胆碱通过肠道菌群影响斑马鱼脂肪肝形成效应及机制

Fatty liver is a common nutritional disease in aquaculture which harming animal health and quality. Choline can prevent fatty liver through promoting fatty transmitssion as phospholipid from liver. Choline can effect the structure of gut microbiota which regulate host lipid metabolism. Study about the structure changes of gut microbiota effected by choline and its action on fatty liver in aquatic animal have not been reported. In this project, healthy and fatty liver zebrafish will be induced by diet control with or without choline supplementation. The structural changes of gut bacteria samples will be analyzed through high throughput sequencing and bioinformatics analysis. The core bacteria groups related to the lipid metabolism will be mined from the data. Then the gut bacteria from treatments dietary with or without choline supplementation will be transmit to the gnotobiotic fish for analyzing the effects on lipid level. High throughput analysis methods-RNA-seq was used to sequence the transcriptome level of liver. Gene ontology analysis (GO) and the KEGG database were used to look for lipid metabolism related genes and signaling pathways. This project is aimed to illuminate the mechanism of choline effect on fatty liver via gut-liver axle in fish which lay a foundation for microbial control of fatty liver in aquatic animal.

脂肪肝病是养殖鱼类常见营养性疾病，严重危害动物健康和品质。胆碱可促进肝内脂肪转变为磷脂而转运至肝外，防止脂肪沉积。饮食中胆碱水平影响宿主肠道菌群结构，而肠道菌群可调节宿主脂肪代谢。关于胆碱通过肠道菌群影响水产动物脂肪肝形成效应及机制未见报道。本项目以模式生物—斑马鱼为对象，通过控制饲料中胆碱水平诱导脂肪肝模型。基于16S rDNA 高通量测序对健康组与诱导组斑马鱼肠道微生物进行比较分析，挖掘差异微生物中与宿主脂代谢相关的菌群因子。利用无菌斑马鱼模型，开展健康组和诱导组斑马鱼肠道微生物移植试验，探究不同菌群对斑马鱼肝脂水平的影响，利用RNA-seq对肝脏测序，进行GO(Gene ontology)分析和KEGG通路富集，寻找微生物对宿主脂代谢相关基因以及信号通路。本研究的目的在于从“肠—肝轴”角度阐释胆碱影响水产动物脂肪肝形成效应及机制，为水产动物脂肪肝微生态防治奠定理论基础。

脂肪肝是世界水产养殖常见的营养性疾病。胆碱可促进肝内脂肪转变为磷脂而转运至肝外，防止肝脏脂肪沉积，同时胆碱也是肠道微生物合成二甲胺和三甲胺的重要原料。因此类似于哺乳动物，胆碱可以影响水生动物肠道微生物结构并且可能与脂肪肝相关。本项目研究了胆碱影响斑马鱼肠道微生物及其与脂肪肝形成关联效应。我们通过饲喂不同胆碱水平（0、0.2%、0.4%、0.6%；C0、C2、C4、C6）的高脂饲料6周诱导脂肪肝模型。在第2、4、6周采取各处理组斑马鱼肝胰腺样品进行脂肪肝检测，结果显示在在6周，各组脂肪肝比例随便胆碱水平升高而降低，胆碱缺乏组（C0）斑马鱼全部生成脂肪肝，C6组几乎都是正常状态。对C0和C6组斑马鱼肠道微生物进行高通量测序发现C6组的鞘脂单胞菌属Sphingomonas、叶杆菌属Phyllobacteira、拟杆菌属Bacteroides和粪杆菌属Faecalibacterium相对丰度增加，鲸杆菌属鲸杆菌属下降，细菌功能预测结果显示C6组微生物功能聚类在脂肪代谢、氨基酸代谢、外源物质生物降解和代谢通路上。然后，我们将C0和C6组斑马鱼肠道微生物转移到无菌斑马鱼，并饲喂无菌高脂饲料7天。油红O染色结果显示接受C6组肠道微生物的无菌鱼（GFTC6）的肝胰腺脂肪含量明显低于接受C0组肠道微生物的无菌鱼（GFTC0）。对GFTC0和GFTC6组斑马鱼进行转录组分析，相对于GFTC0组，GFTC6组与之差异的部分基因与脱磷酸作用、脂肪转运、脂肪定位和脂蛋白代谢进程相关通路。本项目阐述了胆碱通过肠道微生物介导的预防斑马鱼脂肪肝形成的性机制，其中核心变化的微生物菌群值得在后续研究和生产中关注。