FoxO信号通路在异亮氨酸提高鱼肠道抗氧化能力中的作用及机制

Fish intestine is an important site for digestion and absorption of nutrients and also plays a key role in defence against pathogen invasion. Structural integrity of the intestine is the main foundation of its function. Isoleucine (Ile) can enhance antioxidant ability of enterocytes and then improve its structural integrity in fish. However, there is still a lack of deep investigations of the underlying mechanism. In mammals, the FoxO signal pathway play an important role in the regulation of antioxidant ability. However, whether a similar regulatory role of the FoxO pathways also play in fish intestine has not been reported. Thus, this project will firstly investigate the distribution characteristics of key signal molecules in FoxO signal pathway（FoxO, Akt and JNK）in proximal intestine (PI), mid intestine (MI) and distal intestine (DI) using the molecular cloning and immunofluorescence techniques. Second, using an in vitro stress model established by our laboratory and combining with gene silencing, western blot and immunofluorescence methods, whether signal molecules FoxO, Akt and JNK involve in the regulation of antioxidant ability in fish intestine will be studied. Furthermore, we will study whether Ile regulate the fish intestinal antioxidant ability through Akt/FoxO and JNK/FoxO pathways by treating fish enterocytes with Ile. Based on the results, this work will reveal molecular regulative mechanisms of Ile-improved antioxidant ability of enterocytes in fish, which may provide some theoretical supports to the mechanism responsible for Ile-regulated intestinal antioxidant ability in fish.

鱼类肠道在营养物质消化吸收和抵御外界病原方面有重要作用。肠道结构完整是其发挥功能的重要基础。异亮氨酸（Ile）能提高建鲤肠道上皮细胞抗氧化损伤能力，保证肠道结构完整性，但作用机制仍缺乏深入研究。FoxO信号途径在调控哺乳动物抗氧化能力中发挥重要作用，但其在鱼肠道中是否也具有相似的作用未见报道。因此，本项目拟利用分子克隆和免疫荧光技术研究FoxO信号途径关键信号分子FoxO、Akt和JNK亚型在草鱼前、中、后肠分布规律；采用本课题组建立的体外应激反应模型，利用基因沉默、免疫印迹和免疫荧光技术研究信号分子FoxO、Akt和JNK在调控鱼肠细胞抗氧化能力中的作用；进一步用Ile处理，考察Ile是否通过Akt/FoxO和JNK/FoxO信号途径调控鱼肠细胞抗氧化能力，揭示Ile调控鱼肠细胞抗氧化能力的作用机制，为阐明Ile提高鱼肠道抗氧化能力的作用机制提供理论支撑。

鱼类肠道在营养物质消化吸收和抵御外界病原方面具有重要作用。肠道结构完整是其发挥功能的重要基础。异亮氨酸能提高建鲤肠道上皮细胞抗氧化损伤能力，保证肠道结构完整性，但作用机制缺乏深入研究。本研究利用分子克隆、免疫印迹和免疫荧光技术考察异亮氨酸是否通过AKT/FOXO和JNK/FOXO信号途径调控鱼肠细胞抗氧化能力，揭示Ile调控鱼肠细胞抗氧化能力的作用机制。本研究结果表明：日粮添加适宜水平异亮氨酸：（1）增重率、摄饵量、特异性生长率、饲料转化效率、蛋白质转化效率显著提高；（2）全鱼粗蛋白含量、粗脂肪含量、蛋白质沉积率、脂肪沉积率和灰分沉积率显著提高，对全鱼粗水分和粗灰分含量无显著影响；以PWG为标识，二次回归分析确定杂交江鲿异亮氨酸需要量为12.43g/kg饲粮（32.05g/kg饲粮蛋白）；（3）肠长指数显著提高，而对肠重和肠体指数无显著影响；（4）肠皱襞高度、肠皱襞宽度、肠皱襞纵截面积、肠道肌层厚度、肠皱襞杯状细胞数量均显著升高；（5）粘蛋白2、紧密连接蛋白ZO2、claudin3和claudin7 mRNA水平显著上调，紧密连接蛋白ZO1、Occludin、claudin2和肌球蛋白轻链激酶mRNA水平显著下调；（6）ROS、MDA和PC含量显著下降， T-SOD、CAT、GST、GPX和GR活力， ASA、AHR和GSH含量显著提高；（7）肠道GST、Nrf2mRNA水平显著上调，CuZnSOD、CAT、GCLC和Keap1 mRNA水平显著下调；（6）Akt1、Akt2、Akt3、JNK1、JNK2、JNK3和FOXO6mRNA水平显著提高，FOXO1、FOXO3a和FOXO4 mRNA水平显著下降；FOXO3a蛋白水平显著提高（8）下调caspase3和caspase9 mRNA水平；（9）LZM、ACP和AKP活性， C3、C4和IgM含量显著提高；（10）c-type-lectin、c-LZM、g-LZM和hepcidin mRNA水平显著提高；（10）显著上调sirt1、p90rsk和β-defensin mRNA水平， ERK1和ERK2 mRNA水平显著降低。综上所述，适宜量的异亮氨酸能提高杂交江鲿生长性能、促进肠道发育。异亮氨酸可能通过调控Nrf2/Keap1、Akt/FOXO3a和JNK/FOXO4信号分子基因转录，调节抗氧化酶相关基因的表达，