Nrf2-ARE信号通路介导的桑叶黄酮缓解马运动氧化应激损伤的机理研究

With social development and change of production mode in China, the function and application of the horse had the huge changes, result in the rising of horse competition. Horse athletic performance is the most important economic characters. Oxidative stress causes horse muscle damage, leading to decreasing the athletic performance, impairing animal welfare. Therefore, the mechanism of oxidative stress regulation and nutritional mitigation strategies is became an imperative problem for competitive horse industry. Nrf2-ARE signaling pathway is an important antioxidant stress pathways. The antioxidant activity of mulberry leaves flavonoids has been proved and application in animal production. However, the effects mulberry leaves flavonoids on alleviating oxidative stress damage of horse during exercise whether associated with Nrf2-ARE pathways is unclear. The present study establishes the model of oxidative stress in satellite cells of horse muscle and investigates the effects of mulberry leaves flavonoids on activation of Nrf2-ARE signaling pathway and mRNA expression of antioxidant enzyme genes, illuminating the mechanism of decreasing oxidative stress in horse during exercise regulated by mulberry leaf flavonoids through Nrf2-ARE signaling pathway. Then the alleviating effect and mechanism of oxidative stress on horse during exercise were verified through the feeding experiment of mulberry leaf flavonoids supplementation. The results of in vitro and in vivo provide theoretical basis for the regulation of oxidative stress and a reference for using mulberry leaves flavonoids as natural antioxidants to alleviate oxidative stress in horses during exercise.

随着我国社会发展和生产方式的转变，马的功能与用途发生了转变，赛马在我国兴起。马在运动过程中容易遭受氧化应激导致肌肉损伤，降低运动性能。运动性能是马重要的经济性状，因此马运动氧化应激调控机理及策略成为赛马业亟需解决的问题。Nrf2-ARE信号通路是重要的抗氧化应激通路。桑叶黄酮的抗氧化活性在动物生产中有广泛的研究和应用。但是桑叶黄酮缓解马运动氧化应激损伤的作用效果以及是否通过Nrf2-ARE通路介导的机理尚不明确。本项目拟建立马骨骼肌卫星细胞氧化应激模型，研究桑叶黄酮对氧化应激细胞Nrf2-ARE信号通路的激活情况及下游抗氧化酶SOD、CAT、GSH-Px基因表达的影响，揭示桑叶黄酮缓解马肌肉氧化应激损伤的机理，并通过饲喂桑叶黄酮对马运动氧化应激缓解效果及机理进行活体动物验证，为马运动氧化应激调控机理，合理利用桑叶黄酮作为抗氧化剂缓解马运动氧化应激损伤提供理论基础和参考依据。

马业是一个古老的产业，作为生产资料马被广泛应用于运输、邮递、耕作、伐木、采矿等多种生产活动。随着时代的发展，传统马产业正在向集文化、体育、竞技、休闲于一体现代马产业转变。马在运动过程中会产生氧化应激，造成肌肉损伤，降低运动性能，损害动物福利。Nrf2-ARE信号通路是重要的抗氧化应激通路。桑叶黄酮的抗氧化活性在动物生产中有广泛的研究和应用。本项目成功建立了两步酶消化法体外原代培养马骨骼肌卫星细胞，并利用诱导分化、免疫荧光、RT-PCR等方法对肌卫星细胞进行鉴定；利用过氧化氢诱导马骨骼肌卫星细胞氧化损伤，分析细胞凋亡率、ROS含量以及抗氧化性能，结果表明过氧化氢作用时间为6h，作用浓度为600mol/L时，骨骼肌卫星细胞产生了较为明显的氧化损伤，可作为构建骨骼肌卫星细胞氧化损伤模型的最佳条件；H2O2损伤组的骨骼肌卫星细胞的Nrf2表达量及下游调控的抗氧化酶基因SOD1、SOD2、GPX1、GPX3、TrxR1的表达结果均低于对照组细胞，说明H2O2抑制了Nrf2-ARE信号通路导致细胞产生氧化应激；通过响应面模型得到桑叶黄酮提取条件为乙醇浓度（54.75%）、提取时间（17.61 min）、料液比（1：30.24）、温度（50.27℃）；对超声辅助粗提取，AB-8大孔树脂纯化提取以及正丁醇萃取得到3种桑叶黄酮工艺产物进行了化合物鉴定以及对马骨骼肌卫星细胞氧化应激的缓解作用，并阐明桑叶黄酮激活马骨骼肌卫星细胞中Nrf2信号通路正向调控下游TrxR1、SOD1、SOD2、GPX1、GPX3抗氧化基因表达，提高超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶含量，提高抗氧化性能，缓解氧化损伤；动物试验表明蒙古马在运动过程中会产生氧化应激，且在日粮中添加桑叶黄酮同样可以提高Nrf2信号通路调控的下游抗氧化基因表达，提高超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶含量，提升机体总抗氧化能力，缓解运动马氧化应激损伤，为抗氧化剂在缓解运动马氧化应激损伤，提高运动性能，保障动物福利提供科学依据。