Tea polyphenols protect bovine mammary epithelial cells from hydrogen peroxide-induced oxidative damage in vitro by activating NFE2L2/HMOX1 pathways

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Abstract

Periparturient dairy cows are likely subject to altered intracellular reduction-oxidation (redox) balance due to the high metabolic rates and physiological adaptations occurring around parturition. Such conditions could induce oxidative damage. In nonruminants, it is well established that nuclear factor erythroid 2 like 2 (NFE2L2) is a critical transcription factor for maintaining cellular redox balance by inducing adaptive responses against oxidative stress (OS) that can otherwise lead to uncontrolled inﬂammation. Tea polyphenols (TP), the major polyphenolic constituents of green tea, are potent antioxidants that could exert protective effects on bovine mammary epithelial cells (BMEC) by scavenging free radicals. We used NFE2L2 short interfering RNA (siRNA) to downregulate NFE2L2 expression in cultured BMEC to investigate whether TP could inhibit H2O2-induced OS by activating the NFE2L2/heme oxygenase-1 (HMOX1) pathway. Isolated BMEC were exposed to H2O2 (600 μM) for 6 h to induce OS. Optimal doses of TP (0, 60, 80, and 100 μg/mL) were evaluated by pretreatment of BMEC for 0, 2, 4, 6, 8, 12, and 24 h, followed by a H2O2 (600 μM) challenge for 6 h. The BMEC were transfected with NFE2L2-siRNA for 48 h, pretreated with 100 µg/mL of TP for 12 h, then challenged by 600 μM H2O2 for 6 h. Results revealed that after H2O2 exposure a concentration of TP of 100 μg/mL during a 12-h incubation led to greater cell viability, protein, and mRNA abundance of NFE2L2, and lower intracellular reactive oxygen species (ROS) accumulation. In addition, transfection with NFE2L2-siRNA decreased abundance of NFE2L2 and HMOX1 in spite of exogenous TP supplementation, whereas ROS production was increased in response to exogenous H2O2 (600 μM). Overall, TP had beneficial effects on redox balance in BMEC, slowing down cellular OS-related injury through decreasing the production of ROS and enhancing mechanisms controlled at least in part by the NFE2L2/HMOX1 pathway.

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Action of Nrf2 and Keap1 in ARE-mediated NQO1 expression by quercetin.

Shunsuke Tanigawa, Shuang-Xing Hou, Makoto Fujii (2007)

Polyphenols are characterized by the presence of more than one phenolic group and are widely distributed in many fruits and vegetables. They possess antioxidant properties and interact with cellular defense systems through the antioxidant-responsive element/electrophile-responsive element (ARE/EpRE) although the precise mechanism by which polyphenols influence transcription factor complexes to target ARE is poorly understood. In the present study, we chose a typical polyphenol, quercetin, to investigate the mechanism in human HepG2 cells. Quercetin enhanced the ARE binding activity and Nrf2-mediated transcription activity. Molecular evidence revealed that quercetin not only up-regulated the expression of Nrf2 mRNA and protein, but also stabilized Nrf2 protein by inhibiting the ubiquitination and proteasomal turnover of Nrf2. At the same time, quercetin markedly reduced the level of Keap1 protein in posttranslational levels through the formation of modified Keap1 protein, rather than 26S proteasome-dependent degradation mechanisms, without affecting the dissociation of Keap1-Nrf2. Silencing Keap1 using Keap1 siRNA significantly increased the Nrf2-dependent ARE activity, whereas silencing Nrf2 using Nrf2 siRNA markedly reduced the ARE activity under both baseline and quercetin-induced conditions. Thus, we conclude that the pathway of quercetin-induced ARE activity involves up-regulation of Nrf2 through the regulation of both transcription and posttranscription sites and repression of Keap1 by affecting the posttranscription site, revealing some substantial differences between oxidative inducers. Thus, the findings provide an insight into the mechanisms underlying polyphenolic compounds in cytoprotection and cancer chemoprevention.

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Redox-regulated turnover of Nrf2 is determined by at least two separate protein domains, the redox-sensitive Neh2 degron and the redox-insensitive Neh6 degron.

Ken Itoh, Masayuki Yamamoto, Stefani Thomas … (2004)

The Nrf2 transcription factor is more rapidly turned over in cells grown under homeostatic conditions than in those experiencing oxidative stress. The variable turnover of Nrf2 is accomplished through the use of at least two degrons and its redox-sensitive interaction with the Kelch-repeat protein Keap1. In homeostatic COS1 cells, the Neh2 degron confers on Nrf2 a half-life of less than 10 min. Analyses of deletion mutants of a Gal4(HA)mNeh2 fusion protein and full-length mNrf2 indicate that full redox-sensitive Neh2 destabilizing activity depends upon two separate sequences within this N-terminal domain. The DIDLID element (amino acids 17-32) is indispensable for Neh2 activity and appears necessary to recruit a ubiquitin ligase to the fusion protein. A second motif within Neh2, the ETGE tetrapeptide (amino acids 79-82), allows the redox-sensitive recruitment of Nrf2 to Keap1. This interaction, which occurs only in homeostatic cells, enhances the capacity of the Neh2 degron to direct degradation by functioning downstream of ubiquitination mediated by the DIDLID element. By contrast with the situation under homeostatic conditions, the Neh2 degron is neither necessary nor sufficient to account for the characteristic half-life of Nrf2 in oxidatively stressed cells. Instead, the previously uncharacterized, redox-insensitive Neh6 degron (amino acids 329-379) is essential to ensure that the transcription factor is still appropriately turned over in stressed cells, albeit with an increased half-life of 40 min. A model can now be proposed to explain how the turnover of this protein adapts in response to alterations in cellular redox state.

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Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions

Daniela Weber, Michael Davies, Tilman Grune (2015)

Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples.