【作者】 刘宇凝；

【导师】 蒋春雷； 王云霞；

【作者基本信息】 第二军医大学 ， 航空、航天与航海医学， 2012， 硕士

【摘要】 随着社会发展科技进步，竞争日益激烈，以不断提高效率为目的的工作学习生活方式和在现代社会中的不时出现的各种挑战，渐渐成为人们生活的常态。但这也给人类带来了多方面的复杂的压力既应激。持久过强的应激严重地影响着人类的身心健康。应激作为几大神经精神疾病（如抑郁症）的重要诱因引起了研究者们越来越多的关注。中枢神经内分泌系统与免疫系统间是一个双向调节的网络系统。当机体处于应激情况下，大量的应激激素分泌产生并作用于机体的免疫系统，调节和改变免疫系统的功能。研究表明，应激反应可诱发机体产生炎症性反应，较为突出的特点是当应激反应发生时在中枢和机体的其他系统，伴随神经精神及其它系统发生障碍的同时常常伴有一种或是几种炎症介质的释放。近年来，有研究表明炎症与慢性精神障碍相关，并且有研究发现抑郁症患者的血清中细胞因子（如IL-1β和IL-6）升高，且炎症反应的产生先于抑郁症。作为影响现代社会的较为严重的疾病的抑郁症，因其高发性，高致残性，对家庭和社会造成了沉重负担，引起了人们的高度重视。据有关文献报道，抑郁症在世界的发病率约为10%（国内发病率约为3-5%），而自杀人群中高达50%-70%的自杀都与抑郁有关。在世界范围内，抑郁的发病率呈现出不断上升的趋势，并伴随着低龄化的发展。据世界卫生组织推测，就对人类造成的负担而言，抑郁症目前为第4大疾病，到2020年将跃升为第2位。多年来，致力于抑郁症领域研究的医学科学工作者们，取得了重要进展，提出了一些可能与抑郁症发病相关的假说，为抗抑郁药的开发和临床应用起到了很好的指导作用。但是，由于抑郁症的发病机制非常复杂，不同病因所致的抑郁症的发病机制可能不同，所以迄今为止关于抑郁症的发病机制尚未十分明确。不过，可以肯定的一点是，应激性事件是导致抑郁症的一个重要诱因。医学研究表明，抑郁是一种有明确生物学基础的疾病，是一种心理神经免疫紊乱性疾病。从抑郁的研究发展来看，抑郁症的炎性反应学说目前在抑郁症的发生发展及防治中受到人们的极大关注。该学说是基于抑郁症患者存在不同的免疫学的改变而提出的，该学说认为免疫激活，包括急性的免疫激活及慢性的免疫激活，在某些抑郁症的发病机制中起重要作用，这一观点主要依据临床和动物实验的结果得来，比如抑郁症患者常先有免疫系统的激活和炎症反应，与此同时，分别在中枢或外周给予炎性因子或免疫激活剂也可诱发抑郁的产生。另一方面，一直以来生物学介质一氧化氮（NO）对机体的生理作用的重要性，特别是对于中枢神经系统的重要影响受到人们的广泛关注。NO是重要的免疫调质，作为一种自由的气体信号分子，它同时参与着神经和免疫系统两方面的调节。NO是由一氧化氮合酶（NOS）被激活后催化产生的。NOS根据其特点，可分为三种，即nNOS（神经元型）、iNOS（诱导型）和eNOS（内皮型）。nNOS和eNOS又称为cNOS（结构型），为“生理性”酶形成，其活性具有钙离子依赖的特点，并可持续表达。iNOS是非钙离子依赖的，在基态下不起作用，而在炎性因子诱导下则会大量生成，且可合成出较cNOS约高1000倍的NO，据推测iNOS可能是由于“病理性”酶的形成产生。在正常生理状态下，nNOS是中枢NO的主要来源；而在炎症状态下，中枢胶质细胞会产生大量的炎性因子，并进一步激活iNOS。在抑郁症的研究领域，NO的作用也引起了学者的较大兴趣，有研究认为NO在应激所导致的抑郁症中起到了一定的作用。但是，对于NO与抑郁症关系的研究，其结果，在不同的实验室差别也较为悬殊，部分实验结果甚至截然相反，例如有研究提出NO对神经递质等具有双相调节的作用，其中包括对抑郁症的双相作用。虽然NO与抑郁症，NO与免疫炎性反应这几个领域均有人研究。但，NO在抑郁症发病机制中的作用却并不明确，NO与应激以及炎性抑郁症之间是何种因果关系，尤其是在整体动物存在应激反应乃至应激导致体内出现免疫炎性反应或应激状态与免疫炎性反应共存的情况下，NO在其中起到的作用究竟如何，值得探讨。我们课题组的前期工作表明，在急慢性免疫系统激活的情况下（采用内毒素LPS和减毒的结核杆菌BCG急慢性激活免疫系统），对抑郁症的发生和发展起着重要作用。这一观点与其他实验室的报道一致。基于以上抑郁症的炎性反应学说、文献报道以及我们课题组的工作基础，我们推测急慢性应激导致的急慢性免疫系统激活诱发的抑郁症可能与NO有密切的关系，并且iNOS通路可能在此过程中起重要作用。本课题分别选用不可预见的慢性轻度应激（UCMS）方式和急性给予低剂量内毒素两种方式，来模拟急慢性应激条件下机体免疫炎性反应的激活状态，建立动物抑郁症模型（此两种模型均已较成熟并广为接受），并在建模过程中均给予iNOS的特异性拮抗剂，然后观察动物体内NO及其代谢产物的变化，以及这些变化与抑郁样行为之间的因果关系，以确定在机体出现急慢性免疫激活时iNOS对抑郁症的作用，并通过对小鼠皮层神经元细胞尼氏染色进一步直观的观察急慢性应激条件下对神经元的影响并探讨NO在其中的作用。主要研究结果如下：1.慢性不可预计轻度应激（连续处理4周后），所建立的慢性抑郁模型中：1）行为学实验结果：（1）与对照组相比，应激处理组小鼠抑郁样行为显著增多。这些行为包括：蔗糖偏爱实验中蔗糖偏爱值的下降；强迫游泳实验中累积不动时间的延长；自发活动实验中探洞时间明显减少。（2）慢性不可预见轻度应激刺激在例如：活动时间，休息时间，活动次数等项目检测中结果更多的体现在对正常的自发活动无明显影响。2）分子生物学检测结果：（1）与对照组相比，UCMS处理的小鼠脑皮层中iNOS的mRNA表达显著增加。（2）与空白对照组相比，UCMS处理的小鼠血浆中的亚硝酸盐含量均显着增加3）形态学实验检测结果：UCMS处理后，小鼠大脑皮质神经元细胞萎缩并深染，神经元细胞上的尼氏小体大量受损甚至消失。4）在应激第二周开始前，提前给予iNOS抑制剂（1400W）进行药物干预处理：（1）可以有效的缓解上述应激所诱导的抑郁样行为，如：升高糖水偏爱值，减少游泳不动时间。同时1400W逆转了应激所致的亚硝酸盐的增多。（2）可以明显的保护小鼠皮层神经元细胞，减少受应激刺激造成的神经元的损伤。1400W预处理的小鼠皮层神经元细胞尼氏染色实验中，富含尼氏小体的神经元细胞数目显著增加。2.腹腔注射低剂量内毒素（LPS）24小时后，所建立的急性抑郁模型中：与慢性轻度不可预见应激的实验结果一致，小鼠出现了诸如蔗糖偏爱值下降，强迫游泳中的累积不动时间延长，自发活动中探究行为减少等一系列显著的抑郁样行为。而提前LPS30分钟给予iNOS抑制剂预处理的小鼠，能观察到LPS诱导的抑郁行为被显著逆转。在进一步对小鼠神经元细胞进行免疫组化染色后，可以观察到iNOS抑制剂有效保护脑皮层神经元免受LPS引起的损伤。结论：以上所有的实验结果均支持iNOS与急慢性应激或急慢性免疫炎症反应诱导的抑郁的产生有关，从小鼠的抑郁模型中来看，其抑郁样的行为产生的生理基础，可能与小鼠大脑皮层内的神经损伤有关。更多还原

【Abstract】 With the scientific and technologic development, the increasingly competitive challenges-stress-are here and there in modern society. Stress has gradually become popular in people’s life. Research shows that the stress response is actually a pro-inflammatory response, along with neuropsychiatric and other system disorders. In recent years, studies have shown that inflammation ties chronic mental disorders, and cytokines such as IL-1β and IL-6increased in the serum of patients with depression found in the study. In the human body, there exists a bidirectional communication between the central nervous system and the immune system. Cytokines, neurotransmitters, hormones and other chemical information of molecular exchange are the common signal. When the body is in stressful situations, the stress hormones secret to regulate and change the function of the immune system.Depression is a common mental disorder, which causes mental illness and suicide. Depression mainly manifests depressed emotion, mental retardation, the lack of interest in pleasure, despair, and severe cases, accompanied by suicidal tendencies. According to the literature reports, the incidence of depression is about10%, and50%-70%of suicide is related with depression. In the worldwide, the incidence of depression showing a rising trend, and accompanied by the development of a younger age. Speculated that, according to World Health Organization, in2020, the depression, the four major disease emerged as the world’s No.2disease from all the world’s major non-fatal disease, take a huge burden on it. Depression as more serious diseases of modern society, because of its high incidence, high-disability, a heavy burden on families and the community while also attracted great attention. Over the years, medical scientists are committed to research in the field of depression, important progress has been made. However, the pathogenesis of depression is very complex, and the pathogenesis of the different causes of depression may be different, so as yet the mechanism of depression is still not clear. Different to the usual understanding of depression, it is not a general emotional or psychological problem. Medical research shows that it is a clear biological basis of disease, is a psychological neuro-immune disorder. Research on depression, the depression of the inflammatory response doctrine in the development and prevention of depression has attracted much attention. The doctrine is based on depression in patients with immunological change. The main idea of this doctrine is that the activation of the immune system plays an important role in the pathogenesis of depression. Results from clinical and animal experiments have confirmed this idea.The importance of the biological medium of nitric oxide (NO) on the body’s physiological role, especially the central nervous system caused widespread concern. As a freedom of gaseous molecules, it is also involved in the regulation of the nervous and immune systems. Studies suggest that NO play certain roles in the depression caused by stress. NO is an important immune quenched and tempered. In the field of depression, the role of NO has attracted the interest of researchers. However, study showed relatively poor, and even opposite result about the role of NO in depression.NO is catalyzed by nitric oxide synthase (NOS). According to the characteristics, NOS can be divided into three types, namely, nNOS (neuronal), iNOS in the (inducible) and eNOS (endothelial). nNOS and eNOS, also known as the structure type of cNOS, sustained expression of its activity is calcium dependent, for the physiological enzyme formation. iNOS is a non-calcium dependent, in the ground state does not work in the inflammatory cytokine induced a large number of generated, and the synthesis of NO compared with cNOS about1000times higher, presumably as a "pathological" enzyme formation. Under normal physiological conditions, nNOS is the main source of central NO; central glial cells produce large amounts of inflammatory factors in the inflammatory state, and activation of iNOS in it.About the relationship between iNOS and depression, studies have reported different, even the biphasic regulatory role of NO on the neurotransmitters, such as the biphasic effect of depression. Therefore, the role of NO in the pathogenesis of depression has yet to be confirmed. NO depression, NO and KP pathway and NO and the immune inflammatory response in several areas have people study and what kind of causal relationship between NO and stress and inflammatory depression. In particular, exist in the whole animal, the stress response and even, the stress causes the body immune inflammatory response or stress and the immune inflammatory response coexistence of NO which play a important role and in how it is the worth exploring.As previously reported that acute and chronic immune system activation play an important role in the occurrence and development of depression. This view is consistent with reports from other laboratories. Experiences and inflammatory mediators are fundamental in the provocation of major depressive disorders (MDD). We investigated the roles and mechanisms of iNOS in inflammatory depression. Based on the doctrine of the inflammatory response of depression, we hypothesized that acute and chronic stress caused acute and chronic immune system activation-induced depression may be closely related to NO and iNOS pathway, and it may play an important role in the process. We used unpredictable chronic mild stress (UCMS) and acute administration of low doses of endotoxin to simulate the immune inflammatory response as acute and chronic stress conditions, which aims to establish the animal inflammatory model of depression (this two models have been more mature and widely accepted). Depressive-like behaviors, iNOS pathway and neuron damage were observed in the present or absent of iNOS antagonist1400w. We used two mouse model of depressive-like state induced by UCMS and LPS. Depressive-like behaviors were evaluated with sucrose preference, locomotor activity and forced swim test. Immunohistochemistry was used to check the loss of Nissl bodies in cerebral cortex neurons. The levels of iNOS mRNA expression in the brain and nitrites in the plasma were measured with RT-PCR and Griess reagent respectively.The main results were as follows:1. Results of behaviors:Compared with the control group, UCMS induced significant depressive-like behaviors. These depressive-like behaviors were manifested with decreased sucrose preference, increased immobility time in forced swim test and reduced numbers of hole-searching times in spontaneous activity test. In contrast, there were no significant effects on some sickness related behaviors such as resting time, number of activity.1400w pretreatment abrogated these depressive-like behaviors.1400W pretreatment could abrogate these depressive-like behaviors. LPS model had obtained similar results.2. Results of molecular biology:(1) Compared with the control group, a significant upregulation of cortical iNOS mRNA expression was observed in UCMS-treated mice. This increase was completely blocked by1400w pretreatment.(2) Compared with the control group, the plasma nitrite level in UCMS-treated mice significantly increased. This increase was completely blocked by1400w pretreatment.3) Results of morphological experiment:Post-UCMS treatment, cortical neurons were shrunken with dark-staining and lost Nissl body. Pretreatment the mice with1400w protected cortical neurons from damage.We found the similar data in LPS models.更多还原