白藜芦醇介导Nrf2/ARE信号通路缓解热应激肉鸡骨骼肌氧化损伤的机理研究

Heat stress (HS) lead to skeletal muscle oxidative damage and therefore seriously affects broiler production. The applicant’s previous studies found that resveratrol improved antioxidant enzymes activities and alleviated oxidative damage in skeletal muscle of broilers subjected to HS. Many antioxidant enzyme genes have antioxidant response element (ARE), and therefore can be regulated by nuclear factor E2-related factor2 (Nrf2). However, it is still not clear whether resveratrol alleviates oxidative damage in skeletal muscle of heat-stressed broilers through mediating Nrf2/ARE signaling pathway, and its mechanism of action also needs to be clarified. Therefore, using the analysis methods of RT-PCR, Western Blot, etc., the effect of resveratrol on oxidative damage status and Nrf2/ARE signaling pathway-related key factors in skeletal muscle of heat-stressed broilers and in skeletal muscle myoblasts subjected to HS will firstly be investigated to get a preliminary understanding of the inter-relationship among "resveratrol-Nrf2/ARE signaling pathway-HS-oxidative damage"；after that, Nrf2 gene is overexpressed, and then the effect of HS on oxidative damage status and Nrf2/ARE signaling pathway-related key factors in myoblasts will be investigated to clarify the inter-relationship among "HS-Nrf2/ARE signaling pathway-oxidative damage"; furthermore, Nrf2 gene is suppressed, and then the effect of resveratrol on oxidative damage status and Nrf2/ARE signaling pathway-related key factors in myoblasts subjected to HS will be investigated to clarify the inter-relationship among "resveratrol-Nrf2/ARE signaling pathway-HS-oxidative damage". Based on the above researches, the molecular mechanism of the effect of resveratrol on alleviating oxidative damage in skeletal muscle of heat-stressed broilers will be illuminated, and the theoretical foundation will be provided for solving the adverse effect of HS.

热应激（HS）导致骨骼肌氧化损伤，影响肉鸡生产。申请人发现，白藜芦醇（Res）可提高HS肉鸡骨骼肌抗氧化酶活力，缓解氧化损伤。多种抗氧化酶基因具有ARE序列，受Nrf2调控。但Res是否介导Nrf2/ARE信号通路（Nrf2通路）缓解HS诱导的鸡骨骼肌氧化损伤尚不明确，机理亟待阐明。为此，本项目拟采用RT-PCR、Western Blot等技术，分析Res对HS下鸡骨骼肌及其成肌细胞氧化损伤和Nrf2通路的影响，探究“Res-Nrf2通路-HS-氧化损伤”的关系；随后过表达Nrf2，分析HS下成肌细胞氧化损伤状态和Nrf2通路表达情况，揭示“HS-Nrf2通路-氧化损伤”的关系；最后沉默Nrf2，分析Res对HS下成肌细胞氧化损伤和Nrf2通路的影响，明晰“Res-Nrf2通路-HS-氧化损伤”的关系。以上结果将阐明Res缓解HS肉鸡骨骼肌氧化损伤的机理，为解决HS的负面影响提供理论依据。

热应激导致肉鸡骨骼肌氧化损伤，影响肉鸡生产，而饲粮添加白藜芦醇可提高热应激肉鸡肌肉抗氧化能力，缓解氧化损伤，但其机理亟待阐明。本研究旨在考察白藜芦醇对热应激肉鸡肌肉及其成肌细胞抗氧化能力、氧化损伤状态及Nrf2/ARE信号通路相关基因和蛋白表达的影响。主要研究结果如下：（1）饲粮添加白藜芦醇可提高热应激肉鸡肉品质和肌肉抗氧化能力，降低肌肉氧化损伤，同时提高Nrf2/ARE信号通路关键基因和蛋白的表达水平；（2）成功分离鉴定了肉鸡成肌细胞，并发现39.5℃是其最佳培养温度，而44.5℃的温度处理1小时即可构建出成肌细胞热应激模型；（3）细胞培养液中25μmol/L的白藜芦醇浓度不影响成肌细胞活力，但可显著提高细胞中Nrf2/ARE信号通路关键基因和蛋白的表达水平；（4）白藜芦醇可显著降低热应激成肌细胞氧化损伤水平，并显著提高热应激成肌细胞Nrf2/ARE信号通路关键基因和蛋白的表达水平；（5）激活Nrf2基因可缓解热应激导致的成肌细胞氧化损伤；（6）抑制Nrf2基因后，白藜芦醇不能激活Nrf2下游具有ARE序列的抗氧化基因表达，因而不能缓解热应激导致的成肌细胞氧化损伤。综上所述，白藜芦醇通过激活Nrf2表达，从而提高其下游一些列具有ARE序列的抗氧化基因表达，进而提高热应激肉鸡骨骼肌的抗氧化能力，最终缓解热应激导致的氧化损伤。本研究基于Nrf2/ARE信号通路阐明了白藜芦醇缓解热应激肉鸡骨骼肌氧化损伤的机理，为解决热应激的负面影响提供了理论依据。