缩合单宁干预花鲈肠道菌群发挥抗氧化作用的机理研究

The intestine is susceptible to free radical attack which usually causes oxidative damage. Recently, the nutrition regulation of intestinal flora to enhance antioxidation and intestinal health has become one of the research hotspots in the field of animal nutrition. Plant condensed tannins (CT) has antioxidant and immunological activities and thereby is an ideal substitute of antibiotics. Our previous study showed that CT improved the serum antioxidant capacity and immunity of Japanese seabass (Lateolabrax japonicus) and increased the abundance of intestinal antioxidant bacteria. However, the possible molecular mechanism of antioxidation regulated by intervention of condensed tannins on intestinal flora is still unclear. Therefore, the intestinal epithelial cell oxidative damage model will be set up to analyzed the repair effects of CT on the oxidative damage of cells. Effects of CT on the intestinal mucosal barrier, DNA oxidative damage, heat shock protein HSP70 and cell tight junction protein genes expression will be evaluated by adding CT to diets of Japanese seabass. Moreover, the signal transduction mechanism between CT degradation products and intestinal flora will be revealed by using metabolomics and genomics methods combined with fecal in vitro fermentation to verify the regulation of CT on intestinal flora. The molecular mechanism of CT on antioxidation will be elucidated from the perspective of intestinal flora. The present study will help to fully understand the theory of nutritional interventions to regulate fish intestinal health and provide a basis for healthy aquaculture and development of antibiotic alternatives.

肠道易受自由基攻击而发生氧化损伤。近年来，肠道菌群的营养调控对增强机体抗氧化和肠道健康的作用成为动物营养学的研究热点之一。天然植物提取物缩合单宁（CT）具有抗氧化和免疫活性，是理想的抗生素替代品。前期试验表明，CT可提高花鲈血清抗氧化和免疫力，增加肠道抗氧化细菌的丰度。但CT干预肠道菌群调控抗氧化作用的机理尚不清楚。本项目拟通过建立体外花鲈肠上皮细胞氧化损伤模型，分析CT对细胞氧化损伤的修复作用。通过在饲料中添加CT，观察其对肠黏膜屏障、DNA氧化损伤、热休克蛋白HSP70和细胞紧密连接蛋白基因表达的影响。同时运用代谢组学和基因组学手段，揭示CT降解产物和肠道菌群结构与功能的信号转导机制，结合粪便体外发酵验证CT与肠道菌群的互作规律。本项目将从肠道菌群的角度初步阐明CT抗氧化作用的分子机制，有助于全面理解营养干预调控鱼类肠道健康理论，为水产健康养殖和抗生素替代品的开发提供依据。

氧化应激可诱发肠道损伤和疾病，是威胁鱼类生命健康的核心原因。后抗生素时代，肠道菌群的营养调控是增强动物抗氧化能力和肠道健康的重要手段。植物缩合单宁具有较强的抗氧化和抑菌活性，能够缓解氧化应激，促进肠道健康。本项目结合生物和化学方法，从植物中提取和纯化缩合单宁；通过构建体外花鲈肠上皮细胞H2O2应激模型，分析缩合单宁对细胞存活率、活性氧水平及抗氧化酶基因表达的影响；通过开展低氧、高铜应激以及氧化鱼油、黄曲霉毒素B1饲喂试验，分析缩合单宁对花鲈肠道屏障功能、肠道紧密连接蛋白基因表达量的影响；通过基因组学和代谢组学手段，结合花鲈粪便体外培养发酵，分析缩合单宁对肠道细菌菌群的组成与结构、肠道代谢产物的影响。本项目发明了植物缩合单宁的提取、纯化、检测方法，厘清了缩合单宁通过介导Keap1-Nrf2/ARE信号通路发挥抗氧化作用的分子机理，阐明了缩合单宁通过上调细胞紧密连接蛋白ZO-1、Claudin-3、Claudin-15a、Occludin基因表达修复肠绒毛损伤和维持肠道组织结构完整的作用机制，揭示了缩合单宁调控花鲈肠道菌属Bacillus、Plesiomonas、Cetobacterium、Clostridium及肠道短链脂肪酸增强机体抗氧化作用的微生态机理，建议花鲈饲料中缩合单宁的添加量小于或等于1 g/kg。本项目从组织、细胞、基因等不同水平阐明了缩合单宁抗氧化应激及修复肠道损伤的分子机制，有助于全面理解营养干预调控鱼类肠道健康理论，为水产健康养殖和开发具有重大应用价值的饲用缩合单宁资源提供重要支撑。