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.
N1 - Funding Information:
This research was supported by grants from China National Natural Science Foundation of China (no. 31460616 and no. 31601975, Beijing) and from the China Agriculture Research System (no. CARS-36, Beijing). Y. Ma received a China Scholarship Council (Beijing) postdoctoral fellowship to train at the University of Illinois (Urbana).
PY - 2018/6
Y1 - 2018/6
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.
AB - 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.
KW - antioxidant
KW - lactation
KW - mammary gland
KW - oxidative stress
UR - http://www.scopus.com/inward/record.url?scp=85044273975&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85044273975&partnerID=8YFLogxK
U2 - 10.3168/jds.2017-14128
DO - 10.3168/jds.2017-14128
M3 - Article
C2 - 29573798
AN - SCOPUS:85044273975
VL - 101
SP - 5329
EP - 5344
JO - Journal of Dairy Science
JF - Journal of Dairy Science
SN - 0022-0302
IS - 6
ER -