Factor Nrf2 Research Articles

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Objectives Preeclampsia is a pregnancy-associated complex condition associated with inflammation, oxidative stress and angiogenic imbalance. Stress sensitive transcription factors nuclear factor-erythroid 2-like 1 (Nrf1) and nuclear factor-erythroid 2-like 2 (Nrf2) are involved in regulation of angiogenic, inflammatory and oxidative stress pathways. Recent evidence suggests a link between Nrf2 and angiogenic factor balance in preeclampsia though the degree to which maternal and placental DNA methylation contributes to disruption of Nrf pathways among women with preeclampsia is unknown. Our central hypothesis is that distinct patterns of DNA methylation in Nrf pathway and angiogenic genes contribute to altered angiogenic balance in preeclampsia. Methods Prospectively, we collected blood in each of three trimesters of pregnancy, maternal white blood cells in the first trimester of pregnancy and placental tissue at delivery from nulliparous women. We analyzed DNA methylation at individual CpG dinucleotides (Illumina Infinium) in Nrf and angiogenic (VEGFA, VEGFB, VEGC, FLT1, KDR, NRF1, PlGF) genes in maternal peripheral blood cells and placenta and circulating angiogenic factors (Millipore Bioplex) from women who developed preeclampsia and normotensive controls ( n = 6/group). Differences in average methylation beta scores (change in beta score >0.2 indicated significant gain of methylation; >−0.2 indicated significant loss of methylation) determined significant differences between groups. Results DNA methylation patterns in Nrf and angiogenic genes in maternal white blood cells and placenta were not significantly different in cases/controls. Among women with preeclampsia, PlGF was significantly lower across pregnancy and sFLT1 was significantly higher in the third trimester among women with preeclampsia. Conclusions DNA methylation in Nrf and angiogenic genes was not associated with altered angiogenic balance in women who developed late onset preeclampsia. Differences in DNA methylation may occur later in pregnancy when circulating factors differed between groups. Disclosures C.M. Anderson: None. J.L. Ralph: None. J.E. Ohm: None.

Molecular signatures in the prevention of radiation damage by the synergistic effect of N-acetyl cysteine and qingre liyan decoction, a traditional chinese medicine, using a 3-dimensional cell culture model of oral mucositis.

Qingre Liyan decoction (QYD), a Traditional Chinese medicine, and N-acetyl cysteine (NAC) have been used to prevent radiation induced mucositis. This work evaluates the protective mechanisms of QYD, NAC, and their combination (NAC-QYD) at the cellular and transcriptional level. A validated organotypic model of oral mucosal consisting of a three-dimensional (3D) cell tissue-culture of primary human keratinocytes exposed to X-ray irradiation was used. Six hours after the irradiation, the tissues were evaluated by hematoxylin and eosin (H and E) and a TUNEL assay to assess histopathology and apoptosis, respectively. Total RNA was extracted and used for microarray gene expression profiling. The tissue-cultures treated with NAC-QYD preserved their integrity and showed no apoptosis. Microarray results revealed that the NAC-QYD caused the upregulation of genes encoding metallothioneins, HMOX1, and other components of the Nrf2 pathway, which protects against oxidative stress. DNA repair genes (XCP, GADD45G, RAD9, and XRCC1), protective genes (EGFR and PPARD), and genes of the NFκB pathway were upregulated. Finally, tissue-cultures treated prophylactically with NAC-QYD showed significant downregulation of apoptosis, cytokines and chemokines genes, and constrained damage-associated molecular patterns (DAMPs). NAC-QYD treatment involves the protective effect of Nrf2, NFκB, and DNA repair factors.

P56 - Diphenyl diselenide improves the antioxidant response via activation of the Nrf-2 pathway in macrophage cells

Diphenyl diselenide [(PhSe)2] is an organoselenium compound that can mimic endogenous antioxidant enzymes, such as glutathione peroxidase (GPx), or be metabolized by thioredoxin reductase to form selenol intermediate, which can copy the function of the antioxidant selenoenzymes. This compound has shown potential role in preventing atherosclerosis and other oxidative stress-related diseases.The understanding of the underlying mechanism by which (PhSe)2 modulates the glutathione-related antioxidant defenses is a relevant question. Therefore, we tested its ability to promote the nuclear translocation of the nuclear factor (erythroid 2-like)-related factor 2 (Nrf-2), increasing the expression of enzymes related to the antioxidant system, such as heme oxygenase 1 (HO-1) and peroxiredoxin 1 (Prx-1), in addition to the main enzyme in the glutathione synthesis - gamma glutamylcysteine synthetase (?-GCS) - in murine J774 macrophage cells.(PhSe)2 (1µM) was able to promote nuclear translocation and increased the expression of the Nrf-2 factor in the nucleus in a time-dependent manner (1-24hours). In addition, this compound significantly increased the expression of HO-1 and Prx-1 at 24hours and GPx-1 after the first hour. Furthermore, (PhSe)2 was able to enhance GSH levels in a time-dependent manner, as well as GPx and GGCS activities. The increase in GPx and GGCS activities was dependent on the activation of PI3K, JNK, and p38MAPKs signaling pathways that may activate the Nrf2 factor.Altogether, these results show that (PhSe)2 improved the antioxidant defense by increasing the expression of HO-1 and Prx-1 and the synthesis of GSH as a consequence of the activation and nuclear translocation of Nrf-2 factor.

Andrographolide inhibits TNFα-induced ICAM-1 expression via suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression through the PI3K/Akt/Nrf2 and PI3K/Akt/AP-1 pathways in human endothelial cells

Andrographolide, the major bioactive component of Andrographis paniculata, has been demonstrated to have various biological properties including anti-inflammation, antioxidation, and anti-hepatotoxicity. Oxidative stress is considered a major risk factor in aging, inflammation, cancer, atherosclerosis, and diabetes mellitus. NADPH oxidase is a major source of endogenous reactive oxygen species (ROS). In this study, we used EA.hy926 endothelial-like cells to explore the anti-inflammatory activity of andrographolide. Andrographolide attenuated TNFα-induced ROS generation, Src phosphorylation, membrane translocation of the NADPH oxidase subunits p47phox and p67phox, and ICAM-1 gene expression. In the small hairpin RNA interference assay, shp47phox abolished TNFα-induced p65 nuclear translocation, ICAM-1 gene expression, and adhesion of HL-60 cells. Andrographolide induced the gene expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase modifier subunit (GCLM) in a time-dependent manner. Cellular glutathione (GSH) content was increased by andrographolide. shGCLM attenuated the andrographolide-induced increase in GSH content and reversed the andrographolide inhibition of HL-60 adhesion. shHO-1 showed a similar effect on andrographolide inhibition of HL-60 adhesion to shGCLM. The mechanism underlying the up-regulation of HO-1 and GCLM by andrographolide was dependent on the PI3K/Akt pathway, and both the Nrf2 and AP-1 transcriptional factors were involved. Our results suggest that andrographolide attenuates TNFα-induced ICAM-1 expression at least partially through suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression, which is PI3K/Akt pathway-dependent.

Macrophage-activating lipopeptide-2 induces the expression of hemeoxygenase-1 in THP-1 cells and its possible mechanism

Objective To investigate whether macrophage-activating lipopeptide-2 （ MALP-2） induces the expression of hemoxygenase-1 （ HO-1 ） in THP-1 cells and to further elucidate its possible regulatory mechanism for a better understanding of protective response upon mycoplasma infection .Methods THP-1 cells were cultured in vitro and stimulated by MALP-2 at different concentrations for 12 h.THP-1 cells were incubated with TLR 2 or TLR6 neutralizing antibodies , or transfected with their dominant negative plasmids to evaluate the effects of TLR 2 and TLR6 on HO-1 expression .Phosphorylation of Akt was detected by Western blot.PI3K inhibitor LY294002 was used to investigate the role of PI3K in HO-1 expression.Immunofluorescence and electrophoretic mobility shift assay （ EMSA ） were performed to observe the nuclear translocation and DNA-binding activity of nuclear factor Nrf 2.Small interfering RNA （ siRNA） was used to silence the genes encoding Nrf2 and HO-1.Cobalt protoporphyrin （CoPP）, an inducer of HO-1, was used to treat THP-1 cells.The expression of HO-1 was detected by Western blot .The secretion of TNF-αand IL-1βby THP-1 cells were measured by ELISA .Results MALP-2 induced the expression of HO-1 in THP-1 cells.However, the expression of HO-1 was inhibited by TLR2 and TLR6 neutralizing antibodies and expression of their dominant negative plasmids .Moreover, PI3K pathway was activated by MALP-2, and with the use of PI3K inhibitor, the expression of HO-1 was decreased.The translocation of Nrf2 to the nucleus and itsDNA-binding activity were enhanced by MALP-2, but were inhibited by the treatment of PI3K inhibitor.The expression of HO-1 was significantly down-regulated upon the interference of Nrf2 gene expression with siRNA.Silenced expression of HO-1 increased the level of TNF-αand IL-1β, while CoPP treatment decreasedthe secretion of MALP-2-induced cytokines.Conclusion MALP-2 might induce the expression ofHO-1 in THP-1 cells through TLR2,6/PI3K/Nrf2 pathways.The expression of HO-1 could negatively regulatethe hyper-secretion of cytokines. Key words: Macrophage-activating lipopeptide-2 ; Hemoxygenase-1 ; Monocyte

Abstract 355: NOX Deficiency Alters Endothelial Response to Blood Flow--Induced Shear Stress

Increased levels of Reactive oxygen species (ROS) lead to vascular complications like atherosclerosis. Important producers of ROS are the family of NADPH oxidases (NOXes). NOX subtypes have recently been shown to be responsive to blood flow-induced shear stress. Shear stress is also responsible for the focal nature of atherosclerosis by regulating expression of the athero-protective transcription factors KLF2 and Nrf2. These transcription factors inhibit pro-inflammatory and promote antioxidant gene expression. High, pulsatile blood flow in straight vessel segments induces KLF2 and Nrf2. In contrast, disturbed flow at vessel bends and bifurcations inhibits them, thus rendering the vessel wall susceptible to systemic risk factors for vascular disease. Several studies indicate that ROS might influence KLF2/Nrf2 expression and/or activation. In this study we set out to determine the interrelationship between shear stress-induced NOX subtypes and KLF-2/Nrf2 activation. HUVEC were exposed to 1.5 Pascal pulsatile shear stress (athero-protective) for 6 days in IBIDI flow chambers and compared to static conditions (athero-prone). NOX 1 and NOX 5 were expressed at undetectable levels for PCR analyses in static HUVEC. NOX 2 and NOX 4 were expressed in static HUVEC. Upon exposure to shear stress, NOX 2 expression decreased to undetectable levels while NOX 4 was reduced by 50%. This shift in NOX 2/NOX 4 balance resulted in a ROS shift from O 2- to H 2 O 2 , as determined by DHE and CMDCF staining. Subsequently, we silenced NOX 2 and 4 by transducing HUVEC with lenti-viral shRNAs, using non-targeting shRNA and non-transduced cells as controls. Silencing did not affect static or shear stress-induced KLF2 and Nrf2 expression, activation, or target gene expression. However, we did observe an Nrf2-independent effect of NOX silencing on antioxidant expression, which is currently under further investigation. Our preliminary results show a shift in NOX/ROS balance under shear stress, without affecting KLF2 or Nrf2. Together with the anti-oxidant activities of KLF2 and Nrf2, this shift in oxidant balance might be causative in the quiescent, anti-atherogenic effect of high, pulsatile shear stress on ECs.

Relationship between Nrf2-ARE pathway and acute lung injury induced by endotoxic shock in rabbits

Objective To evaluate the relationship between erythroid 2-related factor （Nrf2 ）-antioxidant response element （ARE） pathway and acute lung injury （ALI） induced by endotoxic shock in rabbits .Methods Thirty healthy male New Zealand white rabbits ,aged 2 months ,weighing 1.5-2.0 kg ,were randomly divided into 3 groups （n=10 each） using a random number table ：control group （group C） ,group ALI and all-trans retinoic acid group （group ATRA ） .In group ATRA ,all-trans retinoic acid 6 mg/kg （in filter sterilized vegetable oil 1.2 ml） was injected intraperitoneally once a day for 2 days .ALI was induced by lipopolysaccharide 5 mg/kg （in normal saline 2 ml ） injected via the auricular vein at 10 h after the last injection of ATRA in ALI and ATRA groups .The equal volume of normal saline was injected instead in group C .The rabbits were sacrificed at 6 h after lipopolysaccharide or normal saline administration .The pulmonary specimens were removed for determination of wet/dry lung weight ratio （W/D ratio ） and expression of Nrf2 mRNA and nuclear protein ,and HO-1 mRNA and protein in lung tissues .The pathological changes of lungs were scored .Results Compared with group C ,the pathological score and W/D ratio were significantly increased , and the expression of Nrf2 mRNA and nuclear protein ,and HO-1 mRNA and protein was up-regulated in ALI and ATRA groups （ P 0.05 ） .Conclusion Activation of Nrf2-ARE pathway is the regulatory mechanism of the body adapting to the ALI induced by endotoxic shock in rabbits . Key words: NF-E2-related factor 2; Response elements; Shock,septic; Respiratory distress syndrome,adult

Anti-inflammatory effects of curcumin in experimental spinal cord injury in rats

Antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) has been shown in our previous studies to play an important role in protection against spinal cord injury (SCI) induced inflammatory response. The objective of this study was to test whether curcumin, a novel Nrf2 activator, can protect the spinal cord against SCI-induced inflammatory damage. Adult male Sprague-Dawley rats were subjected to laminectomy at T8-T9 and compression with a vascular clip. The spinal cords spanning the injury site about 0.8 cm were collected for testing. There were three groups: (a) sham group; (b) SCI group; and (c) SCI + curcumin group. We measured Nrf2 and nuclear factor kappa B (NF-κB) binding activities by electrophoretic mobility shift assay, concentrations of tumor necrosis factor-α, interleukin-1β and interleukin-6 by enzyme-linked immunosorbent assay, hindlimb locomotion function by Basso, Beattie, and Bresnahan rating, spinal cord edema by the wet/dry weight method, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis. Induction of the Nrf2 activity by curcumin markedly decreased NF-κB activation and inflammatory cytokines production in the injured spinal cord. Administration of curcumin also significantly ameliorated the secondary spinal cord damage, as shown by decreased severity of locomotion deficit, spinal cord edema, and apoptosis. Post-SCI curcumin administration attenuates the inflammatory response in the injured spinal cord, and this may be a mechanism whereby curcumin improves the outcome following SCI.

Saponins from <i>Panax japonicas</i> Reduces Myocardial Infarction Induced Reactive Oxygen Species Production and Cardiomyocyte Apoptosis via Activation of the Nrf-2 Pathway

Myocardial infarction (MI) increased reactive oxygen species (ROS) and cardiomyocyte apoptosis. Saponins fromPanax japonicus(SPJ) exerted beneficially cardioprotective effects on myocardial ischemia injury and possess antioxidant properties to scavenge the toxic radicals. Nuclear factor erythroid 2-related factor (Nrf2) is a transcription factor that controls the expression of a large pool of anti-oxidant and protects cells from oxidative damage. The data from this study show that SPJ reduced the production of ROS by increase the expression of antioxidants and restore the balance between prooxidants and antioxidants via activation of the Nrf-2 pathway. Furthermore, SPJ might reduce ROS production and apoptosis in hydrogen peroxide-induced H9c2 cells. The effect of MI apoptotic cells induces generation of ROS in abundance. Taken together, SPJ treatment significantly improved MI-induced injury and this may be attributing to inhibiting ROS related apoptosis via activation of the Nrf-2 pathway.

Early modulation of the transcription factor Nrf2 in rodent striatal slices by quinolinic acid, a toxic metabolite of the kynurenine pathway

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor involved in the orchestration of antioxidant responses. Although its pharmacological activation has been largely hypothesized as a promising tool to ameliorate the progression of neurodegenerative events, the actual knowledge about its modulation in neurotoxic paradigms remains scarce. In this study, we investigated the early profile of Nrf2 modulation in striatal slices of rodents incubated in the presence of the toxic kynurenine pathway metabolite, quinolinic acid (QUIN). Tissue slices from rats and mice were obtained and used throughout the experiments in order to compare inter-species responses. Nuclear Nrf2 protein levels and oxidative damage to lipids were compared. Time– and concentration–response curves of all markers were explored. Nrf2 nuclear activation was corroborated through phase 2 antioxidant protein expression. The effects of QUIN on Nrf2 modulation and oxidative stress were also compared between slices of wild-type (Nrf2+/+) and Nrf2 knock-out (Nrf2−/−) mice. The possible involvement of the N-methyl-d-aspartate receptor (NMDAr) in the Nrf2 modulation and lipid peroxidation was further explored in mice striatal slices. In rat striatal slices, QUIN stimulated the Nrf2 nuclear translocation. This effect was accompanied by augmented lipid peroxidation. In the mouse striatum, QUIN per se exerted an induction of Nrf2 factor only at 1h of incubation, and a concentration–response effect on lipid peroxidation after 3h of incubation. QUIN stimulated the striatal content of phase 2 enzymes. Nrf2−/− mice were slightly more responsive than Nrf2+/+ mice to the QUIN-induced oxidative damage, and completely unresponsive to the NMDAr antagonist MK-801 when tested against QUIN. Findings of this study indicate that: (1) Nrf2 is modulated in rodent striatal tissue in response to QUIN; (2) Nrf2−/− striatal tissue was moderately more vulnerable to oxidative damage than the Wt condition; and (3) early Nrf2 up-regulation reflects a compensatory response to the QUIN-induced oxidative stress in course as part of a general defense system, whereas Nrf2 down-regulation might contribute to more intense oxidative cell damage.

Nitric oxide sets off an antioxidant response in adrenal cells: Involvement of sGC and Nrf2 in HO-1 induction

Induction of microsomal heme oxygenase 1 (HO-1) activity is considered a cytoprotective mechanism in different cell types. In adrenal cells, HO-1 induction by ACTH exerts a modulatory effect on steroid production as well. As nitric oxide (NO) has been also regarded as an autocrine/paracrine modulator of adrenal steroidogenesis we sought to study the effects of NO on the induction of HO-1 and the mechanism involved. We hereby analyzed the time and dose-dependent effect of a NO-donor (DETA/NO) on HO-1 induction in a murine adrenocortical cell line. We showed that this effect is mainly exerted at a transcriptional level as it is inhibited by actinomycin D and HO-1 mRNA degradation rates were not affected by DETA/NO treatment. HO-1 induction by NO does not appear to involve the generation of oxidative stress as it was not affected by antioxidant treatment. We also demonstrated that NO-treatment results in the nuclear translocation of the nuclear factor-erythroid 2-related factor (Nrf2), an effect that is attenuated by transfecting the cells with a dominant negative isoform of Nrf2. We finally show that the effects of the NO-donor are reproduced by a permeable analog of cGMP and that a soluble guanylate cyclase specific inhibitor blocked both the induction of HO-1 by NO and the nuclear translocation of Nrf2.

Resveratrol protects against hydrogen peroxide induced injury in human umbilical vein endothelial cells

To investigate the effect of Resveratrol on Hydrogen peroxide-treated human umbilical vein endothelial cells (HUVEC). Oxidative stress in HUVEC was induced by hydrogen peroxide (H2O2). The cells were divided in to 5 groups: A. Control group; B. H2O2 group; C. Res + H2O2 group; D. Res + H2O2 + LY294002 group; E. H2O2 + LY294002 group. The cell viability were measured by cck-8 assay; Malondialdehyde (MDA) and Superoxide dismutase (SOD) were determined by using commercially available kits; Reactive oxygen species (ROS)were detect by flow cytometer; the expression of Nuclear factor-E2-related factor(Nrf2), Protein kinase B(Akt), Mammalian target of rapamycin (mTOR) were determined by Western blot. Compared with control, resveratrol increased the cells viability (36.14 ± 2.20) to (72.05 ± 2.46) (P = 0.004), decreased MDA formation (299.38 ± 12.42) to (159.17 ± 7.30)(P = 0.006), elevated SOD activity(35.61 ± 3.26) to (81.37 ± 4.55) (P = 0.007), inhibited ROS production (177.86 ± 8.98) to (133.96 ± 4.17) (P = 0.003). These changes were accompany with the activation of p-Nrf2 (198.57 ± 6.89) to (371.10 ± 8.41) (P = 0.005), p-Akt (102.93 ± 3.29) to (221.62 ± 6.51) (P = 0.004), p-mTOR (93.26 ± 4.12) to (172.14 ± 5.74) (P = 0.008). However these protective effect of resveratrol were abolished by LY294002. Resvertrol could protects HUVEC cells from H2O2-induced oxidative stress, in which the PI3K-Akt-mTOR pathway may be involved.

Abstract 3679: Modulation of estrogen depurinating DNA adduct by sulforaphane or KEAP1 knockdown in human breast epithelial cells.

Abstract Modulation of estrogen depurinating DNA adduct by sulforaphane or KEAP1 knockdown in human breast epithelial cells Sulforaphane (SFN) is a potent inducer of detoxication enzymes such as NAD(P)H:quinone oxidoreductase (NQO1) and glutathione-S-transferases (GST) via the Kelch-like ECH-associated protein 1 (Keap1) - Nuclear Factor- E2-related factor (Nrf2) signaling pathway. NQO1 reduces the carcinogenic estrogen metabolite, catechol estrogen-3,4-quinone (CE-3,4-Q), while GSTs detoxify it through nucleophilic addition. CE-3,4-Q can bind with DNA to form depurinating DNA adducts. Thus, SFN, a isothiocyanate found in broccoli, may alter estrogen metabolism to protect against estrogen-mediated carcinogenesis. MCF10A cells were treated with either vehicle or SFN (10 μM) and either estradiol (E2) or 4-OHE2 (10 μM). NQO1 activity increased 3.2-fold by SFN treatment compared to vehicle. Estrogen metabolites and depurinating DNA adducts in the cell culture medium were partially purified by solid phase extraction and then analyzed by UHPLC-MS/MS. Following E2 treatment, 4-OHE1/2-1-N3Ade and 4-OHE1/2-1-N7Gua adducts were reduced by 60% in SFN treatment; 4-OHE1/2- glutathione conjugates increased 1.9 while 4-OCH3E1/2 increased 3.0 fold with SFN. Following treatment with the proximate metabolite 4-OHE2, 4-OHE1/2-1-N3Ade and 4-OHE 1/2-1-N7Gua adducts also decreased 60% in SFN treated cells compared to vehicle; while 4-OHE1/2-glutathione-conjugates increased 5.0-fold and 4-OCH3E1/2 levels were 3.4-fold higher. To constitutively enhance the expression of Nrf2-regulated genes including NQO1, cells were treated with either scrambled or siKEAP1 RNA and E2 or 4-OHE2. NQO1 activity increased 2.2-fold with siKEAP1 treatment. Following E2 treatment, 4-OHE1/2-1-N3Ade and 4-OHE1/2-1-N7Gua adducts dropped 70% in siKEAP1 treated cells compared to scrambled; 4-OHE1/2-glutathione conjugates increased 1.3-fold; however, 4-OCH3E1/2 decreased 50% with siKEAP1 treatment. Following 4-OHE2 treatment, the 4-OHE1/2-1-N3Ade and 4-OHE 1/2-1-N7Gua adducts declined 60% in siKEAP1 treated cells compared to scrambled; 4-OHE1/2-glutathione conjugates increased 1.4-fold while 4-OCH3E1/2 declined 60% with siKEAP1 treatment. SFN or siKEAP1 have similar effects on up-regulating NQO1 transcripts, protein expression and activity levels and on diminution of depurinating estrogen DNA adducts following E2 or 4-OHE2 challenge. However, these pharmacologic and genetic approaches have different effects on COMT. siKEAP1 down-regulates COMT expression, which inhibits 4-OCH3E1 but not 4-CH3E2 formation whilst SFN elevates COMT expression and subsequently levels of 4-OCH3E1/2. Thus, activation of the Nrf2 pathway may account for some but not all of the protective effects of SFN against estrogen-mediated DNA damage. Supported by DOD BCRP Postdoctoral Fellowship103928. Citation Format: Li Yang, Muhammad Zahid, Eleanor G. Rogan, Ercole L. Cavalieri, James D, Yager, Kala Visvanathan, John Groopman, Nancy E. Davidson, Thomas W. Kensler. Modulation of estrogen depurinating DNA adduct by sulforaphane or KEAP1 knockdown in human breast epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3679. doi:10.1158/1538-7445.AM2013-3679

Induction of the Cystine/Glutamate Exchanger SLC7A11 in Retinal Pigment Epithelial Cells by the Antipsoriatic Drug Monomethylfumarate

Oxidative stress is a common pathological factor in degenerative retinal diseases; therefore, identifying novel strategies for its limitation is critically important and highly relevant clinically. Along these lines, our present goal was to evaluate the effect(s) of the fumarate ester and antipsoriatic agent monomethylfumarate (MMF) on the expression and functional activity of the cystine/glutamate exchanger SLC7A11 (system xc(-)), a transport system critical to potentiation of antioxidant signaling in retina. ARPE-19 and primary mouse RPE cells were cultured in the presence or absence of varying concentrations of MMF (0-5000 μM) for 0 to 24 hours. MMF (10 mM) was also delivered intravitreally to mouse eyes. RT-PCR, radiolabeled uptake, Western blotting, and glutathione (GSH) assays were then used to evaluate the effects of MMF on endogenous antioxidant machinery. MMF induced system xc(-), Nrf2, and hypoxia-inducible factor 1α (Hif-1α) in cultured RPE cells. Additionally, the compound was recognized as a transportable substrate by the Na(+)-coupled monocarboxylate transporter SLC5A8 (SMCT1). In vivo these factors were evidenced by a significant increase in retinal levels of GSH. MMF stimulates multiple pathways in retinal cells that potentiate cellular events leading to the upregulation of genes/mechanisms that function to protect retina against various forms of insult; upregulation of system xc(-) is one such consequence. To our knowledge, this is the first report that fumarate esters, compounds already employed clinically for other indications, are effective in retina via xc(-) induction. This novel, hitherto unknown mechanism helps to explain the antioxidant feature of these compounds and highlights their therapeutic potential in retina.

Bisphenol A Activates the Nrf1/2-Antioxidant Response Element Pathway in HEK 293 Cells

Bisphenol A (BPA) is used in the production of polycarbonate plastics and epoxy resins for baby bottles, liners of canned food, and many other consumer products. Previously, BPA has been shown to reduce the activity of several antioxidant enzymes, which may contribute to oxidative stress. However, the underlying mechanism of the BPA-mediated effect upon antioxidant enzyme activity is unknown. Antioxidant and phase II metabolizing enzymes protect cells from oxidative stress and are transcriptionally activated by Nrf1 and Nrf2 factors through their cis-regulatory antioxidant response elements (AREs). In this work, we have assessed the effect of BPA on the Nrf1/2-ARE pathway in cultured human embryonic kidney (HEK) 293 cells. Surprisingly, glutathione and reactive oxygen species (ROS) assays revealed that BPA application created a more reduced intracellular environment in cultured HEK 293 cells. Furthermore, BPA increased the transactivation activity of ectopic Nrf1 and Nrf2 and increased the expression of ARE-target genes ho-1 and nqo1 at high (100-200 μM) BPA concentrations only. Our study suggests that BPA activates the Nrf1/2-ARE pathway at high (>10 μM) micromolar concentrations.

The pathological roles of environmental and redox stresses in cardiovascular diseases

Oxidative stress and inflammation are implicated in cardiovascular diseases such as atherosclerosis, reperfusion injury, hypertension, and heart failure. High levels of oxidative stress resulting from increased cardiac generation of reactive oxygen species (ROS) is thought to contribute to contractile and endothelial dysfunction, apoptosis and necrosis of myocytes, and extracellular matrix remodeling in the heart. ROS activate several transcription factors known as redox-regulated transcription factors, and these transcription factors play important roles in the pathophysiology of cardiovascular diseases. This review focuses on the pathological roles of environmental and redox stresses in cardiovascular diseases, especially severe cardiac dysfunction and the transition from compensated hypertrophy to heart failure. The aryl hydrocarbon receptor (AHR) and NF-E2 p45-related factor (Nrf2) are transcription factors involved in the regulation of drug-metabolizing enzymes. AHR has been studied as a receptor for environmental contaminants and as a mediator of chemical toxicity. However, other roles for AHR in cardiac and vascular development have recently been described. Moreover, Nrf2 protects against oxidative stress by increasing the transcription of genes, including those for several antioxidant enzymes. The roles of these transcription factors, AHR and Nrf2 in angiogenesis are also discussed in this review.

Ursolic acid promotes the neuroprotection by activating Nrf2 pathway after cerebral ischemia in mice

BackgroundOxidative and inflammatory damages have been suggested to play an important role in cerebral ischemic pathogenesis, and provide promising therapeutic strategies for stroke. Nuclear factor-erythroid 2-related factor 2 (Nrf2), a pleiotropic transcription factor, has been shown to play a key role in protecting cells against oxidative injury in cerebral ischemia. In this study, we demonstrated the hypothesis that ursolic acid (UA), a natural pentacyclic triterpenoid acid, isolated from edible plants in the Oleaceae family, a well-known anti-oxidative and anti-inflammatory reagent, protects the brain against ischemic injury by activating the Nrf2 pathway. Methods: Nrf2−/− and wild-type (WT) mice were induced into focal cerebral ischemia by transient middle cerebral artery occlusion (MCAO), and received UA treatment immediately after MCAO. The behavioral dysfunction, infarct size, and the expression of Nrf2, HO-1 and inflammatory factors (TLR4 and NF-κB) in ischemic brain were measured at 24h after stroke. Results: UA treatment significantly improved neurological deficit and reduced infarct size in WT mice after MCAO. Administration of UA also decreased the product of lipid peroxidation, promoted the activation of Nrf2 pathway and decreased the expression of TLR4 and NF-KB after stroke in WT mice. However, Nrf2−/− mice demonstrated more severe neurologic deficits, infarct size and inflammatory damage after MCAO, and did not benefit from the protective effect of UA. Conclusion: The results indicated that UA protected the brain against ischemic injury in mice by anti-oxidative and anti-inflammatory effects after MCAO. Activation of the Nrf2 pathway contributes to the neuroprotective effects induced by UA in cerebral ischemia.

RETRACTED: 6-OHDA-induced apoptosis and mitochondrial dysfunction are mediated by early modulation of intracellular signals and interaction of Nrf2 and NF-κB factors

6-Hydroxydopamine (6-OHDA) is a neurotoxin that generates an experimental model of Parkinson's disease in rodents and is commonly employed to induce a lesion in dopaminergic pathways. The characterization of those molecular mechanisms linked to 6-OHDA-induced early toxicity is needed to better understand the cellular events further leading to neurodegeneration. The present work explored how 6-OHDA triggers early downstream signaling pathways that activate neurotoxicity in the rat striatum. Mitochondrial function, caspases-dependent apoptosis, kinases signaling (Akt, ERK 1/2, SAP/JNK and p38) and crosstalk between nuclear factor kappa B (NF-κB) and nuclear factor-erythroid-2-related factor 2 (Nrf2) were evaluated at early times post-lesion. We found that 6-OHDA initiates cell damage via mitochondrial complex I inhibition, cytochrome c and apoptosis-inducing factor (AIF) release, as well as activation of caspases 9 and 3 to induce apoptosis, kinase signaling modulation and NF-κB-mediated inflammatory responses, accompanied by inhibition of antioxidant systems regulated by the Nrf2 pathway. Our results suggest that kinases SAP/JNK and p38 up-regulation may play a role in the early stages of 6-OHDA toxicity to trigger intrinsic pathways for apoptosis and enhanced NF-κB activation. In turn, these cellular events inhibit the activation of cytoprotective mechanisms, thereby leading to a condition of general damage.

P35 Sodium sulfide therapy attenuates myocardial ischemia–reperfusion injury in the type-2 diabetic heart via the activation of Nrf2 signaling

Background Hydrogen sulfide (H2S) therapy protects non-diabetic animals in various models of myocardial injury, including acute myocardial infarction and heart failure. However, it has yet to be determined if H2S therapeutic strategies have similar cardioprotective effects in the setting of type-2 diabetes. Here we sought to examine if H2S therapy could provide cardioprotection in the db/db diabetic mouse model. Methods Diabetic (db/db; 12 weeks of age) mice were subjected to transient myocardial ischemia for a period of 30 min followed by reperfusion. Sodium Sulfide (Na2S) or vehicle (saline) was administered either daily for 7 days before ischemia or 24 h prior to ischemia. Results Na2S therapy significantly decreased myocardial injury in the db/db diabetic mouse, as evidenced by a reduction in infarct size, circulating troponin-I levels, oxidative stress, and cleaved caspase-3 expression. In an effort to evaluate the signaling mechanism responsible for the observed cardioprotection, mice were administered (via tail vein) a single injection of Na2S or daily injections of either vehicle or Na2S for 7 days. Heart tissue was then excised and processed for Western blot analysis. Analysis uncovered a complication in the ability of Na2S to activate a Nuclear factor erythroid 2-related factor (Nrf2) signaling cascade in the diabetic heart. Specifically, the results indicate that the ability of Na2S to increase the nuclear localization of Nrf2 is not altered by diabetes. However, we did find that diabetes impaired Nrf2 transcriptional activity, as evidenced by the finding that it takes 7 days of Na2S therapy to increase Heme oxygenase-1 (HO-1) levels in the diabetic heart. Further analysis revealed that Nrf2 was unable to bind to the antioxidant response element (ARE). This appears to have been caused by Bach1, a known repressor of HO-1 transcription, as evidenced by the increased levels of Bach1 in the nucleus of the diabetic heart and the finding that 7 days of Na2S treatment reduced its nuclear expression. Conclusions Our findings demonstrate that Na2S therapy attenuates myocardial ischemia–reperfusion injury in the db/db diabetic mouse model, confirming the therapeutic potential of using Na2S to treat a heart attack in the setting of diabetes. Our results also demonstrate that Na2S activates Nrf2 signaling in the diabetic heart by removing Bach1 from the nucleus.

Update on the pathogenesis of complex regional pain syndrome: Role of oxidative stress

Complex regional pain syndrome (CRPS) is a chronic inflammatory pain syndrome that affects one or more extremities of the body. It is characterized by burning pain and abnormalities in the sensory, motor, and autonomic nervous systems. This review illustrates how oxidative stress and nuclear factor erythroid 2-related factor (Nrf2) activation might contribute to understanding the etiopathogenesis of CRPS. The precise cause of CRPS remains unclear, and current treatments are not effective in many patients. The mechanism underlying CRPS may differ across patients and even within a single patient over time. Inflammatory and neuronal mechanisms have been suggested as key contributors to CRPS. Recent evidence demonstrates that oxidative stress is associated with clinical symptoms in patients with CRPS-I. Oxidative stress plays a key role in CRPS pathogenesis. The Nrf2 factor is a master regulator of the transcription of multiple antioxidants, which protects against oxidative stress and inflammation by inducing antioxidant and detoxifying genes through binding with an antioxidant response element. It has antinociceptive effects against inflammatory pain in an animal model. This review summarises the effect of oxidative stress and mitochondrial dysfunction in the pathogenesis of CRPS. It also addresses the question of whether there is a potential role for Nrf2 (activated by pharmacological or nutritional activators) in alleviating the clinical features of CRPS or preventing its progression.