慢性束缚应激对大鼠LC-NE系统的影响及逍遥散的干预作用的研究
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摘要
研究背景
近年来,肝郁证模型以其实证性研究方法而被广泛采用。动物模型是肝郁证实验研究的基础,是研究成败与否的关键所在。肝郁证模型以中医辨证论治理论为指导,运用现代科技方法,模拟出具有中医肝郁证候特征的模型,为临床治疗提供理想的实验观察对象。
中医证候模型必须以中医病因学理论为指导来塑造,鉴于情志失调在肝郁证发病中的作用,国、内外相继出现了多种模型,主要从两个方面入手：一是用过量或毒性药物造成急、慢性肝脏中毒损伤来复制模型；二是通过情志刺激改变动物的生理状况来复制模型。药物造模,可造成实质性肝脏的急、慢性毒性损伤,中医肝郁证并不等于肝脏的实质性损伤,同时药物也会影响动物多个器官系统的功能。药物造模法与中医理论证的实质差别太大,故一直未受认可,逐渐被淘汰；情志刺激造模,模拟出的这种肝郁证模型较为常用,因为肝郁证的产生多因消极应激及不良情绪体验所致。考虑到情志失调在肝郁证发病中的关键作用,一些研究者据此采用捆绑、限制活动或激怒等情志刺激方式来诱发动物产生肝郁证,在模型塑造方面取得了可喜的尝试。最早是用细条束缚四肢,限制活动。束缚法是国内更受认可的肝郁证造模方法,但这种束缚可能会造成实验动物躯体伤害。因此相关学者采用模具束缚法,模拟情志不舒的中医病因,避免了对动物躯体的伤害；为避免动物对束缚制动的习惯化,后对造模的时间采取随机化的方式,即每一次刺激,对大鼠而言都是一种新的应激。此类造模方法优点有二：能模拟人类心理郁怒而不得发泄,压抑体内的愤恨,久而成病,这本身就是一种慢性心理应激的状态,不会造成躯体损害；可以排除由于其他因素造成的病因病机方面的干扰,与临床上肝郁证的发病机制较为吻合。此后,以束缚制动作为肝郁证的造模方法被广大学者所认可,国外学者在研究应激、情绪异常等疾病时也多用此类模型。
调节机体的核心机构是五脏,对于情绪变化,尤其在调节情志因素(心理应激)引起的各种变化时,肝脏起着决定性的作用。对于各种刺激所引起的气机变化,主要由肝脏来调整。肝主疏泄,主藏血,有主动主升的生理功能及性喜条达的生理特性。肝主疏泄,即具有疏通、宣泄、调畅之义,包括了肝脏的多项生理功能。肝疏泄功能正常,全身气血才得以调畅,情志才得以条达,饮食的消化与排泄、水谷精微的输布与转归、津液的排泄等功能才得以正常。因此,一旦肝失条达疏畅之性,则会导致肝之疏泄功能失常,首先发生气机阻滞的病理变化,形成肝郁证,肝郁证为病广泛,常见于临床多种疾病中,是中医脏腑证候中常见的证候之一,也是中医肝病发病学的主要环节。因此,研究肝郁证的实质,能为临床多种疾病的诊断和防治提供新的思路和方法。
一般认为,肝郁病证的产生,多是由于情志不遂,饮食失节,劳逸失度,用药失当等多种因素导致。肝郁证的主要表现是精神抑郁,情绪不宁等症。这是由于肝气疏泄不及,肝失条达而致。从心理应激发生的病理机制和中医各脏腑的具体功能特点而言,中医的“肝”是机体调节心理应激反应的核心。在情志异常(心理应激)的状态下,最先受到影响的是人体的气机,进一步发展则可导致气与血的运行及功能的异常。
从肝的现代生理病理学研究成果来看,中医所论肝病证候,多出现神经、内分泌和免疫等系统的功能改变,如肝郁时,去甲肾上腺素、肾上腺素、5-羟色胺和多巴胺等中枢神经递质,皮肤电位,血中儿茶酚胺等激素,肾上腺的组织结构以及CD4+/CD8+等免疫系统多项指标均发生改变；并且多伴有交感神经偏亢。这提示中医所说的肝的功能与西医学的神经一内分泌一免疫网络密切相关。近年来,对肝脏实质的研究进一步表明,肝脏的生理功能和病理变化涉及人体多个系统,并以神经、内分泌、免疫、消化、心血管等系统疾病为多,这表明中医肝脏功能与机体不良情志等心理应激状态下导致的神经内分泌紊乱、免疫功能异常等密切相关。
现代研究认为,肝郁证是在抑郁、焦虑、悲痛等应激状态下,以高级神经中枢调节紊乱为前提,对神经、内分泌、免疫、循环、消化、感觉等多个系统的某些病理改变、病证表现的概括。中医古典医籍中虽然没有应激这一提法,但古代医家很早就已认识到各种不良刺激对机体的影响,非常重视情志因素对机体的影响。根据中医藏象理论,肝主疏泄为肝脏基本的生理功能之一,它对整个机体的气机调畅起着至关重要的作用,其中肝脏的生理、心理功能等方面,都与应激有联系。近年来,对肝脏实质的研究进一步表明,肝脏的生理功能和病理变化涉及人体许多个系统,神经、内分泌、免疫、消化、心血管等系统疾病为最多,这表明中医肝脏功能与机体不良情志等心理应激状态下导致的神经内分泌紊乱、免疫功能异常等密切相关。
国外对应激所涉及的下丘脑-垂体-肾上腺皮质轴(HPA)、蓝斑-去甲肾上腺素能系统(LC-NE)等研究较为全面。虽然肝郁证与神经内分泌系统的关系是研究的热点,但是多集中在HPA轴、下丘脑-垂体-甲状腺轴(HPT)和下丘脑-垂体-性腺轴(HPGA)上,对在慢性束缚应激中,肝郁证物质基础与LC-NE能系统分泌的神经递质相关性方面的研究尚未报道,因此,从应激角度出发,以中医药理论为指导,探讨肝主疏泄的功能对于应激反应的作用具有重要意义。
研究目的
观察肝郁模型大鼠的一般状态、体重、行为学的变化、检测血清中NE水平及组织形态学以验证及评价模型；检测各组大鼠蓝斑中NE合成酶TH、DBH以及神经肽CRF蛋白与基因的表达水平,并观测逍遥散和氟西汀干预后的表达。从LC-NE系统的角度对肝郁证在神经系统的物质基础进行初步探讨,为肝郁证的实质性研究提供一定的依据。
研究方法
1.慢性束缚应激肝郁模型大鼠的建立
动物分组：实验用清洁级雄性SD大鼠72只,鼠龄5周,体重(150士20)g,于造模前适应性饲养1周,随机分为对照组(A组)、肝郁证模型组(B组)、逍遥散治疗组(C组),氟西汀治疗组(D组),每组18只。A组3只1笼,其余3组单笼孤养。
模型建立：根据文献报道模拟中医肝郁证的病因,将B、C、D组大鼠置于特制的束缚制动筒内,固定身体与尾部,使之不能随意活动。每日束缚制动1次,造模时间随机,持续时间从第1天的1h逐渐增至每天6h,连续21d,造模。A、C组大鼠在束缚制动期间禁食、禁水。A组大鼠不予任何干预。
药物干预：A、B组大鼠以生理盐水灌胃,C组大鼠根据正常成人逍遥散每日用量,按体表面积换算成大鼠用药量为10g/kg.d,氟西汀为5mg/kg.d根据大鼠体重每日下午16时经胃灌相应药量。
模型评价：从一般状态、液体消耗、行为学、血清NE水平、逍遥散与氟西汀干预后疗效等方面评价模型的可靠性。
2.用H&E染色、尼氏染色方法,观察大鼠LC形态学改变和尼氏体的变化。
3.用免疫组化法检测LC中TH、DBH以及神经肽CRF的表达变化。用Imagepro plus6.0软件进行分析。
4.蛋白免疫印迹法(western blotting)检测LC中TH、DBH以及神经肽CRF的表达变化。
5.实时荧光定量(real-time PCR)检测LC中TH、DBH以及神经肽CRF的表达变化
6.所有数据采用SPSS17.0软件包进行统计分析。计量资料结果以x±s表示,两组间比较采用t-test。多组间比较采用One way ANOVA,首先对数据进行方差齐性检验,方差齐则用LSD法进间两两比较,如果数据方差不齐,则采用Welch法进行方差分析,应用Dunnett T3法进行两两比较。等级资料资料采用Kruskal-Wallis H非参数检验进行分析,P<0.05为有显著性差异。
研究结果
1.从一般状况来看,B组大鼠造模开始时表现为易激惹、挣扎,随后表现为倦怠少动、喜好贴壁、叫声弱细、反应迟钝、饮食减少,伴有毛色晦暗发黄、大便稀溏等肝郁证的表现。C、D组大鼠上述反应较轻,而A组未见异常行为状态。各组间体重增长存在显著差异(P<0.01),B组生长较为缓慢,C,D组服药后体重增长缓慢的情况得到改善。旷场实验发现B组的直立次数、理毛次数和穿越格数均显著低于A组(P<0.01)。液体消耗实验提示各组间糖水偏好存在显著性差异(P<0.01),B组的糖水偏好率明显小于其它三组。血清NE发现：B组的血清NE浓度明显高于A组(P<0.01)。说明慢性束缚应激复制出了大鼠肝郁证模型,有效的模拟出临床中肝郁证兴趣丧失、快感缺乏等症状。
2.血清NE水平检测显示：B组的血清NE浓度明显高于A组(P<0.01)。
3.H&E染色显示,大鼠LC组织形态未见明显异常,尼氏染色可见LC神经元内尼氏体染色增强、数量增多,但与正常组比较,模型组中LC未见明显异常。
4.免疫组化法检测LC中TH、DBH以及神经肽CRF的表达变化,发现B组TH、DBH和CRF的阳性神经元数目均大于A组(P<0.05),染色强度也明显增强,平均光密度值也明显高于A组(P<0.05)。经逍遥散,氟西汀干预的组显示出有下调二者表达的作用。
5.蛋白免疫印迹法(western blotting)检测LC中TH、DBH以及神经肽CRF的表达变化。与A组比较,B组TH、DBH和CRF蛋白表达显著增高(P<0.05),C、D组TH、DBH和CRF蛋白表达较B组明显降低(P<0.05)。
6.实时荧光定量(Real-time PCR)检测LC中TH、DBH以及神经肽CRF的表达变化。与A组比较,B组TH、DBH和CRFmRNA的表达显著增高(P<0.05),C、D组TH、DBH和CRFmRNA表达较B组明显降低(P<0.05)。
结论
1.本次研究采用慢性束缚应激的方法,复制了出大鼠肝郁证模型。
2.慢性束缚应激过程中,大鼠LC的组织形态未见明显异常；尼氏染色可见LC神经元内尼氏体染色增强、数量增多,模型组形态亦未见明显异常。提示21天的慢性束缚应激不会引起大鼠LC组织的形态学变化。
3.慢性束缚应激激活了大鼠LC-NE系统,使其释放NE儿茶酚胺类神经递质,进而使NE能神经元中合成酶TH、DBH增加；同时应激使神经肽CRF促进LC-NE神经元活性,与LC-NE系统形成交互影响,从而引起机体的兴奋、警觉、紧张及焦虑等情绪反应,而逍遥散起到了有效的干预效应,下调其过表达。
Background
Recently, depression syndrome model, as empirical research has been widely adopted by others. The animal model is on the basis of the experimental study of depression syndrome, is also the key of the success or failure of the reaserch. Depression syndrome model simulate specific TCM (Traditional Chinese medicine) syndrome on base of traditional Chinese medicine diagnosis and treatment theory by modern technology to offer the clinical ideal experimental observation object.
TCM syndrome model must be base on TCM etiology theory.To take into account the critical role of emotional disorders in liver depression, the pathogenesis have appeared a variety of models both at home and abroad, mainly two aspects: First, using excess or toxic drugs to induce acute and chronic liver poisoning injury to simulate depression model; Secondly, to change the physiological status of the animal to replicate the model by emotional stimulation.Drug modeling, acute and chronic toxicity can cause liver substantial damage,TCM depression Syndrome does not mean to substantial damage to the liver, and drugs also affect the animal multiple organ system function. Drug modeling had a significant difference from TCM theory, it has been an unapproved by others, phased out gradually.
The kernel institutions to regulate the body's is five organs. In the psychological aspects:the liver plays a decisive role in this procedure.To adjust the Qi caused by a variety of external stimulation, mainly regulated by the liver. Liver have a catharsis function, control the blood circulation. Liver regulate the catharsis that is it have the physiological characteristics of dispersion, catharsis. The normal function of liver catharsis can circulate systemic blood smoothly, regulate the emotions, digestion and excretion of dietary cereal better, semen excretion and help to reach to whole body. Therefore, once liver fail up to charge of Qi, will lead to liver dysfunction of catharsis, air-block pathological changes will happen first, then hepatic depression syndrome will be formed. Hepatic depression is an extensive syndrome, common disease in clinic and common syndromes in TCM viscera syndrome, also is one of the major aspects of liver disease pathogenesis in TCM. Therefore, explore the substance of hepatic depression syndrome can provide new ideas and methods for clinical diagnosis and prevention of many diseases.
Generally, liver depression disease and syndrome produce many unmoral emotions due to a variety of factors result from diet disorder, maladjustment, drug misconduct.Liver emotional disorders, such as depression, mood restless due to liver catharsis is not sufficient. From the pathological mechanisms of psychological stress and the specific features of various organs in Chinese medicine," liver" is the core which the body regulate the psychological stress response.Under abnormal emotional (psychological stress), Qi of body will be affected firstly, it can cause circulation and functional abnormalities of Qi and blood for further development. From the pathological mechanisms of psychological stress and the specific features of various organs in Chinese medicine," liver" is the core which the body regulate the psychological stress response. Under abnormal emotional (psychological stress), Qi of body will bet firstly affected, it can cause circulation and functional abnormalities of Qi and blood for further development.
Recently, from the physiological pathology of the liver research, the changes of the liver physiological function and pathological involve in the multiple systems, and the nervous,endocrine, immune etc. For example,When stagnation,the central neurotransmitter norepinephrine, epinephrine, serotonin amine,dopamine, skin potential, several catecholamines in the blood, adrenal organizational structure, and CD4+/CD8+in immune system will be changed; and more associated with sympathetic abnormalism.This show that liver function in TCM closely relate to Western medicine neuro-endocrine-immune network. Research on liver further show that the physiological function and pathological changes of the liver is involved in many systems, the nervous, endocrine, immune, digestive, cardiovascular system diseases are be observed frequently, suggesting that TCM liver function are closely related neuroendocrine disorders, immune dysfunction caused by poor emotional state and psychological stress.
Studies suggest that hepatic depression syndrome generalize about the nervous, endocrine, immune, circulatory, digestive, sensory and other systems of certain pathological changes depression, anxiety, grief, stress, premising on nerve center of the adjustment disorder. Although there is no stress formulation in Chinese medicine classical medical books, the ancient physicians had early recognized that a variety of adverse stimuli effects on the body, they attached great importance to the influence of emotional factors on the body. According to the theory of Zang organs,Liver control dispersion is one of a liver basic physiological functions, it plays a crucial role in regulating entire body, the liver of the physiological, psychological functions relate with the flow of Qi. In recent years, studies of the liver parenchyma further show that the physiological functions and pathological changes of the liver relate to many of the body system and the nervous, endocrine, immune, digestive, cardiovascular system diseases, which indicates that liver function are closely related with neuroendocrine disorders, immune dysfunction caused by bad emotional and psychological stress.
The rearch of stress involved in the hypothalamic-pituitary-adrenal cortex system (HPA), the locus coeruleus-sympathetic-adrenal medullary system (LC-NE) and so more comprehensive in abroad than our country. In our country, although the research focus on the relationship of the liver and neuroendocrine system is popular, the research focus on the HPA axis, the hypothalamic-pituitary-thyroid axis (HPT) or the hypothalamic-pituitary gonadal axis (HPGA).The material basis of hepatic depression syndrome relevance of neurotransmitters of LC-NE secretion under chronic restraint stress has not been reported.Thereby,we try to explore liver function of controlling dispersion is of great significance base on the Chinese medicine theory.
Research purposes
To observe the general state of rats in the liver depression model, body weight, behavior, serological NE level and histological evaluation model to validate the model.Detected protein and gene expression of the synthetic enzyme TH and DBH in LC-NE system and neuropeptides CRF, and observing variation level after Xiaoyao powder and fluoxetine intervention. Explore the liver substantiality in the nervous system in LC-NE system, and offer a evidence for liver substantiality research.
Research Methods
(1) Establishment of chronic restraint stress liver depression rats model
Groups:5-weeks-old72SD rats(150±20) g were randomly divided into four groups with18rats in each group after one-week's adaptation living:group A(control group), group B(model group), group C(Xiaoyao Powder treatment group),fluoxetine treatment group (D),The rats from the group B,C and D were housed individually in cages while rats from group A were be housed in two cage.
Model establishment:Rats from group B, C and D were be bound in a specific bound frame, fixed body and tail. Restraint time were be randomly and gradually durated1day1h to6h at day21th.
Drug Intervention:Rats from group C, D were deprived from diet and water during chronic immobilization stress. The rats in group A did not give any other intervention.At16:00pm daily, Rats from group A, B were given2ml physiological saline by intragastric administration. Rats from group C were given Xiaoyao powder with the same ways according to body surface area converted into rat dosage(10g/kg.d), Group D rats were given fluoxetine (5mg/kg.d).
Model evaluation:mode reliability will be evaluated by the observation of behavior, fluid consumption test, serum NE level, drug intervention efficacy.
2. H&E staining to observe the morphological changes in rat LC and the changes of Nissl bodies by Nissl staining.
3. Expression of TH, DBH in LC and neuropeptides CRF detected by immunohistochemical method and analyzed with Imagepro plus6.0.
4. Expression of TH, DBH in LC and neuropeptides CRF detected by Western blotting.
5. Expression of TH, DBH in LC and neuropeptides CRF detected by Real-time fluorescence quantitative (real-time PCR).
6. All data were be statistically analyzed using SPSS17.0software package. The measurement data results show in x±s.Statistics of comparison between the two groups were be used t-test;Statistical comparison among multiple groups will be chose One way ANOVA;The first test for homogeneity of variance of the data, homogeneity of variance with LSD method; if the data is the heterogeneity of variance, the Welch method of variance analysis;Comparison between each two by Dunnett T3method;Hierarchical data using Kruskal-Wallis H nonparametric test analysis.P<0.05was considered significant difference.
Results
1. Group B rat model manifested irritability, struggle, and then manifested as fatigue, lack of exercise preferences adherent sounds weak and thin, unresponsive, eating less, accompanied by the coat dull yellow stool thin pond Syndrome of depressed performance. C and D rats were mild, while no abnormal behavior state in group A. The rats' weight increased and significantly different from other groups (P<0.01). It showed rats in group B grew the slowest, while rats in group C, D has been rectified after durg intervention. In open field test,The erect time,grooming time and traversing grid also reduced in group B(P<0.01).Fluid consumption test revealed that group B lost appetite for sweet water during the whole procession. Statistics showed there were significant differences between these four groups (P<0.01).
2. The serum level of NE was also increased in rat model(P<0.01).
3. H&E staining showed that the LC morphology in rats are normal. The Nissl visible neurons and the number. of Nissl body enhanced, but model group has no obvious morphological abnormalities.
4. Immunohistochemical staining showed positive neurons of the TH, DBH and neuropeptides of CRF in group B were greater than group A (P<0.05), the staining intensity was significantly enhanced, the average optical density values significantly higher than group A (P<0.05). Treatment group showed a downward expression.
5. Western blot showed TH, DBH and neuropeptides of CRF in LC. Compared with group A, TH, DBH and CRF protein expression was significantly higher (P <0.05) in Group B, Group C and D significantly lower than group B (P<0.01).
6. Real-time fluorescence quantitative (real-time PCR) detection TH, DBH and neuropeptides of CRF mRNA in LC. Compared with group A, TH, DBH and neuropeptides of CRF mRNA in group B significantly higher (P<0.05), Group C and D significantly lower than group B (P<0.01).
Conclusion
1.The rat model of liver depression syndrome from chronic constraint stress is reliable and credible.
2. In chronic restraint stress process, no obvious abnormalities in the morphology of the rat LC, no obvious abnormalities; Nissl staining revealed that the LC neurons Nissl body staining increased.But there were no obvious morphological abnormalities in model group.This showed that chronic stress(21days) does not cause morphological changes in rat LC tissue.
3. This project firstly explored the hepatic depression syndrome in Locus coeruleus-noradrenergic system, and provides a theoretical basis for the further study of the molecular mechanisms of hepatic depression syndrome, and also give a beneficial trial for deeper research of the essence of liver depression syndrome.
4. Chronic restraint stress activated rat LC-NE system to release catecholamines NE, thereby TH and DBH level was increased in synthase NE neurons; at the same time, stress activating the neuropeptide CRF promoted LC-NE neurons activity, with the formation of LC-NE system interaction, causing the body's excitement, alertness, tension and anxiety and other emotional responses.However, Xiaoyao powder treatment can effectively reverse the effect.
近年来,肝郁证模型以其实证性研究方法而被广泛采用。动物模型是肝郁证实验研究的基础,是研究成败与否的关键所在。肝郁证模型以中医辨证论治理论为指导,运用现代科技方法,模拟出具有中医肝郁证候特征的模型,为临床治疗提供理想的实验观察对象。
中医证候模型必须以中医病因学理论为指导来塑造,鉴于情志失调在肝郁证发病中的作用,国、内外相继出现了多种模型,主要从两个方面入手：一是用过量或毒性药物造成急、慢性肝脏中毒损伤来复制模型；二是通过情志刺激改变动物的生理状况来复制模型。药物造模,可造成实质性肝脏的急、慢性毒性损伤,中医肝郁证并不等于肝脏的实质性损伤,同时药物也会影响动物多个器官系统的功能。药物造模法与中医理论证的实质差别太大,故一直未受认可,逐渐被淘汰；情志刺激造模,模拟出的这种肝郁证模型较为常用,因为肝郁证的产生多因消极应激及不良情绪体验所致。考虑到情志失调在肝郁证发病中的关键作用,一些研究者据此采用捆绑、限制活动或激怒等情志刺激方式来诱发动物产生肝郁证,在模型塑造方面取得了可喜的尝试。最早是用细条束缚四肢,限制活动。束缚法是国内更受认可的肝郁证造模方法,但这种束缚可能会造成实验动物躯体伤害。因此相关学者采用模具束缚法,模拟情志不舒的中医病因,避免了对动物躯体的伤害；为避免动物对束缚制动的习惯化,后对造模的时间采取随机化的方式,即每一次刺激,对大鼠而言都是一种新的应激。此类造模方法优点有二：能模拟人类心理郁怒而不得发泄,压抑体内的愤恨,久而成病,这本身就是一种慢性心理应激的状态,不会造成躯体损害；可以排除由于其他因素造成的病因病机方面的干扰,与临床上肝郁证的发病机制较为吻合。此后,以束缚制动作为肝郁证的造模方法被广大学者所认可,国外学者在研究应激、情绪异常等疾病时也多用此类模型。
调节机体的核心机构是五脏,对于情绪变化,尤其在调节情志因素(心理应激)引起的各种变化时,肝脏起着决定性的作用。对于各种刺激所引起的气机变化,主要由肝脏来调整。肝主疏泄,主藏血,有主动主升的生理功能及性喜条达的生理特性。肝主疏泄,即具有疏通、宣泄、调畅之义,包括了肝脏的多项生理功能。肝疏泄功能正常,全身气血才得以调畅,情志才得以条达,饮食的消化与排泄、水谷精微的输布与转归、津液的排泄等功能才得以正常。因此,一旦肝失条达疏畅之性,则会导致肝之疏泄功能失常,首先发生气机阻滞的病理变化,形成肝郁证,肝郁证为病广泛,常见于临床多种疾病中,是中医脏腑证候中常见的证候之一,也是中医肝病发病学的主要环节。因此,研究肝郁证的实质,能为临床多种疾病的诊断和防治提供新的思路和方法。
一般认为,肝郁病证的产生,多是由于情志不遂,饮食失节,劳逸失度,用药失当等多种因素导致。肝郁证的主要表现是精神抑郁,情绪不宁等症。这是由于肝气疏泄不及,肝失条达而致。从心理应激发生的病理机制和中医各脏腑的具体功能特点而言,中医的“肝”是机体调节心理应激反应的核心。在情志异常(心理应激)的状态下,最先受到影响的是人体的气机,进一步发展则可导致气与血的运行及功能的异常。
从肝的现代生理病理学研究成果来看,中医所论肝病证候,多出现神经、内分泌和免疫等系统的功能改变,如肝郁时,去甲肾上腺素、肾上腺素、5-羟色胺和多巴胺等中枢神经递质,皮肤电位,血中儿茶酚胺等激素,肾上腺的组织结构以及CD4+/CD8+等免疫系统多项指标均发生改变；并且多伴有交感神经偏亢。这提示中医所说的肝的功能与西医学的神经一内分泌一免疫网络密切相关。近年来,对肝脏实质的研究进一步表明,肝脏的生理功能和病理变化涉及人体多个系统,并以神经、内分泌、免疫、消化、心血管等系统疾病为多,这表明中医肝脏功能与机体不良情志等心理应激状态下导致的神经内分泌紊乱、免疫功能异常等密切相关。
现代研究认为,肝郁证是在抑郁、焦虑、悲痛等应激状态下,以高级神经中枢调节紊乱为前提,对神经、内分泌、免疫、循环、消化、感觉等多个系统的某些病理改变、病证表现的概括。中医古典医籍中虽然没有应激这一提法,但古代医家很早就已认识到各种不良刺激对机体的影响,非常重视情志因素对机体的影响。根据中医藏象理论,肝主疏泄为肝脏基本的生理功能之一,它对整个机体的气机调畅起着至关重要的作用,其中肝脏的生理、心理功能等方面,都与应激有联系。近年来,对肝脏实质的研究进一步表明,肝脏的生理功能和病理变化涉及人体许多个系统,神经、内分泌、免疫、消化、心血管等系统疾病为最多,这表明中医肝脏功能与机体不良情志等心理应激状态下导致的神经内分泌紊乱、免疫功能异常等密切相关。
国外对应激所涉及的下丘脑-垂体-肾上腺皮质轴(HPA)、蓝斑-去甲肾上腺素能系统(LC-NE)等研究较为全面。虽然肝郁证与神经内分泌系统的关系是研究的热点,但是多集中在HPA轴、下丘脑-垂体-甲状腺轴(HPT)和下丘脑-垂体-性腺轴(HPGA)上,对在慢性束缚应激中,肝郁证物质基础与LC-NE能系统分泌的神经递质相关性方面的研究尚未报道,因此,从应激角度出发,以中医药理论为指导,探讨肝主疏泄的功能对于应激反应的作用具有重要意义。
研究目的
观察肝郁模型大鼠的一般状态、体重、行为学的变化、检测血清中NE水平及组织形态学以验证及评价模型；检测各组大鼠蓝斑中NE合成酶TH、DBH以及神经肽CRF蛋白与基因的表达水平,并观测逍遥散和氟西汀干预后的表达。从LC-NE系统的角度对肝郁证在神经系统的物质基础进行初步探讨,为肝郁证的实质性研究提供一定的依据。
研究方法
1.慢性束缚应激肝郁模型大鼠的建立
动物分组：实验用清洁级雄性SD大鼠72只,鼠龄5周,体重(150士20)g,于造模前适应性饲养1周,随机分为对照组(A组)、肝郁证模型组(B组)、逍遥散治疗组(C组),氟西汀治疗组(D组),每组18只。A组3只1笼,其余3组单笼孤养。
模型建立：根据文献报道模拟中医肝郁证的病因,将B、C、D组大鼠置于特制的束缚制动筒内,固定身体与尾部,使之不能随意活动。每日束缚制动1次,造模时间随机,持续时间从第1天的1h逐渐增至每天6h,连续21d,造模。A、C组大鼠在束缚制动期间禁食、禁水。A组大鼠不予任何干预。
药物干预：A、B组大鼠以生理盐水灌胃,C组大鼠根据正常成人逍遥散每日用量,按体表面积换算成大鼠用药量为10g/kg.d,氟西汀为5mg/kg.d根据大鼠体重每日下午16时经胃灌相应药量。
模型评价：从一般状态、液体消耗、行为学、血清NE水平、逍遥散与氟西汀干预后疗效等方面评价模型的可靠性。
2.用H&E染色、尼氏染色方法,观察大鼠LC形态学改变和尼氏体的变化。
3.用免疫组化法检测LC中TH、DBH以及神经肽CRF的表达变化。用Imagepro plus6.0软件进行分析。
4.蛋白免疫印迹法(western blotting)检测LC中TH、DBH以及神经肽CRF的表达变化。
5.实时荧光定量(real-time PCR)检测LC中TH、DBH以及神经肽CRF的表达变化
6.所有数据采用SPSS17.0软件包进行统计分析。计量资料结果以x±s表示,两组间比较采用t-test。多组间比较采用One way ANOVA,首先对数据进行方差齐性检验,方差齐则用LSD法进间两两比较,如果数据方差不齐,则采用Welch法进行方差分析,应用Dunnett T3法进行两两比较。等级资料资料采用Kruskal-Wallis H非参数检验进行分析,P<0.05为有显著性差异。
研究结果
1.从一般状况来看,B组大鼠造模开始时表现为易激惹、挣扎,随后表现为倦怠少动、喜好贴壁、叫声弱细、反应迟钝、饮食减少,伴有毛色晦暗发黄、大便稀溏等肝郁证的表现。C、D组大鼠上述反应较轻,而A组未见异常行为状态。各组间体重增长存在显著差异(P<0.01),B组生长较为缓慢,C,D组服药后体重增长缓慢的情况得到改善。旷场实验发现B组的直立次数、理毛次数和穿越格数均显著低于A组(P<0.01)。液体消耗实验提示各组间糖水偏好存在显著性差异(P<0.01),B组的糖水偏好率明显小于其它三组。血清NE发现：B组的血清NE浓度明显高于A组(P<0.01)。说明慢性束缚应激复制出了大鼠肝郁证模型,有效的模拟出临床中肝郁证兴趣丧失、快感缺乏等症状。
2.血清NE水平检测显示：B组的血清NE浓度明显高于A组(P<0.01)。
3.H&E染色显示,大鼠LC组织形态未见明显异常,尼氏染色可见LC神经元内尼氏体染色增强、数量增多,但与正常组比较,模型组中LC未见明显异常。
4.免疫组化法检测LC中TH、DBH以及神经肽CRF的表达变化,发现B组TH、DBH和CRF的阳性神经元数目均大于A组(P<0.05),染色强度也明显增强,平均光密度值也明显高于A组(P<0.05)。经逍遥散,氟西汀干预的组显示出有下调二者表达的作用。
5.蛋白免疫印迹法(western blotting)检测LC中TH、DBH以及神经肽CRF的表达变化。与A组比较,B组TH、DBH和CRF蛋白表达显著增高(P<0.05),C、D组TH、DBH和CRF蛋白表达较B组明显降低(P<0.05)。
6.实时荧光定量(Real-time PCR)检测LC中TH、DBH以及神经肽CRF的表达变化。与A组比较,B组TH、DBH和CRFmRNA的表达显著增高(P<0.05),C、D组TH、DBH和CRFmRNA表达较B组明显降低(P<0.05)。
结论
1.本次研究采用慢性束缚应激的方法,复制了出大鼠肝郁证模型。
2.慢性束缚应激过程中,大鼠LC的组织形态未见明显异常；尼氏染色可见LC神经元内尼氏体染色增强、数量增多,模型组形态亦未见明显异常。提示21天的慢性束缚应激不会引起大鼠LC组织的形态学变化。
3.慢性束缚应激激活了大鼠LC-NE系统,使其释放NE儿茶酚胺类神经递质,进而使NE能神经元中合成酶TH、DBH增加；同时应激使神经肽CRF促进LC-NE神经元活性,与LC-NE系统形成交互影响,从而引起机体的兴奋、警觉、紧张及焦虑等情绪反应,而逍遥散起到了有效的干预效应,下调其过表达。
Background
Recently, depression syndrome model, as empirical research has been widely adopted by others. The animal model is on the basis of the experimental study of depression syndrome, is also the key of the success or failure of the reaserch. Depression syndrome model simulate specific TCM (Traditional Chinese medicine) syndrome on base of traditional Chinese medicine diagnosis and treatment theory by modern technology to offer the clinical ideal experimental observation object.
TCM syndrome model must be base on TCM etiology theory.To take into account the critical role of emotional disorders in liver depression, the pathogenesis have appeared a variety of models both at home and abroad, mainly two aspects: First, using excess or toxic drugs to induce acute and chronic liver poisoning injury to simulate depression model; Secondly, to change the physiological status of the animal to replicate the model by emotional stimulation.Drug modeling, acute and chronic toxicity can cause liver substantial damage,TCM depression Syndrome does not mean to substantial damage to the liver, and drugs also affect the animal multiple organ system function. Drug modeling had a significant difference from TCM theory, it has been an unapproved by others, phased out gradually.
The kernel institutions to regulate the body's is five organs. In the psychological aspects:the liver plays a decisive role in this procedure.To adjust the Qi caused by a variety of external stimulation, mainly regulated by the liver. Liver have a catharsis function, control the blood circulation. Liver regulate the catharsis that is it have the physiological characteristics of dispersion, catharsis. The normal function of liver catharsis can circulate systemic blood smoothly, regulate the emotions, digestion and excretion of dietary cereal better, semen excretion and help to reach to whole body. Therefore, once liver fail up to charge of Qi, will lead to liver dysfunction of catharsis, air-block pathological changes will happen first, then hepatic depression syndrome will be formed. Hepatic depression is an extensive syndrome, common disease in clinic and common syndromes in TCM viscera syndrome, also is one of the major aspects of liver disease pathogenesis in TCM. Therefore, explore the substance of hepatic depression syndrome can provide new ideas and methods for clinical diagnosis and prevention of many diseases.
Generally, liver depression disease and syndrome produce many unmoral emotions due to a variety of factors result from diet disorder, maladjustment, drug misconduct.Liver emotional disorders, such as depression, mood restless due to liver catharsis is not sufficient. From the pathological mechanisms of psychological stress and the specific features of various organs in Chinese medicine," liver" is the core which the body regulate the psychological stress response.Under abnormal emotional (psychological stress), Qi of body will be affected firstly, it can cause circulation and functional abnormalities of Qi and blood for further development. From the pathological mechanisms of psychological stress and the specific features of various organs in Chinese medicine," liver" is the core which the body regulate the psychological stress response. Under abnormal emotional (psychological stress), Qi of body will bet firstly affected, it can cause circulation and functional abnormalities of Qi and blood for further development.
Recently, from the physiological pathology of the liver research, the changes of the liver physiological function and pathological involve in the multiple systems, and the nervous,endocrine, immune etc. For example,When stagnation,the central neurotransmitter norepinephrine, epinephrine, serotonin amine,dopamine, skin potential, several catecholamines in the blood, adrenal organizational structure, and CD4+/CD8+in immune system will be changed; and more associated with sympathetic abnormalism.This show that liver function in TCM closely relate to Western medicine neuro-endocrine-immune network. Research on liver further show that the physiological function and pathological changes of the liver is involved in many systems, the nervous, endocrine, immune, digestive, cardiovascular system diseases are be observed frequently, suggesting that TCM liver function are closely related neuroendocrine disorders, immune dysfunction caused by poor emotional state and psychological stress.
Studies suggest that hepatic depression syndrome generalize about the nervous, endocrine, immune, circulatory, digestive, sensory and other systems of certain pathological changes depression, anxiety, grief, stress, premising on nerve center of the adjustment disorder. Although there is no stress formulation in Chinese medicine classical medical books, the ancient physicians had early recognized that a variety of adverse stimuli effects on the body, they attached great importance to the influence of emotional factors on the body. According to the theory of Zang organs,Liver control dispersion is one of a liver basic physiological functions, it plays a crucial role in regulating entire body, the liver of the physiological, psychological functions relate with the flow of Qi. In recent years, studies of the liver parenchyma further show that the physiological functions and pathological changes of the liver relate to many of the body system and the nervous, endocrine, immune, digestive, cardiovascular system diseases, which indicates that liver function are closely related with neuroendocrine disorders, immune dysfunction caused by bad emotional and psychological stress.
The rearch of stress involved in the hypothalamic-pituitary-adrenal cortex system (HPA), the locus coeruleus-sympathetic-adrenal medullary system (LC-NE) and so more comprehensive in abroad than our country. In our country, although the research focus on the relationship of the liver and neuroendocrine system is popular, the research focus on the HPA axis, the hypothalamic-pituitary-thyroid axis (HPT) or the hypothalamic-pituitary gonadal axis (HPGA).The material basis of hepatic depression syndrome relevance of neurotransmitters of LC-NE secretion under chronic restraint stress has not been reported.Thereby,we try to explore liver function of controlling dispersion is of great significance base on the Chinese medicine theory.
Research purposes
To observe the general state of rats in the liver depression model, body weight, behavior, serological NE level and histological evaluation model to validate the model.Detected protein and gene expression of the synthetic enzyme TH and DBH in LC-NE system and neuropeptides CRF, and observing variation level after Xiaoyao powder and fluoxetine intervention. Explore the liver substantiality in the nervous system in LC-NE system, and offer a evidence for liver substantiality research.
Research Methods
(1) Establishment of chronic restraint stress liver depression rats model
Groups:5-weeks-old72SD rats(150±20) g were randomly divided into four groups with18rats in each group after one-week's adaptation living:group A(control group), group B(model group), group C(Xiaoyao Powder treatment group),fluoxetine treatment group (D),The rats from the group B,C and D were housed individually in cages while rats from group A were be housed in two cage.
Model establishment:Rats from group B, C and D were be bound in a specific bound frame, fixed body and tail. Restraint time were be randomly and gradually durated1day1h to6h at day21th.
Drug Intervention:Rats from group C, D were deprived from diet and water during chronic immobilization stress. The rats in group A did not give any other intervention.At16:00pm daily, Rats from group A, B were given2ml physiological saline by intragastric administration. Rats from group C were given Xiaoyao powder with the same ways according to body surface area converted into rat dosage(10g/kg.d), Group D rats were given fluoxetine (5mg/kg.d).
Model evaluation:mode reliability will be evaluated by the observation of behavior, fluid consumption test, serum NE level, drug intervention efficacy.
2. H&E staining to observe the morphological changes in rat LC and the changes of Nissl bodies by Nissl staining.
3. Expression of TH, DBH in LC and neuropeptides CRF detected by immunohistochemical method and analyzed with Imagepro plus6.0.
4. Expression of TH, DBH in LC and neuropeptides CRF detected by Western blotting.
5. Expression of TH, DBH in LC and neuropeptides CRF detected by Real-time fluorescence quantitative (real-time PCR).
6. All data were be statistically analyzed using SPSS17.0software package. The measurement data results show in x±s.Statistics of comparison between the two groups were be used t-test;Statistical comparison among multiple groups will be chose One way ANOVA;The first test for homogeneity of variance of the data, homogeneity of variance with LSD method; if the data is the heterogeneity of variance, the Welch method of variance analysis;Comparison between each two by Dunnett T3method;Hierarchical data using Kruskal-Wallis H nonparametric test analysis.P<0.05was considered significant difference.
Results
1. Group B rat model manifested irritability, struggle, and then manifested as fatigue, lack of exercise preferences adherent sounds weak and thin, unresponsive, eating less, accompanied by the coat dull yellow stool thin pond Syndrome of depressed performance. C and D rats were mild, while no abnormal behavior state in group A. The rats' weight increased and significantly different from other groups (P<0.01). It showed rats in group B grew the slowest, while rats in group C, D has been rectified after durg intervention. In open field test,The erect time,grooming time and traversing grid also reduced in group B(P<0.01).Fluid consumption test revealed that group B lost appetite for sweet water during the whole procession. Statistics showed there were significant differences between these four groups (P<0.01).
2. The serum level of NE was also increased in rat model(P<0.01).
3. H&E staining showed that the LC morphology in rats are normal. The Nissl visible neurons and the number. of Nissl body enhanced, but model group has no obvious morphological abnormalities.
4. Immunohistochemical staining showed positive neurons of the TH, DBH and neuropeptides of CRF in group B were greater than group A (P<0.05), the staining intensity was significantly enhanced, the average optical density values significantly higher than group A (P<0.05). Treatment group showed a downward expression.
5. Western blot showed TH, DBH and neuropeptides of CRF in LC. Compared with group A, TH, DBH and CRF protein expression was significantly higher (P <0.05) in Group B, Group C and D significantly lower than group B (P<0.01).
6. Real-time fluorescence quantitative (real-time PCR) detection TH, DBH and neuropeptides of CRF mRNA in LC. Compared with group A, TH, DBH and neuropeptides of CRF mRNA in group B significantly higher (P<0.05), Group C and D significantly lower than group B (P<0.01).
Conclusion
1.The rat model of liver depression syndrome from chronic constraint stress is reliable and credible.
2. In chronic restraint stress process, no obvious abnormalities in the morphology of the rat LC, no obvious abnormalities; Nissl staining revealed that the LC neurons Nissl body staining increased.But there were no obvious morphological abnormalities in model group.This showed that chronic stress(21days) does not cause morphological changes in rat LC tissue.
3. This project firstly explored the hepatic depression syndrome in Locus coeruleus-noradrenergic system, and provides a theoretical basis for the further study of the molecular mechanisms of hepatic depression syndrome, and also give a beneficial trial for deeper research of the essence of liver depression syndrome.
4. Chronic restraint stress activated rat LC-NE system to release catecholamines NE, thereby TH and DBH level was increased in synthase NE neurons; at the same time, stress activating the neuropeptide CRF promoted LC-NE neurons activity, with the formation of LC-NE system interaction, causing the body's excitement, alertness, tension and anxiety and other emotional responses.However, Xiaoyao powder treatment can effectively reverse the effect.
引文
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