虾壳肽GK8靶向Bmal1/Nrf2钟控通路调控中华鳖肌肉氧化应激机制研究

氧化应激严重威胁水产动物健康与品质，生物活性肽可有效缓解氧化应激对机体的危害。我们前期研究发现，克氏原螯虾虾壳肽GK8能显著降低中华鳖肌肉氧化应激水平，并恢复抗氧化关键基因Nrf2的昼夜节律，但其内在机制尚不清楚。本项目拟以中华鳖为对象，分析GK8对高脂饲养中华鳖生长、肌肉氧化应激及核心生物钟昼夜节律的影响，探讨核心生物钟和氧化应激昼夜节律变化规律，明确核心生物钟基因Bmal1和Nrf2的昼夜节律表达模式；采用分子对接、局域表面等离子共振和染色质免疫共沉淀等技术解析GK8、Bmal1和Nrf2分子间的互作关系，揭示Bmal1与GK8互作及对Nrf2基因的调控机制；分别在离体和在体水平通过抑制Bmal1、Nrf2，探究GK8介导的中华鳖肌肉氧化应激机理。本项目将阐明虾壳肽GK8靶向Bmal1/Nrf2钟控通路对中华鳖肌肉氧化应激的调控机制，为水产动物品质调控与健康养殖提供理论依据和技术支撑。

Oxidative stress is a serious threat to the health and quality of aquatic animals. Bioactive peptides can effectively alleviation oxidative stress from harming the organism. Our previous study found that red swamp crayfish shell peptide GK8 significantly reduced the oxidative stress level in muscle of the Chinese soft-shelled turtle and restore the circadian rhythm of antioxidant key gene Nrf2, but its internal mechanism is still unclear. This project plans to take Chinese soft-shelled turtle as the object. The effects of GK8 on growth, muscle oxidative stress and circadian rhythm of core circadian clock of Chinese soft-shelled turtles fed with high-fat diet were analyzed, and to explore the circadian rhythm of core circadian clock and oxidative stress. The circadian rhythmic expression patterns of the core biological clock Bmal1 and Nrf2 were clarified. The binding modes and regulation relationship between GK8, Bmal1 and Nrf2 molecules were analyzed by molecular docking, LSPR, ChIP and other techniques, to reveal the recognition mechanism between GK8 and Bmal1 protein, and the regulation effect of Bmal1 protein on Nrf2 gene. In addition, we will explore the regulation of GK8 on muscle oxidative stress of Chinese soft-shelled turtles by inhibiting the expression of Bmal1 and Nrf2 in vitro and in vivo. Based on the above studies, this project will elucidate the regulation mechanism of red swamp crayfish shell peptide GK8 targeting Bmal1/Nrf2 clock-controlled pathway on muscle oxidative stress of the Chinese soft-shelled turtle. It provides theoretical basis and technical support for the regulation of quality and healthy breeding in aquatic animals.