辣椒素对高脂饮食诱导肥胖小鼠肠道菌群的影响研究

发布时间：2018-01-11 12:19

本文关键词：辣椒素对高脂饮食诱导肥胖小鼠肠道菌群的影响研究　出处：《第三军医大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:肥胖症是由遗传因素和环境因素共同作用导致的营养代谢障碍性疾病,可以增加代谢性疾病、心血管等疾病的发病率。现有的肥胖治疗手段包括饮食调节/干预、运动锻炼、手术减肥,但这些方法都存在耐受性差、副作用大或治疗后反弹等缺点,亟待开发新的肥胖预防和治疗方法。近年来,几种来源于天然植物的生物分子被证实具有减肥的作用,包括辣椒素,低聚果糖,多酚,岩藻依聚糖等。辣椒作为蔬菜或调味品在全球范围内作被广泛食用,其主要成分辣椒素(Capsaicin)在人体和动物上的减肥作用已经被广泛的报道。其中涉及到的机制较为复杂,包括全身性的机体代谢调节,食物摄入量减少等。研究发现辣椒素激活瞬时受体电位香草醛亚家族1(Transient Receptor Potential Vanilloid 1,简称TRPV1)钙离子通道并增强肾上腺髓质中的儿茶酚胺分泌,从而增强产热作用起到减少体重增加的作用。研究发现辣椒素能减少食物摄入,且这种作用依赖于TRPV1。越来越多的证据显示肠道微生态失调在肥胖和其它代谢性综合症中扮演了重要的角色。肠道微生物与宿主之间存在大量的生理学上和病理学上的相互作用,比如宿主能量平衡的调节、糖代谢、慢性炎症状态。研究发现肥胖人群和肥胖小鼠中肠道微生物群落组成发生了巨大的改变。辣椒素通过TRPV1的减肥作用已经被广泛研究,但是辣椒素对肠道微生物群落(肠道菌群)的影响尚未见系统报道。在微生物群落分析中,不管是基于16S r RNA基因的扩增子测序(Amplicon sequencing)还是鸟枪法宏基因组测序(Shotgun metagenomics sequencing),不管是使用何种分析流程,其生物信息分析流程都涉及多个软件,包括大量繁杂的步骤,包括测序原始数据质量控制、分样、格式转换等等。如何准确、高效地完成这些分析步骤是生物信息分析和开发研究工作者迫切需要解决的问题。目的:研究辣椒素对高脂饮食诱导肥胖小鼠肠道菌群的影响;针对一种或多种肠道微生物,研究探索辣椒素改变肠道菌群结构的机制;设计开发微生物群落分析辅助工具。方法与结果:本研究分为三个部分:第一部分:辣椒素对高脂饮食诱导肥胖小鼠肠道菌群结构的影响研究研究喂食辣椒素9周后高脂饮食诱导肥胖C57BL/6J小鼠与不喂食辣椒素的肥胖小鼠之间肠道微生物群落的变化,发现这种辣椒素的减肥作用与肠道微生物群落的改变相关。1、在微生物群落组成上,在门的水平,长期食用辣椒素的高脂饮食肥胖小鼠肠道中变形菌门(Proteobacteria)丰度显著降低。在属的水平,拟杆菌属(Bacteroides)、粪球菌属(Coprococcus)、普氏菌属(Prevotella)和阿克曼氏菌属(Akkermansia)的丰度则显著增加,且这些细菌丰度的增加均对健康有利。2、在微生物群落功能上,与帕金森疾病有关的代谢通路、与细菌毒力相关的四型分泌系统代谢模块丰度降低,而有利于改善宿主糖代谢的糖酵解代谢模块丰度增加。第二部分:辣椒素增加肠道细菌Akkermansia muciniphila丰度的机制研究1、辣椒素在体外直接刺激A.muciniphila结果显示,辣椒素在高浓度下会抑制A.muciniphila的生长,但在低浓度下也不会促进其生长;在黏蛋白(Mucin)存在下,A.muciniphila生长速度提高,在平台期细菌密度更高。2、脂餐诱导肥胖C57BL/6J小鼠喂食9周辣椒素后与不喂食辣椒素的肥胖小鼠相比,没有增加肠道杯状细胞的数量和密度。3、给正常C57BL/6J小鼠灌食辣椒素30分钟后,空肠杯状细胞主要黏蛋白基因Muc2表达上调,并持续到90分钟;肠道抗菌肽基因Reg3g的表达在结肠中稳步上升。第三部分:设计开发微生物群落分析辅助工具应对微生物群落分析的可重复性、多样性和高效性要求,用Go语言开发多个微生物群落分析辅助工具,提供针对Windows,Linux,OS X,Free BSD,Open BSD等主流操作系统的单一可执行二进制文件,无需安装和配置,下载即用,极大降低微生物和生物信息工作者的安装和学习成本,已受到生物信息和开源社区广大用户的欢迎和认可。其中Seq Kit和Taxon Kit均被邀请录入英文书籍《The Biostar Handbook:A Beginner's Guide to Bioinformatics》中。1、设计、开发、高度优化FASTA/Q序列处理软件包Seq Kit,提供20个实用的子命令和丰富的文档,提高测序数据的处理效率。2、设计、开发CSV/TSV格式处理软件包csvtk,提供25个易用和实用的子命令,用于处理微生物群落分析中的数据文件。3、设计、开发NCBI分类学数据处理软件包Taxon Kit,用于高效查询、转换NCBI生物分类学数据,用于应对不同微生物群落分析软件的格式需求。4、设计、开发作业并行软件rush,提供高实用性的功能,用于简化和加速对大量处理样品的包含微生物群落分析在内的生物信息分析。结论:1、高脂饮食诱导肥胖小鼠长期食用辣椒素能减轻体重,改善葡萄糖稳态。这种改善作用与肠道菌群的改变有关。肠道菌群的改变及肠道细菌Akkermansia muciniphila的丰度增加可能在这种保护性作用中起到关键作用。因此我们推测辣椒素可能通过改变肠道菌群从而起到减肥作用。2、辣椒素增加A.muciniphila的可能机制包括:(1)辣椒素刺激杯状细胞分泌更多的黏蛋白,为其提供更多的食物来源,从而使得A.muciniphila绝对丰度增加;(2)肠道抗菌蛋白Reg3g分泌增加,抑制其它细菌,从而使得A.muciniphila相对丰度增加;(3)小鼠食物摄入量减少,其它细菌因食物来源减少而丰度降低,而A.muciniphila可以以黏蛋白作为唯一的氮源和氮源,不受影响从而相对丰度增加。3、本研究设计开发多个跨平台、易用、实用、高效的微生物群落分析辅助软件,以应对微生物群落分析的可重复性、多样性和高效性要求,满足日益增长的测序大数据的分析需求。FASTA/Q格式处理工具Seq Kit、CSV/TSV格式处理软件包csvtk、NCBI分类学数据处理软件Taxon Kit和作业并行软件rush能极大提高微生物和生物信息工作者的效率,简化和加速对大量样品的包含微生物群落分析在内的生物信息分析,已受到广大用户的欢迎和好评。
[Abstract]:Background: obesity is a nutritional and metabolic disorder caused by genetic and environmental factors that can increase metabolic diseases, the incidence of cardiovascular disease. The existing obesity treatment methods including diet / intervention, exercise, weight loss surgery, but these methods have poor tolerance, side effects or after treatment, the rebound and other shortcomings, the need to develop new methods of prevention and treatment of obesity. In recent years, several biological molecules derived from natural plants was confirmed to have a slimming effect, including capsaicin, fructooligosaccharides, polyphenols, chitosan and so on. According to the rock algae as a vegetable or chili seasoning in global range is widely consumed, the main ingredient capsaicin (Capsaicin) in human and animal weight loss has been widely reported. The mechanism involves more complex, including systemic metabolic regulation of food intake. Reduced. Study found that capsaicin activates transient receptor potential subfamily (Transient Receptor 1 with Potential Vanilloid 1, referred to as TRPV1) calcium channel and increase catecholamine secretion in the adrenal medulla, thereby enhancing thermogenesis to reduce weight gain for study found that capsaicin can reduce food intake, and this effect is dependent on TRPV1. more and more evidences show that intestinal microflora imbalance plays an important role in obesity and other metabolic syndrome. There are a large number of physiological and pathological studies on the interaction between gut microbiota and host host, such as the regulation of energy balance and glucose metabolism and chronic inflammation. The study found that intestinal microflora of obese people and in the composition of obese mice changed. Capsaicin effect of TRPV1 by weight loss has been widely studied, but the chili The intestinal microbial community (gut flora) effects have not been reported. In the analysis of microbial community, whether amplicon sequencing 16S R gene based on RNA (Amplicon sequencing) or shotgun metagenome sequencing (Shotgun metagenomics sequencing), which is not the analysis process, the biological information analysis process involves a number of software, including a large number of complex steps, including the original sequencing data quality control, sample, format conversion and so on. How to accurately and efficiently complete the analysis steps as researchers of biological information analysis and development of the urgent problem to be solved. Objective: To study the effect of capsaicin on high fat diet induced obesity affects the intestinal flora of mice; according to one or several microorganisms, exploring the change mechanism of capsaicin of intestinal microflora; design and development of microbial community analysis tool. Methods and results: This study is divided into three parts: the first part: the changes of intestinal microbial community between capsaicin on high fat diet induced obese mice intestinal flora structure influence research on the feeding of capsaicin after 9 weeks of high fat diet induced obese C57BL/6J mice and obese mice fed capsaicin, capsaicin found that the weight loss effect and the intestinal microbial community is related to the change of.1 in the microbial community composition, on the level of mouse intestinal bacteria door deformation of high fat diet and long-term consumption of capsaicin (Proteobacteria) was significantly lower in abundance. At the genus level, Bacteroides (Bacteroides), coprococcus (Coprococcus), P. Copri (Prevotella) and Ackerman's genus (Akkermansia) abundance was significantly increased, and the increase of the bacterial abundance are beneficial to health in.2, microbial community function, associated with Parkinson disease The metabolic pathways associated with bacterial virulence, type four secretion system metabolic module abundance was reduced, and is conducive to the improvement of glucose metabolism of host glycolytic module abundance increased. The second part: the mechanism of capsaicin increased intestinal bacteria Akkermansia muciniphila abundance of 1 capsaicin in vitro direct stimulation of A.muciniphila results showed that capsaicin could inhibit A.muciniphila in under the high concentration of growth, but at low concentrations will not promote its growth; mucin (Mucin) in the presence of A.muciniphila growth rate increase in plateau bacterial density higher.2, compared with the fat meal induced obese C57BL/6J mice were fed 9 weeks after feeding and capsaicin capsaicin in obese mice, no increase in intestinal goblet the cell number and density of.3 to normal C57BL/6J mice fed capsaicin after 30 minutes, jejunal goblet cells mainly mucin gene expression of Muc2 was up-regulated, and Continued to 90 minutes; the expression of intestinal antibacterial peptide gene Reg3g increased steadily in the colon. The third part: the analysis of auxiliary tools to cope with the microbial community reproducibility of the design and development of microbial community diversity and efficiency requirements, the development of a microbial community analysis tool using Go language, for Windows, Linux, OS X, Free BSD, Open BSD and other mainstream operating system of the single executable binary file, no need to install and configure, download and use, greatly reducing the microbial and biological information workers and the installation cost of the study, has received the biological information and the open source community recognized and welcomed by the customers. The Seq Kit and Taxon Kit were .1, design, development, highly optimized FASTA/Q sequence processing software package Seq Kit, provides 20 practical sub commands And the rich design documents, improve the processing efficiency of.2 sequencing data, the development of CSV/TSV format processing software package csvtk 25 is easy to use and practical command, for the treatment of microbial community analysis in.3 data files, design, development of NCBI taxonomy data processing software package Taxon Kit, used for efficient query conversion, NCBI taxonomic data, used to deal with different microbial community analysis software format requirements of.4 design, development, operation of parallel software rush, providing high availability features, for the analysis of biological information to simplify and accelerate the amount of processing samples containing microbial community analysis,. Conclusion: 1, long-term consumption of capsaicin can reduce the body weight induced by high fat diet obese mice, improve the glucose homeostasis. The improvement of intestinal flora and changes. The abundance of intestinal flora and intestinal bacteria Akkermansia muciniphila. May play a key role in the protective effect. So we speculate that capsaicin may alter the intestinal flora and thus play a slimming effect including.2, capsaicin increased the possible mechanism of A.muciniphila (1): capsaicin stimulates secretion of mucin more goblet cells, provide more sources of food, which makes the A.muciniphila absolute abundance increased; (2) intestinal Reg3g protein secretion, inhibition of other bacteria, so that the relative abundance of A.muciniphila increased; (3) to reduce food intake in mice, because other bacterial food source and reduce the abundance decreased, while A.muciniphila with mucin as nitrogen source and nitrogen source only, which is not affected by the increase in relative abundance.3, the research design and development of multiple cross platform, easy to use, practical, efficient microbial community analysis software, to deal with the microbial community analysis repeatability, Diversity and efficiency requirements, meet the growing of the sequencing data analysis needs.FASTA/Q format processing tool of Seq Kit, CSV/TSV format software package csvtk, NCBI taxonomy data processing software Taxon Kit and rush parallel software can greatly improved the efficiency of microbial and biological information workers, including simplified and accelerated biological information analysis of microbial community the analysis of a large number of samples, and has been welcomed by the majority of users and praise.

【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】：R589.2

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