硒代蛋氨酸与无机硒调控肉仔鸡抗氧化功能差异及Nrf2-ARE信号通路解析

The most important function of selenium (Se) is antioxidation. Our previous studies showed that the antioxidant status in kidney, liver, pancreas and muscle of broilers was significantly improved by organic Se selenomethionine (Se-Met) in comparison with inorganic Se sodium selenite (SS). To better understand the mechanism that Se-Met has higher antioxidant capability than SS, cell culture system of chicken embryo kidney (CEK) will be established to study the antioxidant effects of Se-Met and SS on CEK cells as well as the protective effects of Se-Met and SS on oxidative damage induced by hydrogen peroxide in CEK cells; the methods of Real-time PCR, Western-blotting, immunofluorescence and electrophoretic mobility shift assay will be used to study the effects of Se-Met and SS on Nrf2-ARE signal pathway activation and expression of key antioxidant and Phase II enzymes; combining with the RNA interference technology, to analyze the association of Nrf2-ARE signal pathway activation with the antioxidant function of Se-Met and SS in CEK cells. Besides, the oxidative stress model of broilers induced by sodium fluoride also will be used to study the effects of Se-Met and SS on Nrf2-ARE signal pathway activation and expression of important antioxidant and Phase II enzymes in kidney of broilers. This study will elucidate the signal transduction regulatory mechanism that involved the different antioxidant function regulated by Se-Met and SS，consequently to provide a theoretical basis for the industrial development and scientific applications in the food, medicine and feed of the Se-Met.

硒最为重要的是它的抗氧化功能。与无机硒亚硒酸钠(SS)相比，有机硒硒代蛋氨酸(Se-Met)具有更好的抗氧化效果，可更好的提高肉鸡肝、肾、胰和肌肉组织抗氧化功能，但Se-Met优于SS的抗氧化作用机理仍然不明。本项目通过构建过氧化氢诱导鸡胚肾细胞和氟化钠诱导肉仔鸡肾脏氧化应激模型，比较分析Se-Met与SS对鸡胚肾细胞和肉仔鸡肾脏抗氧化调节效应及氧化损伤的保护作用；通过RT-PCR、Western-blotting、EMSA和免疫荧光等方法研究Se-Met与SS对Nrf2-ARE信号通路激活的影响；结合关键信号分子RNA干扰技术，解析硒源抗氧化调节Nrf2-ARE通路关键信号分子；旨在查明Se-Met优于SS的抗氧化调节效应及其形成的信号转导调节机制，为Se-Met的产业化开发及在食品、医药和饲料中的科学应用提供理论依据，具有重要的理论意义和应用价值。

多数研究报道，有机硒-硒代蛋氨酸（SM）在提高动物抗氧化功能方面优于无机硒-亚硒酸钠（SS）。通常情况下，硒的抗氧化功能主要是通过硒蛋白来发挥，但我们的前期研究结果显示，SM与SS对肉仔鸡抗氧化硒蛋白基因表达的影响并无显著差异。为此，本项目通过构建过氧化氢（H2O2）诱导鸡胚肾细胞和氟化钠诱导肉仔鸡肾脏氧化应激模型，研究氧化应激信号通路Nrf2-ARE在SM和SS调控肉仔鸡抗氧化功能中的作用，探讨SM和SS是否可以通过调控Nrf2，激活Nrf2-ARE信号通路，进而诱导下游相关基因的表达而发挥作用，旨在探明SM优于SS的抗氧化机制。 研究结果表明：（1）培养液中添加SM与SS均可提高鸡胚肾细胞的抗氧化能力；H2O2诱导的氧化应激可使鸡胚肾细胞ROS含量提高及抗氧化能力下降，进而导致细胞生物大分子氧化损伤和细胞调亡；而SM和SS均可减弱H2O2诱导的鸡胚肾细胞氧化损伤程度，且SM的效果优于SS。（2）饲粮中加氟可导致肉仔鸡氧化应激，并引起生物大分子氧化损伤及肝肾组织损伤，进而降低肉仔鸡生产性能；而饲粮补硒能显著提高肉仔鸡日增重并降低料重比，减轻氧化应激对肉仔鸡生长性能的不良影响，且SM的效果优于SS。（3）同比SS，SM能显著上调肉仔鸡肾脏和鸡胚肾细胞Nrf2的基因表达，激活Nrf2-ARE信号通路，增强下游抗氧化酶和二相解毒酶的基因表达，进而提高机体的抗氧化能力，降低氧化应激对肾细胞结构及生物大分子的损伤作用，维护细胞结构的完整性。本结果可为SM的产业化开发及在食品、医药和饲料中的科学应用提供理论依据，具有重要的理论意义和应用价值。