulate HPA axis effectively, so we could think the mechanism of it have close relationship with HPA axis.
Third, chronic psychological stress displayed the obvious change in different sexes. the hypothalamus-pituitarium-thyroid gland axis and hypothalamus-pituitarium-sex gland axis were involved, but it need to be study further.
Fourth, immune function were restrained obviously in state of chronic psychological stress, and JWSNS can improve it effectively.
Last, hippocampus neurone apoptosis rate of chronic psychological stress rats increase obviously, we can think the regulation of HPAA were influenced by the increase death of hippocampus neurone, and JWSNS can improve it effectively, the mechanism of it have close relationship with hippocampus.
Part Four Deficiency and Prospect
Because of the limitation of time and fund, the model's duplication and evaluation as well as the whole regulation mechanism on neuro-endocrine-immune system need to be consummated, especially the relative pathes. Another, hippocampus neurone apoptosis is only the start of hippocampus hurt. In the future, we will focus on the validation section of JWSNS against chronic psychological stress.
引文
1．严灿，徐志伟．肝主疏泄调畅情志功能的中枢神经生物学机制探讨．中国中西医结合杂志，2005；(05)：459-462
2．George P Ch rousOs，MD，Philip W Gold，MD．应激与应激系统紊乱的概念概述身体的和行为的内环境稳定．美国医学会杂志中文版，1992；2(5)：290-298
3．韩晓春．下丘脑-垂体-肾上腺在慢性应激过程中的调节作用．医学综述，2006；12(2)：73-75
4．李凌江，李则宣．精神应激的生物学致病机制研究-Ⅱ：神经内分泌机制．中国行为医学科学杂志，2005；14(2)：156-158
5. Bessel A, Vander Kolk. The psychobiology and psychopharmacology of PTSD. Human Psychopharmacology, 2001; 16: 49-64.
6. Hageman I, Andersen HS, JorgcnsenMB. Posttraumatic stress disorder: a review of psychobiology and pharmacotherapy. Acta sychiatr Scand, 2001; 104: 411-422.
7．李泽桂．大鼠室旁核CRF神经元在烫伤应激中的变化．第三军医大学学报，1999；21(3)：160-163
8．韩良辅．次声作用引起大鼠下丘脑c-fos mRNA与CRF mRNA的表达．第四军医大学学报，1999；20(1)：1-3
9. Kam K, Park Y, Cheon M, et al, Effects of immobilization stress on est rogen2induced surges of luteinizing hormone and pro lactin in ova riectomized rats [J]. Endocrine, 2000; 12 (3): 279-287.
10. Kalanta ridou S N, Makrigianna kis A, Zouma kis E, et al. Rep roductive functions of corticot ropin2releasing hormone: Research and potential clinical utility of antala rmins [J]. American Jour nal of Rep roductive Immunology, 2004; 51 (4): 269-274.
11. Murase M, Uemura T, Kondoh Y, et al. Role of corticot ropin2releasing hormone in ovarian steroidogenesis [J ]. Endocrine, 2002; 18 (3): 255-260.
12. Breen K M, Billings H J, Wagenmaker E R, et al. Endocrinebasis for disruptive effects of cortisol on preovulatory events[J] Endocrinology, 2005; 146 (4): 2107-2015.
13．黎萍，郭锡永．应激影响女性生殖内分泌功能机制的研究进展．中国妇幼健康研究，2006；17(3)：215-217
14. Tsigos C, Chrousos GP. Hypothalamic-pituitary-adrenal axis, neuroendocrine factors and stress[J]. J Psychosom Res, 2002; 53 (4): 865-871.
15. Venditti P, De Rosa R, Caldarone G, et al. Functional and biochemical characteristics of mitochondrial fractions from rat liver in cold2induced oxidative stress[J]. Cell Mol Life Sci, 2004; 61 (24): 310423116.
16．赵福东，董竟成．应激时内分泌变化对免疫功能影响的研究进展．国外医学内分泌学分册，2005；25(4)：241-243
17. Sapolsky RM, Uno H, Tarara R, et al. Hippocampal damage associated with prologned and fatal stress in primates [J]. JNeurosci, 1989; 9: 1705-1711.
18. Goldapple K, Segal Z, Garson C, et al. Modulation of cortieal2limbicpathways in major depression; treatment specific effects of cognitive be2havior therapy[J]. Arch Gen Psychiatry, 2004; 61 (1): 34241.
19．郑红，南路．男人、女人与应激反应[J]．世界科学，1992，(03)：23-25
20．李则宣，李凌江．应激动物模型设计的成败与思考．中国行为医学科学，2002；11(1)：105-110
21．吴振宇，杨军平，吴广均，等．小站台水环境不同应激时间对组织中5-羟色胺的影响．中国行为医学科学，2004；13(1)：11-13．
22．周萍，朱文锋．七情学说与应激理论．医学与哲学，1995；16(9)：484-485
23．刘晓伟，曲宏达．关于中医七情学说研究的思考．天津中医，2002；19(6)：44-45
24．赵平，冯前进．情志致病学说与心因性应激反应理论．山西中医学院学报，2000；1(1)：7-9
25．罗家发．情志医学研究的思考[J]．安徽中医学院学报，2002；21(2)：7-9
26．武刚．情志学说研究思路探析[J]．安徽中医学院学报，2001；10(4)：4-6
27．严灿，李艳，史亚飞．七情学说实验研究相关问题探讨．中国中医基础医学杂志，2001；(10)：11-13
28．中医现代化科技发展战略研究课题组．情志疾病的中医药防治研究与养生保健理论与方法的研究．世界科学技术—中药现代化，2000；4(2)：19-22
29．岳文浩，付文青，芦宗玉，等．“怒伤肝”机制研究[J]．医学与哲学，1995；16(9)：481-4831
30．王文勋，郑洪新，王继伟，等．怒伤肝与神经-内分泌-免疫系统失调探析[J]．辽宁中医杂志，1997；24(5)：205-2061
31．赵晓林，李恩，贾漪涛，等．滋补肝肾方药对慢性激怒应激大鼠下丘脑肝脏核糖体聚态的影响[J]．中国中医基础杂志，1997；3(5)：28～301
32．刘晓伟，张红梅，曲宏达，等．“怒伤气”大鼠血ACTH、CORT、IL-2、IL-8的含量变化[J]．中国全科医学，2004；7(22)：1653～16541
33．刘雁峰，王天芳，杨维益，等．复合应激因素致“慢性疲劳”模型大鼠脾脏变化的研究[J]．中国中医基础杂志，2000；6(6)：372～3731
34．王爱成，王玉来，尹岭，等．肝气郁结证的PET影像学研究[J]．中医研究，2005；18(3)：24～251
35．严灿，徐志伟，史亚飞．加减四逆散对慢性心理应激大鼠胸腺糖皮质激素受体作用的研究．中国中西医结合杂志．2002；22(9)：691-694
36．沈雁，匡调元，张伟荣，等．“恐伤肾”的实验研究[J]．中国医药学报，1991；6(1)：13～161
37．王米渠，马向东，段光周，等．“肾为先天之本”行为遗传中关于怒伤肾的表征[J]．中国中医基础杂志，1997；3(4)：23-25
38．王米渠．“恐伤肾”母子两代的生理研究．福建中医药．2003；34(1)：3-4
39．朱凌凌，童瑶，陈慧娟，张挺．脾的中西医学比较研究．浙江中医杂志，2006；41(1)：1-5
40．岳广欣，陈家旭，王竹风．中医心肝肾三脏在应激反应中的作用．辽宁中医杂志，2005；32(6)：528-530
41．乔明琦，张惠云，陈雨振，等．肝郁证动物模型研究的理论思考[J]．中国医学学报，1997；12(5)：42-44
42．温建余，祝丽霞，等．肝郁证大鼠单胺类递质变化与性行为关系的实验研究[J]．中国男科学杂志，2002；16(3)：205-207
43．叶林，叶向荣，等．肝郁证大鼠中枢神经递质变化的观察[J]．福建中医药，2002；33(2)：171-173
44．罗欣拉，熊振芳．柴胡疏肝散对慢性束缚应激性肝郁证大鼠下丘脑-垂体-肾上腺轴的调节作用[J]．湖北中医杂志，2003；25(11)：71-73
45．秦雪梅，王明军．逍遥散和柴胡对慢性束缚应激肝郁模型大鼠脑内单胺类神经递质的影响[J]．中药药理与临床，2005；21(2)：6～7
46．严灿，徐志伟．加减四逆散对慢性心理应激大鼠胸腺糖皮质激素受体作用的研究[J]．中国中西医结合杂志，2002；22(9)：691-693．
47．徐志伟，严灿．加减四逆散对慢性心理应激大鼠的神经内分泌和行为学的调整作用[J]．广州中医药大学学报，2002；19(2)：127-130
48．李艳，严灿．反复心理应激大鼠血浆和下丘脑单胺神经递质的变化及加减四逆散的影响[J]．中药药理与临床，2002；18(1)：1-4．
49．陈松，李家邦．肝郁证T、B淋巴细胞活性改变及疏肝治疗影响的实验研究[J]．湖南中医学院学报，2001；21(2)：7-9．
50．金光亮，南睿，郭耀珍．慢性应激肝郁证大鼠模型的建立[J]．北京中医药大学学报，2003；26(2)：18-20．
51．熊振芳，朱清静．柴胡疏肝散对慢性束缚应激性肝郁证大鼠的影响[J]．中国中西医结合消化杂志，2004；12(4)：220～221
52．赵益业．肝郁证的免疫学探讨[J]．山东中医药大学学报，1997；21(1)：28-30
53．刘莲，臧佩林．保神开郁冲剂对肝郁证大鼠免疫学机制的影响[J]．辽宁中医学院学报，2001；3(2)：139-141
54．许辉，陈松，李加邦，等．特异性免疫应答在肝郁证与脾虚证及相互关系的初步研究[J]．中国医师杂志，2002；4(8)：829-831
55．杨冬花，李家邦，郑爱国，等．肝气郁结证模型大鼠Th1／Th2细胞因子变化及柴胡疏肝敞的干预作用[J]．湖南中医学院学报，2003；23(5)：4-7
56．李加邦，杨冬花，蒋荣鑫，等．NFOATc在肝气郁结、脾气虚证模型大鼠的表达以及柴胡疏肝散、四君子汤的干预作用[J]．中国现代医学杂志，2005；15(10)：1515-15171
57．陈泽奇，陈国林，金益强，等．肝气郁结证病理生理学基础研究[J]．中国现代医学杂志，2000；(10)：211
58．陈泽奇，陈国林，胡随瑜，等．肝气郁结证辅助实验诊断指标的初步研究[J]．中国现代医学杂志，2001；11(12)：81
59．陈泽奇，金益强，陈国林，等．肝气郁结证血浆去甲肾上腺素和肾上腺素测定结果分析[J]．中医药学报，1997；(5)：471
60．陈泽奇，陈国林，石林阶，等．肝气郁结证患者血浆LOENK、AVP、ANP含量分析[J]．湖南中医学院学报，1997；17(3)：37-39
61．胡随瑜，龙劲松，张宏耕．肝气郁结证、肝火上炎证与5OHTT、TPH基因多态性相关研究[J]．中国中医基础医学杂志，2004；10(3)：49-52
62．王爱成，王玉来，尹岭，等．肝气郁结证的PET影像学研究[J]．中医研究，2005；18(3)：24-251
63．陈建伟，李祥，陈进，等．正常人与肝气郁结证人指甲无机元素的比较分析[J]．广东微量元素科学，2001；8(11)：34-37．
64．马渊，周文霞，程军平．六味地黄汤对悬吊应激小鼠下丘脑—垂体—卵巢轴激素的影响[J]．中国实验方剂学杂志，2002；8(6)：18-20．
65．韩双红，王玉芬．君肾舒冲剂药理作用的研究[J]．中草药，1999；30(4)：282-284．
66．赵长鹰，沈英森．金水六君煎抗应激作用的实验研究[J]．新中医，1997；29(10)：35-37．
67．孙莉，甄铁梅．加味保元汤对束缚应激小鼠免疫功能影响的研究[J]．中国行为医学科学，1999；8(2)：99-101．
68．樊蔚虹，岳广欣．肝肾阴虚证大鼠下丘脑—垂体—甲状腺轴的变化及中药对其调节作用[J]．中国中医药信息杂志，2001；8(10)：21-22．
69．王蕾．不老丸对小鼠应激反应的影响[J]．中医研究，1998；11(5)：13-15．
70．缪硕斌．力康胶囊的抗应激作用研究[J]．沈阳药科大学学报，1998；15(1)：5-7．
71．唐已婷，陈家旭．三种中药复方对慢性束缚应激大鼠下丘脑—垂体—肾上腺轴的调节[J]．北京中医药大学学报，2002；25(3)：23-25．
72．钱瑞琴，孟勇．补益中药调整应激模型动物免疫细胞受抑及其可能机制[J]．中国中药杂志，2000；25(3)：169-170．
73．韩娟．慢性应激对大鼠下丘脑神经递质的影响及中药的调节作用[J]．吉林中医药，2002；22(4)：54．
74．李敏，赵法仍．生脉饮对热应激大鼠血浆锌、铜、铁等微量元素含量的影响[J]．卫生研究，1999；29(1)：45．
75．俞昌琪，彭小冰．炙甘草汤抗衰老等作用的实验观察[J]．贵阳中医学院学报，1999；21(1)：59．
76．张有志，聂惠民．柴胡加龙骨牡蛎汤等经方治疗抑郁症的动物行为学研究[J]．中国中医基础医学杂志，2001；7(7)：30．
77．肖延龄，钱令嘉，王万银．颐心宁对束缚应激大鼠心电图、血清皮质醇的影响[J]．中草药，2002；33(6)：526．
78．李红，蒋孟良．开胃理脾口服液抗应激及体重增长的实验研究[J]．湖南中医学院学报，2000；20(4)：23．
79．王苏静，郭艳幸．加味益气丸对小鼠抗应激作用的实验研究[J]．中医正骨，1998；10(6)：7．
80．徐书显，赵进沛．复方黄芪口服液对小鼠应激实验的影响[J]．现代中西医结合杂志，2000；9(23)：2341．
81．王琳，王天芳．消疲怡神口服液对应激大鼠下丘脑—垂体—肾上腺—胸腺轴影响的实验研究[J]．中国中西医结合杂志，2000；20(2)：116．
1．金惠铭．病理生理学[M]．北京：人民卫生出版社，2000
2．张明岛，陈福国．医学心理学[M]．上海：上海科学技术出版社，1998
3．胡随瑜，中医肝病常见证型的植物神经功能状态与研究．湖南中医杂志，1996，12(1)：11—14．
4．金益强．肝阳上亢本质的研究．中西医结合杂志，1988，8(8>：13ti—140．
5．金益强，刘爱平，张翔，等．肝风内动让病人血浆儿茶酚胺测定及其临床意义．湖南医科大学学报，1992，77(4)：356-358．
6．朱崇学，金益强，张翔，等．肝火上炎证和肝胆湿热证患者血浆几茶酚胺测定．湖南医科大学学报，1996，21(4)：308--310．
7．刘湘华，胡随瑜．肝阳上亢证患者个性、情绪特征及血浆精氨酸加压素水平的初步研究．湖南医科大学学报，1998，23(1)：31—34．
8．陈家旭．肝气虚证人格特征探讨．中国行为医学杂志，1993．2(1)：26—27．
9．徐志伟，严灿，李艳，等．加味四逆散对慢性心理应激大鼠的神经内分泌和行为学的调整作用．广州中医药大学学报2002；19(2)：127-129．
10．严灿，徐志伟，李艳，等．调肝、补肾、健脾方药对慢性心理应激大鼠单胺类神经递质影响的比较研究．中国中西医结合杂志2002；22(12)：925-927．
11．李艳，严灿，徐志伟，等．调肝、补肾、健脾治法及人参总皂甙对反复心理应激大鼠下丘脑及海马氨基酸含量的影响．中药药理与临床2003；19(2)：1-3．
12．严灿，徐志伟，吴丽丽等．慢性心理应激大鼠下丘脑环核昔酸系统和不同脑区核团c-fos蛋白表达的变化及中药复方JWSNS的影响．中国行为医学科学，2004；
13．严灿，徐志伟．肝主疏泄调畅情志功能的中枢神经生物学机制探讨．中国中西医结合杂志．2005，(05)：459-462
14．元晓丽，林文娟．焦虑和抑郁动物模型的研究方法和策略．心理科学进展 2005，13 (3)：327-332
15. Yamamoto T, Une T. Animal models of psychiatric disorder and their validity - from the perspective of behavioral pharmacology. Japanese Journal of Pharmacology, 2002, 120: 173～180
16. Paul Willner, David Benton, Emma Brown, et. al.. Depression, increases. craving. for sweet rewardsin animal and human models of depression and craving. Psychopharmacology, 1998; 136: 272-283.
17. Keeney A J, Hogg S. Behavioral consequences of repeated social defeat in the mouse: preliminary evaluation of a potential animal model of depression. Behavioural Pharmacology, 1999, 10: 753-764
1. Paul Willner, David Benton, Emma Brown, et. al.. Depression, increases. craving. for sweet rewardsin animal and human models of depression and craving. Psychopharmacology, 1998; 136: 272-283.
2. Keeney A J, Hogg S. Behavioral consequences of repeated social defeat in the mouse: preliminary evaluation of a potential animal model of depression. Behavioural Pharmacology, 1999, 10: 753-764
3．元晓丽，林文娟．焦虑和抑郁动物模型的研究方法和策略．心理科学进展2005，13 (3)：327-332
4. Yamamoto T, Une T. Animal models of psychiatric disorder and their validity - from the perspective of behavioral pharmacology. Japanese Journal of Pharmacology, 2002, 120: 173～180
5. Katz RJ: 1981; Animal models and human depressive disorders. Neurosci biobehav Rev 16: 525-534
6. Winner P, Towell A, Sampson D, Muscat. Sophokleous S: Reduction of sucrose preference by chronic mild stress and its restoration by a tricyclic antidepressant. Psychopharmacotogy 1987; 93: 358-364
1. Mastorakos G, Pavlatou MG, Mizamtsidi M. The hypotha-lamic-pituitary-adrenal and the hypothalamic-pituitary-go-nadal axes interplay [J]. Pediatr Endocrinol Rev, 2006, 3 (Suppl 1): 172-181.
2．洪枫，奚耕思．雌激素及应激与人类认知功能的关系．现代生物医学进展．2006，6(9)：99-104
1．邵枫，林文娟．应激对免疫作用机制的研究进展．心理学动态，1999，7(3)：1-6
2．庄洁：陈佩杰：张剑．心理应激对不同特质焦虑运动员T淋巴细胞亚群和糖皮质激素及其受体的影响．中国运动医学杂志，200，16(2)：174-177
1. Sapolsky RM, Zola Morgan S, Squire LR. Inhibition of glucocorticoid secretion by thehippocampal formation in the primate. J-Neurosci. 1991; 11(12): 3695-3704
2. Feldman S, Weidenfeld J. Glucocorticoid receptor antagonists in the hippoeampus modify the negative feedback following neural stimuli. Brain-Res. 1999; 821(1): 33-37
3. McEwen BS. Ann Rew Neurosei, 1999; 22: 105-109
4. Magarinos AM, Verdugo JM, MeEwen BS. Proe-natI-Acad-Sei-U-S-A, 1997; 94(25): 14002-14007
5. McEwen BS. Biol Psychatry, 2000; 48: 721-724
6. Gould E, et al. Biol Psychiatry, 1999; 46: 1472
7. Zhan RZ, Fujiwara N, Endoh H et al. Thiopental inhibits increases in [Ca2+]i induced by membrance depolarization, NMDA receptor activition, and ischemia in rat hippocampal and cortical slices. A nesthesiology, 1998; 89 (2) :456-66
8. Zhan RZ, Qi S, Wu C et al. Intravenous anesthetics differentially reduce neurotransmission damage caused by oxygenglucose feprivation in rat hippocarnpal slices in correlation with N-methyl-D-aspartate receptor inhibition. Crit Care Med, 2001; 29 (4): 808-813
9. Preston E, Webster J. Spectrophotometric measurement of experimental brain injury. J NeurosciMethods, 2000; 94 (2): 187-92
10．严乐勒，魏尔清，胡海涛．一种定量评价离体海马脑片缺氧／缺糖损伤的新方法．浙江大学学报(医学版)．2002；31(2)：81-85
1．夏军，叶慧，周义成．慢性应激大鼠抑郁模型的建立及其有效性的探讨，华中科技大学学报(医学版)．2005；34(4)：493-495
2. Lazarus RS, Folkman S. Stress, Appraisal and Coping. NewYork: Springer, 1984
3．不同群体的应激、应对与心理健康．许清鹏，陈龙，裴华．中国心理卫生杂志．2005；19(2)：94-96
4. LP, McEwen BS. Controversies surrounding glueocorticoid-mediated cell death in the hippocampus. J Chem Neuroanat, 1997; 13(3): 149-167.
5. Goldberg J. L. Barres B. A. The relationship between neuronal survival and reration. Annu. Rev. Neurosci. 2000; 23: 579-612.
6. Duman R. S, Malberg J, Nalcagawa S et al. Neuronal plasticity and survival in mood disorders. Biol Psychatry, 2000; 48: 732-739.
7. Bredt D. S., Snyder S. H. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. PROC. NATL. ACAD. SCI. U. S. A, 1989; 86(22): 9030-9033.
8. Stepien A, Chalimoniuk M, Strosznajder J. Serotonin SHT1B/1D receptor agonists abolish NMDA receptor evoked enhancement of nitric oxide synthase activity and cGMP concentration in brain cortex slices. Cephalalgia. 1999; 19(10): 859-865.
2．George P Ch rousOs，MD，Philip W Gold，MD．应激与应激系统紊乱的概念概述身体的和行为的内环境稳定．美国医学会杂志中文版，1992；2(5)：290-298
3．韩晓春．下丘脑-垂体-肾上腺在慢性应激过程中的调节作用．医学综述，2006；12(2)：73-75
4．李凌江，李则宣．精神应激的生物学致病机制研究-Ⅱ：神经内分泌机制．中国行为医学科学杂志，2005；14(2)：156-158
5. Bessel A, Vander Kolk. The psychobiology and psychopharmacology of PTSD. Human Psychopharmacology, 2001; 16: 49-64.
6. Hageman I, Andersen HS, JorgcnsenMB. Posttraumatic stress disorder: a review of psychobiology and pharmacotherapy. Acta sychiatr Scand, 2001; 104: 411-422.
7．李泽桂．大鼠室旁核CRF神经元在烫伤应激中的变化．第三军医大学学报，1999；21(3)：160-163
8．韩良辅．次声作用引起大鼠下丘脑c-fos mRNA与CRF mRNA的表达．第四军医大学学报，1999；20(1)：1-3
9. Kam K, Park Y, Cheon M, et al, Effects of immobilization stress on est rogen2induced surges of luteinizing hormone and pro lactin in ova riectomized rats [J]. Endocrine, 2000; 12 (3): 279-287.
10. Kalanta ridou S N, Makrigianna kis A, Zouma kis E, et al. Rep roductive functions of corticot ropin2releasing hormone: Research and potential clinical utility of antala rmins [J]. American Jour nal of Rep roductive Immunology, 2004; 51 (4): 269-274.
11. Murase M, Uemura T, Kondoh Y, et al. Role of corticot ropin2releasing hormone in ovarian steroidogenesis [J ]. Endocrine, 2002; 18 (3): 255-260.
12. Breen K M, Billings H J, Wagenmaker E R, et al. Endocrinebasis for disruptive effects of cortisol on preovulatory events[J] Endocrinology, 2005; 146 (4): 2107-2015.
13．黎萍，郭锡永．应激影响女性生殖内分泌功能机制的研究进展．中国妇幼健康研究，2006；17(3)：215-217
14. Tsigos C, Chrousos GP. Hypothalamic-pituitary-adrenal axis, neuroendocrine factors and stress[J]. J Psychosom Res, 2002; 53 (4): 865-871.
15. Venditti P, De Rosa R, Caldarone G, et al. Functional and biochemical characteristics of mitochondrial fractions from rat liver in cold2induced oxidative stress[J]. Cell Mol Life Sci, 2004; 61 (24): 310423116.
16．赵福东，董竟成．应激时内分泌变化对免疫功能影响的研究进展．国外医学内分泌学分册，2005；25(4)：241-243
17. Sapolsky RM, Uno H, Tarara R, et al. Hippocampal damage associated with prologned and fatal stress in primates [J]. JNeurosci, 1989; 9: 1705-1711.
18. Goldapple K, Segal Z, Garson C, et al. Modulation of cortieal2limbicpathways in major depression; treatment specific effects of cognitive be2havior therapy[J]. Arch Gen Psychiatry, 2004; 61 (1): 34241.
19．郑红，南路．男人、女人与应激反应[J]．世界科学，1992，(03)：23-25
20．李则宣，李凌江．应激动物模型设计的成败与思考．中国行为医学科学，2002；11(1)：105-110
21．吴振宇，杨军平，吴广均，等．小站台水环境不同应激时间对组织中5-羟色胺的影响．中国行为医学科学，2004；13(1)：11-13．
22．周萍，朱文锋．七情学说与应激理论．医学与哲学，1995；16(9)：484-485
23．刘晓伟，曲宏达．关于中医七情学说研究的思考．天津中医，2002；19(6)：44-45
24．赵平，冯前进．情志致病学说与心因性应激反应理论．山西中医学院学报，2000；1(1)：7-9
25．罗家发．情志医学研究的思考[J]．安徽中医学院学报，2002；21(2)：7-9
26．武刚．情志学说研究思路探析[J]．安徽中医学院学报，2001；10(4)：4-6
27．严灿，李艳，史亚飞．七情学说实验研究相关问题探讨．中国中医基础医学杂志，2001；(10)：11-13
28．中医现代化科技发展战略研究课题组．情志疾病的中医药防治研究与养生保健理论与方法的研究．世界科学技术—中药现代化，2000；4(2)：19-22
29．岳文浩，付文青，芦宗玉，等．“怒伤肝”机制研究[J]．医学与哲学，1995；16(9)：481-4831
30．王文勋，郑洪新，王继伟，等．怒伤肝与神经-内分泌-免疫系统失调探析[J]．辽宁中医杂志，1997；24(5)：205-2061
31．赵晓林，李恩，贾漪涛，等．滋补肝肾方药对慢性激怒应激大鼠下丘脑肝脏核糖体聚态的影响[J]．中国中医基础杂志，1997；3(5)：28～301
32．刘晓伟，张红梅，曲宏达，等．“怒伤气”大鼠血ACTH、CORT、IL-2、IL-8的含量变化[J]．中国全科医学，2004；7(22)：1653～16541
33．刘雁峰，王天芳，杨维益，等．复合应激因素致“慢性疲劳”模型大鼠脾脏变化的研究[J]．中国中医基础杂志，2000；6(6)：372～3731
34．王爱成，王玉来，尹岭，等．肝气郁结证的PET影像学研究[J]．中医研究，2005；18(3)：24～251
35．严灿，徐志伟，史亚飞．加减四逆散对慢性心理应激大鼠胸腺糖皮质激素受体作用的研究．中国中西医结合杂志．2002；22(9)：691-694
36．沈雁，匡调元，张伟荣，等．“恐伤肾”的实验研究[J]．中国医药学报，1991；6(1)：13～161
37．王米渠，马向东，段光周，等．“肾为先天之本”行为遗传中关于怒伤肾的表征[J]．中国中医基础杂志，1997；3(4)：23-25
38．王米渠．“恐伤肾”母子两代的生理研究．福建中医药．2003；34(1)：3-4
39．朱凌凌，童瑶，陈慧娟，张挺．脾的中西医学比较研究．浙江中医杂志，2006；41(1)：1-5
40．岳广欣，陈家旭，王竹风．中医心肝肾三脏在应激反应中的作用．辽宁中医杂志，2005；32(6)：528-530
41．乔明琦，张惠云，陈雨振，等．肝郁证动物模型研究的理论思考[J]．中国医学学报，1997；12(5)：42-44
42．温建余，祝丽霞，等．肝郁证大鼠单胺类递质变化与性行为关系的实验研究[J]．中国男科学杂志，2002；16(3)：205-207
43．叶林，叶向荣，等．肝郁证大鼠中枢神经递质变化的观察[J]．福建中医药，2002；33(2)：171-173
44．罗欣拉，熊振芳．柴胡疏肝散对慢性束缚应激性肝郁证大鼠下丘脑-垂体-肾上腺轴的调节作用[J]．湖北中医杂志，2003；25(11)：71-73
45．秦雪梅，王明军．逍遥散和柴胡对慢性束缚应激肝郁模型大鼠脑内单胺类神经递质的影响[J]．中药药理与临床，2005；21(2)：6～7
46．严灿，徐志伟．加减四逆散对慢性心理应激大鼠胸腺糖皮质激素受体作用的研究[J]．中国中西医结合杂志，2002；22(9)：691-693．
47．徐志伟，严灿．加减四逆散对慢性心理应激大鼠的神经内分泌和行为学的调整作用[J]．广州中医药大学学报，2002；19(2)：127-130
48．李艳，严灿．反复心理应激大鼠血浆和下丘脑单胺神经递质的变化及加减四逆散的影响[J]．中药药理与临床，2002；18(1)：1-4．
49．陈松，李家邦．肝郁证T、B淋巴细胞活性改变及疏肝治疗影响的实验研究[J]．湖南中医学院学报，2001；21(2)：7-9．
50．金光亮，南睿，郭耀珍．慢性应激肝郁证大鼠模型的建立[J]．北京中医药大学学报，2003；26(2)：18-20．
51．熊振芳，朱清静．柴胡疏肝散对慢性束缚应激性肝郁证大鼠的影响[J]．中国中西医结合消化杂志，2004；12(4)：220～221
52．赵益业．肝郁证的免疫学探讨[J]．山东中医药大学学报，1997；21(1)：28-30
53．刘莲，臧佩林．保神开郁冲剂对肝郁证大鼠免疫学机制的影响[J]．辽宁中医学院学报，2001；3(2)：139-141
54．许辉，陈松，李加邦，等．特异性免疫应答在肝郁证与脾虚证及相互关系的初步研究[J]．中国医师杂志，2002；4(8)：829-831
55．杨冬花，李家邦，郑爱国，等．肝气郁结证模型大鼠Th1／Th2细胞因子变化及柴胡疏肝敞的干预作用[J]．湖南中医学院学报，2003；23(5)：4-7
56．李加邦，杨冬花，蒋荣鑫，等．NFOATc在肝气郁结、脾气虚证模型大鼠的表达以及柴胡疏肝散、四君子汤的干预作用[J]．中国现代医学杂志，2005；15(10)：1515-15171
57．陈泽奇，陈国林，金益强，等．肝气郁结证病理生理学基础研究[J]．中国现代医学杂志，2000；(10)：211
58．陈泽奇，陈国林，胡随瑜，等．肝气郁结证辅助实验诊断指标的初步研究[J]．中国现代医学杂志，2001；11(12)：81
59．陈泽奇，金益强，陈国林，等．肝气郁结证血浆去甲肾上腺素和肾上腺素测定结果分析[J]．中医药学报，1997；(5)：471
60．陈泽奇，陈国林，石林阶，等．肝气郁结证患者血浆LOENK、AVP、ANP含量分析[J]．湖南中医学院学报，1997；17(3)：37-39
61．胡随瑜，龙劲松，张宏耕．肝气郁结证、肝火上炎证与5OHTT、TPH基因多态性相关研究[J]．中国中医基础医学杂志，2004；10(3)：49-52
62．王爱成，王玉来，尹岭，等．肝气郁结证的PET影像学研究[J]．中医研究，2005；18(3)：24-251
63．陈建伟，李祥，陈进，等．正常人与肝气郁结证人指甲无机元素的比较分析[J]．广东微量元素科学，2001；8(11)：34-37．
64．马渊，周文霞，程军平．六味地黄汤对悬吊应激小鼠下丘脑—垂体—卵巢轴激素的影响[J]．中国实验方剂学杂志，2002；8(6)：18-20．
65．韩双红，王玉芬．君肾舒冲剂药理作用的研究[J]．中草药，1999；30(4)：282-284．
66．赵长鹰，沈英森．金水六君煎抗应激作用的实验研究[J]．新中医，1997；29(10)：35-37．
67．孙莉，甄铁梅．加味保元汤对束缚应激小鼠免疫功能影响的研究[J]．中国行为医学科学，1999；8(2)：99-101．
68．樊蔚虹，岳广欣．肝肾阴虚证大鼠下丘脑—垂体—甲状腺轴的变化及中药对其调节作用[J]．中国中医药信息杂志，2001；8(10)：21-22．
69．王蕾．不老丸对小鼠应激反应的影响[J]．中医研究，1998；11(5)：13-15．
70．缪硕斌．力康胶囊的抗应激作用研究[J]．沈阳药科大学学报，1998；15(1)：5-7．
71．唐已婷，陈家旭．三种中药复方对慢性束缚应激大鼠下丘脑—垂体—肾上腺轴的调节[J]．北京中医药大学学报，2002；25(3)：23-25．
72．钱瑞琴，孟勇．补益中药调整应激模型动物免疫细胞受抑及其可能机制[J]．中国中药杂志，2000；25(3)：169-170．
73．韩娟．慢性应激对大鼠下丘脑神经递质的影响及中药的调节作用[J]．吉林中医药，2002；22(4)：54．
74．李敏，赵法仍．生脉饮对热应激大鼠血浆锌、铜、铁等微量元素含量的影响[J]．卫生研究，1999；29(1)：45．
75．俞昌琪，彭小冰．炙甘草汤抗衰老等作用的实验观察[J]．贵阳中医学院学报，1999；21(1)：59．
76．张有志，聂惠民．柴胡加龙骨牡蛎汤等经方治疗抑郁症的动物行为学研究[J]．中国中医基础医学杂志，2001；7(7)：30．
77．肖延龄，钱令嘉，王万银．颐心宁对束缚应激大鼠心电图、血清皮质醇的影响[J]．中草药，2002；33(6)：526．
78．李红，蒋孟良．开胃理脾口服液抗应激及体重增长的实验研究[J]．湖南中医学院学报，2000；20(4)：23．
79．王苏静，郭艳幸．加味益气丸对小鼠抗应激作用的实验研究[J]．中医正骨，1998；10(6)：7．
80．徐书显，赵进沛．复方黄芪口服液对小鼠应激实验的影响[J]．现代中西医结合杂志，2000；9(23)：2341．
81．王琳，王天芳．消疲怡神口服液对应激大鼠下丘脑—垂体—肾上腺—胸腺轴影响的实验研究[J]．中国中西医结合杂志，2000；20(2)：116．
1．金惠铭．病理生理学[M]．北京：人民卫生出版社，2000
2．张明岛，陈福国．医学心理学[M]．上海：上海科学技术出版社，1998
3．胡随瑜，中医肝病常见证型的植物神经功能状态与研究．湖南中医杂志，1996，12(1)：11—14．
4．金益强．肝阳上亢本质的研究．中西医结合杂志，1988，8(8>：13ti—140．
5．金益强，刘爱平，张翔，等．肝风内动让病人血浆儿茶酚胺测定及其临床意义．湖南医科大学学报，1992，77(4)：356-358．
6．朱崇学，金益强，张翔，等．肝火上炎证和肝胆湿热证患者血浆几茶酚胺测定．湖南医科大学学报，1996，21(4)：308--310．
7．刘湘华，胡随瑜．肝阳上亢证患者个性、情绪特征及血浆精氨酸加压素水平的初步研究．湖南医科大学学报，1998，23(1)：31—34．
8．陈家旭．肝气虚证人格特征探讨．中国行为医学杂志，1993．2(1)：26—27．
9．徐志伟，严灿，李艳，等．加味四逆散对慢性心理应激大鼠的神经内分泌和行为学的调整作用．广州中医药大学学报2002；19(2)：127-129．
10．严灿，徐志伟，李艳，等．调肝、补肾、健脾方药对慢性心理应激大鼠单胺类神经递质影响的比较研究．中国中西医结合杂志2002；22(12)：925-927．
11．李艳，严灿，徐志伟，等．调肝、补肾、健脾治法及人参总皂甙对反复心理应激大鼠下丘脑及海马氨基酸含量的影响．中药药理与临床2003；19(2)：1-3．
12．严灿，徐志伟，吴丽丽等．慢性心理应激大鼠下丘脑环核昔酸系统和不同脑区核团c-fos蛋白表达的变化及中药复方JWSNS的影响．中国行为医学科学，2004；
13．严灿，徐志伟．肝主疏泄调畅情志功能的中枢神经生物学机制探讨．中国中西医结合杂志．2005，(05)：459-462
14．元晓丽，林文娟．焦虑和抑郁动物模型的研究方法和策略．心理科学进展 2005，13 (3)：327-332
15. Yamamoto T, Une T. Animal models of psychiatric disorder and their validity - from the perspective of behavioral pharmacology. Japanese Journal of Pharmacology, 2002, 120: 173～180
16. Paul Willner, David Benton, Emma Brown, et. al.. Depression, increases. craving. for sweet rewardsin animal and human models of depression and craving. Psychopharmacology, 1998; 136: 272-283.
17. Keeney A J, Hogg S. Behavioral consequences of repeated social defeat in the mouse: preliminary evaluation of a potential animal model of depression. Behavioural Pharmacology, 1999, 10: 753-764
1. Paul Willner, David Benton, Emma Brown, et. al.. Depression, increases. craving. for sweet rewardsin animal and human models of depression and craving. Psychopharmacology, 1998; 136: 272-283.
2. Keeney A J, Hogg S. Behavioral consequences of repeated social defeat in the mouse: preliminary evaluation of a potential animal model of depression. Behavioural Pharmacology, 1999, 10: 753-764
3．元晓丽，林文娟．焦虑和抑郁动物模型的研究方法和策略．心理科学进展2005，13 (3)：327-332
4. Yamamoto T, Une T. Animal models of psychiatric disorder and their validity - from the perspective of behavioral pharmacology. Japanese Journal of Pharmacology, 2002, 120: 173～180
5. Katz RJ: 1981; Animal models and human depressive disorders. Neurosci biobehav Rev 16: 525-534
6. Winner P, Towell A, Sampson D, Muscat. Sophokleous S: Reduction of sucrose preference by chronic mild stress and its restoration by a tricyclic antidepressant. Psychopharmacotogy 1987; 93: 358-364
1. Mastorakos G, Pavlatou MG, Mizamtsidi M. The hypotha-lamic-pituitary-adrenal and the hypothalamic-pituitary-go-nadal axes interplay [J]. Pediatr Endocrinol Rev, 2006, 3 (Suppl 1): 172-181.
2．洪枫，奚耕思．雌激素及应激与人类认知功能的关系．现代生物医学进展．2006，6(9)：99-104
1．邵枫，林文娟．应激对免疫作用机制的研究进展．心理学动态，1999，7(3)：1-6
2．庄洁：陈佩杰：张剑．心理应激对不同特质焦虑运动员T淋巴细胞亚群和糖皮质激素及其受体的影响．中国运动医学杂志，200，16(2)：174-177
1. Sapolsky RM, Zola Morgan S, Squire LR. Inhibition of glucocorticoid secretion by thehippocampal formation in the primate. J-Neurosci. 1991; 11(12): 3695-3704
2. Feldman S, Weidenfeld J. Glucocorticoid receptor antagonists in the hippoeampus modify the negative feedback following neural stimuli. Brain-Res. 1999; 821(1): 33-37
3. McEwen BS. Ann Rew Neurosei, 1999; 22: 105-109
4. Magarinos AM, Verdugo JM, MeEwen BS. Proe-natI-Acad-Sei-U-S-A, 1997; 94(25): 14002-14007
5. McEwen BS. Biol Psychatry, 2000; 48: 721-724
6. Gould E, et al. Biol Psychiatry, 1999; 46: 1472
7. Zhan RZ, Fujiwara N, Endoh H et al. Thiopental inhibits increases in [Ca2+]i induced by membrance depolarization, NMDA receptor activition, and ischemia in rat hippocampal and cortical slices. A nesthesiology, 1998; 89 (2) :456-66
8. Zhan RZ, Qi S, Wu C et al. Intravenous anesthetics differentially reduce neurotransmission damage caused by oxygenglucose feprivation in rat hippocarnpal slices in correlation with N-methyl-D-aspartate receptor inhibition. Crit Care Med, 2001; 29 (4): 808-813
9. Preston E, Webster J. Spectrophotometric measurement of experimental brain injury. J NeurosciMethods, 2000; 94 (2): 187-92
10．严乐勒，魏尔清，胡海涛．一种定量评价离体海马脑片缺氧／缺糖损伤的新方法．浙江大学学报(医学版)．2002；31(2)：81-85
1．夏军，叶慧，周义成．慢性应激大鼠抑郁模型的建立及其有效性的探讨，华中科技大学学报(医学版)．2005；34(4)：493-495
2. Lazarus RS, Folkman S. Stress, Appraisal and Coping. NewYork: Springer, 1984
3．不同群体的应激、应对与心理健康．许清鹏，陈龙，裴华．中国心理卫生杂志．2005；19(2)：94-96
4. LP, McEwen BS. Controversies surrounding glueocorticoid-mediated cell death in the hippocampus. J Chem Neuroanat, 1997; 13(3): 149-167.
5. Goldberg J. L. Barres B. A. The relationship between neuronal survival and reration. Annu. Rev. Neurosci. 2000; 23: 579-612.
6. Duman R. S, Malberg J, Nalcagawa S et al. Neuronal plasticity and survival in mood disorders. Biol Psychatry, 2000; 48: 732-739.
7. Bredt D. S., Snyder S. H. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. PROC. NATL. ACAD. SCI. U. S. A, 1989; 86(22): 9030-9033.
8. Stepien A, Chalimoniuk M, Strosznajder J. Serotonin SHT1B/1D receptor agonists abolish NMDA receptor evoked enhancement of nitric oxide synthase activity and cGMP concentration in brain cortex slices. Cephalalgia. 1999; 19(10): 859-865.