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Background: Bovine mammary epithelial cells after calving undergo serious metabolic challenges and oxidative stress both of which could compromise autophagy. Transcription factor EB (TFEB)-mediated autophagy is an important cytoprotective mechanism against oxidative stress. However, effects of TFEB-mediated autophagy on the oxidative stress of bovine mammary epithelial cells remain unknown. Therefore, the main aim of the study was to investigate the role of TFEB-mediated autophagy in bovine mammary epithelial cells experiencing oxidative stress. Results: H2O2 challenge of the bovine mammary epithelial cell MAC-T increased protein abundance of LC3-II, increased number of autophagosomes and autolysosomes while decreased protein abundance of p62. Inhibition of autophagy via bafilomycin A1 aggravated H2O2-induced reactive oxygen species (ROS) accumulation and apoptosis in MAC-T cells. Furthermore, H2O2 treatment triggered the translocation of TFEB into the nucleus. Knockdown of TFEB by siRNA reversed the effect of H2O2 on protein abundance of LC3-II and p62 as well as the number of autophagosomes and autolysosomes. Overexpression of TFEB activated autophagy and attenuated H2O2-induced ROS accumulation. Furthermore, TFEB overexpression attenuated H2O2-induced apoptosis by downregulating the caspase apoptotic pathway. Conclusions: Our results indicate that activation of TFEB mediated autophagy alleviates H2O2-induced oxidative damage by reducing ROS accumulation and inhibiting caspase-dependent apoptosis. [ABSTRACT FROM AUTHOR]

Background: 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). Results: 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. Conclusions: The data suggested that NFE2L2-mediated autophagy is an important antioxidant mechanism in bovine mammary epithelial cells experiencing increased FFA loads. [ABSTRACT FROM AUTHOR]