Resveratrol alleviates oxidative stress caused by Streptococcus uberis infection via activating the Nrf2 signaling pathway.

Due to its antioxidant properties, resveratrol may relieve the cellular oxidative injury induced by Streptococcus uberis (S. uberis) infection. However, the underlying molecular mechanisms remain unknown. Herein, we used S. uberis to challenge C57BL/6 mice or a mouse mammary epithelial cell line (EpH4-Ev), and the regulatory molecular mechanism of resveratrol on hosts' oxidative injury were investigated. The results showed that gavage of resveratrol alleviate the inflammatory responses and oxidative injury of mammary gland tissues induced by S. uberis infection via activating Nrf2 signaling pathways. To further understand the molecular mechanism, inhibitor of Nrf2 (ML385) and siRNA targeting p62 were used in mammary epithelial cells. The findings indicated that resveratrol mediates Keap1 degradation by activating p62, induces the expression of Nrf2 and its downstream antioxidant signaling pathways, and ameliorates oxidative damage during S. uberis infection. Collectively, these outcomes suggested that resveratrol can function as an activator of the p62-Keap1/Nrf2 signaling pathway to improve oxidative injury caused by S. uberis in mammary glands as well as in EpH4-Ev cells. Therefore, resveratrol may be useful to prevent and control S. uberis-induced bovine mastitis by relieving oxidative stress.

Effects of heat stress on the liver of the Chinese giant salamander Andrias davidianus: Histopathological changes and expression characterization of Nrf2-mediated antioxidant pathway genes.

Nuclear factor E2-related factor 2 (Nrf2) is a crucial transcription factor that regulates the basal and inducible expression of many antioxidant-relevant genes, and the Nrf2-mediated antioxidant pathway has been regarded as a critical switch in the initiation of cellular defence systems against oxidative damages. In this study, Nrf2 was first identified and characterized in the Chinese giant salamander (Andrias davidianus). A. davidianus was exposed to a high ambient temperature of 30 °C for various periods of time (0, 3, 6, 12, 24, 48 and 72 h). We investigated the effects of heat stress on alterations of the hepatic malondialdehyde (MDA) concentration, the activities of lactic acid dehydrogenase (LDH), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), the histology of the liver, and the mRNA expression patterns of 11 genes involved in the Nrf2-mediated antioxidant pathway in A. davidianus. The results showed that both the hepatic LDH activity and MDA content significantly increased after heat exposure, indicating that heat stress could induce cell injury and oxidative damage. Histological analysis of the liver showed that heat stress caused hepatocyte abnormalities, fat accumulation and ultrastructural alterations of the hepatocytes, endoplasmic reticulum and nuclei. The expression patterns of genes involved in the Nrf2-mediated antioxidant pathway in the liver were distinct when A. davidianus was exposed to heat stress. To the best of our knowledge, this study is the first on the characterization of Nrf2 in A. davidianus and even in amphibians. The results indicated that heat stress could induce oxidative damage, and the Nrf2 antioxidant pathway might play a critical role in the resistance against heat stress in A. davidianus. These findings will deepen and enrich the current knowledge on the evolutionary conserved antioxidant roles and mechanisms of Nrf2 in A. davidianus, or even in amphibians, in the antioxidant defence against heat stress.

Activation of Nrf2 by H2O2: de novo synthesis versus nuclear translocation.

The most common mechanism described for the activation of the transcription factor Nrf2 is based on the inhibition of its degradation in the cytosol followed by its translocation to the nucleus. Recently, Nrf2 de novo synthesis was proposed as an additional mechanism for the rapid upregulation of Nrf2 by hydrogen peroxide (H2O2). Here, we describe a detailed protocol, including solutions, pilot experiments, and experimental setups, which allows exploring the role of H2O2, delivered either as a bolus or as a steady state, in endogenous Nrf2 translocation and synthesis. We also show experimental data, illustrating that H2O2 effects on Nrf2 activation in HeLa cells are strongly dependent both on the H2O2 concentration and on the method of H2O2 delivery. The de novo synthesis of Nrf2 is triggered within 5min of exposure to low concentrations of H2O2, preceding Nrf2 translocation to the nucleus which is slower. Evidence of de novo synthesis of Nrf2 is observed only for low H2O2 steady-state concentrations, a condition that is prevalent in vivo. This study illustrates the applicability of the steady-state delivery of H2O2 to uncover subtle regulatory effects elicited by H2O2 in narrow concentration and time ranges.

Expression, purification, and refolding of active Nrf2 transcription factor fused to protein transduction TAT tag.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor responsible for activation of diverse genes that protect the cell against xenobiotics and oxidative stress. The Nrf2-dependent transcription is tightly controlled by cytoplasmic interaction of Nrf2 with its inhibitor, Kelch ECH-associating protein 1 (Keap1). The Keap1-mediated inhibition can be overcome by addition of xenobiotics or by overexpression of Nrf2 protein. The overexpressed Nrf2 overwhelms the Keap1 inhibition, translocates into the nucleus and activates antioxidant response element (ARE)-dependent gene transcription that protects the cell against oxidative stress. We expressed and purified recombinant mouse Nrf2 protein fused to a protein transduction domain (TAT), derived from transactivator of transcription protein from HIV-1. Full-length TAT-Nrf2 was expressed in Escherichia coli in insoluble inclusion bodies and purified to homogeneity using denaturing size exclusion chromatography. Optimal refolding conditions were determined through the use of a light scattering-based refolding assay and analytical size exclusion chromatography. The results demonstrate that the refolded TAT-Nrf2 could transduce into cultured human neuroblastoma cells. The transduced TAT-Nrf2 activated transcription of ARE-dependent genes and conferred protection against intracellular reactive oxygen species (ROS) generated by hydrogen peroxide exposure.

Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model.

The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central alpha-helix followed by a mini antiparallel beta-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the beta-propeller structure of Keap1. In contrast, the central alpha-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two beta-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central alpha-helix in a correct position to benefit ubiquitin signaling.