PSYCH OpenIR  > 健康与遗传心理学研究室
肠道共生菌对宿主身心健康的影响及机制研究
其他题名The impact of intestinal microbiota on physiological and psychological health of the host and the mechanisms of action
罗佳
学位类型博士
导师金锋
2014-05
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业心理学
关键词肠道共生菌群 肠–脑轴 焦虑 抑郁 认知 鞭毛蛋白 Toll 样受体
摘要【研究背景】肠道内定植了数量众多、种类丰富的肠道菌群,它们和宿主形成了互利共生的关系,对宿主的健康具有重大影响。近年来,随着对肠道菌群调控作用研究的不断深入,我们发现肠道菌群不仅调控胃肠道活动(比如调控胃肠道运动和分泌、促进营养物质的消化和吸收、维持肠粘膜正常免疫反应和上皮屏障的完整性等),还影响宿主脑功能和行为。肠道菌群通过肠–脑轴调控宿主行为,而肠–脑轴是由免疫、神经内分泌和迷走神经途径构成的肠道和脑之间的交流系统。动物研究(无菌动物、肠道病原菌感染以及抗生素和益生菌处理动物)和临床观测的结果表明,肠道菌群通过肠–脑轴对宿主的应激反应、焦虑、抑郁和认知功能产生重要影响。平衡的肠道菌群促进宿主的身心健康,而肠道菌群失调则可能引发胃肠–脑疾病,比如肠易激综合征(IBS)、功能性消化不良(FD)、炎性肠道疾病(IBS)和肝性脑病(HE)。深入了解肠道菌群对宿主行为的影响,能够帮助我们更好地理解IBS 等的发病机理,并使得我们认识到调节和恢复正常肠道菌群的安全有效措施(补充益生菌)是治疗精神心理疾病的重要组成部分。益生菌维持肠道菌群平衡,是一种具有良好前景治疗IBS、FD 和HE 的安全有效方法。其中,乳酸菌是普遍使用的一种益生菌。相关的动物和临床研究表明,益生菌能够改善胃肠道环境,降低焦虑和抑郁、提高认知能力,有益于人类的身心健康。虽然已有不少的临床研究显示益生菌能够改善IBS、FD、IBD 和HE 患者的消化道症状和提高生活质量,然而目前还没有研究探索益生菌对这些患者情绪和神经精神症状的影响。
【研究目的】我们的主要研究目的是探讨益生菌对胃肠–脑疾病神经精神症状的改善作用。我们首先造模了胃肠–脑疾病的动物模型,以观测胃肠道疾病能否引发脑功能和行为异常。在此基础之上,给疾病动物服用益生菌,观测益生菌能否改善这样的行为异常,以及其中的可能机制。本研究中,我们选用的益生菌是瑞士乳杆菌NS8,因为已有研究报道瑞士乳杆菌具有抗炎功能,能够调控中枢神经系统活动,并改善脑功能和行为。
【研究方法】首先,我们探讨了胃肠疾病能否引发行为异常,以及益生菌对脑功能和行为的影响。我们选择了肠易激综合征(IBS)、肝性脑病(HE)和功能性消化不良(FD)的胃肠–脑疾病模型。在开展动物研究前,我们在临床上观测益生菌(瑞士乳杆菌NS8)对IBS 患者焦虑和抑郁情绪的影响。IBS 患者被随机分为安慰剂组和益生菌组,每日早晚服用两粒安慰剂或益生菌胶囊。分别于胶囊服用前和服用一个月后采用Zung 自评焦虑量表、罗森伯格信心量表和Zung自评抑郁量表评估被试的焦虑、信心和抑郁状态。在动物研究中,高血氨大鼠(HE动物模型)和胃炎大鼠(FD 动物模型)的造模方法分别是腹腔注射醋酸铵和服用碘乙酰胺;益生菌的给予方式是服用浓度为109CFU/ml 的益生菌溶液。高架十字和旷场测试评估大鼠的焦虑行为,糖水偏好实验测试大鼠的抑郁行为,Morris 水迷宫测试大鼠的认知功能。
其次,我们探讨了益生菌调控脑功能和行为的可能机制,即它是否通过肠–脑轴调控行为。相关研究表明神经炎和5-HT 代谢异常可能是导致HE 认知功能减退和焦虑症状的重要原因,因此我们检测了大鼠小脑、前额叶和海马(参与认知和情绪调控的脑区)的炎性标记物(诱导型一氧化氮合酶、前列腺素E2 和白介素1β)、5-HT 和其代谢物5-HIAA 的水平,以及血浆色氨酸及其代谢物犬尿氨酸和犬尿喹咛酸的浓度。而血浆中促炎细胞因子白介素-6、干扰素-γ 和肿瘤坏死因子-α 的水平代表肠–脑轴免疫途径的活动;下丘脑表达的C-fos mRNA(神经元激活标记物)代表肠–脑轴迷走神经途径的活动;下丘脑表达的糖皮质激素受体(Gr)和促肾上腺皮质激素释放因子(Crf)mRNA 以及血浆皮质酮水平代表肠–脑轴神经内分泌(HPA 轴)途径的活动。此外,由于FD 的发病具有明显的性别差异,在女性中的发病率远高于男性。而性激素可能是导致FD 性别差异的重要原因,因为研究发现性激素在两性的生理和行为差异中扮演了重要角色。因此,我们在FD 大鼠的研究中同时考察了雄性和雌性大鼠的行为、肠–脑轴活动、性激素及其受体的表达。
第三,我们初步探讨了益生菌被宿主识别的分子机制,因为这是益生菌发挥调控作用的前提。宿主对肠道菌群的识别主要依赖于免疫系统,大量的证据表明免疫系统和肠道菌群的相互识别维护着机体正常的免疫活动,具体表现为免疫系统对病原菌进行免疫清除而对益生菌耐受。其中,免疫系统的Toll 样受体(Toll-like receptors,TLRs)和肠道菌群的微生物相关的分子模型(Microorganismassociated molecular patterns, MAMPs)被认为在宿主免疫系统对病原菌和益生菌的区分中发挥了重要作用,因为TLRs 对MAMPs 的识别能够激活先天性免疫并诱导获得性免疫做好准备。在TLRs 对MAMPs 的识别中,只有TLR5 对细菌鞭毛蛋白的识别是基于蛋白–蛋白的相互作用,因此比较容易对其结合方式进行研究。我们想要确定TLR5 和鞭毛蛋白的相互作用是如何影响宿主区分病原菌和益生菌。因此,我们构建了多种胃肠道细菌(包括益生菌和病原菌)鞭毛蛋白的系统发育树、序列比对了它们的鞭毛蛋白序列在TLR5 识别位点的差异,并探讨了TLRs 是如何对益生菌和病原菌作出不同反应。
【研究结果】益生菌显著地降低了IBS 患者的焦虑和抑郁水平,有效地改善高血氨大鼠的认知功能和焦虑行为,并改善胃炎雌鼠的焦虑和抑郁行为。伴随着行为的改变,益生菌能够显著地降低高血氨大鼠的神经炎程度和5-HT代谢。益生菌显著地降低了胃炎雌鼠过度的HPA 轴活动,但对血浆中细胞因子浓度以及下丘脑C-fos mRNA 表达量没有明显影响。此外,益生菌显著提高了胃炎雌鼠雌二醇水平和下丘脑ER ß 的表达量,降低了胃炎雄鼠睾丸酮的浓度和增加下丘脑ER α 的表达量。此外,我们发现病原菌和益生菌的鞭毛蛋白序列有所不同,尤其是TLR5 结合并识别的鞭毛蛋白位点。TLRs 在肠上皮细胞的分布具有顶端和基侧面的两极性,能够分别引发对病原菌的免疫反应和对益生菌的免疫耐受。
【结论】胃肠道疾病能够引发脑功能和行为异常,而益生菌能够显著改善胃肠―脑疾病大鼠的认知功能、焦虑和抑郁行为。益生菌改善高血氨大鼠行为的作用是由肠-脑轴的免疫和神经内分泌(5-HT 系统)途径,改善胃炎大鼠行为是由神经内分泌(HPA 轴)介导。胃炎导致雌鼠而非雄鼠出现焦虑和抑郁行为,这种行为上的性别差异可能是由于性激素的不同改变造成。这些结果说明,益生菌瑞士乳杆菌可能是治疗IBS、HE 和FD 认知功能下降以及焦虑和抑郁的安全有效方法。益生菌发挥有益调控功能的前提是被宿主识别。对TLR5–鞭毛蛋白相互作用的分析结果表明,病原菌和益生菌的鞭毛蛋白序列在TLR5 识别位点的差异,以及TLRs 的极性分布引发的对益生菌和病原菌截然不同的反应可能是宿主区分病原菌和益生菌的重要机制,产生对病原菌免疫防御而与益生菌互利共生。
其他摘要Backgroud: The complex communities of enteric flora that colonise the human gastrointestinal (GI) tract play an important role in human health. Within the GI tract, the microbiota have a mutually beneficial relationship with their host that modulate gut motility and secretion, maintain normal mucosal immune function, epithelial barrier integrity and nutrient absorption. Although many people are aware of the impact of enteric flora on regulating GI physiological activity, fewer know about the ability of microbiota to influence the brain function and behavior. Accumulating data now indicate that the intestinal microbiota communicate with the central nervous system (CNS) possibly through the immune, neuroendocrine and neural pathwaysthe gut-brain axis. Studies in germ-free animals and in animals exposed to pathogenic bacterial infections, probiotic bacteria or antibiotic drugs suggest a role for the intestinal microbiota in the regulation of stress response, anxiety, depression and cognition. Homeostasis of enteric flora composition improves the health of the host, while disruption of this composition alters gut-brain function and increases disease susceptibility, such as irritable bowel syndrome (IBS), functional dyspepsia (FD), inflammatory bowel disease (IBD), hepatic encephalopathy (HE), autism, Alzheimer’s disease (AD), Mutiple Sclerosis (MS) and chronic fatigue syndrome (CFS). In support of this proposition, growing data from clinical studies reveal that the enteric flora compositions in these patients are altered compared to healthy individuals, and microbiota dysbiosis can induce similar symptoms in animals. Gaining a better understanding of the impact of microbiota on behavior could provide insight into the pathogenesis of GI-brain dysfunction (IBS, FD, IBD and HE), and promote further exploration for development of any microbiota-based and microbiota-specific therapeutic strategies for CNS diseases. Probiotics, such as the Lactobacillus species, are living microorganisms that contribute to the balance of intestinal microbiota, thereby improving the health of the host. A growing body of studies have shown a beneficial role of Lactobacillus in the regulation of gut and brain functions. Although data from clinical studies suggested probiotics may be an efficient treatment for gastrointestinal symptom in HE, IBS, FD and IBD patients, few studies evaluated the impact of probiotics on psychological symptom in these patients. Lactobacillus helveticus (L. helveticus) strain NS8 was used in our study, because it has been shown to inhibit pro-inflammatory responses and improve murine brain function and behavior.
Objective: The overall aim of this study was to test for a possible impact of GI inflammation on psychological behavior, a potential therapeutic role of probiotics and evaluated potential mechanisms of action.
Methods:Firstly, we assessed the impact of GI diseases on brain and the effect of probiotic treatment on brain function and behaviors in irritable bowel syndrome (IBS), hepatic encephalopathy (HE) and functional dyspepsia (FD), since these diseases that are known as GI-brain diseases have the symptoms of gastrointestinal disorder and neurological alteration. A preliminary clinical study conducted before the animal study evaluated the impact of probiotics Lactobacillus helveticus (L. helveticus) strain NS8 on anxiety and depression in IBS patients. The IBS patients were randomly divided into probiotic and placebo groups. All patients received two capsules a time, two times a day. The Zung self-rating anxiety scale, Rosenberg self-esteem scale, and Zung self-rating depression scale evaluated the anxiety, esteem and depression levels in patients prior to and at the one-month treatment. The rats models of HE and FD were induced by intraperitoneal injection of ammonium acetate and adding 0.1 % (w/v) iodoacetamide (IAA) to the sterile drinking water respectively, and then were treated with probiotics. Sucrose preference test assessed the depression-like behavior, open field test and elevated plus maze evaluated the anxiety-like behavior, and the Morris water maze task assessed cognitive function. Secondly, we evaluated the potential mechanisms of the impact of gastrointestinal diseases and probiotics on the brain function, whether they influence the brain via the gut-brain axis. Evidence suggests that the hyperammonemia (HA)-induced neuroinflammation and alterations in the serotonin (5-HT) system may contribute to cognitive decline and anxiety disorder during HE. Neuroinflammation was assessed by measuring the inflammatory markers (inducible nitric oxide synthase, prostaglandin E2, and interleukin-1 β), and the 5-HT system activity was evaluated by measuring 5-HT and its metabolite, 5-HIAA, and the 5-HT precursor, tryptophan in the cerebellum, hippocampus and prefrontal cortex (brain regions are crucially involved in cognitive function and emotional state). The activities of the three GI-brain axis pathways through which gastric inflammation may modulate the brain function and behavior were also evaluated. The plasma pro-inflammatory cytokines including interleukin-6 (IL-6), interferon-gama (INF-), and tumor necrosis factor-alpha (TNF-α) assessed the immune pathway activity of GI-brain axis. The hypothalamic C-fos mRNA (marker of neuronal activity) expression indicated the vagus nervers pathway activity of GI-brain axis. The mRNA expression of corticotropin-releasing factor (Crf) and glucocorticoid receptor (Gr) in hypothalamus indicated the neuroendocrine pathway (HPA axis) activity of GI-brain axis. In addition, since FD have a considerably higher prevalence in women than in men, we tested both male and female rats in order to reveal any gender difference in the possible impact of gastritis on psychological behavior. Thirdly, we test for the mechanism by which probiotics interact with and enable their protective effects to host following the exploration for the effect of probiotics on host behavior. The interaction between the host and the commensal microbiota is crucially dependent on a cross-talk between host immune system and enteric flora in which the host recognizes and responses distinctively to probiotic and pathogenic bacteria. The toll-like receptors (TLRs) and microorganism associated molecular patterns (MAMPs) may play a major role in the host discrimination between probiotics and pathogens, as the recognition of MAMPs by TLRs can activate innate immune response and prime the adaptive immune system. In the TLRs family, TLR5 that responds to flagellin is the only protein-binding TLR, it’s much easier to study the flagellin-TLR5 interaction structurally and functionally. The aim of this study was to test for a possible contribution of the flagellin-TLR5 crosstalk to the host discrimination between probiotic and pathogenic bacteria. This is achieved by constructing phylogenetic trees of flagellin protein sequences, sequence alignment of flagellin proteins between probiotic and pathogenic bacteria, and clarify the possible mechanism by which TLRs discriminately respond to pathogens and probiotics.
Results: Probiotic treatment significantly improved anxiety, depression and esteem in IBS patients. Probiotic treatment of HA rats can significantly restored cognitive function and improved anxiety-like behavior, while probiotic treatment of female gastritis rats significantly reduced anxiety and depression-like behaviors. In addition, probiotic treatment also significantly decreased the levels of inflammatory markers and 5-HT metabolism in HA rats, reduced the hyperactivity of HPA axis in female gastritis rats. Furthermore, in the protein-protein interaction of TLR5-flagellin, we found that probiotic and pathogenic bacteria differed in flagellin protein sequence, particularly in the TLR5 recognition sites. Stimulation of basolaterally expressed TLRs results in inflammatory response, but activation of apically expressed TLRs leads to inhibition of the proinflammatory response.
Conclusion: These results indicate that GI inflammation can impair normal brain function and induce changes in psychological behavior in a gender-related manner, and probiotic L. helveticus strain NS8 may be beneficial for the treatment of cognitive decline, anxiety and depression-like behaviors in HE, IBS and FD. The behavioral effects of probiotics are mediated by the gut-brain axis. This findings of different recognition sites on flagellin between probiotics and pathogens as well as the polarized localization of TLRs may provide preliminary but encouraging evidence for the existence of crosstalk between flagellin and TLR5 which may be one of the mechanisms for the host discrimination between probiotics and pathogens.
学科领域行为遗传学
语种中文
文献类型学位论文
条目标识符http://ir.psych.ac.cn/handle/311026/19634
专题健康与遗传心理学研究室
作者单位中国科学院心理研究所
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罗佳. 肠道共生菌对宿主身心健康的影响及机制研究[D]. 北京. 中国科学院研究生院,2014.
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