高糖饮食干预下AD模型小鼠肠道菌群结构和血浆代谢谱变化的研究
发布时间：2018-09-06 08:55
【摘要】：阿尔茨海默症(Alzheimer’s disease,AD)是一种表现为记忆和认知功能退化的进行性神经退行疾病。大量研究表明AD与2型糖尿病存在联系,共同致病机理有慢性炎症、胰岛素抵抗和线粒体功能异常。另一方面,肠道菌群能调节宿主的营养、代谢、免疫,并影响远端的中枢神经系统。而高脂高糖的西式饮食可诱导肠道菌群失衡引起多种代谢性疾病。因此,本研究的目的是研究长期高糖饮食对AD发生发展,肠道菌群结构和血浆代谢谱的影响,并探讨它们背后的可能联系。首先,我们以10%的蔗糖饮水(高糖组)和普通饲料饮水(正常饮食组)分别给予野生型和AD模型APP/PS1小鼠。6个月后,Morris水迷宫显示高糖饮食促进空间学习记忆能力的下降。免疫组化和免疫印迹分析发现APP/PS1鼠高糖组大脑的可溶性Aβ含量和沉积增多。进一步实验显示高糖组出现胰岛素抵抗,同时血液中促炎因子(TNF-α和IL-6)及脂多糖结合蛋白水平上升,提示高糖饮食可能导致内毒素血症和慢性炎症。另外,RT-qPCR检测到高糖组小鼠结肠两种紧密连接蛋白claudin-5和occludin mRNA转录水平下降。以上结果提示高糖饮食可能导致肠道通透性增加和内毒素入血从而引发慢性炎症和胰岛素抵抗。而根据文献报道,上述变化可能与肠道菌群失衡有关。随后,我们对盲肠细菌进行16S rRNA基因测序,有效序列聚类得到可操作分类单元(OTUs)。基于各样品OTU组成的PCA、PLS-DA等模型显示高糖饮食明显改变肠道菌群落的整体结构。OTUs的物种注释结果显示:硬壁菌门/拟杆菌门的比值和疣微菌门相对丰度在高糖组下降,变形菌门则上升;属水平上,多种有益菌Allobaculum,Akkermansia和乳杆菌属丰度在高糖组下降,而拟杆菌属,螺杆菌属,Alloprevotella和Alistipes则上升。根据文献报道,上述不同种类肠道菌含量的变化可能与肠道内短链脂肪酸的产生异常,肠道屏障功能破坏,能量代谢失衡,慢性炎症,胰岛素抵抗和衰老等密切相关。此外,我们还通过UPLC-Q-TOF-MS代谢组学平台,研究了高糖饮食对血浆代谢谱的影响,PLS-DA得分图显示高糖组和普通饮食组血浆代谢谱有明显差异。经鉴定的差异代谢物中,多种磷脂类、必需的多不饱和脂肪酸含量在APP/PS1高糖组中下降,而脂酰肉毒碱类和三种胆汁酸则上升。此外两种肠道菌相关的代谢物p-cresol sulfate和p-cresol glucuronide在高糖组上升,它们是蛋白结合尿毒症毒素。这些生物标志物的变化表明:血脂异常、细胞膜破坏,以及肠道菌的有毒产物进入体内可能是高糖饮食促进AD发展的致病机制之一。总之,本研究发现长期高糖饮食可促进AD模型小鼠空间学习记忆能力下降和大脑淀粉样变性。相关机制为高糖饮食可能导致肠壁屏障功能损坏,慢性炎症,胰岛素抵抗和血浆代谢谱异常,并可能与肠道菌群结构失衡有关,但需进一步验证。本研究可能有助于为AD的致病机制研究提供新的视角,为疾病的预防和治疗提供新的途径。
[Abstract]:Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory and cognitive impairment. A large number of studies have shown that AD is associated with type 2 diabetes mellitus. The common pathogenesis of AD is chronic inflammation, insulin resistance and mitochondrial dysfunction. On the other hand, intestinal flora can regulate the nutrition and metabolism of the host. The aim of this study was to investigate the effects of a long-term high-glucose diet on the occurrence and development of AD, intestinal flora structure and plasma metabolic profiles, and to explore the possible links between them. Six months later, Morris water maze showed that high-sugar diet promoted the decline of spatial learning and memory. Immunohistochemistry and Western blot analysis showed that the soluble A beta content and deposition in the brain of APP/PS1 mice were increased in high-sugar diet group. In addition, RT-qPCR detected the transcriptional levels of claudin-5 and occludin mRNA in colon of mice in high glucose group. These results suggest that high-glucose diet may lead to increased intestinal permeability and endotoxin inflammation leading to chronic inflammation and insulin resistance. According to the literature, these changes may be related to the imbalance of intestinal flora. Subsequently, 16S rRNA gene sequencing of cecum bacteria, effective sequence clustering obtained operable taxon. (OTUs). PCA and PLS-DA models based on OTU composition of each sample showed that high-sugar diet significantly altered the overall structure of the intestinal bacterial community. The results of OTUs species annotation showed that the ratio of scleroderma to Bacteroides and the relative abundance of verrucous microflora decreased in the high-sugar group, while that of Proteus increased in the high-sugar group. The abundance of Lactobacillus spp. and Lactobacillus spp. decreased in the high glucose group, but increased in Bacillus spp., Helicobacter spp., Alloprevotella and Allistipes. In addition, we also studied the effects of high-sugar diet on plasma metabolic profiles using UPLC-Q-TOF-MS metabonomics platform. PLS-DA scores showed that there were significant differences in plasma metabolic profiles between high-sugar diet group and normal diet group. In addition, two intestinal bacteria-related metabolites, p-cresol sulfate and p-cresol glucuronide, increased in the high glucose group. They are protein-bound uremic toxins. Changes in these biomarkers indicate abnormal blood lipids, destruction of cell membranes, and entry of toxic products from intestinal bacteria into the body. In conclusion, this study found that long-term high-glucose diet could promote the decline of spatial learning and memory ability and amyloidosis in AD model mice. The mechanism is that high-glucose diet may lead to intestinal barrier dysfunction, chronic inflammation, insulin resistance and abnormal plasma metabolic profiles. This study may provide a new perspective for the pathogenesis of AD and provide a new way for the prevention and treatment of diseases.
【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16;R-332
本文编号：2225842
[Abstract]:Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory and cognitive impairment. A large number of studies have shown that AD is associated with type 2 diabetes mellitus. The common pathogenesis of AD is chronic inflammation, insulin resistance and mitochondrial dysfunction. On the other hand, intestinal flora can regulate the nutrition and metabolism of the host. The aim of this study was to investigate the effects of a long-term high-glucose diet on the occurrence and development of AD, intestinal flora structure and plasma metabolic profiles, and to explore the possible links between them. Six months later, Morris water maze showed that high-sugar diet promoted the decline of spatial learning and memory. Immunohistochemistry and Western blot analysis showed that the soluble A beta content and deposition in the brain of APP/PS1 mice were increased in high-sugar diet group. In addition, RT-qPCR detected the transcriptional levels of claudin-5 and occludin mRNA in colon of mice in high glucose group. These results suggest that high-glucose diet may lead to increased intestinal permeability and endotoxin inflammation leading to chronic inflammation and insulin resistance. According to the literature, these changes may be related to the imbalance of intestinal flora. Subsequently, 16S rRNA gene sequencing of cecum bacteria, effective sequence clustering obtained operable taxon. (OTUs). PCA and PLS-DA models based on OTU composition of each sample showed that high-sugar diet significantly altered the overall structure of the intestinal bacterial community. The results of OTUs species annotation showed that the ratio of scleroderma to Bacteroides and the relative abundance of verrucous microflora decreased in the high-sugar group, while that of Proteus increased in the high-sugar group. The abundance of Lactobacillus spp. and Lactobacillus spp. decreased in the high glucose group, but increased in Bacillus spp., Helicobacter spp., Alloprevotella and Allistipes. In addition, we also studied the effects of high-sugar diet on plasma metabolic profiles using UPLC-Q-TOF-MS metabonomics platform. PLS-DA scores showed that there were significant differences in plasma metabolic profiles between high-sugar diet group and normal diet group. In addition, two intestinal bacteria-related metabolites, p-cresol sulfate and p-cresol glucuronide, increased in the high glucose group. They are protein-bound uremic toxins. Changes in these biomarkers indicate abnormal blood lipids, destruction of cell membranes, and entry of toxic products from intestinal bacteria into the body. In conclusion, this study found that long-term high-glucose diet could promote the decline of spatial learning and memory ability and amyloidosis in AD model mice. The mechanism is that high-glucose diet may lead to intestinal barrier dysfunction, chronic inflammation, insulin resistance and abnormal plasma metabolic profiles. This study may provide a new perspective for the pathogenesis of AD and provide a new way for the prevention and treatment of diseases.
【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16;R-332
【参考文献】
相关期刊论文 前1条
1 赵虎;周庭银;;脂多糖结合蛋白的结构和功能[J];国外医学(微生物学分册);2000年03期
，本文编号：2225842
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