肠道菌群在辣椒素代谢调节效应中的作用及机制研究

发布时间：2018-05-26 09:15

本文选题：辣椒素 + 肠道菌群　；参考：《第三军医大学》2017年博士论文

【摘要】：肥胖是威胁人类健康的主要疾病之一,发病率呈逐年上升趋势。深入探讨肥胖的发病机制,并制定有效的防治策略,具有重要的现实意义。人体共生的肠道菌群在维持机体正常的代谢、消化和免疫功能等方面具有不可替代的作用。肠道菌群紊乱与肥胖、糖尿病、心血管疾病和神经退行性疾病密切相关。肠道菌群具有一定的可塑性,而膳食是影响和改变肠道菌群的结构和功能最重要的因素。高脂膳食诱导下肠道菌群紊乱,革兰氏阴性菌增多,肠道通透性增加,导致大量脂多糖(Lipopolysaccharide,LPS)入血,甚至细菌入血出现细菌易位(Bacterial Translocation)现象,形成代谢性内毒素血症(Metabolic Endotoxemia),促发全身低度慢性炎症反应(Chronic Low-grade Inflammation),最终导致胰岛素抵抗和肥胖等症状。因此,肠道菌群被认为可能是在膳食与人体自身基因的相互作用下导致疾病发生的重要环节,而以肠道菌群为靶点的膳食营养干预受到越来越多的关注。辣椒素(Capsaicin,CAP)是辣椒中含有的生物碱类植物化合物,具有调节能量代谢、调节血脂、抗炎、增强胰岛素敏感性等生物学效应。然而以往评价辣椒素对人群控制、改善胰岛素抵抗以及降低体内炎症等方面的研究较少,且结果不一。此外,辣椒素对环境中存在致病性细菌有抑制作用,而促进乳酸杆菌等有益菌的生长。通过培养皿培养法发现辣椒素可影响大肠杆菌、乳杆菌属和双歧杆菌属的生长;通过q PCR分析发现辣椒素可以增加大鼠肠道细菌Prevotella、Lactobacillus和Akkermansia相对丰度。然而关于辣椒素对于肠道菌群的作用研究多停留在传统的体外培养和q PCR等技术方法,远远不能深入了解辣椒素引起肠道菌群的真实变化情况,而辣椒素引起的肠道菌群变化又是如何影响其代谢调节作用,具体作用机制也有待于进一步探讨。基于上述研究背景,我们提出“辣椒素通过重塑肠道菌群进而发挥其代谢调节作用”的研究假说。本课题首先开展了为期6周的膳全膳食人群干预研究,应用当前流行的16S r RNA基因测序技术结合多元统计分析,深入分析辣椒素对人体肠道菌群的影响,以及肠道菌群在辣椒素发挥健康代谢调节中的作用。然后通过高脂膳食诱导肥胖小鼠模型,利用无菌小鼠(Germ Free mice)技术平台和广谱抗生素清除等实验方法,进一步探讨肠道菌群在辣椒素发挥改善炎症状态、增强胰岛素敏感性以及降低体脂等健康效应中的作用及因果关系。本研究的主要实验结果和结论如下:(1)不同肠型人群对膳食辣椒素表现出差异性应答效应本研究通过全膳食人群干预发现辣椒素可促进体内ghrelin、GIP和GLP-1等胃肠激素的分泌;通过16S r RNA基因测序发现经过10mg/d的膳食辣椒素干预2周后,人群肠道产丁酸细菌Faecalibacteria的相对丰度增加(P0.05);此外。本研究中人群在肠道菌群属水平上可分为Bacteriodes(E1)和Prevotella(E2)两种肠型,而E1亚型人群在5或10mg/d膳食辣椒素期下呈现出更好的代谢调节效应。(2)膳食辣椒素通过改善肠道屏障功能进而预防肥胖的发生发展高脂膳食诱导肥胖小鼠肠道屏障连接蛋白ZO-1和Occludin基因表达下降,肠道通透性增加,同时伴随较高的血浆LPS水平、体内慢性炎症水平、体内糖耐量受损等症状;高脂膳食补充辣椒素后可增加肠道屏障连接蛋白ZO-1和Occludin基因和降低肠道通透性,降低血浆LPS水平、改善体内慢性炎症水平、体内糖耐量受损情况,以及降低体重等代谢调节作用。(3)膳食辣椒素通过重塑肠道菌群发挥其健康代谢调节作用膳食辣椒素可显著增加小鼠肠道中产SCFAs细菌Lachnospiraceae和Ruminococcaceae的相对丰度,降低肥胖小鼠肠道菌群中高丰度S24-7以及Lipopolysaccharide biosynthesis的相关功能。通过广谱抗生素干预后发现辣椒素带来的健康代谢调节作用减弱,而利用无菌小鼠技术,发现辣椒素的代谢调节效应可以通过菌群移植方式,传递到子代小鼠。结果表明肠道菌群是辣椒素发挥健康代谢效应的关键靶点。(4)内源性大麻素系统参与膳食辣椒素对肠道屏障功能的改善膳食辣椒素可显著增加结肠大麻素受体1(Cannabinoid receptor type 1,CB1)的表达。应用CB1激动剂可阻止膳食辣椒素带来的代谢调节效应,重新诱导小鼠出现肠道屏障连接蛋白ZO-1和Occludin基因表达下降、肠道通透性增加、血浆LPS水平升高、体内慢性炎症,糖耐量受损情况以及体重增加等肥胖表型。结果表明抑制CB1是肠道菌群介导的辣椒素发挥减肥等健康代谢效应中的重要机制。综上所述,本课题通过全膳食人群干预试验,首次发现膳食辣椒素可影响人体肠道菌群的组成,人群肠型的差异是导致膳食辣椒素干预效果的不同的原因之一。通过16S r RNA基因测序发现辣椒素可影响肠道菌群的组成,增加产SCFAs细菌,降低革兰氏阴性菌(S24-7)以及LPS合成功能;采用广谱抗生素干预和菌群移植实验进一步证实了肠道菌群是膳食辣椒素发挥减肥等健康代谢调节作用的重要靶点,而抑制CB1则是其中的重要机制。本课题从人群和动物水平上深入系统研究了肠道菌群在膳食辣椒素发挥健康代谢效应的作用和机制,为个性化膳食辣椒素干预提供了重要实验依据。
[Abstract]:Obesity is one of the major diseases that threaten human health. The incidence of obesity is increasing year by year. It is of great practical significance to explore the pathogenesis of obesity and formulate effective prevention and control strategies. The intestinal microflora of human body has an irreplaceable role in maintaining the normal metabolism of the body, digestive and immune functions. Group disorders are closely related to obesity, diabetes, cardiovascular disease and neurodegenerative diseases. Intestinal microflora has a certain plasticity, and diet is the most important factor affecting and changing the structure and function of intestinal flora. The disorder of intestinal flora, the increase of gram negative bacteria, the increase of intestinal permeability, and a large amount of fat in the high fat diet. Sugar (Lipopolysaccharide, LPS) into the blood, even bacterial entry into the bacterial translocation (Bacterial Translocation) phenomenon, the formation of metabolic endotoxemia (Metabolic Endotoxemia), the promotion of systemic low chronic inflammatory response (Chronic Low-grade Inflammation), the final cause of insulin resistance and obesity and other symptoms. Therefore, the intestinal flora is considered to be Capsaicin (CAP) is the alkaloid plant compound contained in Capsicum, with regulating energy metabolism, regulating blood lipid, anti-inflammatory and enhancing islets of pancreas. However, the previous evaluation of capsaicin for population control, improvement of insulin resistance, and reduction of inflammation in the body was less, and the results were different. In addition, capsaicin inhibited the existence of pathogenic bacteria in the environment and promoted the growth of Lactobacillus and other beneficial bacteria. Capsaicin can affect the growth of Escherichia coli, Lactobacillus and Bifidobacterium, and Q PCR analysis shows that capsaicin can increase the relative abundance of Prevotella, Lactobacillus and Akkermansia in intestinal bacteria in rats. However, the study of capsaicin's effect on intestinal flora is mostly in traditional culture in vitro and Q PCR and other technical methods. It is far from understanding the true changes in the intestinal flora caused by capsaicin, and how capsaicin induced changes in intestinal flora affect its metabolic regulation, and the specific mechanism of action needs to be further explored. Based on the above research, we propose "capsaicin overweight plastic intestinal microflora and then play its metabolic regulation." Using the "research hypothesis", the subject first carried out a 6 week diet whole diet group intervention study, the current popular 16S R RNA gene sequencing technology combined multivariate statistical analysis, in-depth analysis of the effects of capsaicin on the human intestinal flora, and the role of intestinal flora in the regulation of capsaicin's volatile metabolism. The fat diet induced obesity mice model, using the Germ Free mice technology platform and the broad-spectrum antibiotic scavenging methods to further explore the role and causality of the intestinal flora in the improvement of the inflammatory state, the enhancement of insulin sensitivity and the reduction of body fat. The main experimental results of this study. And the conclusions are as follows: (1) the difference in response to dietary capsaicin in different enteric populations was found in the study. This study found that capsaicin could promote the secretion of gastrointestinal hormones such as ghrelin, GIP and GLP-1 through the whole diet group intervention. The intestinal production of butyric acid bacteria in the intestinal tract after 2 weeks after the 10mg/d dietary capsaicin intervention was found by the 16S R RNA gene sequencing. The relative abundance of Faecalibacteria increased (P0.05); in addition. In this study, the population could be divided into two intestinal types at the level of intestinal flora, Bacteriodes (E1) and Prevotella (E2), while the E1 subtypes showed better metabolic regulation effect under the 5 or 10mg/d dietary capsaicin. (2) dietary capsaicin was prevented by improving the intestinal barrier function and then prevented. The development and development of fat diet induced the decrease of intestinal barrier connexin ZO-1 and Occludin gene expression, the increase of intestinal permeability, higher plasma LPS levels, chronic inflammatory levels in the body, and impaired glucose tolerance in the body, and the increase of intestinal barrier connexin ZO-1 and Occlu after the high fat diet supplemented with peppericin Din gene, reducing intestinal permeability, reducing plasma LPS level, improving the level of chronic inflammation in the body, impaired glucose tolerance in the body, and reducing the metabolic regulation of body weight. (3) dietary capsaicin can significantly increase the SCFAs bacterial Lachnospir in the intestinal tract of mice by remolding the intestinal microflora. The relative abundance of aceae and Ruminococcaceae reduces the related functions of high abundance S24-7 and Lipopolysaccharide biosynthesis in the intestinal microflora of obese mice. After broad-spectrum antibiotics intervention, it is found that the healthy metabolic regulation of capsaicin is weakened, and the metabolic regulation effect of capsaicin can be found through aseptic mice technology. The results showed that the intestinal flora is the key target for the healthy metabolism of capsaicin. (4) endogenous cannabinoid system participates in the improvement of dietary capsaicin on the intestinal barrier function, the dietary capsaicin can significantly increase the expression of Cannabinoid receptor type 1, CB1. CB1 agonists could prevent the metabolic regulation effect of dietary capsaicin, re induced the decrease of intestinal barrier connexin ZO-1 and Occludin gene expression, the increase of intestinal permeability, the increase of plasma LPS level, chronic inflammation in the body, impaired glucose tolerance and weight gain and other obesity phenotypes. The results showed that the inhibition of CB1 was intestinal bacteria. The group mediated capsaicin plays an important mechanism in the healthy metabolic effect of weight loss. To sum up, this subject is first found that the dietary capsaicin can affect the composition of the intestinal flora in the human body. The difference in the intestinal type of the population is one of the different reasons leading to the intervention of capsaicin in the diet by the 16S R RNA gene. Capsaicin can affect the composition of intestinal flora, increase the production of SCFAs bacteria, reduce the Gram-negative bacteria (S24-7) and the function of LPS synthesis. The broad-spectrum antibiotic intervention and bacterial group transplantation experiments further confirm that the intestinal flora is an important target for the dietary capsaicin to play the role of regulating the weight loss and so on, and the inhibition of CB1 is one of the important targets. The role and mechanism of intestinal flora in dietary capsaicin, which provide important experimental basis for individualized dietary capsaicin intervention, are systematically studied from the level of population and animal.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R151

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