Upregulated HO-1 Expression Research Articles

KCHO-1, a Novel Antineuroinflammatory Agent, Inhibits Lipopolysaccharide-Induced Neuroinflammatory Responses through Nrf2-Mediated Heme Oxygenase-1 Expression in Mouse BV2 Microglia Cells.

The brain is vulnerable to oxidative stress and inflammation that can occur as a result of aging or neurodegenerative diseases. Our work has sought to identify natural products that regulate heme oxygenase (HO)-1 and to determine their mechanism of action in neurodegenerative diseases. KCHO-1 is a novel herbal therapeutic containing 30% ethanol (EtOH) extracts from nine plants. In this study, we investigated the antineuroinflammatory effects of KCHO-1 in lipopolysaccharide- (LPS-) treated mouse BV2 microglia. KCHO-1 inhibited the protein expression of inducible nitric oxide synthase (iNOS), iNOS-derived nitric oxide (NO), cyclooxygenase- (COX-) 2, and COX-2-derived prostaglandin E2 (PGE2) in LPS-stimulated BV2 microglia. It also reduced tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 production. This effect was correlated with the suppression of inhibitor of nuclear factor kappa B-α (IκB-α) phosphorylation and degradation and nuclear factor kappa B (NF-κB) translocation and DNA binding. Additionally, KCHO-1 upregulated HO-1 expression by promoting nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) in mouse BV2 microglia. Tin protoporphyrin (SnPP), an HO activity inhibitor, was used to verify the inhibitory effects of KCHO-1 on proinflammatory mediators and proteins associated with HO-1 expression. Our data suggest that KCHO-1 has therapeutic potential in neurodegenerative diseases caused by neuroinflammation.

Inhibitory effect of 9-hydroxy-6,7-dimethoxydalbergiquinol from Dalbergia odorifera on the NF-κB-related neuroinflammatory response in lipopolysaccharide-stimulated mouse BV2 microglial cells is mediated by heme oxygenase-1

The heartwood of Dalbergia odorifera T. Chen (Leguminosae) is an important source of traditional Korean and Chinese medicines. 9-Hydroxy-6,7-dimethoxydalbergiquinol (HDDQ), a compound isolated from D. odorifera, has various biological activities. The aim of this study was to determine the efficacy of HDDQ in modulating the regulation of anti-inflammatory activity through the upregulation of heme oxygenase (HO)-1 in BV2 microglia. HDDQ inhibited the protein expression of inducible nitric oxide synthase (iNOS), iNOS-derived nitric oxide (NO), and the production of cyclooxygenase (COX)-2 and COX-2-derived prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated mouse BV2 microglia. HDDQ also reduced tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production, and suppressed the phosphorylation and degradation of IκB-α and the nuclear translocation of p65 in mouse BV2 microglia in response to LPS. Furthermore, HDDQ upregulated HO-1 expression via nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) in mouse BV2 microglia. Using tin protoporphyrin (SnPP), an HO activity inhibitor, we verified that the inhibitory effects of HDDQ on the proinflammatory mediators NO, PGE2, TNF-α, and IL-1β, and nuclear factor kappa B (NF-κB) DNA-binding activity are associated with the induction of HO-1 expression. Our data suggest that HDDQ has therapeutic potential against neurodegenerative diseases caused by neuroinflammation.

Z-ligustilide activates the Nrf2/HO-1 pathway and protects against cerebral ischemia–reperfusion injury in vivo and in vitro

Z-ligustilide (LIG), the main lipophilic component of Radix Angelica sinensis, has been shown to protect against brain ischemic damage in rodents by oral and intra-peritoneal treatments. The present study aimed to confirm the therapeutic effect of LIG administered intravenously on 2h middle cerebral artery occlusion (MCAO) and 22h reperfusion injury in rats since oral administration has low bioavailability, slow absorption and distribution. Moreover, whether LIG activated the NF-E2-related factor 2/ heme oxygenase-1 (Nrf2/HO-1) pathway was also investigated in vivo and in vitro to further elucidate the precise protective mechanisms. In vivo, rats treated intravenously with LIG immediately after the surgery was finished had less neurological dysfunction and smaller infarct volume than that of the vehicle-treated rats. Additionally, LIG promoted Nrf2 nuclear translocation, and further remarkably increased Nrf2 and HO-1 protein expression. In vitro, LIG induced Nrf2 nuclear translocation and up-regulated HO-1 expression in a time-dependent and concentration-dependent manner. Furthermore, LIG treatment reduced cell death induced by OGD, however, the protective action was abolished while Nrf2/HO-1 expression was knockdown by RNA interference. These results noted that intravenous post-treatment with LIG exhibits noticeable neuroprotective properties against brain damage by ischemia–reperfusion and the ability of LIG to activate Nrf2/HO-1 pathway may be partly responsible for it.

Celecoxib induces heme oxygenase-1 expression in macrophages and vascular smooth muscle cells via ROS-dependent signaling pathway

The multiple cytoprotective mechanisms of heme oxygenase (HO)-1 make it a promising therapeutic target. This study investigated whether the selective cyclooxygenase (COX)-2 inhibitor, celecoxib, can upregulate HO-1 expression. Murine J774 macrophages and rat aortic vascular smooth muscle cells (VSMCs) were used to study the effect of celecoxib on HO-1 expression. A signal transduction pathway involving reactive oxygen species (ROS) was also investigated. We found that celecoxib can upregulate HO-1 gene and protein expressions in J774 macrophages and VSMCs. This effect was not diminished by prostaglandin E(2) or 15dPGJ(2), while it was additive to hypoxia-induced HO-1 expression, suggesting an event independent of COX-2 activity or hypoxia-inducible factor-1α. Moreover, celecoxib activated ERK, p38, Akt, and Nrf2 as well as increased ROS production. All these events contributed to the increase in the expression of HO-1 caused by celecoxib. In this study, we also, for the first time, demonstrated that AMP-activated protein kinase (AMPK) can mediate HO-1 expression via the downstream activation of p38 and Akt. However, the HO-1-inducing actions of celecoxib and hypoxia were not associated with AMPK. This study demonstrates a COX-2-independent action of celecoxib in upregulating HO-1 in macrophages and VSMCs. This action is dependent on ROS, Akt, ERK, p38, and Nrf2 activation. These findings provide new insights into the action mechanism of celecoxib with broad implications for anti-inflammation therapy.

Studies on Heme Oxygenase 1 During Erythroid Differentiation

AbstractAbstract 4254Heme is indispensable for the function of all aerobic cells as a prosthetic group of innumerable proteins. However, “free heme” (uncommitted) can initiate the formation of free radicals and cause lipid peroxidation, which can lead to cellular damage and tissue injury. Therefore, the rate of heme biosynthesis and catabolism must be well balanced by tight control mechanisms. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC), whose precursors synthesize heme with rates that are at least one order of magnitude higher (on the per cell basis) than those in the liver – the second most active heme producer in the body. The degradation of heme is exclusively carried out by heme oxygenases 1 and 2 (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using both a murine erythroleukemia cell line (MEL) and primary erythroid cells isolated from mouse fetal livers, we have demonstrated that during erythroid differentiation HO1 is up-regulated at both mRNA and protein levels. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase. These data suggest that in developing RBC, in addition to the continuous assembly of heme with globin chains, there is an increase in levels of uncommitted heme, which upregulates HO1 expression. Additionally, we have shown that down-regulation of HO1 via siRNA increased hemoglobinization in differentiating MEL cells. In contrast, induction of HO1 expression by NaAsO2 reduced the hemoglobinization of MEL cells. This effect could be reversed to control levels by the addition of HO1 inhibitor tin-protophorphyrin (SnPP). These results show that in differentiating erythroid cells the balance between levels of heme and HO1 have to be tightly regulated to maintain hemoglobinization at appropriate levels. Our results lead us to propose that disturbances in HO1 expression could play a role in some pathophysiological conditions such as thalassemias. Disclosures:No relevant conflicts of interest to declare.

Heme oxygenase-1 participates in the anti-inflammatory activity of taurine chloramine

Interleukin-6 (IL-6) and interleukin-8 (IL-8) are implicated in the pathogenesis of rheumatic diseases. In affected joints fibroblast-like synoviocytes (FLS) are the major source of these pro-inflammatory cytokines. We have previously found that production of both cytokines is inhibited in vitro by taurine chloramine (Tau-Cl). Heme oxygenase (HO-1) activity was also reported to restrict synthesis of various inflammatory mediators, including IL-6 and IL-8. The aim of present study was to investigate whether this enzyme activity is implicated in the mechanism of Tau-Cl suppressive effect. We have shown that in rheumatoid FLS both hemin (known HO-1 inducer) and Tau-Cl significantly up-regulate HO-1 expression at the mRNA and protein levels and simultaneously inhibit IL-1 beta-triggered production of pro-inflammatory cytokines. However, the inhibitory potency of these compounds differs, because hemin is more potent inhibitor of IL-8 than IL-6 production, while Tau-Cl exerts opposite effect. Importantly, pretreatment of the cells with HO-1 inhibitor completely reverses the inhibitory effect of hemin on both cytokines production. However, in Tau-Cl treated cells this inhibitor fully restores only IL-8 secretion but has weaker effect on IL-6 response. Thus, the present results: (i) support HO-1 activity to be relevant to negatively control production of pro-inflammatory cytokines, and (ii) underline implication of HO-1 in mediating Tau-Cl inhibitory action.

Upregulation of heme oxygenase-1 by brazilin via the phosphatidylinositol 3-kinase/Akt and ERK pathways and its protective effect against oxidative injury

Heme oxygenase (HO)-1 is a cytoprotective enzyme that is activated by various phytochemicals. We examined the ability of brazilin to upregulate HO-1 expression in auditory cells. We found that brazilin induced the expressions of HO-1 mRNA and protein in concentration- and time-dependent manners. Brazilin induced nuclear factor-E2-related factor 2 (Nrf2) nuclear translocation, and dominant-negative Nrf2 attenuated brazilin-induced expression of HO-1. Brazilin induced a temporary increase in the phosphorylation of Akt. While LY294002, a non-selective phosphotidylinositol 3-kinase (PI3K) inhibitor, was able to reduce brazilin-induced phosphorylation of Akt and the subsequent induction of HO-1. Brazilin activated the extracellular signal-regulated kinase (ERK) and p38 pathways, and the ERK pathway played an important role in HO-1 expression. Brazilin protected the cells against t-butyl hydroperoxide ( t-BHP)-induced cell death. The protective effect of brazilin was abrogated by anti-sense oligodeoxynucleotides (ODN) against the HO-1 gene. These results demonstrate that the expression of HO-1 by brazilin is mediated via the PI3K/Akt and ERK pathways, and this expression inhibits t-BHP-induced cell death in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells.

Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria

Cerebral malaria claims more than 1 million lives per year. We report that heme oxygenase-1 (HO-1, encoded by Hmox1) prevents the development of experimental cerebral malaria (ECM). BALB/c mice infected with Plasmodium berghei ANKA upregulated HO-1 expression and activity and did not develop ECM. Deletion of Hmox1 and inhibition of HO activity increased ECM incidence to 83% and 78%, respectively. HO-1 upregulation was lower in infected C57BL/6 compared to BALB/c mice, and all infected C57BL/6 mice developed ECM (100% incidence). Pharmacological induction of HO-1 and exposure to the end-product of HO-1 activity, carbon monoxide (CO), reduced ECM incidence in C57BL/6 mice to 10% and 0%, respectively. Whereas neither HO-1 nor CO affected parasitemia, both prevented blood-brain barrier (BBB) disruption, brain microvasculature congestion and neuroinflammation, including CD8(+) T-cell brain sequestration. These effects were mediated by the binding of CO to hemoglobin, preventing hemoglobin oxidation and the generation of free heme, a molecule that triggers ECM pathogenesis.

The Mechanism of Hepatic Heme Oxygenase-1 Expression by Limb Remote Ischemic Preconditioning

In vitro studies (1–3) have found the molecular signaling pathways of heme oxygenase (HO)-1 induction included MAPKs, Nrf2, and Bach1. However, the cellular mechanism of HO-1 induction in the target tissue (liver) by remote ischemic preconditioning (limb) is still under investigation. It has been proposed that either neurogenic and/or humoral factors were responsible for the induction of HO-1. The blocking assays were the commonly used methods to prove these theories. For example, ganglion blockers (4, 5) or mitochondria KATP channel blocker (6) have been shown to inhibit the protective effect of remote preconditioning. In our study, though we demonstrated the HO-1 was not induced in the peripheral lymphocytes but in the liver, we did not find the exact mechanism of remote HO-1 induction. Kanoria et al. (7) has indicated nitric oxide (NO) as an important mediator of the protective effect of limb remote ischemic preconditioning (RIPC). Is it possible that NO also is an important inducer of HO-1 expression? Choi et al. had shown that NO donor upregulated HO-1 expression in hepatocyte cultures (8, 9). The endothelium of limb vessels might produce NO derivatives in the process of limb ischemia-reperfusion. The study of Kanoria et al. (7) showed increased nitrate/nitrite levels in hepatic vein and systemic arterial plasma after RIPC. However, there existed another puzzle that how the short-lived NO derivatives transfer signals to induce HO-1 in remote liver. Of course, remote hepatic HO-1 induction might simply reflect secondary liver ischemic stress originating from limb ischemia-reperfusion. To solve this problem, we have to investigate the perfusion dynamics of liver during limb ischemia-reperfusion. I-Rue Lai King-Jen Chang Department of Surgery National Taiwan University Hospital National Taiwan University College of Medicine Taipei, Taiwan Chau-Fong Chen Department of Physiology National Taiwan University College of Medicine Taipei, Taiwan

PROTECTIVE ROLE OF HEME OXYGENASE-1 INDUCTION AGAINST ISCHEMIA/REPERFUSIONINDUCED LIVER DAMAGE IN RATS

Heme oxygenase (HO)-1 has been shown to be beneficial in a variety of ischemia/reperfusion (I/R) models. However, there are no studies examining the relationship between the upregulation of HO-1 expression and the development of I/R injury in the reperfused liver. Therefore, the purpose of this study was to determine the protective role of upregulated HO-1 expression during hepatic I/R. Rats were subjected to 60 min of hepatic ischemia, liver and blood samples were taken 0, 1, 2, 4, 6, 8, 12 and 24 hr after reperfusion. HO activity in the liver increased significantly at 1 hr after reperfusion and reached maximum at 6 hr after reperfusion, and then declined gradually. Rats were pretreated twice with ZnPP (6.74 mg/kg, an inhibitor of HO activity) or with hemin (30 mg/kg, an inducer of HO expression and activity) at 16 and 3 hr prior to ischemia. The serum ALT level increased markedly immediately following reperfusion and peaked between 4 and 6 hr and then declined gradually. This increase was significantly augmented by ZnPP but was attenuated by hemin. iNOS protein and iNOS mRNA were upregulated markedly at 4 hr after reperfusion, which was suppressed by hemin. Conversely, in the ZnPP-treated group, the iNOS induction resulting from I/R was markedly enhanced. Immediately after reperfusion, the COX-2 mRNA significantly increased in I/R group and this increase remained until at 6 hr after reperfusion. The increase in the COX-2 mRNA expression was potentiated by ZnPP but attenuated by hemin at all time points. In the rats pretreated with ZnPP and subjected to hepatic I/R, the plasma TNF-α content and its mRNA level were significantly higher than that observed in the serum of control rats subjected to I/R. However, the TNF-α content in rats pretreated with hemin and subjected to I/R was attenuated. Consequently, prior induction of HO-1 expression may provide a new strategy to protect livers against injury caused by ischemia/reperfusion which occur in liver transplantation.

Regulation of CO production by glutamate in cultured astrocytes from the cerebral cortex

Carbon monoxide (CO), a potent dilator in cerebral circulation, is a product of heme degradation by heme oxygenase (HO) that is highly expressed in the brain as a constitutive HO-2 isoform, whereas inducible HO-1 is not detectable. In newborn pigs, brain CO production is increased during glutamatic seizures without HO-1 induction. Astrocytes that account for majority of the cell population in the cerebral cortex may contribute to overall brain CO production. We addressed the hypothesis that glutamate increases CO production in astrocytes via activation of constitutive HO-2. Primary cultures of astrocytes from cerebral cortex of newborn piglets were grown on Costar plates or Cytodex microcarrier beads and were identified by immunostaining for glial fibrillary acidic protein and aquaporin-4. CO production by cultured astrocytes was detected by gas chromatography/mass spectrometry. HO-2 is the major isoform in quiescent astrocytes, although HO-1 is also immunodetectable. Glutamate (100 mkM, 1h) increased basal CO production from 13±2 to 45±8 pmol/mg protein and stimulated HO catalytic activity (astrocyte CO formation from exogenous heme) 2-fold. We upregulated HO-1 expression ~ 10-fold by a heme analog cobalt protoporphyrin (CoPP). In CoPP-treated astrocytes, basal CO production was increased to 139±20 pmol CO/mg protein consistent with HO-1 overexpression. In HO-1 overexpressing astrocytes, glutamate did not stimulate HO catalytic activity and only mildly enhanced CO formation. We conclude that glutamate rapidly increases astrocyte CO production via posttranslational activation of HO-2 but not HO-1.

Hydrogen sulfide and carbon monoxide are in synergy with each other in the pathogenesis of recurrent febrile seizures.

1. The purpose of the present study was to investigate the interaction between hydrogen sulfide (H(2)S) and carbon monoxide (CO) during recurrent febrile seizures (FS) 2.H(2)S and CO are important intra- and intercellular messengers, regulating various brain functions. Our recent studies showed that both of them alleviate the hippocampal damage induced by recurrent FS. In the present study, on a rat model of recurrent FS, we found that hydroxylamine (an inhibitor of cystathionine b-synthase, CBS) reduced CO level and down regulated heme oxygenase (HO-1) expression, while NaHS (a donor of H(2)S) elevated CO level and upregulated HO-1 expression. ZnPP-IX (an inhibitor of HO-1) decreased H(2)S formation and down regulated CBS expression, while hemin (which increases the production of endogenous CO) enhanced H(2)S formation and elevated CBS expression. 3. Our data demonstrate that endogenous H(2)S and CO are in synergy with each other in recurrent FS.

Thymidine Phosphorylase Gene Transfer Inhibits Vascular Smooth Muscle Cell Proliferation by Upregulating Heme Oxygenase-1 and p27 KIP1

Thymidine phosphorylase (TP) reportedly promotes endothelial cell migration and induces heme oxygenase (HO)-1 expression. However, its effect on vascular smooth muscle cells (VSMCs) is poorly understood. In this study, we examined the effect of TP on VSMCs in vitro and in vivo. Phagemid vector encoding human TP gene was transfected into rat VSMCs, and a clone overexpressing TP was selected (C2). C2 showed a slower migration and proliferation than VSMCs cloned with empty vector (pC) under basal, serum-stimulated, and hypoxic conditions. This decrease in proliferation correlated with TP-induced HO-1 expression and was reversed by inhibitors of either TP or HO activity. Furthermore, in C2, the cyclin-dependent kinase inhibitor (p27KIP1) was much more abundant than in pC, and the cell cycle was arrested at the G1 phase. TP or HO activity inhibitors decreased p27(KIP1) expression in C2 to the level seen in pC. Adventitial TP gene delivery significantly reduced neointimal VSMC migration and neointima formation in balloon-injured rat carotid arteries. TP overexpression upregulated HO-1 expression and consequently increased p27(KIP1) in cultured VSMCs, and inhibited VSMC migration and proliferation in vitro and in vivo. TP represents a promising target for treating vascular obstructive disease.

UPREGULATION OF HEME OXYGENASE-1 EXPRESSION IS ASSOCIATED WITH IMPROVEMENT OF CARDIAC FUNCTION AFTER HUMAN BONE MARROW MESENCHYMAL CELLS TRANSPLANTATION IN INFARCTED RAT

P1101 Aim: Heme oxygenase (HO)-1, a stress response and cytoprotective heat shock protein, was shown to protect cells from apoptosis. The aim of this study was to investigate the effects of transplantation of human bone marrow mesenchymal cells (hMSCs) on the expression of HO-1 and cardiac function in myocardiac infarction (MI) rats. Methods: A total of 5×106 hMSCs were injected into the ischemic zone in MI rats. Fibroblast or equal volume saline were served as controls. Cardiac function was evaluated by echocardiography. The expression of HO-1 was assessed by real-time RT-PCR and Western blot. The localization of HO-1 protein was determined under fluorescence microscopy. The infarct size and the number of apoptosis cells were examined by histology. Results: Real-time RT-PCR results showed that the mRNA expression of HO-1 was enhanced in hMSC group at 1, 3 and 7 days post transplantation and peaked at 3 days (P<0.01). Consistent with this result, Western blot also revealed markedly elevated HO-1 expression peaking at 3 days (P<0.01) post-transplantation in hMSCs group. HO-1 activity was significantly increased in hMSCs group compared to control groups at each time point and peaked at day 3 (P<0.01). Transplantation of hMSCs also reduced infarct size, 36% reduction in hMSCs group versus 44% and 43% in control groups on day 7 post transplantation, respectively. The number of apoptosis cardiac myocytes was reduced and cardiac function was improved in hMSCs group ((P<0.01, vs control groups). Conclusion: Transplantation of hMSCs could upregulate HO-1 expression, which might play an important role in protecting transplanted hMSCs and myocardiocytes from apoptosis. The upregulated expression of HO-1 might associate with cardiac function improvement during the early stage after myocardial infarction.