In vitro–In vivo Hybrid Approach for Studying Modulation of NRF2 in Immortalized Bovine Mammary Cells

Abstract

Nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role in the response to oxidative stress. Diets containing known NRF2 modulators could be used to minimize oxidative stress in dairy cows. Currently, studies evaluating the activity of NRF2 in bovine have used the classical in vitro approach using synthetic media, which is very different than in vivo conditions. Furthermore, studies carried out in vivo cannot capture the short-term and dynamic response of NRF2. Thus, there is a need to develop new approaches to study NRF2 modulation. The aim of the present study was to establish an in vitro–in vivo hybrid system to investigate activation of NRF2 in bovine cells that can serve as an intermediate model with results closer to what is expected in vivo. To accomplish the aim, we used a combination of a gene reporter assay in immortalized bovine mammary cells, synthetic NRF2 modulators, and blood serum from periparturient cows. Synthetic agonist tert-butylhydroquinone and sulforaphane confirmed to be effective activators of bovine NRF2 with acute and large effect at 30 and 5 μM, respectively, with null response after the above doses due to cytotoxicity. When the agonists were added to blood serum the response was more linear with maximum activation of NRF2 at 100 and 30 μM, respectively, and the cytotoxicity was prevented. High concentration of albumin in blood serum plays an important role in such an effect. Brusatol (100 nM) was observed to be an effective NRF2 inhibitor while also displaying general protein synthesis inhibition and cytotoxicity when added to synthetic media. A consistent inhibition of NRF2 was observed when brusatol was added to the blood serum but the cytotoxicity was reduced. The synthetic inhibitor ML385 had no effect on modulation of bovine NRF2. Hydrogen peroxide activates NRF2 in bovine mammary cells starting from 100 μM; however, strong cytotoxicity was detected starting at 250 μM when cells were cultivated in the synthetic media, while blood serum prevented cytotoxicity. Overall, our data indicated that the use of synthetic media can be misleading in the study of NRF2 in bovine and the use of blood serum appears necessary.

Effects of Pasture Type on Metabolism, Liver and Kidney Function, Antioxidant Status, and Plant Secondary Compounds in Plasma of Grazing, Jersey Dairy Cattle During Mid-lactation

“…This effect was somewhat surprising considering that serum from the cows grazing in those pastures contains a higher concentration of secondary compounds that are known to activate NRF2 in the monogastric animals, such as formononetin (Aladaileh et al, 2019), equol (Lin et al, 2016), and kaempferol (Yuan et al, 2021); however, all the above data were obtained in monogastric and there is a lack of information specific to bovine or ruminants. Our data also confirmed prior observation of reduced activation of NRF2 when the cells are cultivated in blood serum compared with when cultivated in DMEM with 10% fetal bovine serum (Ford et al, 2021). The reason is unclear for such an effect with possibly some effects by the presence of albumin, as previously demonstrated (Ford et al, 2021).…”

“…Our data also confirmed prior observation of reduced activation of NRF2 when the cells are cultivated in blood serum compared with when cultivated in DMEM with 10% fetal bovine serum (Ford et al, 2021). The reason is unclear for such an effect with possibly some effects by the presence of albumin, as previously demonstrated (Ford et al, 2021). The ex-vivo method we used is not a perfect representation of the in vivo situation but can somewhat reflect the response in vivo of the cells that are reached by the serum from the circulating blood since the cells in our model are affected by all the compounds in serum present in the extracellular space surrounding the cells.…”

“…The bovine mammary alveolar cells (MACT) were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) with sodium pyruvate (Cat #25-500, Genesee Scientific, San Diego, CA, USA) and 10% fetal bovine serum (Cat #F4135, Genesee Scientific) and grown and sub-cultured as previously described (Osorio and Bionaz, 2017). The cells were passed into separate white 96-well-plates (Cat#655098, Corning, Germany) and cotransfected with the plasmids pgl4.37 (luc2P/ARE/Hygro) (Cat #E3641, Promega Corporation, Madison, WI, USA) and pRL-TK (Cat #E2241, Promega Corporation) plasmids at a 40:1 ratio in OptiMEM (Cat #11058021, Genesee Scientific) using Lipofectamine 3,000 (Cat #L3000001, Thermo Fisher Scientific, Carlsbad, CA, USA) in accordance with a previously validated method (Ford et al, 2021). Then, 24 h after transfection, the growth medium was removed and replaced with serum or DMEM for another 24 h before reading the plate.…”

“…These antioxidant effects are likely driven by the upregulation of the nuclear factor erythroid 2related factor 2 (NRF2), (Zhai et al, 2013;Wang et al, 2016), a transcription factor that plays a key role in activating the endogenous antioxidant response (Tonelli et al, 2018). Of interest, the NRF2 activity can be modulated in bovine mammary cells by both natural and synthetic agonists (Ford et al, 2021).…”

Effects of PUFA-Rich Dietary Strategies on Ruminants’ Mammary Gland Gene Network: A Nutrigenomics Review

“…Nonetheless, the target genes of NFE2L2 , although well-established in monogastric animals, have not been fully confirmed in ruminants. Nevertheless, there is a need for more precise experimental approaches and new techniques to further understand the mechanism of this TF and its dynamic in the lessening of oxidative stress [ 92 ].…”