Nrf2 Response Research Articles

Epigallocatechin-3-gallate enhances key enzymatic activities of hepatic thioredoxin and glutathione systems in selenium-optimal mice but activates hepatic Nrf2 responses in selenium-deficient mice

Selenium participates in the antioxidant defense mainly through a class of selenoproteins, including thioredoxin reductase. Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechin in green tea. Depending upon the dose and biological systems, EGCG may function either as an antioxidant or as an inducer of antioxidant defense via its pro-oxidant action or other unidentified mechanisms. By manipulating the selenium status, the present study investigated the interactions of EGCG with antioxidant defense systems including the thioredoxin system comprising of thioredoxin and thioredoxin reductase, the glutathione system comprising of glutathione and glutathione reductase coupled with glutaredoxin, and the Nrf2 system. In selenium-optimal mice, EGCG increased hepatic activities of thioredoxin reductase, glutathione reductase and glutaredoxin. These effects of EGCG appeared to be not due to overt pro-oxidant action because melatonin, a powerful antioxidant, did not influence the increase. However, in selenium-deficient mice, with low basal levels of thioredoxin reductase 1, the same dose of EGCG did not elevate the above-mentioned enzymes; intriguingly EGCG in turn activated hepatic Nrf2 response, leading to increased heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 protein levels and thioredoxin activity. Overall, the present work reveals that EGCG is a robust inducer of the Nrf2 system only in selenium-deficient conditions. Under normal physiological conditions, in selenium-optimal mice, thioredoxin and glutathione systems serve as the first line defense systems against the stress induced by high doses of EGCG, sparing the activation of the Nrf2 system.

A21 Development of an Nrf2-KEAP1 protein-protein disruptor for proof of concept

Mechanisms that activate innate antioxidant responses, as a way to mitigate oxidative stress at the site of action, hold much therapeutic potential in diseases, such as the Huntington’s disease, where the use of frank antioxidants has not yielded positive results. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor whose activity upregulates the expression of antioxidant and cell detoxifying enzymes such as oxidoreductase, catalases and oxygenases in response to oxidative stress. NRF2 activation has a strong interplay with other cellular pathways such as apoptosis, autophagy and cell death, thus regulating cell fate when an oxidative insult cannot be resolved. NRF2 levels are tightly regulated by KEAP1, a sensor of oxidative stress or reactive electrophilic compounds. KEAP1 binds NRF2 and facilitates its ubiquitination and subsequent degradation. Under ROS or reactive electrophiles, modification of KEAP1 cysteines leads to conformational changes that release NRF2 for nuclear translocation and transcription of the antioxidant enzyme cascade. To date, most NRF2 activating compounds published modulate NRF2 levels via covalent binding to KEAP1 cysteines. As a consequence, these present several issues in terms of off-target effects, toxicity and inappropriate pharmacokinetics at the desired site of action. Recently, compounds that reversibly disrupt the NRF2-KEAP1 interaction have been described, opening the field to a new era of safer NRF2 activators. Here we describe the design and early phases of a drug discovery program aimed at identifying new chemical classes able to induce the NRF2 response via reversible binding of KEAP1. With this regards, new and more informative biochemical assays were developed that enabled a better prioritisation and characterisation of tool compounds and hit molecules. In addition, the monitoring of cellular events, starting from target engagement down to protection from reactive oxygen species insults, led to the validation of non-covalent KEAP1 binders on primary neurons and astrocytes derived from a mouse model of the Huntington’s disease.

Different effects of low- and high-dose waterborne zinc on Zn accumulation, ROS levels, oxidative damage and antioxidant responses in the liver of large yellow croaker Pseudosciaena crocea.

The aim of the present study was to assess survival rate, Zn accumulation, reactive oxygen species (ROS) levels, oxidative damage and antioxidant responses after Zn exposure (2 and 8mgL-1 Zn) at different exposure times (6, 12, 24, 48 and 96h) in the liver of large yellow croaker. Survival rate was reduced at 96h, and hepatic Zn content increased during 24-96 by 8mgL-1 Zn. In the 2mgL-1 Zn group, no fish died and the increase in Zn content merely occurred at 96h. Exposure to 8mgL-1 Zn induced accumulation of ROS, lipid peroxidation and protein carbonylation during the late stage of exposure. In contrast, exposure to 2mgL-1 Zn did not result in oxidative damage, which may result from the up-regulation of antioxidant defenses. Although exposure to 8mgL-1 Zn increased activities and mRNA levels of antioxidant enzymes during the early stage of exposure, including Cu/Zn-SOD, Mn-SOD, CAT, GPx and GR, the activities of these enzymes except Cu/Zn-SOD were inhibited at 96h. Furthermore, a sharp increase in Nrf2 expression was observed in fish exposed to 8mgL-1 at 6 and 12h, and 2mgL-1 at 12h and 24h, suggesting that Nrf2 was required for the protracted induction of these genes. The late increase in Keap1 expression may support its role in switching off the Nrf2 response. In conclusion, the present study demonstrated different effects of low- and high-dose waterborne Zn on antioxidant responses, which could contribute to the understanding of antioxidant and toxic roles of zinc on a molecular level.

Functionalized Mesoporous Silica Nanoparticle with Antioxidants as a New Carrier That Generates Lower Oxidative Stress Impact on Cells.

Mesoporous silica nanoparticles (MSNs) were covalently coated with antioxidant molecules, namely, caffeic acid (MSN-CAF) or rutin (MSN-RUT), in order to diminish the impact of oxidative stress induced after transfection into cells, thus generating safer carriers used for either drug delivery or other applications. Two cellular models involved in the entry of NPs in the body were used for this purpose: the intestinal Caco-2 and the epidermal HaCaT cell lines. Rutin gave the best results in terms of antioxidant capacities preservation during coupling procedures, cellular toxicity alleviation, and decrease of ROS level after 24 h incubation of cells with grafted nanoparticles. These protective effects of rutin were found more pronounced in HaCaT than in Caco-2 cells, indicating some cellular specificity toward defense against oxidative stress. In order to gain more insight about the Nrf2 response, a stable transfected HaCaT cell line bearing repeats of the antioxidant response element (ARE) in front of a luciferase reporter gene was generated. In this cell line, both tBHQ and quercetin (Nrf2 agonists), but not rutin, were able to induce, in a dose-dependent fashion, the luciferase response. Interestingly, at high concentration, MSN-RUT was able to induce a strong Nrf2 protective response in HaCaT cells, accompanied by a comparable induction of HO-1 mRNA. The level of these responses was again less important in Caco-2 cells. To conclude, in keratinocyte cell line, the coupling of rutin to silica nanoparticles was beneficial in term of ROS reduction, cellular viability, and protective effects mediated through the activation of the Nrf2 antioxidant pathway.

Nrf2 expression is increased in peripheral blood mononuclear cells derived from mild-moderate ex-smoker COPD patients with persistent oxidative stress.

Inadequacy of antioxidant nuclear factor-E2-related factor 2 (Nrf2) and endoplasmic reticulum stress-mediated unfolded protein response has been implicated in severe chronic obstructive pulmonary disease (COPD) and cigarette smoking-induced emphysema. As evidence suggests that the ability to upregulate Nrf2 expression may influence the progression of COPD and no data exist up to now in ex-smokers with mild–moderate COPD, this study was first aimed to evaluate Nrf2 and unfolded protein response expression in peripheral blood mononuclear cells (PBMC) of mild–moderate ex-smokers with COPD compared to smoking habit-matched non-COPD subjects. Then, we tested whether oxidative stress persists after cigarette smoking cessation and whether the concentrations of oxidized phospholipids (oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine [oxPAPC]) in the PBMC of the same subjects may have a causative role in determining the upregulation of Nrf2. The expression (mRNA and protein) of Nrf2 and of its related gene heme oxygenase-1 was significantly increased in COPD group without differences in the unfolded protein response. Plasma malondialdehyde, the circulating marker of oxidative stress, and oxPAPC in PBMC were significantly higher in COPD than in non-COPD subjects. The fact that the expression of p47phox, a subunit of NADPH oxidase, was increased in PBMC of COPD patients and that it was directly correlated with oxPAPC may indicate that oxPAPC may be one of the determinants of oxidative stress-induced Nrf2 upregulation. Finally, we also demonstrated that lung function inversely correlated with plasma malondialdehyde and with Nrf2 and heme oxygenase-1 mRNA expression in all subjects. Our results indicate that mild–moderate ex-smokers with COPD may be able to counteract oxidative stress by increasing the expression of Nrf2/antioxidant-response elements. Because Nrf2 failure significantly contributes to the development of COPD, our findings suggest that the possibility to prevent Nrf2 reduction may open a new scenario in helping to prevent the oxidative stress-associated lung function decline.

The role of Nrf2/Keap1 signaling in inorganic mercury induced oxidative stress in the liver of large yellow croaker Pseudosciaena crocea

The aim of the present study was to evaluate the effects of acute inorganic Hg exposure (0, 32 and 64μgHgL−1) on lipid peroxidation, activities and gene expression of antioxidant enzymes (Cu/Zn-SOD, CAT, GPx, GR and GST), and mRNA levels of the Keap1-Nrf2 signaling molecules at different exposure times (6h, 12h, 24h, 48h, and 96h) in the liver of large yellow croaker Pseudosciaena crocea. The results showed that lipid peroxidation was sharply reduced by 32μg Hg L−1 during 6–12h before returning to control levels. Similarly, lipid peroxidation was significantly reduced during 6–12h followed by a sharp increase towards the end of the exposure in the 64μgHgL−1 group. There was a negative relationship between lipid peroxidation and antioxidant enzyme activities, and positive relationship between activities and gene expression of antioxidant enzymes, suggesting that the changes at a molecular level may underlie enzymatic level and accordingly affect hepatic lipid peroxidation. Obtained results also showed a coordinated transcriptional regulation of antioxidant genes, suggesting that Nrf2 is required for the protracted induction of these genes. Furthermore, a negative relationship between the mRNA levels of Nrf2 and Keap1 indicated that Keap1 may play an important role in switching off the Nrf2 response. In conclusion, this is the first study to elucidate effects of waterborne Hg on antioxidant system in large yellow croaker through the Keap1-Nrf2 pathway, which will aid our understanding of the molecular mechanisms of waterborne heavy metal on antioxidant responses in fish.

Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration

The selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4−/− mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4−/− livers manifested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4−/− pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4−/− mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation.

A click chemistry approach identifies target proteins of xanthohumol.

Many phytochemicals with beneficial pharmacological properties contain electrophilic sites, e.g. α,β-unsaturated carbonyl (enone) groups. There is increasing evidence that many biological effects of electrophilic compounds depend on covalent conjugation to reactive protein thiols. For example, the reaction of electrophiles with cysteinyl residues of the sensor protein Keap1 activates the cell-protective Nrf2 response. Thus it is of interest to identify more generally the proteins to which small molecule electrophiles bind covalently. Here we use a Click chemistry approach to identify target proteins of the chemopreventive phytochemical xanthohumol (XN), an enone-containing chalcone from hops (Humulus lupulus L.). Using an alkynylated analog of XN (XN-alkyne), we purified covalent protein-electrophile conjugates from cell lysates. We confirm the previously described conjugation of XN to Keap1. One of the newly identified candidate target proteins is glucose-6-phosphate dehydrogenase (G6PDH). We confirm that XN attenuates intracellular G6PDH activity at low micromolar concentrations. We find support for the notion that XN modulates multiple pathways and processes by covalent modification of proteins with reactive cysteines.

Bixin protects mice against ventilation-induced lung injury in an NRF2-dependent manner.

Mechanical ventilation (MV) is a therapeutic intervention widely used in the clinic to assist patients that have difficulty breathing due to lung edema, trauma, or general anesthesia. However, MV causes ventilator-induced lung injury (VILI), a condition characterized by increased permeability of the alveolar-capillary barrier that results in edema, hemorrhage, and neutrophil infiltration, leading to exacerbated lung inflammation and oxidative stress. This study explored the feasibility of using bixin, a canonical NRF2 inducer identified during the current study, to ameliorate lung damage in a murine VILI model. In vitro, bixin was found to activate the NRF2 signaling pathway through blockage of ubiquitylation and degradation of NRF2 in a KEAP1-C151 dependent manner; intraperitoneal (IP) injection of bixin led to pulmonary upregulation of the NRF2 response in vivo. Remarkably, IP administration of bixin restored normal lung morphology and attenuated inflammatory response and oxidative DNA damage following MV. This observed beneficial effect of bixin derived from induction of the NRF2 cytoprotective response since it was only observed in Nrf2+/+ but not in Nrf2−/− mice. This is the first study providing proof-of-concept that NRF2 activators can be developed into pharmacological agents for clinical use to prevent patients from lung injury during MV treatment.

Zinc acclimation mitigated high zinc induced oxidative stress by enhancing antioxidant defenses in large yellow croaker Pseudosciaena crocea

The hypothesis tested in the present study was that Zn acclimation will alleviate high Zn induced oxidative stress in large yellow croaker Pseudosciaena crocea. To the end, fish were pre-exposed to 0 and 2mgZnL−1 for 48h and then exposed to 0 and 10mgZnL−1 for 48h. Lipid peroxidation, activities and mRNA levels of antioxidant enzyme genes (Cu/Zn-SOD, CAT, GPx and GR), and gene expressions of Nrf2–Keap1 signaling molecules at different exposure time (12h, 24h and 48h) were determined in the liver and spleen of large yellow croaker. 10mgZnL−1 exposure alone enhanced lipid peroxidation in the liver during 12–48h and in the spleen during 24–48h. Although 2mgZnL−1 pre-exposure did not affect lipid peroxidation, 2mgZnL−1 pre-exposure mitigated high Zn induced oxidative stress. The positive effect of Zn acclimation could be attributed to the up-regulated expression and activities of antioxidant enzyme genes under high Zn stress. Obtained results also showed a coordinated transcriptional regulation of antioxidant genes, suggesting that Nrf2 is required for the protracted induction of these genes. Besides, the sharp increase in Keap1 expression levels would support its role in switching off Nrf2 response. In conclusion, Zn acclimation mitigated high Zn-induced oxidative stress in large yellow croker, emphasizing a central role of transcription factor Nrf2 in the process.

Genetic and Pharmacologic Targeting of Glycogen Synthase Kinase 3β Reinforces the Nrf2 Antioxidant Defense against Podocytopathy.

Evidence suggests that the glycogen synthase kinase 3 (GSK3)-dictated nuclear exclusion and degradation of Nrf2 is pivotal in switching off the self-protective antioxidant stress response after injury. Here, we examined the mechanisms underlying this regulation in glomerular disease. In primary podocytes, doxorubicin elicited cell death and actin cytoskeleton disorganization, concomitant with overactivation of GSK3β (the predominant GSK3 isoform expressed in glomerular podocytes) and minimal Nrf2 activation. SB216763, a highly selective small molecule inhibitor of GSK3, exerted a protective effect that depended on the potentiated Nrf2 antioxidant response, marked by increased Nrf2 expression and nuclear accumulation and augmented production of the Nrf2 target heme oxygenase-1. Ectopic expression of the kinase-dead mutant of GSK3β in cultured podocytes reinforced the doxorubicin-induced Nrf2 activation and prevented podocyte injury. Conversely, a constitutively active GSK3β mutant blunted the doxorubicin-induced Nrf2 response and exacerbated podocyte injury, which could be abolished by treatment with SB216763. In murine models of doxorubicin nephropathy or nephrotoxic serum nephritis, genetic targeting of GSK3β by doxycycline-inducible podocyte-specific knockout or pharmacologic targeting by SB216763 significantly attenuated albuminuria and ameliorated histologic signs of podocyte injury, including podocytopenia, loss of podocyte markers, podocyte de novo expression of desmin, and ultrastructural lesions of podocytopathy (such as foot process effacement). This beneficial outcome was likely attributable to an enhanced Nrf2 antioxidant response in glomerular podocytes because the selective Nrf2 antagonist trigonelline abolished the proteinuria-reducing and podocyte-protective effect. Collectively, our results suggest the GSK3β-regulated Nrf2 antioxidant response as a novel therapeutic target for protecting podocytes and treating proteinuric glomerulopathies.

Differential expression of the Nrf2-linked genes in pediatric septic shock.

IntroductionExperimental data from animal models of sepsis support a role for a transcription factor, nuclear erythroid-related factor 2 p45-related factor 2 (Nrf2), as a master regulator of antioxidant and detoxifying genes and intermediary metabolism during stress. Prior analysis of a pediatric septic shock transcriptomic database showed that the Nrf2 response is a top 5 upregulated signaling pathway in early pediatric septic shock.MethodsWe conducted a focused analysis of 267 Nrf2-linked genes using a multicenter, genome-wide expression database of 180 children with septic shock 10 years of age or younger and 53 healthy controls. The analysis involved RNA isolated from whole blood within 24 h of pediatric intensive care unit admission for septic shock and a false discovery rate of 5 %. We compared differentially expressed genes from (1) patients with septic shock and healthy controls and (2) across validated gene expression–based subclasses of pediatric septic shock (endotypes A and B) using several bioinformatic methods.ResultsWe found upregulation of 123 Nrf2-linked genes in children with septic shock. The top gene network represented by these genes contained primarily enzymes with oxidoreductase activity involved in cellular lipid metabolism that were highly connected to the peroxisome proliferator activated receptor and the retinoic acid receptor families. Endotype A, which had higher organ failure burden and mortality, exhibited a greater downregulation of Nrf2-linked genes than endotype B, with 92 genes differentially regulated between endotypes.ConclusionsOur findings indicate that Nrf2-linked genes may contribute to alterations in oxidative signaling and intermediary metabolism in pediatric septic shock.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-1052-0) contains supplementary material, which is available to authorized users.

Suppression in PHLPP2 induction by morin promotes Nrf2-regulated cellular defenses against oxidative injury to primary rat hepatocytes

Recent advances indicate a possible role of phytochemicals as modulatory factors in signaling pathways. We have previously demonstrated PHLPP2-mediated suppression of Nrf2 responses during oxidant attack. The present study was designed to explore Nrf2-potentiating mechanism of morin, a flavonol, via its possible role in intervening PHLPP2-regulated Akt/GSK3β/Fyn kinase axis. Efficacy of morin was evaluated against oxidative stress-mediated damage to primary hepatocytes by tert-butyl hydroperoxide (tBHP) and acetaminophen. The anti-cytotoxic effects of morin were found to be a consequence of fortification of Nrf2-regulated antioxidant defenses since morin failed to sustain activities of redox enzyme in Nrf2 silenced hepatocytes. Morin promoted Nrf2 stability and its nuclear retention by possibly modulating PHLPP2 activity which subdues cellular Nrf2 responses by activating Fyn kinase. Pull-down assay using morin-conjugated beads indicated the binding affinity of morin towards PHLPP2. Molecular docking also revealed the propensity of morin to occupy the active site of PHLPP2 enzyme. Thus, dietary phytochemical morin was observed to counteract oxidant-induced hepatocellular damage by promoting Nrf2-regulated transcriptional induction. The findings support the novel role of morin in potentiating Nrf2 responses by limiting PHLPP2 and hence Fyn kinase activation. Therefore, morin may be exploited in developing novel therapeutic strategy aimed at enhancing Nrf2 responses.

Structural and Dynamic Characterization of Mutated Keap1 for Varied Affinity toward Nrf2: A Molecular Dynamics Simulation Study.

Keap1 is an adaptor protein that regulates Nrf2 in response to oxidative stress. Under basal conditions, Nrf2 is negatively regulated through ubiquitination by Keap1. However, upon exposure to oxidative stress, the ubiquitination of Nrf2 is inhibited, resulting in an increased steady-state level of Nrf2 in the nucleus and increased transcription of cytoprotective genes. A gene variant G364C and somatic mutation G430C on Keap1 have recently been reported to substantially impair the Keap1-Nrf2 interaction and to be associated with lung cancer. By contrast, alanine scanning experiments have shown that the mutations S363A, S508A, S555A, and S602A do not affect the ability of Keap1 to bind to Nrf2, regardless of the fact that G364 and G430 are not in contact with Nrf2 whereas the four serine residues are involved in the accommodation of Nrf2 with their hydroxy groups. In this study, molecular dynamics simulations were performed to investigate the structural and dynamic variances among wild-type (WT) Keap1 and the six mutants in unbound form. Principal component analysis of the collected MD trajectories was performed to provide dynamic diversity. Our dynamic and structural observations suggest that the G364C and G430C mutants possess a mobile D385 that moves toward R380, an anchor residue to accommodate an acidic residue in Nrf2, thereby hampering the Keap1-Nrf2 recognition of an electrostatic nature. By contrast, none of the four serine-to-alanine mutants alters the H-bond network formed by the serine backbone to its partner; accordingly, these mutants are almost as intact as the WT structurally and dynamically.

PP2 - Modulation of oxidative stress in human hippocampal progenitor cells: a model to study underlying mechanisms of depression

Background: Recent findings suggest that oxidative stress (OS) plays an important role in the pathophysiology of depression. Excessive reactive oxygen species (ROS) alter neural cytoarchitecture and function and could contribute to lower neuroplasticity and reduced neurogenesis, both of which have been linked to lower mood. This study aimed to explore the effects of OS on human hippocampal progenitor cells capable of neurogenesis. Methods: We induced OS in human hippocampal progenitor cells by treatment with tert-butylhydroperoxide (T-BHP) in different concentrations. Cell viability was assessed by the MTS assay. Level of malondialdehyde (MDA) was measured in cells supernatant using the TBARS assay. Activation of Nf-kB and Nrf2 transcription factors were followed by immunostaining and TransAM kit respectively. After differentiation cells were labeled with DCX and MAP2 – markers of young and mature neurons and assessed using CellInsight platform. Results: At 100 μM, 200 μM and 500 μM, T-BHP caused a reduction of cell viability for 23%, 54%, and 74% respectively, compared with vehicle, with a concomitant increase in levels of MDA. At 50 μM and 100 μM of T-BHP, Nf-κB positive cells increased by 16% and 17% respectively, while Nrf2 dose-dependently translocated to the nucleus. At 1 μM, 10 μM and 25 μM, T-BHP increased MAP2 and DCX positive cells by 34%, 33% and 24% for MAP2 and 38% and 30% for at 1 μM and 10 μM for DCX. Conclusions: Our results show that cell damage and cell death of human hippocampal progenitor cells caused by T-BHP occur in a dose-dependent manner. We found activation of transcription factors Nf-κB and Nrf2 in response to OS stimuli. Our results also indicate higher levels of neurogenesis in cells treated with low doses of T-BHP. Thus, we conclude that ROS serve as secondary messengers and facilitate various signaling pathways regulating fundamental neurobiological processes known to be affected in depression.

Morin mitigates acetaminophen-induced liver injury by potentiating Nrf2 regulated survival mechanism through molecular intervention in PHLPP2-Akt-Gsk3β axis.

Acetaminophen (APAP) is frequently taken to relieve pain. Staggered APAP overdoses have been reported to cause acetaminophen-induced liver injury (AILI). Identification of efficacious therapeutic modalities to address complications imposed by accidental/intentional long-term APAP ingestion is needed. Morin, a plant-derived phytochemical, possesses a multitude of pharmacological properties including hepatoprotective action; however, the underlying mechanisms have been inadequately explored. Our present report demonstrates significant attenuation of APAP-mediated liver injury by morin supplementation in vivo as indicated by reduction in histological and serum markers of hepatotoxicity. Morin not only limited necroinflammation as revealed by reduced HMGB1 release, NALP3 and caspase-1 maturation, but also suppressed oxidative stress and mitochondrial dysfunction. This suggests that morin may have exerted its cytoprotective role by way of early intervention in the pathway leading to perpetuation of AILI. Morin reinforced cellular defenses by suppressing Nrf2 ubiquitination and promoting nuclear Nrf2 retention as well as ARE-Nrf2 binding affinity. The effects were observed to be a result of molecular intervention in the activity of PHLPP2, a phosphatase previously reported by us to subdue cellular Nrf2 responses via Fyn kinase activation. Morin was observed to inhibit APAP-induced increase in PHLPP2 activity ex vivo as well as its association with cellular target Akt1. As a result, morin prevented oxidative stress induced deactivation of Akt (Ser473) leading to suppression in GSK3β and Fyn kinase activation. The study supports the inhibitory action of morin against PHLPP2-regulated Nrf2-suppression and hence indentifies Nrf2-potentiating property of morin that may be exploited in developing novel therapeutic strategy to address AILI.

Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways.

In most tissues, cells are exposed to frequent changes in levels of oxidative stress and inflammation. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and nuclear factor-κB (NF-κB) are the two key transcription factors that regulate cellular responses to oxidative stress and inflammation respectively. Pharmacological and genetic studies suggest that there is functional cross-talk between these two important pathways. The absence of Nrf2 can exacerbate NF-κB activity leading to increased cytokine production, whereas NF-κB can modulate Nrf2 transcription and activity, having both positive and negative effects on the target gene expression. This review focuses on the potentially complex molecular mechanisms that link the Nrf2 and NF-κB pathways and the importance of designing more effective therapeutic strategies to prevent or treat a broad range of neurological disorders.

Activation of the Nrf2 response by intrinsic hepatotoxic drugs correlates with suppression of NF-κB activation and sensitizes toward TNFα-induced cytotoxicity.

Drug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions are oxidative stress and cytokine signaling based on the activation of the transcription factors Nrf2 and NF-κB, respectively. Here, we systematically investigated Nrf2 and NF-κB signaling upon DILI-related drug exposure. Transcriptomics analyses of 90 DILI compounds in primary human hepatocytes revealed that a strong Nrf2 activation is associated with a suppression of endogenous NF-κB activity. These responses were translated into quantitative high-content live-cell imaging of induction of a selective Nrf2 target, GFP-tagged Srxn1, and the altered nuclear translocation dynamics of a subunit of NF-κB, GFP-tagged p65, upon TNFR signaling induced by TNFα using HepG2 cells. Strong activation of GFP-Srxn1 expression by DILI compounds typically correlated with suppression of NF-κB nuclear translocation, yet reversely, activation of NF-κB by TNFα did not affect the Nrf2 response. DILI compounds that provided strong Nrf2 activation, including diclofenac, carbamazepine and ketoconazole, sensitized toward TNFα-mediated cytotoxicity. This was related to an adaptive primary protective response of Nrf2, since loss of Nrf2 enhanced this cytotoxic synergy with TNFα, while KEAP1 downregulation was cytoprotective. These data indicate that both Nrf2 and NF-κB signaling may be pivotal in the regulation of DILI. We propose that the NF-κB-inhibiting effects that coincide with a strong Nrf2 stress response likely sensitize liver cells to pro-apoptotic signaling cascades induced by intrinsic cytotoxic pro-inflammatory cytokines.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-015-1536-3) contains supplementary material, which is available to authorized users.

4-Hydroxynonenal induces Nrf2-mediated UCP3 upregulation in mouse cardiomyocytes

4-Hydroxy-2-nonenal (HNE) is a highly cytotoxic product of lipid peroxidation. Nevertheless, at low concentrations, it is able to mediate cell signaling and to activate protective pathways, including that of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). In addition, HNE activates uncoupling proteins (UCPs), mitochondrial inner membrane proteins that mediate uncoupling of oxidative phosphorylation and have been proposed to protect against oxidative stress. It is not known, however, whether HNE might induce UCP expression via Nrf2 to cause mitochondrial uncoupling. We investigated the effects of HNE on UCP3 expression in mouse cardiomyocytes and the involvement of Nrf2. HNE induced the nuclear accumulation of Nrf2 and enhanced UCP3 expression, effects prevented by the antioxidant N-acetylcysteine. ChIP assays indicated that Nrf2 bound to the Ucp3 promoter after HNE treatment, increasing its expression. Cardiomyocytes treated with Nrf2- or UCP3-specific siRNA were less tolerant to HNE as reflected by increased cell death, and Nrf2 siRNA prevented HNE-induced UCP3 upregulation. The treatment with HNE greatly altered cardiomyocyte bioenergetics, increasing the proton leak across the inner mitochondrial membrane and severely decreasing the maximal respiratory capacity and the respiratory reserve capacity. These findings confirm that low HNE doses activate Nrf2 in cardiomyocytes and provide the first evidence of Nrf2 binding to the Ucp3 promoter in response to HNE, leading to increased protein expression. These results suggest that the upregulation of UCP3 mediated by Nrf2 in response to HNE might be important in the protection of the heart under conditions of oxidative stress such as ischemia–reperfusion.

Green tea polyphenol (−)-epigallocatechin-3-gallate triggered hepatotoxicity in mice: Responses of major antioxidant enzymes and the Nrf2 rescue pathway

(−)-Epigallocatechin-3-gallate (EGCG), a constituent of green tea, has been suggested to have numerous health-promoting effects. On the other hand, high-dose EGCG is able to evoke hepatotoxicity. In the present study, we elucidated the responses of hepatic major antioxidant enzymes and nuclear factor erythroid 2-related factor 2 (Nrf2) rescue pathway to high-dose levels of EGCG in Kunming mice. At a non-lethal toxic dose (75mg/kg, i.p.), repeated EGCG treatments markedly decreased the levels of superoxide dismutase, catalase, and glutathione peroxidase. As a rescue response, the nuclear distribution of Nrf2 was significantly increased; a battery of Nrf2-target genes, including heme oxygenase 1 (HO1), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and those involved in glutathione and thioredoxin systems, were all up-regulated. At the maximum tolerated dose (45mg/kg, i.p.), repeated EGCG treatments did not disturb the major antioxidant defense. Among the above-mentioned genes, only HO1, NQO1, and GST genes were significantly but modestly up-regulated, suggesting a comprehensive and extensive activation of Nrf2-target genes principally occurs at toxic levels of EGCG. At a lethal dose (200mg/kg, i.p.), a single EGCG treatment dramatically decreased not only the major antioxidant defense but also the Nrf2-target genes, demonstrating that toxic levels of EGCG are able to cause a biphasic response of Nrf2. Overall, the mechanism of EGCG-triggered hepatotoxicity involves suppression of major antioxidant enzymes, and the Nrf2 rescue pathway plays a vital role for counteracting EGCG toxicity.