In early lactation, bovine mammary epithelial cells undergo serious metabolic challenges and oxidative stress both of which could be alleviated by activation of autophagy. Nuclear factor erythroid 2 related factor 2 (NFE2L2), a master regulator of cellular redox homeostasis, plays an important role in the regulation of autophagy and oxidative stress. Thus, the objective of this study was to investigate the role of NFE2L2-mediated autophagy on oxidative stress of bovine mammary epithelial cells in response to exogenous free fatty acids (FFA). Exogenous FFA induced linear and quadratic decreases in activities of glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD), and increases in the contents of reactive oxygen species (ROS) and malondialdehyde (MDA). Protein abundance of LC3-phosphatidylethanolamine conjugate (LC3-II) and the number of autophagosomes and autolysosomes decreased in a dose-dependent manner, while protein abundance of p62 increased in cells challenged with FFA. Activation of autophagy via pre-treatment with Rap attenuated the FFA-induced ROS accumulation. Importantly, FFA inhibited protein abundance of NFE2L2 and the translocation of NFE2L2 into the nucleus. Knockdown of NFE2L2 by siRNA decreased protein abundance of LC3-II, while it increased protein abundance of p62. Furthermore, sulforaphane (SFN) pre-treatment attenuated the FFA-induced oxidative stress by activating NFE2L2-mediated autophagy. The data suggested that NFE2L2-mediated autophagy is an important antioxidant mechanism in bovine mammary epithelial cells experiencing increased FFA loads.

中文翻译：

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抑制核因子红细胞 2 相关因子 2 介导的牛乳腺上皮细胞自噬诱导对外源性脂肪酸反应的氧化应激

在泌乳早期，牛乳腺上皮细胞经历严重的代谢挑战和氧化应激，这两者都可以通过激活自噬来缓解。核因子红细胞2相关因子2（NFE2L2）是细胞氧化还原稳态的主要调节因子，在调节自噬和氧化应激中起重要作用。因此，本研究的目的是研究 NFE2L2 介导的自噬对牛乳腺上皮细胞对外源性游离脂肪酸 (FFA) 的氧化应激的作用。外源性 FFA 导致谷胱甘肽过氧化物酶 (GSH-Px)、过氧化氢酶 (CAT) 和超氧化物歧化酶 (SOD) 的活性呈线性和二次下降，并增加活性氧 (ROS) 和丙二醛 (MDA) 的含量。LC3-磷脂酰乙醇胺偶联物 (LC3-II) 的蛋白质丰度以及自噬体和自溶酶体的数量以剂量依赖性方式降低，而在 FFA 攻击的细胞中 p62 的蛋白质丰度增加。通过用 Rap 预处理激活自噬减弱了 FFA 诱导的 ROS 积累。重要的是，FFA 抑制了 NFE2L2 的蛋白质丰度和 NFE2L2 易位到细胞核中。siRNA 敲低 NFE2L2 会降低 LC3-II 的蛋白质丰度，同时增加 p62 的蛋白质丰度。此外，萝卜硫素 (SFN) 预处理通过激活 NFE2L2 介导的自噬来减弱 FFA 诱导的氧化应激。数据表明，NFE2L2 介导的自噬是牛乳腺上皮细胞中 FFA 负荷增加的重要抗氧化机制。