Nrf2-ARE通路介导的奶牛乳腺上皮细胞抗氧化应激作用机制及其营养调控研究

Dairy cows undergo rapid metabolic and physiological adaptations during calving and lactation. It is becoming clear that dairy cows, especially in their mammary glands, suffer an oxidative stress during this transition period. Food antioxidant supplements can provide health benefits to protect cells against oxidative stress due to their ability to either directly scavenge free radicals or indirectly increase the endogenous cellular antioxidant potential, such as through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 is a transcription factor which regulates the expression of several antioxidant/detoxifying enzymes through its interaction with the antioxidant responsive element (ARE) in gene promoters and plays a central role in protection of the cells against cytotoxicity caused by oxidative stress. However, the effects of oxidative stress and antioxidants have not been well studied in bovine mammary epithelial cells (bMECs). Therefore, the objectives of this project are to investigate the molecular and cellular mechanisms of Nrf2-ARE-mediated antioxidation and evaluate the antioxidative effects of several natural antioxidants targeting Nrf2-ARE signaling in bMECs. Knowledge generated from this study will provide new insights into oxidative stress of dairy cows and provide scientific basis for the application of antioxidant in dairy production.

奶牛乳腺是乳汁合成的主要场所，代谢旺盛的泌乳阶段极易发生氧化应激。抗氧化剂因其改善动物健康功能已被广泛接受和认可，然而其在奶牛乳腺氧化应激中的效果评价和分子靶点研究却刚刚起步。鉴于Nrf2-ARE信号通路是迄今为止发现的最为重要的抗氧化应激通路，本课题拟以此为切入点和分子靶标, 利用奶牛乳腺上皮细胞体外培养模型，结合奶牛生产实验和活体取样技术，在细胞、组织和个体多水平系统研究奶牛乳腺细胞氧化应激中细胞反应及Nrf2-ARE信号通路的作用机制，探讨Nrf2-ARE信号通路作为抗氧化剂分子靶点的可能性。该研究有利于揭示奶牛乳腺氧化应激的分子机制，阐明Nrf2-ARE信号通路与乳腺健康的关系，为奶牛乳腺健康高效泌乳提供理论基础，并为反刍动物天然抗氧化添加剂的开发应用提供参考。

乳腺上皮细胞是乳汁合成的主要场所，与乳产量和乳品质密切相关。乳腺上皮细胞在生理及病理阶段，易发生氧化应激。抵御乳腺上皮细胞的氧化应激和增强其抗氧化能力是维持乳腺健康和高效泌乳的有效手段。白藜芦醇是一种高效的抗氧化剂，大量研究发现了它的保健和治疗功效，然尚无研究将其用于健康的乳腺抗氧化研究。本研究首先采用H2O2刺激MAC-T细胞构建了奶牛乳腺上皮细胞氧化应激模型，评价了白藜芦醇抵御乳腺上皮细胞氧化应激的效果。然后利用分子生物学手段探索了白藜芦醇通过Nrf2-ARE信号通路、MAPK及PI3K/AKT等信号通路对乳腺上皮细胞抵御氧化应激的保护作用机理。最后通过向哺乳期ICR小鼠日粮中添加低剂量及高剂量的白藜芦醇检测白藜芦醇在哺乳期饲喂的安全性，并明确其对乳腺的氧化还原平衡及对Nrf2-ARE信号通路的调控作用。研究成功构建了奶牛乳腺上皮细胞氧化应激模型，利用此模型研究了白藜芦醇在乳腺上皮细胞中的抗氧化损伤作用及其机制，并采用在体实验证实了白藜芦醇的乳腺保护作用及其对Nrf2-ARE-抗氧化链的调节作用。其中Nrf2-ARE-抗氧化链在乳腺抗氧化应激和细胞存活中发挥重要作用，白藜芦醇可能通过PI3K/AKT、ERK通路影响Nrf2-ARE-抗氧化链从而调控乳腺中的氧化还原平衡。本研究也为白藜芦醇在奶牛乳腺保护中的应用提供研究基础及提示。