目的 探讨富含不同脂肪酸高脂饲料通过肠道菌群影响小鼠认知功能。方法 3 w龄雄性SPF级C57BL/6J小鼠,喂养基础饲料2 w后随机分为9组,每组10只;除对照-0（control-0, CON-0）组外,所有小鼠无菌处理4w后,CON-0及CON-1组灌胃生理盐水,其余组灌胃不同饲料喂养的供体小鼠粪菌,包括基础饲料（+CON）组、猪油（+long-chain saturated fatty acid, +LCSFA）组、椰子油（+medium-chain saturated fatty acid, +MCSFA）组、亚麻籽油（+n-3 polyunsaturated fatty acid, +n-3 PUFA）组、大豆油（+n-6 PUFA）组及反式脂肪酸（+trans fatty acid,+TFA）组,连续10 w;21 w龄时通过Morris水迷宫检测学习记忆能力;23 w龄时,麻醉解剖,留取血样、远端结肠、大脑组织和内脏脂肪,采用ELISA法检测内毒素（lipopolysaccharide,LPS）、D-乳酸（D-lactate,D-LA）、二胺氧化酶（diamine oxidase,DAO）及肠型脂肪酸结合蛋白（intestinal fatty acid binding protein,I-FABP）,HE染色结肠组织切片,免疫组化染色测定结肠组织中白介素-1β（interleukin-6, IL-1β）、转化生长因子-β1（transforming growth factor, TGF-β1）含量,并用全自动蛋白质印迹法测定脑组织中Toll样受体-4（toll-like receptor-4,TLR-4）、启动髓系分化初级反应蛋白88（myeloid differentiation factor 88,MyD88）、核因子κB（nuclear factor-κB,NF-κB） p65等炎症因子的蛋白表达。结果 较CON-0、CON-1及+LCSFA组,+MUFA和+TFA组肥胖指数（adiposity index, AI）显著升高;较+MCSFA、+n-3 PUFA及+MUFA组,水迷宫检测第4 d+LCSFA组逃避潜伏期延长;+LCSFA、+MCSFA、+MUFA及+TFA组血浆DAO、I-FABP和LPS表达升高;+LCSFA、+MCSFA、+MUFA和+n-6 PUFA组结肠组织隐窝结构不同程度改变,杯状细胞数量减少,炎性细胞浸润,上皮细胞坏死、脱落;+MCSFA及+n-6 PUFA组小鼠结肠组织中IL-1β显著升高,+n-3 PUFA及+TFA组明显降低;+n-3 PUFA组结肠组织中TGF-β1显著升高,+LCSFA及+MCSFA组明显降低;+MCSFA、+n-6 PUFA、+MUFA及+TFA组大脑组织中p65蛋白表达水平上调;+MCSFA、+n-6 PUFA及+MUFA组大脑组织中TLR-4蛋白表达水平上调。以上差异均有统计学意义（P<0.05）。结论 富含LCSFA、MCSFA、MUFA、n-6 PUFA及TFA的高脂饲料可能通过肠道菌群,提高肠道屏障通透性,促进脑组织炎症状态,影响小鼠的认知功能,而n-3 PUFA可能对上述过程起抑制作用。
Abstract
Objective To investigate the effects of high-fat diets containing different types of fatty acids on cognitive function in mice via gut microbiota. Methods 3-week-old male C57BL/6J mice were randomly assigned into 9 groups. Except for the control-0 (CON-0) group, all mice underwent 4-week aseptic treatment. Thereafter, the CON-0 and CON-1 groups were intragastrically administered normal saline, while the remaining groups received fecal microbiota from donor mice fed different experimental diets for 10 consecutive weeks. The fecal microbiota transplantation groups included +CON group, +long-chain saturated fatty acid (+LCSFA) (lard-enriched) group, +medium-chain saturated fatty acid (+MCSFA) (coconut oil-enriched) group, +n-3 polyunsaturated fatty acid (+n-3 PUFA) (linseed oil-enriched) group, +n-6 PUFA group, and +trans fatty acid-enriched (+TFA) soybean oil-enriched group. The learning and memory ability of 21-week-old mice was evaluated by Morris water maze test. Mice aged 23-week were anesthetized and blood samples, distal colon, brain tissue, and visceral fat were collected. Lipopolysaccharide (LPS), D-lactate (D-LA), diamine oxidase (DAO), and intestinal fatty acid binding protein (I-FABP) were detected by ELISA. HE staining was used for colon tissues. The expression of IL-1β and TGF-β1 in colon tissues was detected by immunohistochemistry. Additionally, fully automated Western blotting was employed to detect the expressions of inflammatory factors including toll-like receptor-4 (TLR-4), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) p65 in brain tissues. Results The adiposity index (AI) of the +MUFA and +TFA groups were higher than that of the CON-0、CON-1 and +LCSFA groups. Compared with the +MCSFA, +n-3 PUFA, and +MUFA groups, escape latency was significantly prolonged in the +LCSFA group on the fourth day of water maze test. Plasma levels of DAO, I-FABP and LPS were elevated in the +LCSFA, +MCSFA, +MUFA and +TFA groups. In the +LCSFA, +MCSFA, +MUFA and +n-6 PUFA groups, colonic crypt structures were altered to varying degrees, accompanied by a reduction in goblet cells, inflammatory cells, as well as epithelial cell necrosis and shedding. In the colon, IL-1β was significantly elevated in the +MCSFA and +n-6 PUFA groups, while it was significantly reduced in the +n-3 PUFA and +TFA groups. TGF-β1 was significantly elevated in the +n-3 PUFA group, while it was significantly reduced in the +LCSFA and +MCSFA groups. In the brain, TLR-4 and p65 expressions were elevated in the +MCSFA, +n-6 PUFA and +MUFA groups, and the p65 expression was elevated in the +TFA group. All of these differences were statistically significant (P<0.05). Conclusion Gut microbiota modulated by high-fat diets enriched in LCSFA, MCSFA, MUFA, n-6 PUFA and TFA might increase the intestinal barrier permeability, which in turn promotes inflammatory states in the brain, leading to altered cognitive function in mice. Conversely, n-3 PUFAs may exert inhibitory effects on these processes.
关键词
膳食脂肪酸 /
高脂饮食 /
菌群移植 /
炎症因子 /
认知功能
Key words
dietary fatty acid /
high-fat diet /
fecal microbiota transplantation /
inflammatory factor /
cognitive function
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基金
国家自然科学基金（No.82373556,81773406）; 北京市卫健委高层次公共卫生技术人才建设项目（No.学科骨干-03-31）
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