Nrf2/ARE介导的草鱼抗氧化应激作用机制及营养调控研究

In intensive aquaculture, fish often suffer from all kinds of adverse environmental factors stress, leading to oxidative stress injury. In this project, grass carp (Ctenopharyngodon idellus), an important freshwater species cultured in China, is used as a research object to establish oxidative stress models of cells and in vivo of grass carp stimulated by hydrogen peroxide (H2O2), herbicide diquat, respectively. Using liver cells from oxidative stress model to screen nutrients species, which can effectively activate Nrf2 and its downstream antioxidant genes expression. Then these nutrients are prepared semi-purified diets for feed trials of grass carp, so as to study antioxidant stress mechanism of different nutrient regulating Nrf2/ARE pathway in vivo level of grass carp, and comprehensively survey antioxidant-related physiological and biochemical indices (ROS, MDA, T-AOC, SOD, CAT, GSH-Px, ALT, AST and Cortisol), and growth performance of fish. This project will discuss mechanisms of Nrf2/ARE signal pathway against oxidative stress of grass carp and nutritional regulation, screen nutrients species and their applied doses, which can effectively regulate Nrf2/ARE pathway, and enhance oxidative stress resistance of grass carp. The research results will deepen the understanding of the mechanisms of fish against oxidative stress through the nutritional regulation way, improve economic benefits of aquaculture, and ensure the safety of aquatic products.

本项目针对养殖鱼类容易遭受外界不良环境因子胁迫而诱发氧化应激损伤情况，拟以我国重要的养殖鱼类--草鱼为研究对象，通过过氧化氢（H2O2）、敌草快（Diquat）分别刺激诱导建立草鱼在细胞、活体水平上的氧化应激模型。利用肝细胞氧化应激模型筛选可有效激活Nrf2及其下游抗氧化基因表达的营养素种类，再用这些营养素分别配制半精制饲料进行草鱼的养殖试验，综合考察鱼体的抗氧化指标以及生长性能，在活体水平上研究不同营养素调控Nrf2/ARE通路在草鱼抗氧化应激中的作用机制。本研究将探讨Nrf2/ARE信号通路在草鱼抗氧化应激中的作用机制及营养调控机理，筛选可有效调控Nrf2/ARE通路，增强鱼体抗氧化应激能力的营养素种类及其使用剂量，为研制复合型功能饲料配方打下基础。

本项目针对养殖鱼类容易遭受外界不良环境因子胁迫而诱发氧化应激损伤情况，以我国重要的养殖鱼类--草鱼为研究对象，首次克隆了草鱼转录因子Nrf2全长的cDNA序列，已上传至GeneBank，登录号为KP411008，并进行了序列分析以及该蛋白质结构与功能的预测；用4 mmol/L过氧化氢（H2O2）处理草鱼肝细胞2 h，构建了草鱼肝细胞氧化应激模型；草鱼幼鱼腹腔注射敌草快10 mg/kg鱼体重24 h，建立了草鱼活体水平的急性氧化应激模型。通过本研究建立的草鱼肝细胞氧化应激模型，筛选了还原型谷胱甘肽（GSH）、姜黄素、大黄素、茶多酚、褪黑素和维生素C等多种物质，主要对GSH、姜黄素进行了深入的研究。研究表明，这2种物质可提高氧化损伤肝细胞的活力和SOD活性，抑制肝细胞中ROS、GPT、GOT、LDH和MDA的水平，其中以5 mmol/L GSH和30 μmol/L姜黄素的效果较佳。通过草鱼活体水平上的急性氧化应激模型，主要研究了GSH和姜黄素调控草鱼Nrf2/ARE信号通路的抗氧化应激作用机制。研究表明，GSH、姜黄素可提高Nrf2及其下游抗氧化酶基因SOD、CAT、GSH-Px及HSP70的表达水平，从而增强草鱼的抗氧化应激能力，提高对外界不良环境因子胁迫的耐受性。其中，当饲料GSH含量为407.45 mg/kg或姜黄素添加量为400-600 mg/kg时，可有效调控草鱼Nrf2/ARE信号通路，增强草鱼的抗氧化应激能力，促进草鱼的生长。研究结果为鱼类抗氧化应激的营养调控提供了理论依据，这对于通过营养学途径来缓解鱼类的氧化应激状态，提高水产养殖业的经济效益，确保水产品安全等方面均具有重要的理论和现实意义。