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Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro.
J Dairy Sci. 2018 Jun; 101(6):5329-5344.JD

Abstract

The experiment was conducted to determine the role of nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) antioxidant response element (ARE) pathway in protecting bovine mammary epithelial cells (BMEC) against H2O2-induced oxidative stress injury. An NFE2L2 small interfering RNA (siRNA) interference or a pCMV6-XL5-NFE2L2 plasmid fragment was transfected to independently downregulate or upregulate expression of NFE2L2. Isolated BMEC in triplicate were exposed to H2O2 (600 μM) for 6 h to induce oxidative stress before transient transfection with scrambled siRNA, NFE2L2-siRNA, pCMV6-XL5, and pCMV6-XL5-NFE2L2. Cell proliferation, apoptosis and necrosis rates, antioxidant enzyme activities, reactive oxygen species (ROS) and malondialdehyde (MDA) production, protein and mRNA expression of NFE2L2 and downstream target genes, and fluorescence activity of ARE were measured. The results revealed that compared with the control, BMEC transfected with NFE2L2-siRNA3 had proliferation rates that were 9 or 65% lower without or with H2O2, respectively. These cells also had apoptosis and necrosis rates that were 27 and 3.5 times greater with H2O2 compared with the control group, respectively. In contrast, transfected pCMV6-XL5-NFE2L2 had proliferation rates that were 64.3% greater or 17% lower without or with H2O2 compared with the control group, respectively. Apoptosis rates were 1.8 times lower with H2O2 compared with the control. In addition, compared with the control, production of ROS and MDA and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione-S-transferase (GST) increased markedly in cells transfected with pCMV6-XL5-NFE2L2 and without H2O2. However, compared with the control, production of ROS and MDA and activity of CAT and GSH-Px increased markedly, whereas activities of SOD and GST decreased in cells transfected with pCMV6-XL5-NFE2L2 and incubated with H2O2. Compared with the control, cells transfected with NFE2L2-siRNA3 with or without H2O2 had lower production of ROS and MDA and activity of SOD, CAT, GSH-Px, and GST. Cells transfected with pCMV6-XL5-NFE2L2 with or without H2O2 had markedly higher protein and mRNA expression of NFE2L2, heme oxygenase-1 (HMOX-1), NADH quinone oxidoreductase 1, glutamate cysteine ligase catalytic subunit, and glutamyl cystine ligase modulatory subunit compared with the control incubations. Cells transfected with NFE2L2-siRNA3 without or with H2O2 had markedly lower protein and mRNA expression of NFE2L2, HMOX-1, NADH quinone oxidoreductase 1, glutamyl cystine ligase modulatory subunit, and glutamate-cysteine ligase catalytic subunit compared with the control incubations. In addition, expression of HMOX-1 was 5.3-fold greater with H2O2 compared with the control. Overall, results indicate that NFE2L2 plays an important role in the NFE2L2-ARE pathway via the control of HMOX-1. The relevant mechanisms in vivo merit further study.

Authors+Show Affiliations

Institute of Animal Nutrition and Feed, Inner Mongolia Academy of Agriculture and Animal Husbandry Sciences, Huhhot 010031, P. R. China.College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, P. R. China.Institute of Animal Nutrition and Feed, Inner Mongolia Academy of Agriculture and Animal Husbandry Sciences, Huhhot 010031, P. R. China. Electronic address: gmyh1588@126.com.Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana 61801. Electronic address: jloor@illinois.edu.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

29573798

Citation

Ma, Y F., et al. "Nuclear Factor Erythroid 2-related Factor 2 Antioxidant Response Element Pathways Protect Bovine Mammary Epithelial Cells Against H2O2-induced Oxidative Damage in Vitro." Journal of Dairy Science, vol. 101, no. 6, 2018, pp. 5329-5344.
Ma YF, Wu ZH, Gao M, et al. Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro. J Dairy Sci. 2018;101(6):5329-5344.
Ma, Y. F., Wu, Z. H., Gao, M., & Loor, J. J. (2018). Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro. Journal of Dairy Science, 101(6), 5329-5344. https://doi.org/10.3168/jds.2017-14128
Ma YF, et al. Nuclear Factor Erythroid 2-related Factor 2 Antioxidant Response Element Pathways Protect Bovine Mammary Epithelial Cells Against H2O2-induced Oxidative Damage in Vitro. J Dairy Sci. 2018;101(6):5329-5344. PubMed PMID: 29573798.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro. AU - Ma,Y F, AU - Wu,Z H, AU - Gao,M, AU - Loor,J J, Y1 - 2018/03/21/ PY - 2017/11/12/received PY - 2018/02/17/accepted PY - 2018/3/27/pubmed PY - 2018/10/20/medline PY - 2018/3/26/entrez KW - antioxidant KW - lactation KW - mammary gland KW - oxidative stress SP - 5329 EP - 5344 JF - Journal of dairy science JO - J Dairy Sci VL - 101 IS - 6 N2 - The experiment was conducted to determine the role of nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) antioxidant response element (ARE) pathway in protecting bovine mammary epithelial cells (BMEC) against H2O2-induced oxidative stress injury. An NFE2L2 small interfering RNA (siRNA) interference or a pCMV6-XL5-NFE2L2 plasmid fragment was transfected to independently downregulate or upregulate expression of NFE2L2. Isolated BMEC in triplicate were exposed to H2O2 (600 μM) for 6 h to induce oxidative stress before transient transfection with scrambled siRNA, NFE2L2-siRNA, pCMV6-XL5, and pCMV6-XL5-NFE2L2. Cell proliferation, apoptosis and necrosis rates, antioxidant enzyme activities, reactive oxygen species (ROS) and malondialdehyde (MDA) production, protein and mRNA expression of NFE2L2 and downstream target genes, and fluorescence activity of ARE were measured. The results revealed that compared with the control, BMEC transfected with NFE2L2-siRNA3 had proliferation rates that were 9 or 65% lower without or with H2O2, respectively. These cells also had apoptosis and necrosis rates that were 27 and 3.5 times greater with H2O2 compared with the control group, respectively. In contrast, transfected pCMV6-XL5-NFE2L2 had proliferation rates that were 64.3% greater or 17% lower without or with H2O2 compared with the control group, respectively. Apoptosis rates were 1.8 times lower with H2O2 compared with the control. In addition, compared with the control, production of ROS and MDA and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione-S-transferase (GST) increased markedly in cells transfected with pCMV6-XL5-NFE2L2 and without H2O2. However, compared with the control, production of ROS and MDA and activity of CAT and GSH-Px increased markedly, whereas activities of SOD and GST decreased in cells transfected with pCMV6-XL5-NFE2L2 and incubated with H2O2. Compared with the control, cells transfected with NFE2L2-siRNA3 with or without H2O2 had lower production of ROS and MDA and activity of SOD, CAT, GSH-Px, and GST. Cells transfected with pCMV6-XL5-NFE2L2 with or without H2O2 had markedly higher protein and mRNA expression of NFE2L2, heme oxygenase-1 (HMOX-1), NADH quinone oxidoreductase 1, glutamate cysteine ligase catalytic subunit, and glutamyl cystine ligase modulatory subunit compared with the control incubations. Cells transfected with NFE2L2-siRNA3 without or with H2O2 had markedly lower protein and mRNA expression of NFE2L2, HMOX-1, NADH quinone oxidoreductase 1, glutamyl cystine ligase modulatory subunit, and glutamate-cysteine ligase catalytic subunit compared with the control incubations. In addition, expression of HMOX-1 was 5.3-fold greater with H2O2 compared with the control. Overall, results indicate that NFE2L2 plays an important role in the NFE2L2-ARE pathway via the control of HMOX-1. The relevant mechanisms in vivo merit further study. SN - 1525-3198 UR - https://www.unboundmedicine.com/medline/citation/29573798/Nuclear_factor_erythroid_2_related_factor_2_antioxidant_response_element_pathways_protect_bovine_mammary_epithelial_cells_against_H2O2_induced_oxidative_damage_in_vitro_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-0302(18)30270-4 DB - PRIME DP - Unbound Medicine ER -