大豆球蛋白调控鱼肠ROS产生的NOX信号通路作用机制研究

Soybean protein is an excellent plant protein source for fish feed. However, high levels of soybean protein could cause damage to the structural integrity of the intestine, resulting in its dysfunction. These negative influences are partially related to the anti-nutritional factor glycinin present in soybean protein. Our previous study firstly showed that soybean glycinin could induce reactive oxygen species (ROS) production to damage the structural integrity of the intestine, but the mechanisms are still unknown. Previous researches in humans revealed that NADPH oxidase (NOX) isoform signaling plays an important role in ROS production, but this signaling pathway is large difference in various species. Unfortunately, there lacks of information on this signaling pathways in fish. Therefore, this project will be the first time to investigate the NOX isoform signaling pathways-regulated the ROS production in fish. Firstly, we will identify the NOX isoforms and the related signaling factors in the intestine of fish. Base on this, we will be the first time to study the roles of NOX isoforms to regulation of intestinal ROS production by using gene silence technology. Then, we will further explore whether soybean glycinin-regulated ROS production is related to the promotion of p22phox binding NOX to formation of a complex located in the cell membrane by using a fluorescent labeling technology. After that, we will use recombinant gene, fluorescence labeling, gene mutation / deletion technologies to further study whether glycinin influences PKCs isoforms to regulate the phosphorylation of p47phox (NOXO1), promoting p47phox (NOXO1) combined with and p67phox (NOXA1) and then translocation to the cell membrane, further binding and activating the cell membrane p22phox/NOX isoform complexes to promote ROS production. Through a series of studies, we will reveal the possible mechanism of glycinin regulation of intestinal ROS production through regulation of p47phox (NOXO1) /p67phox (NOXA1, DUOXA)/p22phox/NOX isoform signaling pathway. These results will give an important theoretical foundation for the mechanism of glycinin-damaged the structural integrity of the intestine.

大豆蛋白是鱼类优质的植物蛋白源，但易引起鱼肠结构破坏、功能异常，这与其含有的大豆球蛋白（11S）有关；11S能引起ROS产生破坏鱼肠结构，但作用机制不清楚。NOX在调控人ROS产生中起关键作用，但调控机制在不同物种间有较大差异，而鱼上缺乏研究。因此，本项目拟在鉴定鱼肠NOX亚型及相关信号分子的基础上，利用基因沉默技术率先探明NOX亚型在调控鱼肠ROS产生中的作用；利用荧光标记技术探索11S是否通过促进p22phox结合NOX亚型形成复合物调控ROS产生；利用基因重组等技术进一步研究11S是否通过PKCs亚型调控p47phox（NOXO1）磷酸化后与p67phox（NOXA1）结合进行细胞膜转位后激活p22phox/NOX亚型复合物调控ROS产生；通过系列研究揭示11S调控鱼肠ROS产生的p47phox（NOXO1）/p67phox（NOXA1）/p22phox/NOX亚型信号通路作用机制。

本项目针对抗营养因子大豆球蛋白（11S）引起鱼类肠道ROS产生导致氧化损伤破坏鱼类肠道健康的科学问题开展了NOX亚型信号途径在11S调控草鱼等主要淡水鱼肠道ROS产生中的作用和机制研究。项目组首次揭示NOX是调控鱼类肠道ROS产生的关键信号分子，率先发现草鱼肠道仅存在NOX1、NOX2、NOX4和DUOX共4种亚型（人上有7种亚型）。进一步通过体内、体外研究结合抑制研究和SiRNA技术首次研究发现水生动物饲料重要的植物蛋白源——豆粕中主要抗营养因子大豆球蛋白（Glycinin，11S）在鱼类中肠仅通过NOXO1b（而不是NOXO1a）/ NOXA1 /NOX1以及p47phox/67phox/NOX2亚型信号途径（不通过NOX4、DUOX）上调了ROS的产生，提高了中肠ROS的含量，后肠仅通过NOXO1b（而不是NOXO1a）/ NOXA1/p22phox/NOX1调控ROS的产生，肠上皮细胞仅通过NOX2亚型信号途径调控ROS的产生；同时，主要通过抑制Keap1b（而非Keap1a）-Nrf2信号通路来降低草鱼肠细胞的酶性和非酶性抗氧化能力，导致脂质过氧化和蛋白质氧化损伤，从而破坏鱼类肠道/肠细胞的细胞和细胞间完整性以及免疫屏障功能的作用。进一步动因分析发现：11S在鱼类肠道被消化为可降解部分和不可降解部分，可降解部分能在鱼类前肠迅速降解产生氨基酸和小肽从而保证了前肠的细胞结构完整性和功能正常，而在中肠可降解部分比前肠少，后肠则比中肠更少，因此11S引起鱼类中肠和后肠大部分氨基酸（如Leu、Met、Thr等）缺乏，同时干扰了维生素（如维生素A、维生素B6）的吸收，引起鱼类肠道结构完整性和屏障功能的破坏。同类物质β-伴大豆球蛋白的研究表明，NOX信号途径调控的ROS产生与细胞骨架的变化有一定关系。部分研究结果已发表SCI收录论文18篇，获国家科技进步二等奖1项、四川省科技进步一等奖1项，获授权国家发明专利2项。