Increases In Heme Oxygenase-1 Activity Research Articles

Preconditioning with the BKCa channel activator NS-1619 prevents ischemia-reperfusion-induced inflammation and mucosal barrier dysfunction: roles for ROS and heme oxygenase-1.

Activation of large-conductance Ca2+-activated K+ (BKCa) channels evokes cell survival programs that mitigate intestinal ischemia and reperfusion (I/R) inflammation and injury 24 h later. The goal of the present study was to determine the roles of reactive oxygen species (ROS) and heme oxygenase (HO)-1 in delayed acquisition of tolerance to I/R induced by pretreatment with the BKCa channel opener NS-1619. Superior mesentery arteries were occluded for 45 min followed by reperfusion for 70 min in wild-type (WT) or HO-1-null (HO-1-/-) mice that were pretreated with NS-1619 or saline vehicle 24 h earlier. Intravital microscopy was used to quantify the numbers of rolling and adherent leukocytes. Mucosal permeability, tumor necrosis factor-α (TNF-α) levels, and HO-1 activity and expression in jejunum were also determined. I/R induced leukocyte rolling and adhesion, increased intestinal TNF-α levels, and enhanced mucosal permeability in WT mice, effects that were largely abolished by pretreatment with NS-1619. The anti-inflammatory and mucosal permeability-sparing effects of NS-1619 were prevented by coincident treatment with the HO-1 inhibitor tin protoporphyrin-IX or a cell-permeant SOD mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), in WT mice. NS-1619 also increased jejunal HO-1 activity in WT animals, an effect that was attenuated by treatment with the BKCa channel antagonist paxilline or MnTBAP. I/R also increased postischemic leukocyte rolling and adhesion and intestinal TNF-α levels in HO-1-/- mice to levels comparable to those noted in WT animals. However, NS-1619 was ineffective in preventing these effects in HO-1-deficient mice. In summary, our data indicate that NS-1619 induces the development of an anti-inflammatory phenotype and mitigates postischemic mucosal barrier disruption in the small intestine by a mechanism that may involve ROS-dependent HO-1 activity.NEW & NOTEWORTHY Antecedent treatment with the large-conductance Ca2+-activated K+ channel opener NS-1619 24 h before ischemia-reperfusion limits postischemic tissue injury by an oxidant-dependent mechanism. The present study shows that NS-1619-induced oxidant production prevents ischemia-reperfusion-induced inflammation and mucosal barrier disruption in the small intestine by provoking increases in heme oxygenase-1 activity.

Mechanisms of the antihypertensive effects of Nigella sativa oil in L-NAME-induced hypertensive rats

OBJECTIVESThis study was conducted to determine whether the blood pressure-lowering effect of Nigella sativa might be mediated by its effects on nitric oxide, angiotensin-converting enzyme, heme oxygenase and oxidative stress markers.METHODS:Twenty-four adult male Sprague-Dawley rats were divided equally into 4 groups. One group served as the control (group 1), whereas the other three groups (groups 2-4) were administered L-NAME (25 mg/kg, intraperitoneally). Groups 3 and 4 were given oral nicardipine daily at a dose of 3 mg/kg and Nigella sativa oil at a dose of 2.5 mg/kg for 8 weeks, respectively, concomitantly with L-NAME administration.RESULTSNigella sativa oil prevented the increase in systolic blood pressure in the L-NAME-treated rats. The blood pressure reduction was associated with a reduction in cardiac lipid peroxidation product, NADPH oxidase, angiotensin-converting enzyme activity and plasma nitric oxide, as well as with an increase in heme oxygenase-1 activity in the heart. The effects of Nigella sativa on blood pressure, lipid peroxidation product, nicotinamide adenine dinucleotide phosphate oxidase and angiotensin-converting enzyme were similar to those of nicardipine. In contrast, L-NAME had opposite effects on lipid peroxidation, angiotensin-converting enzyme and NO.CONCLUSION:The antihypertensive effect of Nigella sativa oil appears to be mediated by a reduction in cardiac oxidative stress and angiotensin-converting enzyme activity, an increase in cardiac heme oxygenase-1 activity and a prevention of plasma nitric oxide loss. Thus, Nigella sativa oil might be beneficial for controlling hypertension.

Dimethyl Fumarate Induces Cytoprotection and Inhibits Inflammation and Vaso-Occlusion in Transgenic Sickle Mice

Patients with sickle cell disease have unrelenting hemolysis leading to the release of hemoglobin and heme into the vasculature that promote oxidative stress, inflammation, vaso-occlusive pain crises, ischemia-reperfusion injury and organ damage. Induction of the cytoprotective heme metabolizing enzyme heme oxygenase-1 (HO-1), the iron-binding protein ferritin heavy chain or administration of the HO-1 metabolite CO induces cytoprotective responses that inhibit oxidative stress, inflammation, vaso-occlusion and organ damage in transgenic sickle mice expressing human βS globins. The master regulator of these anti-oxidative and cytoprotective responses is nuclear factor erythroid 2-related factor (Nrf2). Nrf2 activity is controlled, in part, by the cytosolic protein, kelch-like ECH-associated protein 1 (Keap1). Nrf2 is anchored in the cytoplasm through binding with Keap1 which results in its ubiquitination and subsequent proteosomal degradation. Upon exposure to stress stimuli, such as reactive oxygen species and electrophiles, Nrf2 is stabilized and able to translocate to the nucleus where it trans-activates target genes that possess an antioxidant responsive element (ARE) in their promoter regions. Recently the FDA and their European and Canadian counterparts approved dimethyl fumarate (Tecfidera) for the treatment of relapsing multiple sclerosis. Dimethyl fumarate (DMF) and its metabolite monomethyl fumarate alkylate a critical reactive thiol Cys-151 on Keap1 causing release of Nrf2, nuclear localization and activation of cellular anti-oxidant and cytoprotective responses. Based on our previous results showing cytoprotection through HO-1 and it products, we evaluated DMF responses in NY1DD transgenic sickle mice. DMF (15mg/kg) or vehicle (0.08% methyl cellulose) was administered by oral gavage BID X 3 days to NY1DD mice. On the first day of treatment, mice were implanted with dorsal skin-fold chambers. One hour after the last treatment, 20-30 flowing subcutaneous venules were selected and mapped in the dorsal skin-fold chamber window followed by infusion of heme (3.2 µmols/kg) into the tail vein. All of the selected venules were re-examined at 1 and 4 hours post-infusion and the number of static (no flow) venules were counted and expressed as percent stasis. After the 4 hour stasis measurement, blood was collected and organs were removed and flash frozen. In sickle mice treated with vehicle, microvascular stasis was 29% and 24% at 1 and 4 hours, respectively (Fig. 1). In contrast, in sickle mice treated with DMF, stasis was 6% and 4% at 1 and 4 hours (p<0.001). There also was a marked increase in nuclear Nrf2 in liver nuclear extracts of DMF-treated sickle mice on Western blots. Analysis of liver mRNA by qRT-PCR revealed that there was a 2.4 to 4.4-fold enrichment of mRNA coding for putative Nrf2-responsive antioxidant proteins in DMF-treated sickle mice compared to vehicle treated mice (Table 1). This was accompanied by a 3.3-fold increase in HO-1 activity (p<0.05), a distinct increase in cytoplasmic ferritin heavy chain on Western blot and a marked decrease in nuclear NF-κB phospho-p65 activation on Western blot of liver nuclear extracts of DMF treated sickle mice versus vehicle controls. Red blood cells and white blood cells isolated from DMF-treated sickle mice had significantly decreased adhesion to resting and heme- and TNF-activated HUVEC compared to blood cells from mice treated with vehicle. We conclude that DMF may be an effective agent to prevent sickle crises through induction of HO-1, ferritin and other antioxidant proteins, and inhibition of vaso-occlusion. These data combined with prior evidence that monomethyl fumarate can induce hemoglobin F make it an idea candidate for clinical trials in SCD patients.Table 1Enrichment of mRNA for Antioxidant Proteins in DMF-treated LiversmRNAFold enrichmentP valueHO-12.40.08Hemopexin2.50.023Ferritin Heavy Chain2.6<0.001Haptoglobin2.70.046Nrf22.70.004Ferroportin3.1<0.001Glutamate#Cysteine Ligase Catalytic Subunit3.3<0.001Glutathione S-Transferase A23.40.032NAD(P)H Dehydrogenase [Quinone] 13.80.008Glutathione S-Transferase Mu 13.8<0.001Multidrug Resistance-associated Protein 24.4<0.001 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Physiological cyclic strain promotes endothelial cell survival via the induction of heme oxygenase-1.

Endothelial cells (ECs) are constantly subjected to cyclic strain that arises from periodic change in vessel wall diameter as a result of pulsatile blood flow. Application of physiological levels of cyclic strain inhibits EC apoptosis; however, the underlying mechanism is not known. Since heme oxygenase-1 (HO-1) is a potent inhibitor of apoptosis, the present study investigated whether HO-1 contributes to the antiapoptotic action of cyclic strain. Administration of physiological cyclic strain (6% at 1 Hz) to human aortic ECs stimulated an increase in HO-1 activity, protein, and mRNA expression. The induction of HO-1 was preceded by a rise in reactive oxygen species (ROS) and Nrf2 protein expression. Cyclic strain also stimulated an increase in HO-1 promoter activity that was prevented by mutating the antioxidant responsive element in the promoter or by overexpressing dominant-negative Nrf2. In addition, the strain-mediated induction of HO-1 and activation of Nrf2 was abolished by the antioxidant N-acetyl-l-cysteine. Finally, application of cyclic strain blocked inflammatory cytokine-mediated EC death and apoptosis. However, the protective action of cyclic strain was reversed by the HO inhibitor tin protoporphyrin-IX and was absent in ECs isolated from HO-1-deficient mice. In conclusion, the present study demonstrates that a hemodynamically relevant level of cyclic strain stimulates HO-1 gene expression in ECs via the ROS-Nrf2 signaling pathway to inhibit EC death. The ability of cyclic strain to induce HO-1 expression may provide an important mechanism by which hemodynamic forces promote EC survival and vascular homeostasis.

H-Ferritin Ferroxidase Activity Induces Cytoprotective Pathways and Inhibits Microvascular Stasis in Transgenic Sickle Mice

AbstractAbstract 378Hemolysis, oxidative stress, inflammation, vaso-occlusion and organ infarction are hallmarks of sickle cell disease (SCD). We hypothesize that intravascular heme, liberated from hemoglobin S derived from hemolyzed sickle red blood cells, is fundamental to inflammation and vaso-occlusion in SCD. Heme can be degraded by heme-oxygenase-1 (HO-1), releasing Fe2+, carbon monoxide (CO), and biliverdin/bilirubin. We have previously shown that increases in HO-1 activity inhibit vascular inflammation and vaso-occlusion in transgenic mouse models of SCD. HO-1 products, CO and biliverdin/bilirubin, also are protective in SCD, but Fe2+ released from the heme ring requires further processing. The released Fe2+ from heme is oxidized by ferritin heavy chain (FHC) ferroxidase activity and safely stored as catalytically-inactive Fe3+ inside ferritin clusters. FHC overexpression has been shown to be cytoprotective in response to inflammation and oxidative stress in vivo and in vitro. In this study, we hypothesize that overexpression of FHC with its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic sickle mice in response to stroma-free hemoglobin. We utilized a Sleeping Beauty transposase plasmid to deliver a human wt-ferritin heavy chain (wt-FHC) transposable element by hydrodynamic tail vein injections to NY1DD SCD mice. Control mice were infused with the same volume of lactated Ringer’s solution LRS) or a triple missense (ms-) human FHC plasmid encoding no ferroxidase enzyme activity. Eight weeks after injection, the mice were implanted with dorsal skin-fold chambers to access microvascular blood flow. LRS-treated mice had 40% microvascular stasis (% non-flowing venules) when infused with stroma-free hemoglobin, while wt-FHC overexpressing mice had only 5% stasis (p<0.05), suggesting vascular protection by wt-FHC. Ferroxidase activity was critical in this protection as mice overexpressing ms-FHC were not protected from microvascular stasis (33%). The wt-FHC SCD mice had marked increases in FHC mRNA and protein, light chain ferritin, 5-aminolevulinic acid synthase (5-ALA-synthase), cellular heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear FHC and microsomal HO-1 activity and protein, and a decrease in activated phosho- and total nuclear factor-kappa B (NF-κB) p65 in the nuclei of the livers. We conclude that wt-FHC ferroxidase activity enhances 5-ALA-synthase activity and cytoprotective Nrf2-regulated proteins including HO-1 resulting in decreased NF-κB-activation, inflammation and microvascular stasis in transgenic SCD mice. Therapies directed at increasing ferritin heavy chain production and decreasing reactive iron availability may be beneficial in limiting sickle cell crises and end organ damage. Disclosures:Vercellotti:Sangart Inc: Consultancy, Research Funding. Chen:Sangart Inc: Research Funding. Hebbel:Sangart Inc: Consultancy, Research Funding. Belcher:Sangart Inc: Research Funding.

Epigallocatechin gallate induces expression of heme oxygenase-1 in endothelial cells via p38 MAPK and Nrf-2 that suppresses proinflammatory actions of TNF-α

Epigallocatechin gallate (EGCG), the major polyphenol in green tea, acutely stimulates production of nitric oxide (NO) from vascular endothelium to reduce hypertension and improve endothelial dysfunction in spontaneously hypertensive rats. Herein, we explored additional mechanisms whereby EGCG may mediate beneficial cardiovascular actions. When compared with vehicle-treated controls, EGCG treatment (2.5 μM, 8 h) of human aortic endothelial cells (HAEC) caused a ∼three-fold increase in heme oxygenase-1 (HO-1) mRNA and protein with comparable increases in HO-1 activity. This was unaffected by pretreatment of cells with wortmannin, LY294002, PD98059 or L-NAME (PI 3-kinase, MEK and NO synthase inhibitors, respectively). Pretreatment of HAEC with SB203580 (p38 MAPK inhibitor) or siRNA knockdown of p38 MAPK completely blocked EGCG-stimulated induction of HO-1. EGCG treatment also inhibited tumor-necrosis-factor-α-stimulated expression of vascular cell adhesion molecule (VCAM)-1 and decreased adhesion of monocytes to HAEC. siRNA knockdown of HO-1, p38 MAPK or Nrf-2 blocked these inhibitory actions of EGCG. In HAEC transiently transfected with a human HO-1 promoter luciferase reporter (or an isolated Nrf-2 responsive region), luciferase activity increased in response to EGCG. This was inhibitable by SB203580 pretreatment. EGCG-stimulated expression of HO-1 and Nrf-2 was blocked by siRNA knockdown of Nrf-2 or p38 MAPK. Finally, liver from mice chronically treated with EGCG had increased HO-1 and decreased VCAM-1 expression. Thus, in vascular endothelium, EGCG requires p38 MAPK to increase expression of Nrf-2 that drives expression of HO-1, resulting in increased HO-1 activity. Increased HO-1 expression may underlie anti-inflammatory actions of EGCG in vascular endothelium that may help mediate beneficial cardiovascular actions of green tea.

Heme and the Vasculature: How the Endothelium Protects Itself Against Toxic Iron

AbstractAbstract SCI-25Iron-derived reactive oxygen species (ROS) are involved in the pathogenesis of numerous vascular disorders. Heme-derived iron plays an instrumental role in the pathology of intravascular hemolytic diseases including malaria, sickle cell anemia, transfusion reactions, DIC and PNH. Heme catalyzed oxidative stress promotes a pro-inflammatory/prothrombogenic endothelium, diminution of bio-available nitric oxide (NO) and attraction of leukocytes and platelets. The vasculature is protected against heme-catalyzed injury by plasma proteins including haptoglobin, hemopexin, albumin, alpha-1-microglobulin and by scavenger receptors for heme complexes including CD163 and CD91. Heme and its concomitant oxidative stress induces the cytoprotective and rate-limiting enzyme in heme catabolism, heme oxygenase-1 (HO-1). In the process, HO-1 releases three enzymatic byproducts: carbon monoxide (CO), biliverdin/bilirubin, and iron, which stimulates ferritin synthesis. These HO-1 by-products have established anti-oxidant and anti-inflammatory properties. Human patients and mouse models elevate HO-1 in response to chronic hemolysis. Of all sites in the body, the endothelium may be at greatest risk of exposure to heme. Heme greatly potentiates endothelial cell killing mediated by leukocytes and other sources of ROS. As a defense against heme, endothelial cells upregulate HO-1 and ferritin. If cultured endothelial cells are briefly pulsed with heme and are then incubated for a prolonged period (16 h), the cells become highly resistant to oxidant-mediated injury and to the accumulation of endothelial lipid peroxidation products. This protection is associated with induction of both HO-1 and ferritin. H-ferritin with its ferroxidase activity is especially cytoprotective. In animal models, increased expression of HO-1 has been shown to protect tissues against ischemia-reperfusion injury, oxidative stress, inflammation, transplant rejection, apoptosis, and cell proliferation. Conversely, HO-1 null mice (hmox-1−/−) and human patients deficient in HO-1 are especially prone to oxidative stress and inflammation. Sickle cell anemia is an archetypal example of heme-induced oxidative stress and cytoprotective adaptation. The sickle patient and sickle mouse models defend and adapt to hemolysis by increasing their defenses against heme. HO-1 plays an essential role in the inhibition and resolution of vaso-occlusion in sickle cell anemia. HO-1 and its products, carbon monoxide and biliverdin, modulate vaso-occlusion through multiple mechanisms including reducing oxidative stress, inhibiting NF-kB, down-regulating endothelial cell adhesion molecules, decreasing red blood cell hemolysis and altering vascular tone. However, sickle cell patients often have adaptive increases in HO-1 activity which are insufficient to completely handle the excessive heme burden, particularly during acute bouts of hemolysis. HO-1 gene therapy in sickle mice using Sleeping Beauty-mediated transposition of an HO-1 transgene provides a promising non-viral approach to significantly enhance HO-1 expression in sickle cell anemia. Strategies to minimize heme-iron activation of the vasculature including increasing HO-1 and its products, anti-oxidants, iron chelators, increasing haptoglobin, hemopexin and/or their receptors CD163/CD91 should be explored in hemolytic disease states. Disclosures:Vercellotti:Sangart: Consultancy, Research Funding. Belcher:Sangart: Research Funding.

Hemeoxygenase-1 expression in response to arecoline-induced oxidative stress in human umbilical vein endothelial cells

BackgroundArecoline, the most abundant areca alkaloid, has been reported to stimulate reactive oxygen species (ROS) production in several cell types. Overproduction of ROS has been implicated in atherogenesis. Hemeoxygenase-1 (HO-1) has cytoprotective activities in vascular tissues. This study investigated the effect of arecoline on adhesion molecule expression and explored the role of HO-1 in this process. MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with arecoline, then ROS levels and the expression of adhesion molecules and HO-1 were analyzed and potential signaling pathways investigated. ResultsAfter 2h of arecoline treatment, ROS production was stimulated and reached a maximum at 12h. Expression of the adhesion molecules ICAM and VCAM was also induced. Glutathione pretreatment completely blocked arecoline-stimulated ROS production and VCAM expression, but not ICAM expression. Arecoline also induced HO-1 expression and this effect was partly due by ROS stimulation. Inhibition of c-jun N-terminal kinase (JNK) by SP600125, p38 by SB 203580, or tyrosine kinase by genistein reduced arecoline-induced HO-1 expression. In contrast, inhibition of ERK (extracellular signal-related MAP kinase) by PD98059 had no effect. Transfection of HUVECs with the GFP/HO-1 gene, which resulted in a 5-fold increase in HO-1 activity, markedly, but not completely, inhibited the decrease in cell viability caused by arecoline. ConclusionsThis study demonstrates that, in HUVECs, arecoline stimulates ROS production and ICAM and VCAM expression. HO-1 expression is also upregulated through the ROS, tyrosine kinase, and MAPK (JNK and p38) signaling pathways.

Heme oxygenase-1 gene delivery by Sleeping Beauty inhibits vascular stasis in a murine model of sickle cell disease

Increases in heme oxygenase-1 (HO-1) and administration of heme degradation products CO and biliverdin inhibit vascular inflammation and vasoocclusion in mouse models of sickle cell disease (SCD). In this study, an albumin (alb) promoter-driven Sleeping Beauty (SB) transposase plasmid with a wild-type rat hmox-1 (wt-HO-1) transposable element was delivered by hydrodynamic tail vein injections to SCD mice. Eight weeks after injection, SCD mice had three- to five-fold increases in HO-1 activity and protein expression in liver, similar to hemin-treated mice. Immunohistochemistry demonstrated increased perinuclear HO-1 staining in hepatocytes. Messenger RNA transcription of the hmox-1 transgene in liver was confirmed by quantitative real-time polymerase chain reaction restriction fragment length polymorphism (qRT-PCR RFLP) with no detectible transgene expression in other organs. The livers of all HO-1 overexpressing mice had activation of nuclear phospho-p38 mitogen-activated protein kinase (MAPK) and phospho-Akt, decreased nuclear expression of nuclear factor-kappa B (NF-kappaB) p65, and decreased soluble vascular cell adhesion molecule-1 (sVCAM-1) in serum. Hypoxia-induced stasis, a characteristic of SCD, but not normal mice, was inhibited in dorsal skin fold chambers in wt-HO-1 SCD mice despite the absence of hmox-1 transgene expression in the skin suggesting distal effects of HO activity on the vasculature. No protective effects were seen in SCD mice injected with nonsense (ns-) rat hmox-1 that encodes carboxy-truncated HO-1 with little or no enzyme activity. We speculate that HO-1 gene delivery to the liver is beneficial in SCD mice by degrading pro-oxidative heme, releasing anti-inflammatory heme degradation products CO and biliverdin/bilirubin into circulation, activating cytoprotective pathways and inhibiting vascular stasis at sites distal to transgene expression.

Heme Oxygenase-1 Gene Therapy in a Murine Model of Sickle Cell Disease.

Abstract 1527Poster Board I-550We previously demonstrated that increases in heme oxygenase-1 (HO-1) and its products, carbon monoxide and biliverdin, inhibit inflammation and vasoocclusion in mouse models of sickle cell disease (SCD). In this study, an albumin promoter driven Sleeping Beauty transposase plasmid with a wild type rat hmox-1 gene (wt-HO-1) transposable element (289 amino acids) was delivered by hydrodynamic tail vein injections to SCD mice. Eight weeks after injection, SCD mice had 3-fold increases in HO-1 activity in their livers, similar to hemin-treated mice. mRNA transcription of the transposable element was confirmed by qRT-PCR RFLP analysis. HO-1 overexpressing mice had marked activation of the phospho-p38 MAPK and phospho-Akt proteins and decreased NF-kB p65 in liver nuclear extracts. Hypoxia-induced stasis, a characteristic of SCD but not normal mice, was inhibited in dorsal skin fold chambers in wt-HO-1 SCD mice. None of these effects were seen in SCD mice injected with Sleeping Beauty containing a nonsense rat hmox-1 gene (ns-HO-1) that encodes for amino acids 1-241 with little or no enzymatic activity. Immunohistochemistry of HO-1 in livers demonstrated perinuclear HO-1 staining in both hepatocytes and sinusoidal Kupffer cells in wt- and ns-HO-1-treated mice. We conclude HO-1 gene therapy targeted to the liver is beneficial in SCD by activating cytoprotective signaling pathways and inhibiting vascular stasis at sites distal to transgene expression. DisclosuresNo relevant conflicts of interest to declare.

Protective effect of sulforaphane on indomethacin‐induced cytotoxicity via heme oxygenase‐1 expression in human intestinal Int 407 cells

Sulforaphane is known to be an indirect antioxidant that acts by inducing NF-E2-related factor 2 (Nrf2)-dependent phase II enzymes. In the present study, we investigated the effect of sulforaphane on the expression of heme oxygenase-1 (HO-1) in human intestinal Int 407 cells. RT-PCR and Western blot data revealed that sulforaphane induced an increase in HO-1 expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in HO-1 activity. Actinomycin D (an RNA synthesis inhibitor) and cycloheximide (a protein synthesis inhibitor) inhibited sulforaphane-responsive HO-1 mRNA expression, indicating that sulforaphane is a requirement for transcription and de novo protein synthesis. Moreover, sulforaphane increased the nuclear levels of Nrf2 and increased the binding activity of nuclear proteins to the antioxidant responsive element consensus sequence. We also found that U0126, an ERK kinase inhibitor, suppressed the sulforaphane-induced HO-1 expression and nuclear translocation of Nrf2. Moreover, the cytoprotective effect of sulforaphane on indomethancin-induced cytotoxicity was partially blocked by ERK and HO-1 inhibitors, further demonstrating that sulforaphane attenuated oxidative stress through a pathway that involved ERK and HO-1. Taken together, this study gives additional support to the possible use of sulforaphane as a dietary preventive agent against oxidative stress-induced intestinal injury.

Heme oxygenase‐1 serves as an important effector in the postischemic anti‐inflammatory phenotype induced by antecedent ethanol ingestion

Since the catalytic products of heme oxygenase-1 (HO-1) activity exert powerful anti-adhesive and anti-oxidant properties, we postulated that ethanol (EtOH)-induced HO-1 expression serves as an effector of the postischemic anti-inflammatory effects of this preconditioning stimulus. EtOH was administered to C57BL/6 mice by gavage at a dose that produced a peak plasma concentration of 45 mg/dl 30 min after ingestion. I/R was induced 24 hr later followed by assessment of leukocyte- and platelet-endothelial interactions, HO-1 activity (bilirubin assay), and HO-1 expression (Western). I/R induced significant leukocyte and platelet adhesion, effects that were abolished by antecedent EtOH. Treatment with a HO-1 inhibitor (SnPP-IX) during I/R prevented the beneficial effects of EtOH-PC. Alternatively, upregulation of HO-1 with hemin mimicked the effects of antecedent EtOH. Although jejunal HO-1 expression was not increased 24 hr after EtOH consumption, HO-1 activity was increased during I/R. Pharmacologic inhibition studies indicated that EtOH-induced increases in HO-1 activity occur by an adenosine A2-receptor/eNOS-dependent mechanism. The latter results are confirmed in WT mice and in eNOS-/- mice. Taken together, our data indicate that HO-1 functions as an effector of EtOH-PC. Our work also suggests that a novel class of agents, HO-1 inducers, may represent a new avenue for therapeutic intervention in I/R. Supported by DK 43785.