逍遥散对肝郁脾虚模型大鼠海马体内单胺类神经递质及其代谢产物的影响

【作者】 敖俪元；

【导师】 陈家旭；

【作者基本信息】 北京中医药大学 ， 中医诊断学， 2011， 硕士

【摘要】 焦虑是一种情绪状态,患者基本的内心体验是害怕,如提心吊胆、忐忑不安,甚至极端恐慌或恐惧；这种情绪是不快和痛苦的,可以有一种迫在眉睫或马上就要虚脱昏倒的感觉。焦虑症主要分为惊恐障碍和广泛性焦虑(GAD)两种。GAD其典型症状包括警觉性高、担心、易激惹、肌紧张增高、震颤和心悸等自主活动改变。现代研究发现,应激和焦虑是密切相关的现象,大多数焦虑症患者都曾经历过严重的应激事件,因此应激事件可以成为焦虑症发病的原因和诱因。较新的应激定义为：机体的不协调状态,或内环境稳定受到威胁。近年来应激研究者在深入探索应激的有效治疗方法,西药抗焦虑药物主要为苯二氮卓类,实验表明抗焦虑药物的中枢作用似与兴奋脑内GABA系统及降低5-HT系统的活性相关。近年来发现脑内存在特异性的苯二氮卓类药物受体,并大量分布于海马等部位,但西药抗焦虑药对神经有很大的毒副作用。中药在抗应激领域已凸显出极大潜能。中医古籍中虽没有“应激”这一术语,但古代医学家很早就已认识到各种不良的环境刺激对机体的影响。古代的医家们认为各种环境因素及心理因素强烈或持久地刺激人体可造成人体气机紊乱,脏腑气血阴阳失调,机体内环境稳定失衡,从而导致疾病的发生。而现代心理应激理论以神经内分泌免疫网络为核心,认为在神经内分泌与免疫系统之间,存在着多种神经递质、神经肽、激素以及免疫因子所介导的相互作用与调节,在整体水平上构成了神经内分泌网络,神经内分泌免疫网络是维持机体内环境及生理功能平衡和稳定的根本基础。中医“肝主疏泄”的功能在机体应激中起着重要的作用,中医的肝脏是机体调节应激反应的核心。因此,结合现代医学有关应激理论的研究成果,进一步探讨肝主疏泄在应激中的主导地位及中药复方制剂在防治慢性应激时所产生的作用机理,不仅有利于中医基础理论和临床的研究发展,而且对于许多疾病的预防和治疗具有重要的意义。逍遥散以疏肝解郁、健脾养血为组方原则,自创制以来,历代医家对其进行了大量的阐发和推广应用,至今仍被广泛应用于临床各科。海马是边缘系统的重要组成部分,和情绪及行为动机的产生以及和脑的高级神经活动如学习记忆等有关,该部位神经功能的失调在恶劣心境障碍的发病中可能有重要作用。可以抑制HPA轴的应激反应,而且可以促进应激状态下亢进的HPA轴恢复到基础水平。然而长时间的慢性应激可以引起海马组织的损伤,引起结构和功能的改变,最终导致情感障碍性疾病的发生。单胺递质包括5-羟色胺(5-HT)、去甲肾上腺素(NE)和多巴胺(DA),它们无论是在精神类疾病的发病机制,还是在精神类药物的作用机制中,都发挥着重要的作用。研究目标：所以本课题主要以海马为研究对象,以慢性应激引起焦虑样行为,再通过高效液相色谱法检测单胺神经递质在慢性束缚应激肝郁脾虚证模型大鼠海马的表达变化,结合行为学的动态变化、推测和揭示逍遥散调节慢性应激状态的作用机制。为中医肝脏生理功能肝主疏泄理论提供实验依据。研究方法：1用SPF级成年雄性SD大鼠25只(16 0±20)g。适应性饲养2天后,根据旷场试验评分随机分为正常对照组(A组)、7天模型组(B组)、7天逍遥散组(C组)、21天模型组(D组)、21天逍遥散组(E组),每组各5只。以慢性束缚方法制作应激大鼠模型,将大鼠束缚于特制的束缚架上,每日3小时,连续21天。自造模第1日始,C、E组每日在束缚前1h灌服逍遥散,灌胃容积为1mL/100g体重。A、B、D组则灌服等体积的蒸馏水2用录像监控大鼠的活动,观察大鼠的精神状态、束缚反应、粪便质地等3用高效液相色谱法检测单胺神经递质的表达变化。在实验中,笔者以海马作为主要的研究对象。因为西药中主要抗焦虑药物为苯二氮卓类,有实验表明脑内存在特异性的苯二氮卓类药物受体,并大量分布于海马等部位。而且本实验只采用左侧海马作为研究对象,应为笔者在文献查阅的过程中发现有研究者采用局部一致性(ReHo)作为海马功能的测量,其结果提示应激海马左侧ReHo值较正常对照组的差异有统计学意义。而右侧海马与正常对照组则无明显变化。提示了应激所致海马功能的异常可能存在侧化的现象。同时又有研究者指出,焦虑症患者左侧海马的氨基酸递质显著高于正常人,而右侧海马神经递质在正常人与焦虑患者间未见差异产生；并发现焦虑患者左侧海马和海马旁区葡萄糖代谢显著增强,而右侧亦无明显变化。证实了焦虑患者的发病过程中存在着左右海马功能活动的不对称性。以上两个研究结果提示了焦虑症的左侧海马有可能参与了该病的病理过程,焦虑患者可能存在左侧海马神经元活性、神经细胞代谢及神经递质传导的异常。所以在实验研究对象中,本实验选择海马—左海马作为研究对象。结果：21天药物组与正常对照组相比较,21天药物组大鼠海马内HIAA含量有显著身高,差异(P<0.01)。21天模型组与正常对照组相比较,在海马内HIAA含量有显著升高(P<0.01),5-HT含量也存在统计学差异(P<0.05)。7天药物组与正常对照组比较在5-HT的含量上升高显著,有统计学差异(P<0.01)；在NE的含量上7天药物组较正常对照组升高,存在显著的统计学差异(P<0.01)。用7天模型组与正常对照组比较,在海马内5-HT及NE的含量上均有所升高,存在显著的差异(P<0.01),具有统计学意义。另外,用21天模型组与21天药物组相比较,两组在5-HT的含量上存在显著差异(P<0.01),具有统计学意义。其次,21天药物组与7天的药物组及7天模型组对比,在HIAA、5-HT及NE的含量上均存在显著差异(P<0.01),具有统计学意义。结论：肝郁脾虚证模型大鼠海马内5-HT、NE均有明显升高。用逍遥散治疗后5-HT、NE值均有所下降,5-HIAA含量增高,提示逍遥散防治焦虑的机制可能与提高5-HT与NE的功能及其转换率有关。并且从实验研究的结果中可以看出,7天组与21组确实存在差异性,在没有药物干预的情况下21天模型组较7天模型组的5-HT与NE含量均有下降,这可能就是慢性应激障碍的原因之一,也可能是大脑习惯性反映的结果。而多巴胺的产物HVA在各组间没有统计学差异。更多还原

【Abstract】 Anxiety is an emotional state, the patient’s inner experience is the basic fear, such as fear, trembling, and even extreme panic or fear; this emotion is unpleasant and painful, can have an imminent collapse or faint feeling soon. Divided into panic disorder anxiety disorder and generalized anxiety (GAD) both. Typical symptoms of GAD include heightened vigilance, worry, irritability, increased muscle tension, tremor and heart palpitations and other independent activities change. Modern research has found that stress and anxiety are closely related phenomena, most patients with anxiety disorders had experienced severe stress events, so events can be anxiety Stress causes disease and incentives. Newer stress is defined as:the body of the uncoordinated, or the environmental stability is threatened. In recent years, researchers stress the in-depth exploration of effective treatment for stress, anti-anxiety drugs, primarily for the western benzodiazepine class, experiments show that the pivotal role of anti-anxiety drugs and excitement like GABA brain system and reduce the 5-HT system related activity. In recent years, found that there is brain specific benzodiazepine receptors drugs and a large number located in the hippocampus and other parts of western anti-anxiety drugs on the nerves but a lot of side effects. Anti-stress medicine in the field has highlighted the great potential. Ancient Chinese medicine, although not in the "stress" the term, but the ancient physicians had long recognized the environmental stimuli of various adverse effects on the body. Ancient physicians believe that environmental factors and strong or lasting psychological factors can cause the body to stimulate the body air-disorders, blood and viscera imbalance of yin and yang, balance and stable internal environment, causing diseases. The modern theory of psychological stress to the core of neuroendocrine-immune network, that in between the neuroendocrine and the immune system, there are a variety of neurotransmitters, neuropeptides, hormones and immune factors mediated interaction and regulation, in the overall level constitutes the neuroendocr ine network, neuroendocrine-immune network is the internal environment to maintain balance and stability and physiological function of the fundamental basis. Chinese medicine, "Liver Shuxie" function in the body stress plays an important role in regulating traditional Chinese medicine the liver is the body’s core stress response. Therefore, the combination of modern medical research related to stress theory to explore further in the liver controlling dispersion and stress the leading role of traditional Chinese medicine in the prevention of chronic stress arising from the mechanism, not only conducive to the basic theory and clinical medicine research and development, but also for the prevention and treatment of many diseases of great importance. Xiaoyaosan to liver qi stagnation, spleen, nourishing the principle of the group side, since the self-created, its ancient physicians a lot of exposition and application, is still widely used in clinical subjects.Hippocampus is an important part of the limbic system, and the generation of emotion and motivation, and the higher nervous activity and the brain, such as learning and memory, respectively, and the parts of nerve function disorders in the pathogenesis of severe mood disorders may have an important role. Can inhibit the HPA axis stress response, and stress can contribute to hyperactivity of the HPA axis status returned to baseline levels. However, chronic stress can cause prolonged damage to the hippocampus, causing structural and functional changes, leading to the occurrence of affective disorders. Monoamine neurotransmitters, including:5-hydroxytryptamine (5-HT), norepinephrin e (NE) and dopamine (DA), they are both in the spirit of the classDisease pathogenesis, or the role of drugs in the spirit of the mechanism, have played an important role. Research Objectives:Therefore, this issue mainly in the hippocampus as the research object to chronic stress-induced anxiety-like behavior, and through high performance liquid chromatography of monoamine neurotransmitters in chronic liver Syndrome Model of restraint stress the hippocampus changes, combined with the dynamic changes of behavior, suggesting that chronic stress, and reveal the state of Xiaoyaosan regulation mechanism. Physiological function of liver and liver of TCM theory to provide experimental basis for controlling dispersion.Methods:1 adult male with a SPF level 25 SD rats (160±20) g.2 days feeding adaptation, according to open field test scores were divided into normal control group (A group),7 day group (B group),7 days Xiaoyaosan group (C group),21 day group (D group),21 days Xiaoyaosan group (E group),5 rats in each group. Approach to chronic restraint stress in rats made, the rats were specially bound in the shackles of the shelves,3 hours per day for 21 days. Since the beginning of mo deling 1, C, E groups before daily restraint 1h fed Xiaoyaosan, intragastric volume 1mL/100g weight. A, B, D group were fed with an equal volume of distilled water.2 activities in rats with a video monitor to observe the rat’s mental state, binding reaction, stool texture.3 high performance liquid chromatography monoamine neurotransmitter expression.In the experiment, the author of the hippocampus as the main object of study.Because the main anti-anxiety drugs in the Western benzodiazepine class, there exist experimental results show that specific brain benzodiazepine receptor drugs, and a large number located in the hippocampus and other parts. And the left hippocampus in this experiment used only as research subjects, should be accessible in the literature the author found that the researchers used the process of local consistency (ReHo) as a measurement of hippocampal function, the results suggest that stress in the hippocampus than the normal value of the left ReHo control group, the difference was statistically significant. The right hippocampus with the control group did not change significantly. Tip of the stress caused by the dysfunction of hippocampus may exist side of the phenomenon. The researchers have also pointed out that the left hippocampus in patients with anxiety disorders were significantly higher than the normal amino acid neurotransmitters, and the right hippocampus in normal human neurotransmitter did not differ between patients with anxiety generated; and found that the left hippocampus in patients with anxiety and parahippocampal area significantly increased glucose metabolism, but no significant change in the right side. Confirmed in the pathogenesis of anxiety in patients about the existence of functional activity of hippocampal asymmetry. These two results suggest that anxiety disorders are left hippocampus may be involved in the pathogenesis of the disease, anxiety, there may be left hippocampus in patients with neuronal activity, metabolism of nerve cells and neurotransmitters conduction abnormalities. Therefore, experimental research subjects in this experiment, select the hippocampus-the left hippocampus of the study.Results:21 days treatment group compared with the control group,21 days of drug HIAA levels in hippocampus were significantly height difference (P<0.01).21 days, the model group compared with the control group, HIAA levels in the hippocampus were significantly higher (P <0.01),5-HT content also significantly different (P<0.05).7 days drug group compared with the control group at 5-HT levels increased significantly higher, with statistical difference (P<0.01); in the NE content of 7 days on the drug group increased compared with normal group, statistically significant differences (P<0.01).7-day group compared with the control group,5-HT in the hippocampus and both are on the NE content increased, there were significant differences (P<0.01), with statistical significance. In addition, with 21 days of model group and drug group compared to 21 days, two groups on the content of 5-HT there were significant differences (P<0.01), with statistical significance. Second, the 21-day group and 7 days of drug treatment group and 7 day group comparison, in the HIAA,5-HT and NE levels were significantly different on both (P<0.01), with statistical significance.Conclusion:Liver Syndrome Model hippocampus 5-HT, NE significantly increased. After treatment with Xiaoyaosan 5-HT, NE values were decreased,5-HIAA levels increased, suggesting that prevention and treatment of anxiety Xiaoyaosan mechanism may increase 5-HT and NE function and its conversion rate related. From the experimental results of the study and can be seen 7 days group and 21 group differences do exist, in the absence of drug intervention group compared with 21 days, 7 days a model model group,5-HT and NE content were decreased, which Chronic stress disorder may be one of the reasons may be the result of the brain reflect customary. The product of dopamine HVA in each group was not statistically significant.更多还原