Inhibiting nuclear factor erythroid 2 related factor 2-mediated autophagy in bovine mammary epithelial cells induces oxidative stress in response to exogenous fatty acids

Abstract

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.

Sulforaphane prevents LPS-induced inflammation by regulating the Nrf2-mediated autophagy pathway in goat mammary epithelial cells and a mouse model of mastitis

“…This was accompanied by an enhancement of IHC staining of Nrf2 and amplitude of LC3 puncta in the mammary gland, also a result consistent with our findings in GMECs. Similar to previous studies [ 39 , 59 ], our data confirm that SFN exerts antioxidant and anti-inflammatory functions by promoting Nrf2-mediated autophagy, which may improve prevention strategies for mastitis in dairy goats.…”

Unraveling the mechanisms underlying drug-induced cholestatic liver injury: identifying key genes using machine learning techniques on human in vitro data sets

“…While downregulated in esophageal adenocarcinoma, it was upregulated in lung squamous cell carcinoma (Zhou et al 2022 ). VSIG10L shares structural similarity with VSIG10 (Zhou et al 2022 ), a gene regulated by NFE2 like BZIP transcription factor 2 ( NFE2L2 ) (Qian et al 2015 ), which protects against oxidative stress (Wolf et al 2016 ) and activates autophagy in epithelial cells (Chang et al 2022 ). As such, VSIG10L may also have antioxidant properties, which may explain its upregulation in non-DIC-treated cells.…”

Free Fatty Acids Induce Apoptosis of Mammary Epithelial Cells of Ketotic Dairy Cows via the Mito-ROS/NLRP3 Signaling Pathway

“…The MAC-T cells were cultured in a lactogenic medium (1 g/L BSA [4240GR500, BioFROXX, Beijing, China] and 2.5 mg/L of prolactin [100-07, Peprotech, RockyHill, NJ] in DMEM/F-12) before treatment as described elsewhere. 22 In Exp. 2, different FFA concentrations were used to simulate hyperlipidemia in ketotic cows.…”

“…The cells were maintained in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 medium (DMEM/F-12, Grand Island, New York) containing 1% of antibiotic–antimycotic mix (Hyclone, South Logan, UT) (100 μg/mL streptomycin and 100 U/mL penicillin), 10 μg/L of insulin (Sigma-Aldrich, St. Louis, MO), growth-promoting hormones (0.05% lactalbumin, 1 mg/L progesterone, and 0.05% α lactose), and 10% of fetal bovine serum (Hyclone, Grand Island, New York). The MAC-T cells were cultured in a lactogenic medium (1 g/L BSA [4240GR500, BioFROXX, Beijing, China] and 2.5 mg/L of prolactin [100-07, Peprotech, RockyHill, NJ] in DMEM/F-12) before treatment as described elsewhere …”