原花青素对高脂饮食诱导肥胖大鼠肠道内毒素产生和移位的调节及其机制

Recent studies revealled that high-fat diet induced marked change in gut microflora, resulting in increased endotoxin level, leading to obesity and impaired glucose tolerance in subhealth people. Procyanidins are natural polyphenols widely present in fruits and vegetables, and capable of improving these subhealth. Our previous studies showed that procyanidins could lower the endotoxin level in high-fat-diet-induced rats, and may act by modulating gut microflora. However, the specific mechanisms remain unknown. So by using such obese rat model, and using PCR-DGGE, RT-PCR, Western-blot, immunofluorescence methods and multivariable analysis, this project will study the effects of procyanidins on the fingerprint and dominating bacteria species in gut microflora, the endotoxin level, and the effect of procyanidins on Bifidobacterium spp. and lactic acid bacteria, two important bacteria forming the bio-barrier of intestinal tract, the effect of procyanidins on the expression and intergrity of occludin and ZO-1, proteins belonging to the tight junction protein family forming the intestinal mechanical barrier. The above studies attempt to systematically reveal the procyanidins effects, and mechanisms on the production and translocation of endotoxin, to help understand the effect of interaction between procyanidins and gut microflora on human gut health, and to provide thereotical evidence for earlier nutritional intervention to subhealth people.

近年来，人们发现高脂膳食诱导肠道菌群结构显著变化，导致机体内毒素水平升高，诱发肥胖和糖耐量受损。原花青素是一种在果蔬中广泛存在的天然多酚类物质，可改善这些亚健康状态。本课题组前期研究发现，原花青素可显著降低高脂饮食诱导肥胖模型大鼠体内的内毒素水平，其可能通过调节肠道菌群来发挥作用,但具体机制尚不清楚。因此本项目拟以此肥胖模型大鼠为对象，运用PCR-DGGE、RT-PCR、免疫印迹法和免疫荧光法等技术和多变量分析方法，研究原花青素对肠道菌群指纹图谱、优势菌群的影响，对内毒素水平的影响，对构成肠道生物屏障的双歧杆菌和乳酸菌的影响，对构成肠道机械屏障的肠道上皮细胞间紧密连接蛋白Occludin和ZO-1表达量，组织形态完整性的影响。尝试通过以上研究，系统揭示原花青素对肠道内毒素的产生和移位的影响及其机制，明确原花青素对人体肠道健康的影响，为亚健康人群早期营养干预提供依据。

高脂膳食诱导肠道菌群结构显著变化，导致机体内毒素水平升高，诱发肥胖和糖耐量受损。原花青素是一种在果蔬中广泛存在的天然多酚类物质，可改善这些亚健康状态。本项目拟以肥胖模型大鼠为对象，运用PCR–DGGE、RT–PCR和免疫荧光法等技术和多变量分析方法，研究葡萄籽原花青素对肠道菌群结构、肠道生物屏障和肠道机械屏障的影响，揭示原花青素对肠道内毒素的产生和移位的影响及其机制。.大鼠肠道菌群共检测到144个属的微生物，丰度较高的主要属有32个，9个门，主要的门有Bacteroidetes， Firmicutes， Proteobacteria。主成分分析显示，高脂膳食诱导的营养肥胖模型大鼠（DIO大鼠）肠道菌群结构显著改变，而原花青素灌胃能促进DIO模型大鼠肠道菌群结构向正常大鼠转变，合适剂量为162.5和250 mg/Kg·bw·d。.后续实验以200 mg/Kg·bw·d原花青素灌胃DIO大鼠，研究原花青素作用于肠道菌群的时间效应关系。基于DGGE和扩增子测序的结果均表明，高脂膳食和原花青素导致的肠道菌群结构变化需要较长时间。高脂膳食喂养大鼠2个月后，大鼠肠道菌群结构在PCA得分图上与正常膳食组分离；原花青素灌胃6周后，灌胃组肠道菌群结构在PCA得分图上与模型组分离，向正常组靠拢。.灌胃原花青素显著增加了Bifidobacterium、Bacteroides、Akkermansia的相对丰度。灌胃原花青素6周，Akkermansia丰度比灌胃前增加70倍。Akkermansia是一种粘液层降解菌，该菌在肥胖和2型糖尿病人肠道菌群中含量降低，低聚果糖则促进该菌增殖，提示原花青素可能通过恢复粘液层促进该菌增殖。.原花青素能显著恢复因高脂膳食而异常升高的肠道壁通透性至正常水平，可能与原花青素保护肠粘膜结构完整性有关。原花青素显著提高回肠绒毛高度与隐窝深度的比值，显著抑制结肠黏膜上皮细胞间杯状细胞数减少，显著提高ZO-1和Occludin这两种细胞间紧密连接蛋白的表达量。.综上所述，原花青素能修复肠粘膜通透性，促进DIO大鼠肠道菌群结构向正常大鼠转变，显著促进Bifidobacterium、Bacteroides、Akkermansia等微生物的增殖。肠道菌群结构的修复和部分微生物的增殖，与肠粘膜通透性有相关关系，但因果关系尚需进一步研究。