Antioxidant Response Element Genes Research Articles

Novel potential pharmacological applications of dimethyl fumarate-an overview and update.

Dimethyl fumarate (DMF) is an FDA-approved drug for the treatment of psoriasis and multiple sclerosis. DMF is known to stabilize the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. It has also been shown that DMF influences autophagy and participates in the transcriptional control of inflammatory factors by inhibiting NF-κB and its downstream targets. DMF is receiving increasing attention for its potential to be repurposed for several diseases. This versatile molecule is indeed able to exert beneficial effects on different medical conditions through a pleiotropic mechanism, in virtue of its antioxidant, immunomodulatory, neuroprotective, anti-inflammatory, and anti-proliferative effects. A growing number of preclinical and clinical studies show that DMF may have important therapeutic implications for chronic diseases, such as cardiovascular and respiratory pathologies, cancer, eye disorders, neurodegenerative conditions, and systemic or organ specific inflammatory and immune-mediated diseases. This comprehensive review summarizes and highlights the plethora of DMF's beneficial effects and underlines its repurposing opportunities in a variety of clinical conditions.

Free radicals, antioxidants, nuclear factor-E2-related factor-2 and liver damage.

The liver performs various biochemical and molecular functions. Its location as a portal to blood arriving from the intestines makes it susceptible to several insults, leading to diverse pathologies, including alcoholic liver disease, viral infections, nonalcoholic steatohepatitis, and hepatocellular carcinoma, which are causes of death worldwide. Illuminating the molecular mechanism underlying hepatic injury will provide targets to develop new therapeutic strategies to fight liver maladies. In this regard, reactive oxygen species (ROS) are well-recognized mediators of liver damage. ROS induce nuclear factor-κB and the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 inflammasome, which are the main proinflammatory signaling pathways that upregulate several proinflammatory and profibrogenic mediators. Additionally, oxygen-derived free radicals induce hepatic stellate cell activation to produce exacerbated quantities of extracellular matrix proteins, leading to fibrosis, cirrhosis and eventually hepatocellular carcinoma. Exogenous and endogenous antioxidants counteract the harmful effects of ROS, preventing liver necroinflammation and fibrogenesis. Therefore, several researchers have demonstrated that the administration of antioxidants, mainly derived from plants, affords beneficial effects on the liver. Notably, nuclear factor-E2-related factor-2 (Nrf2) is a major factor against oxidative stress in the liver. Increasing evidence has demonstrated that Nrf2 plays an important role in liver necroinflammation and fibrogenesis via the induction of antioxidant response element genes. The use of Nrf2 inducers seems to be an interesting approach to prevent/attenuate hepatic disorders, particularly under conditions where ROS play a causative role.

Oxidative Stress, Genomic Integrity, and Liver Diseases

Excess reactive oxygen species production and free radical formation can lead to oxidative stress that can damage cells, tissues, and organs. Cellular oxidative stress is defined as the imbalance between ROS production and antioxidants. This imbalance can lead to malfunction or structure modification of major cellular molecules such as lipids, proteins, and DNAs. During oxidative stress conditions, DNA and protein structure modifications can lead to various diseases. Various antioxidant-specific gene expression and signal transduction pathways are activated during oxidative stress to maintain homeostasis and to protect organs from oxidative injury and damage. The liver is more vulnerable to oxidative conditions than other organs. Antioxidants, antioxidant-specific enzymes, and the regulation of the antioxidant responsive element (ARE) genes can act against chronic oxidative stress in the liver. ARE-mediated genes can act as the target site for averting/preventing liver diseases caused by oxidative stress. Identification of these ARE genes as markers will enable the early detection of liver diseases caused by oxidative conditions and help develop new therapeutic interventions. This literature review is focused on antioxidant-specific gene expression upon oxidative stress, the factors responsible for hepatic oxidative stress, liver response to redox signaling, oxidative stress and redox signaling in various liver diseases, and future aspects.

Qualitative and quantitative analysis of Arnebiae Radix and Dictamni Cortex and efficacy study of herbal extracts on allergic contact dermatitis using 3D human reconstructed epidermis

ObjectiveTo evaluate the quality of Arnebiae Radix (AR) and Dictamni Cortex (DC) and study the efficacy of herbal extracts of these two herbs on the treatment of allergic contact dermatitis (ACD). MethodsQualitative and quantitative analysis of effective components was performed using High Performance Thin Layer Chromatography (HPTLC), High Performance Liquid Chromatography (HPLC), and HPLC-Quadrupole Time of Flight-Mass Spectrometry (HPLC-QTOF-MS). In vitro allergic ACD 3D model was established by incubating 3D reconstructed human epidermis (RHE) with skin sensitizer, potassium dichromate. A total of 65 gene expression that were associated with ACD, which included 24 antioxidant responsive element (ARE) and 41 SENS-IS genes were quantified by qRT-PCR. More than or equal to 10 ARE genes and 18 SENN-IS genes were induced by 1.3-fold, demonstrating the successful establishment of in vitro ACD model. Oil extracts of AR and DC were applied on the in vitro ACD model to study the efficacy. ResultsBatch 3 of AR and batch 2 of DC showed presence of all active ingredients with the highest concentrations. Active ingredients of the herbs were extracted using a special oil and formulated into herbal oil extracts. The herbal oil extracts were able to down regulate the induced genes in the in-vitro ACD skin model, bringing the tissue back to homeostatic status. ConclusionThe oil extracts showed the potent efficacy of using AR and DC in ACD treatment. The combination study will be done to optimize the formulation ratio which will be developed into a topical cream.

Modulation of NRF2/ARE pathway- and cell death-related genes during drug-induced liver injury.

Transcriptional factor NRF2 is an emerging tool in reviewing mechanistic behavior of drug-specific injury pathways. Drug-induced liver injury (DILI) represents a major clinical concern that often manifests oxidative stress and cell death. Despite the pivotal role of NRF2 pathway in liver pathologies, it is questioned whether NRF2 activation or regulatory efficiency could be hindered in by the severity of DILI and progression of cell death. In this study, we evaluate NRF2 as a biomarker to DILI in comparison to severity of injury as well as explore stress mediating factors affecting Nrf2 expression. In vivo DILI model was established in C57BL/6 mice by acetaminophen (APAP) at different toxic doses, confirmed by dose-dependent liver pathological changes and accompanied with in vitro time- and dose-dependent depletion of GSH and SOD in isolated primary mouse hepatocytes. Increase in liver NRF2 translocation and cytosolic content was observed in 70 mg/kg APAP-treated mice. At this subtoxic dose, liver Nrf2 transcription was increased in mice by 18.3-fold, a prominent downregulation was seen in ARE (antioxidant response element) genes; Hmox1, Nqo1 and Glcm, and apoptotic Bcl2 regulating genes. In addition, upregulation in necrosis inducer Parp2 was associated to downregulation in Hmgb1. Collectively, expression of genes related to cell survival were regulated at mild APAP hepatotoxicity. By increasing APAP dose, hemorrhagic necrosis and impaired genetic transcription in both Nrf2 and several other genes were evident. In conclusion, NRF2/ARE system and cell death modulation is halted by the increase of chemical stress and found directly associated with DILI severity.

Akt / GSK3β / Nrf2 / HO-1 pathway activation by flurbiprofen protects the hippocampal neurons in a rat model of glutamate excitotoxicity

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates redox homeostasis of the cell through regulation of the antioxidant response element genes transcription. Nrf2 also regulates the antiapoptotic Bcl-2 gene. Nrf2 degradation and nuclear translocation is regulated by upstream kinases Akt and GSK3β. Glutamate excitotoxicity is a process of neuronal cells death due to excessive activation of glutamate receptors. Glutamate excitotoxicity participates in the pathophysiology of several acute and chronic neurological conditions. In addition, glutamate excitotoxicity interrupts the PI3K/Akt prosurvival pathway so GSK3β remains active. Active GSK3β increases Nrf2 degradation, decreases Nrf2 nuclear translocation and increases Nrf2 nuclear export which decreases the ARE genes transcription such as, SOD, GSH synthesis enzyme and HO-1. Also, Bcl-2 transcription decreases. Flurbiprofen is a COX inhibitor. Previous studies showed that it has a neuroprotective effect in neurodegeneration and in focal cerebral ischemia/reperfusion model. In our research we aimed to test the hypothesis that flurbiprofen may have a neuroprotective effect in a rat model of glutamate-induced excitotoxicity and this neuroprotection may occur through modulation of (Akt/GSK3β/Nrf2/HO-1) pathway. Rats were divided into 4 groups; control, MSG (2.5 g/Kg, i.p), low dose FB (5 mg/kg, i.p) and high dose FB (10 mg/kg, i.p). We found that low and high doses FB decreased COX-2, PGE2, NO and MDA and increased SOD and GSH in brain compared to MSG group. High dose was more effective than low dose. Western blotting analysis in hippocampus tissue showed that high dose FB increased p-Akt, p-GSK3β, nuclear Nrf2 and HO-1 and decreased cytosolic Nrf2 level in comparison with MSG group. Immunohistochemical analysis in hippocampus and cerebral cortex showed that high dose FB increased Bcl-2 and decreased Bax compared to MSG group. In addition, FB increased the number of intact neurons in hippocampus areas and cerebral cortex neurons and showed an anxiolytic-like action in OF and EPM tests. These findings suggest that FB has a neuroprotective effect in glutamate-induced excitotoxicity model through reduction of the glutamate excitotoxicity damage and activation of the survival pathway. These may occur due to modulation the survival pathway (Akt/GSK3β/Nrf2/HO-1) and inhibition of COX-2.

Friend or Foe: Xenobiotic Activation of Nrf2 in Disease Control and Cardioprotection.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that governs a highly conserved pathway central to the protection of cells against various oxidative stresses. However, the biological impact of xenobiotic intervention of Nrf2 in physiological and pathophysiological conditions remains debatable. Activation of Nrf2 in cancer cells has been shown to elevate drug resistance and increase cell survival and proliferation, while inhibition of Nrf2 sensitizes cancer cells to drug treatment. On the other hand, activation of Nrf2 in normal healthy cells has been explored as a rather successful strategy for cancer chemoprevention. Selective activation of Nrf2 in off-target cells has recently been investigated as an approach for protecting off-target tissues from untoward drug toxicity. Specifically, induction of antioxidant response element genes via Nrf2 activation in cardiac cells is being explored as a means to limit the well-documented cardiotoxicity accompanied by cancer treatment with commonly prescribed anthracycline drugs. In addition to cancers, Nrf2 has been implicated in many other diseases including Alzheimer's and Parkinson's Diseases, diabetes, and cardiovascular disease. In this review, we discuss the roles of Nrf2 and its downstream target genes in the treatment of various diseases, and its recently explored potential for increasing the benefit: risk ratio of commonly utilized cancer treatments.

Neuroprotective Effects of Triterpenoids from Camellia japonica against Amyloid β-Induced Neuronal Damage.

Alzheimer's disease (AD), a neurocognitive impairment affecting human mental capacity, is related to the accumulation of amyloid-β peptide (Aβ) and the hyperphosphorylation of tau protein. In addition to modern therapies approved for AD treatment, natural products with antioxidant and anti-inflammatory properties have been studied for their potential to prevent AD pathogenesis. Six new noroleanane triterpenoids from the fruit peels of Camellia japonica were isolated, and their structures were determined by diverse spectroscopic methods. The neuroprotective effects of the six new compounds were tested against Aβ-induced neurotoxicity and neuroinflammation in mouse hippocampal and microglial cells. In the model of HT22-transfected cells, compounds 1-4 showed strongly neuroprotective effects via antioxidant response element gene activation and decreased the level of glutamate uptake. Compounds 1-4 also appeared to have strong inhibitory effects on NO production in Aβ1-42-transfected BV2 microglial cells. A docking simulation study was used to explain the inhibitory effects of compounds 1-4 on β-secretase 1 (BACE1). Noroleanane triterpenoids 1-4 had potential neuroprotective and anti-inflammatory effects against Aβ-induced neuronal damage. The structure-activity relationships of the 30 oleanane triterpenoids from C. japonica were assessed in a model of Aβ1-42-transfected HT22 cells.

Centella Asiatica Improves Memory and Promotes Antioxidative Signaling in 5XFAD Mice.

Centella asiatica (CA) herb is a traditional medicine, long reputed to provide cognitive benefits. We have reported that CA water extract (CAW) treatment improves cognitive function of aged Alzheimer’s disease (AD) model Tg2576 and wild-type (WT) mice, and induces an NRF2-regulated antioxidant response in aged WT mice. Here, CAW was administered to AD model 5XFAD female and male mice and WT littermates (age: 7.6 +/ 0.6 months), and object recall and contextual fear memory were tested after three weeks treatment. CAW’s impact on amyloid-β plaque burden, and markers of neuronal oxidative stress and synaptic density, was assessed after five weeks treatment. CAW antioxidant activity was evaluated via nuclear transcription factor (erythroid-derived 2)-like 2 (NRF2) and NRF2-regulated antioxidant response element gene expression. Memory improvement in both genders and genotypes was associated with dose-dependent CAW treatment without affecting plaque burden, and marginally increased synaptic density markers in the hippocampus and prefrontal cortex. CAW treatment increased Nrf2 in hippocampus and other NRF2 targets (heme oxygenase-1, NAD(P)H quinone dehydrogenase 1, glutamate-cysteine ligase catalytic subunit). Reduced plaque-associated SOD1, an indicator of oxidative stress, was observed in the hippocampi and cortices of CAW-treated 5XFAD mice. We postulate that CAW treatment leads to reduced oxidative stress, contributing to improved neuronal health and cognition.

Identification of Novel Protein Targets of Dimethyl Fumarate Modification in Neurons and Astrocytes Reveals Actions Independent of Nrf2 Stabilization

The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.

Carbon monoxide releasing molecule A-1 attenuates acetaminophen-mediated hepatotoxicity and improves survival of mice by induction of Nrf2 and related genes

Acute liver injury is frequently associated with oxidative stress. Here, we investigated the therapeutic potential of carbon monoxide releasing molecule A-1 (CORM A-1) in oxidative stress-mediated liver injury. Overnight-fasted mice were injected with acetaminophen (APAP; 300 mg/kg; intraperitoneally) and were sacrificed at 4 and 12 h. They showed elevated levels of serum transaminases, depleted hepatic glutathione (GSH) and hepatocyte necrosis. Mice injected with CORM A-1 (20 mg/kg) 1 h after APAP administration, had reduced serum transaminases, preserved hepatic GSH and reduced hepatocyte necrosis. Mice that received a lethal dose of APAP (600 mg/kg), died by 10 h; but those co-treated with CORM A-1 showed a 50% survival. Compared to APAP-treated mice, livers from those co-treated with CORM A-1, had upregulation of Nrf2 and ARE genes (HO-1, GCLM and NQO-1). APAP-treated mice had elevated hepatic mRNA levels of inflammatory genes (Nf-κB, TNF-α, IL1-β and IL-6), an effect blunted in those co-treated with CORM A-1. In tert-butyl hydroperoxide (t-BHP)-treated HepG2 cells, CORM A-1 augmented cell viability, reduced oxidative stress, activated the nuclear factor erythroid 2–related factor 2 (Nrf2) and anti-oxidant response element (ARE) genes. The molecular docking profile of CO in the kelch domain of Keap1 protein suggested that CO released from CORM A-1 mediated Nrf2 activation. Collectively, these data indicate that CORM A-1 reduces oxidative stress by upregulating Nrf2 and related genes, and restoring hepatic GSH, to reduce hepatocyte necrosis and thus minimize liver injury that contributes to an overall improved survival rate.

Boric Acid Activation of eIF2α and Nrf2 Is PERK Dependent: a Mechanism that Explains How Boron Prevents DNA Damage and Enhances Antioxidant Status.

Boron is abundant in vegetables, nuts, legumes, and fruit and intake is associated with reduced risk of cancer and DNA damage and increased antioxidant status. Blood boric acid (BA) levels are approximately 10μM BA in men at the mean US boron intake. Treatment of DU-145 human prostate cancer cells with 10μM BA stimulates phosphorylation of elongation initiation factor 2α (eIF2α) at Ser51 leading to activation of the eIF2α/ATF4 pathway which activates the DNA damage-inducible protein GADD34. In the present study, we used MEF WT and MEF PERK (±) cells to test the hypothesis that BA-activated eIF2α phosphorylation requires protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activates Nrf2 and the antioxidant response element (ARE). BA (10μM) increased phosphorylation of eIF2α Ser51 in MEF WT cells at 1h, but not in MEF Perk -/- cells exposed for as long as 6h. GCN2 kinase-dependent phosphorylation of eIF2α Ser51 was activated in MEF PERK -/- cells by amino acid starvation. Nrf2 phosphorylation is PERK dependent and when activated is translocated from the cytoplasm to the nucleus where it acts as a transcription factor for ARE. DU-145 cells were treated with 10μM BA and Nrf2 measured by immunofluorescence. Cytoplasmic Nrf2 was translocated to the nucleus at 1.5-2h in DU-145 and MEF WT cells, but not MEF PERK -/- cells. Real-time PCR was used to measure mRNA levels of three ARE genes (HMOX-1, NQO1, and GCLC). Treatment with 10μM BA increased the mRNA levels of all three genes at 1-4h in DU-145 cells and HMOX1 and GCLC in MEF WT cells. These results extend the known boric acid signaling pathway to ARE-regulated genes. The BA signaling pathway can be expressed using the schematic [BA + cADPR → cADPR-BA → [[ER]i Ca2+↓] → 3 pathways: PERK/eIF2αP → pathways ATF4 and Nrf2; and [[ER]i Ca2+↓] → ER stress → ATF6 pathway. This signaling pathway provides a framework that links many of the molecular changes that underpin the biological effects of boron intake.

Abstract B097: NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy

Abstract Aberrant activation of the PI3K pathway leading to increases in AKT activity is seen in many prostate cancer patients refractory to androgen blockade therapy. A number of small-molecule inhibitors of PI3K, including BKM120 (Novartis), have entered clinical trials. As with PI3K-AKT activation, highly expressed Pim kinase has been shown to drive the growth of prostate cancer. Recent studies have demonstrated that Pim kinase plays a critical role in promoting resistance of breast cancer to PI3K inhibition. Similarly, when the Pim-1 protein kinase is overexpressed, prostate cancer cell lines become resistant to PI3K inhibitor. In both tissue culture and animal models, the combination of a Pim inhibitor, PIM447, and BKM120 acted synergistically in blocking the survival and growth of prostate cancer cells. We therefore investigated the biochemical mechanism by which Pim-1 mediates cell resistance to the PI3K inhibitor. Previously, we have demonstrated that knockout of Pim kinases led to accumulation of reactive oxygen species (ROS) accompanied by a lower level of expression of ROS scavengers. To explore whether Pim-1 overexpression reverses this phenotype in prostate epithelial cancer, using lentiviral transduction, we expressed Pim-1 in mouse prostate epithelial cancer cells derived from spontaneous prostate adenocarcinomas that arose in a mouse model in which Tp53 and Rb1 are deleted in prostate epithelium. Pim-1 overexpression promoted anchorage-independent growth and formation of mouse prostate epithelial tumors in vivo. Gene expression arrays demonstrated that Pim-1 induced an Nrf2 antioxidant response element gene signature, including increases in Heme oxygenase-1 (Hmox1), NAD(P)H quinone oxidoreductase 1 (Nqo1), and glutathione S-transferase kappa1 (Gstk1) genes. Similarly, overexpression of Pim-1 in human prostate cancer LNCaP cells results in increased expression of Nrf2 and Hmox1 protein. As expected, depletion of Nrf2 with shRNA prevented Pim-1-mediated increases in Hmox1 expression, indicating that Nrf2 activation is required for the Pim-1 induction of ROS scavengers. In line with this result, in Pim-1 overexpressing cell lines knockdown of either Hmox1 or Nrf2 restored sensitivity to BKM120 treatment. A vital role of ROS scavengers in mediating the resistance to BKM120 was further confirmed by the treatment of cells and xenografted tumors with buthionine sulfoximine (inhibitor of glutathione synthesis), which resensitized the Pim-1 overexpressing cells to BKM120 via ROS induction. Our data further demonstrated that Pim-1 overexpression decreases the ubiquitination of Nrf2 protein, thus preventing its degradation, and increasing its half-life. Conversely, inhibition of Pim kinase promoted Nrf2 ubiquitination and proteasomal degradation. Taken together, our results show that the increased expression of Pim kinases in prostate cancer blocks therapeutic efficacy of PI3K inhibitor by inducing Nrf2 and antioxidant scavengers that inhibit the induction of ROS. Citation Format: Jin H. Song, Libia A. Leuvano, Neha Singh, Andrew S. Kraft. NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B097.

Melatonin enhances antioxidant molecules in the placenta, reduces secretion of soluble fms-like tyrosine kinase 1 (sFLT) from primary trophoblast but does not rescue endothelial dysfunction: An evaluation of its potential to treat preeclampsia.

Preeclampsia is one of the most serious complications of pregnancy. Currently there are no medical treatments. Given placental oxidative stress may be an early trigger in the pathogenesis of preeclampsia, therapies that enhance antioxidant pathways have been proposed as treatments. Melatonin is a direct free-radical scavenger and indirect antioxidant. We performed in vitro assays to assess whether melatonin 1) enhances the antioxidant response element genes (heme-oxygenase 1, (HO-1), glutamate-cysteine ligase (GCLC), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), thioredoxin (TXN)) or 2) alters secretion of the anti-angiogenic factors soluble fms-like tyrosine kinase-1 (sFLT) or soluble endoglin (sENG) from human primary trophoblasts, placental explants and human umbilical vein endothelial cells (HUVECs) and 3) can rescue TNF-α induced endothelial dysfunction. In primary trophoblast melatonin treatment increased expression of the antioxidant enzyme TXN. Expression of TXN, GCLC and NQO1 was upregulated in placental tissue with melatonin treatment. HUVECs treated with melatonin showed an increase in both TXN and GCLC. Melatonin did not increase HO-1 expression in any of the tissues examined. Melatonin reduced sFLT secretion from primary trophoblasts, but had no effect on sFLT or sENG secretion from placental explants or HUVECs. Melatonin did not rescue TNF-α induced VCAM-1 and ET-1 expression in endothelial cells. Our findings suggest that melatonin induces antioxidant pathways in placenta and endothelial cells. Furthermore, it may have effects in reducing sFLT secretion from trophoblast, but does not reduce endothelial dysfunction. Given it is likely to be safe in pregnancy, it may have potential as a therapeutic agent to treat or prevent preeclampsia.

Nrf2-Mediated HO-1 Induction and Antineuroinflammatory Activities of Halleridone.

Nuclear factor E2-related factor 2 (Nrf2) is the master regulator of antioxidant enzymes and is known to act on the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway. Few studies have examined the bioactivity of halleridone. Herein, we investigated whether halleridone, which was isolated from the stems of the plant Cornus walteri, could regulate Nrf2-mediated heme oxygenase (HO)-1 expression and prevent intramicroglial inflammation induced by amyloid beta (Aβ)1-42 overexpression. Biochemical and molecular experiments, such as real-time polymerase chain reaction, Western blot analysis, immunocytochemistry, immunofluorescence, and luciferase reporter gene assays, were performed. The results demonstrated that halleridone promoted the upregulation of Nrf2 expression and its translocation to the nucleus, thereby activating antioxidant response element gene transcription and HO-1 expression in murine hippocampal HT22 cells. Additionally, halleridone removed intramicroglial Aβ1-42 and suppressed the production of inflammatory mediators such as interleukin (IL)-1β, IL-6, prostaglandin E2, and nitric oxide (NO) induced by artificially overexpressed Aβ1-42 and decreased pNF-κB accumulation in the nucleus and the expression of inducible NO synthase and cyclooxygenase II in BV-2 cells. In conclusion, halleridone activated Nrf2-mediated HO-1 expression and inhibited Aβ1-42-overexpressed microglial BV-2 cell activation. These observations suggest that halleridone may have therapeutic potential for targeting neurodegeneration through neuroinflammation.

Drug-Repositioning Screening for Keap1-Nrf2 Binding Inhibitors using Fluorescence Correlation Spectroscopy

The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 μM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.

Reduction of facial wrinkles by hydrolyzed water-soluble egg membrane associated with reduction of free radical stress and support of matrix production by dermal fibroblasts.

ObjectiveThe aim of this study was to evaluate the effects of water-soluble egg membrane (WSEM) on wrinkle reduction in a clinical pilot study and to elucidate specific mechanisms of action using primary human immune and dermal cell-based bioassays.MethodsTo evaluate the effects of topical application of WSEM (8%) on human skin, an open-label 8-week study was performed involving 20 healthy females between the age of 45 years and 65 years. High-resolution photography and digital analysis were used to evaluate the wrinkle depth in the facial skin areas beside the eye (crow’s feet). WSEM was tested for total antioxidant capacity and effects on the formation of reactive oxygen species by human polymorphonuclear cells. Human keratinocytes (HaCaT cells) were used for quantitative polymerase chain reaction analysis of the antioxidant response element genes Nqo1, Gclm, Gclc, and Hmox1. Evaluation of effects on human primary dermal fibroblasts in vitro included cellular viability and production of the matrix components collagen and elastin.ResultsTopical use of a WSEM-containing facial cream for 8 weeks resulted in a significant reduction of wrinkle depth (P<0.05). WSEM contained antioxidants and reduced the formation of reactive oxygen species by inflammatory cells in vitro. Despite lack of a quantifiable effect on Nrf2, WSEM induced the gene expression of downstream Nqo1, Gclm, Gclc, and Hmox1 in human keratinocytes. Human dermal fibroblasts treated with WSEM produced more collagen and elastin than untreated cells or cells treated with dbcAMP control. The increase in collagen production was statistically significant (P<0.05).ConclusionThe topical use of WSEM on facial skin significantly reduced the wrinkle depth. The underlying mechanisms of this effect may be related to protection from free radical damage at the cellular level and induction of several antioxidant response elements, combined with stimulation of human dermal fibroblasts to secrete high levels of matrix components.

Sulforaphane Reduces Inflammatory Gene Expression in Microglia from Aged Mice

Increased oxidative stress resulting from heightened microglial activation is associated with age‐related cognitive impairment, neurodegenerative disease, and response to immune stimulus. The primary compensatory mechanism for neutralizing oxidative stress is through the nuclear factor E2‐related factor 2 (Nrf2) pathway which regulates transcription of antioxidant response element (ARE) genes. Here, BV2 cells and brain CD11b+ microglial cells isolated from young adult and aged mice were used to investigate the effects of sulforaphane (SFN), a potent Nrf2‐inducing bioactive, on ARE and inflammatory gene expression associated with increased age. We hypothesized that SFN would upregulate ARE genes in primary microglial cells and that this would attenuate elevated proinflammatory cytokine expression in microglia from aged mice. In support of our hypothesis, SFN upregulated antioxidant genes NAD(P)H dehydrogenase, quinone 1 (NQO1), heme oxygenase‐1 (HMOX1), and the modulatory subunit of glutamate‐cysteine ligase, modifier subunit (GCLM) in BV2 cells and primary microglia. Primary microglia from aged mice had higher expression of interleukin (IL‐)1β, IL‐6, and inducible nitric oxide synthase (iNOS) than adult controls, and SFN reduced IL‐1β and iNOS in microglia from aged mice. Taken together these data indicate that SFN is a potential therapeutic supplement that may be beneficial for reducing microglia‐mediated neuroinflammation and oxidative stress associated with aging.Supported by NIH RO1AG16710

EGFR mediates astragaloside IV-induced Nrf2 activation to protect cortical neurons against in vitro ischemia/reperfusion damages

In this study, we tested the potential role of astragaloside IV (AS-IV) against oxygen and glucose deprivation/re-oxygenation (OGD/R)-induced damages in murine cortical neurons, and studied the associated signaling mechanisms. AS-IV exerted significant neuroprotective effects against OGD/R by reducing reactive oxygen species (ROS) accumulation, thereby attenuating oxidative stress and neuronal cell death. We found that AS-IV treatment in cortical neurons resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by Nrf2 Ser-40 phosphorylation, and its nuclear localization, as well as transcription of antioxidant-responsive element (ARE)-regulated genes: heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO-1) and sulphiredoxin 1 (SRXN-1). Knockdown of Nrf2 through lentiviral shRNAs prevented AS-IV-induced ARE genes transcription, and abolished its anti-oxidant and neuroprotective activities. Further, we discovered that AS-IV stimulated heparin-binding-epidermal growth factor (HB-EGF) release to trans-activate epidermal growth factor receptor (EGFR) in cortical neurons. Blockage or silencing EGFR prevented Nrf2 activation by AS-IV, thus inhibiting AS-IV-mediated anti-oxidant and neuroprotective activities against OGD/R. In summary, AS-IV protects cortical neurons against OGD/R damages through activating of EGFR-Nrf2 signaling.

Abstract 4888: The novel cytokine IL-17D at the intersection of cellular stress and tumor immunity

Abstract Our laboratory has recently identified a novel role for the cytokine IL-17D as an important mediator of the antitumor immune response; and, the loss of IL-17D as a key component of tumor progression. Our data show for the first time that overexpression of the cytokine IL-17D is sufficient to induce tumor rejection or growth delay. Tumors expressing IL-17D displayed an increased infiltration of natural killer (NK) cells, which gave way to a commensurate increase of M1-type macrophages. While examining the regulation of IL-17D, we came across the surprising finding that IL-17D can be induced by oxidative stress signals via the transcription factor nrf2. Nrf2, responsible for regulating the expression of the anti-oxidant response element genes, is known as the primary cellular responder to oxidative stress. Although the role of nrf2 in cancer biology has been well studied, the role of nrf2 in the regulation of immune responses (particularly via IL-17D) has yet to be defined. We find that nrf2 is necessary to induce IL-17D expression in a variety of tumor and non-tumor cells, and that this regulation is active in tumor progression. These studies constitute an intriguing and novel connection between oxidative cellular stress and immune activation. Citation Format: Robert Saddawi-Konefka, Jack Bui. The novel cytokine IL-17D at the intersection of cellular stress and tumor immunity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4888. doi:10.1158/1538-7445.AM2014-4888