Tin Protoporphyrin Research Articles

Haem oxygenase-1 gene transfer protects retinal ganglion cells from ischaemia/reperfusion injury

RGC (retinal ganglion cell) death following ischaemic insult is the major cause of a number of vision-threatening diseases, including glaucoma. The aim of the present study was to evaluate the role of HO-1 (haem oxygenase-1) in the retina against IR (ischaemia/reperfusion) injury. Adenovirus-mediated HO-1 gene transfer (Adv-HO-1) was carried out by injection into the vitreous body to induce HO-1 overexpression. At 3 weeks after transfection, levels of HO-1 expression, as measured by Western blot analysis, immunohistochemical staining and activity assay, were drastically up-regulated. Transient retinal ischaemia was induced by raising the intraocular pressure to 150 mmHg for 60 min. Untreated IR caused a significant decrease in RGC numbers at 3 and 7 days after reperfusion (76.1 and 67.2% of control eyes with sham IR respectively; P<0.001). Eyes pretreated with Adv-HO-1 had less RGC loss on day 3 and 7 following reperfusion compared with control eyes injected with Adv-GFP (adenovirus containing a gene for green fluorescent protein; 94.3 and 88.2% respectively; P=0.007 and 0.001). SnP (tin protoporphyrin), an HO-1 inhibitor, counteracted the effects of Adv-HO-1. In conclusion, these findings provide evidence that augmentation of HO-1 enzyme overexpression by intravitreal injection is able to protect RGCs against IR-induced damage.

Heme Oxygenase-1 Inhibits the Proliferation of Pancreatic Stellate Cells by Repression of the Extracellular Signal-Regulated Kinase1/2 Pathway

Activation of pancreatic stellate cells (PSCs) is the key process in the development of pancreatic fibrosis, a common feature of chronic pancreatitis and pancreatic cancer. In recent studies, curcumin has been shown to inhibit PSC proliferation via an extracellular signal-regulated kinase (ERK)1/2-dependent mechanism. In addition, curcumin is a potent inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1) in other cell types. Therefore, the aims of this study were to 1) characterize the effect of curcumin on HO-1 gene expression in PSCs, 2) explore whether HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation, and 3) clarify the involvement of the mitogen-activated protein kinase (MAPK) family in this context. Cultured rat PSCs were incubated with curcumin and assessed for HO-1 up-regulation by Northern blot analysis, immunoblotting, and activity assays. The effect of HO-1 on platelet-derived growth factor (PDGF)-induced PSC proliferation and MAPK activation was determined by immunoblotting, cell proliferation assays, and cell count analyses. Curcumin induced HO-1 gene expression in PSCs in a time- and dose-dependent manner and inhibited PDGF-mediated ERK1/2 phosphorylation and PSC proliferation. These effects were blocked by treatment of PSCs with tin protoporphyrin IX, an HO inhibitor, or transfection of HO-1 small interfering RNA. Our data provide evidence that HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation. Therefore, therapeutic up-regulation of HO-1 could represent a mode for inhibition of PSC proliferation and thus may provide a novel strategy in the prevention of pancreatic fibrosis.

Activation of the oxygen-sensing signal cascade prevents mitochondrial injury after mouse liver ischemia-reperfusion

The mitochondrial permeability transition (MPT) plays an important role in hepatocyte death caused by ischemia-reperfusion (IR). This study investigated whether activation of the cellular oxygen-sensing signal cascade by prolyl hydroxylase inhibitors (PHI) protects against the MPT after hepatic IR. Ethyl 3,4-dihyroxybenzoate (EDHB, 100 mg/kg ip), a PHI, increased mouse hepatic hypoxia-inducible factor-1alpha and heme oxygenase-1 (HO-1). EDHB-treated and untreated mice were subjected to 1 h of warm ischemia to approximately 70% of the liver followed by reperfusion. Mitochondrial polarization, cell death, and the MPT were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. EDHB largely blunted alanine aminotransferase (ALT) release and necrosis after reperfusion. In vehicle-treated mice at 2 h after reperfusion, viable cells with depolarized mitochondria were 72%, and dead cells were 2%, indicating that depolarization preceded necrosis. Mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. NIM811, a specific inhibitor of the MPT, blocked mitochondrial depolarization after IR, further confirming that mitochondrial depolarization was due to MPT onset. EDHB decreased mitochondrial depolarization to 16% and prevented the MPT. Tin protoporphyrin (10 micromol/kg sc), an HO-1 inhibitor, partially abrogated protection by EDHB against ALT release, necrosis, and mitochondrial depolarization. In conclusion, IR causes the MPT and mitochondrial dysfunction, leading to hepatocellular death. PHI prevents MPT onset and liver damage through an effect mediated partially by HO-1.

Up-regulation of heme oxygenase-1 by isoflurane preconditioning during tolerance against neuronal injury induced by oxygen glucose deprivation

Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme to produce bile pigments and carbon monoxide. The HO-1 isozyme is induced by a variety of factors such as heat, heme, ischemia, and hydrogen peroxide. In recent years, mounting findings have suggested that HO-1 has a neuroprotective activity against ischemic injury. The neuroprotective role of isoflurane, a commonly used anesthetic, has been well documented, but little is known about the underlying mechanisms involved. Recently, isoflurane has been shown to up-regulate HO-1 in the liver. In this study, we show that isoflurane preconditioning promotes the survival of cultured ischemic hippocampal neurons by increasing the number of surviving neurons and their viability. Further study by reverse transcription-polymerase chain reaction and Western blot analysis showed that isoflurane preconditioning significantly increases HO-1 expression in oxygen glucose deprivation (OGD)-induced neuronal injury. Furthermore, inhibition of HO activity by tin protoporphyrin partially abolishes isoflurane preconditioning's protective effect as measured by lactate dehydrogenase release in OGD neurons. These findings indicated that the neuroprotective role of isoflurane preconditioning against OGD-induced injury might be associated with its role in up-regulating HO-1 in ischemic neurons.

Cudratricusxanthone A Protects Mouse Hippocampal Cells against Glutamate-Induced Neurotoxicity via the Induction of Heme Oxygenase-1

Cudratricusxantone A (CTXA), isolated from the roots of Cudrania tricuspidata Bureau (Moraceae), has potent hepatoprotective, antiproliferative, and monoamine oxidase inhibitory effects. In this study, we examined whether CTXA could protect HT22-immortalized hippocampal cells against glutamate-induced oxidative stress through the induction of heme oxygenase (HO)-1 expression. CTXA induced the expression of HO-1 and increased HO activity dose- and time-dependently. CTXA also suppressed glutamate-induced ROS generation in HT22 cells. Interestingly, treatment of neuronal cells with CTXA enhanced cellular resistance to glutamate oxidative stress. The protective effect of CTXA was abrogated by tin protoporphyrin IX, an HO inhibitor. In addition, treatment with the HO-1 inducer, cobalt protoporphyrin IX, and bilirubin, one of the enzymatic products of HO-1, produced comparable protection. These results demonstrate that CTXA protects neuronal cells from glutamate-induced oxidative stress via the induction of HO-1.

Effects of Heme Oxygenase 1 on Brain Edema and Neurologic Outcome after Cardiopulmonary Resuscitation in Rats

Heme oxygenase 1 (HO-1) has been shown to attenuate neuronal injury. Therefore, the authors examined whether HO-1 would reduce the brain damage caused by cardiac arrest. Rats anesthetized with halothane were subjected to 8 min of cardiac arrest by asphyxia without any pretreatment (control group) or pretreated with an inducer of HO-1 (hemin group) or with an inhibitor of HO-1 (tin protoporphyrin group). Then, the animals were resuscitated with a standardized method. Brain water content (1 and 6 h after resuscitation), neurologic deficit score, viable neuronal counts, and caspase-3 immunostaining in the hippocampus (4 and 14 days) were evaluated. Water content in the hemin group was significantly reduced (mean +/- SD: 83.1 +/- 1.9% for 1 h after resuscitation; P = 0.03) and significantly greater in the tin protoporphyrin group (91.1 +/- 2.0% for 1 h after resuscitation; P = 0.035) when compared with the control group (88.2 +/- 2.4%). Water content of the cortex was nearly the same as that of the hippocampus. Neurologic deficit scores and neuronal survival were significantly better in the hemin group than in the control group on the 4th and 14th days. In rats that survived for 4 days, the amount of caspase 3-positive neurons was 27 +/- 7 in the control group, whereas the value was 14 +/- 6 in the hemin group (P < 0.05). In rats resuscitated from cardiac arrest, induction of HO-1 by hemin reduced brain edema, improved neurologic outcome, and protected neurons against apoptosis.

Inhibition of vascular smooth muscle cell proliferation by chronic hemin treatment

Hemin, an oxidized form of heme, is an essential regulator of gene expression and cell cycle progression. Our laboratory previously reported (34) that chronic hemin treatment of spontaneously hypertensive rats reversed the eutrophic inward remodeling of small peripheral arteries. Whether long-term treatment of cultured vascular smooth muscle cells (VSMCs) with hemin alters the proliferation status of these cells has been unknown. In the present study, hemin treatment at 5 muM for 4, 7, 14, and 21 days significantly inhibited the proliferation of cultured rat aortic VSMCs (A-10 cells) by arresting cells at G0/G1 phases so as to decelerate cell cycle progression. Heme oxygenase (HO) activity and inducible HO-1 protein expression were significantly increased by hemin treatment. HO inhibitor tin protoporphyrin IX (SnPP) abolished the effects of hemin on cell proliferation and HO activity. Interestingly, hemin-induced HO-1 expression was further increased in the presence of SnPP. Hemin treatment had no significant effect on the expression of constitutive HO-2. Expression of p21 protein and the level of reactive oxygen species (ROS) were decreased by hemin treatment, which was reversed by application of SnPP. After removal of hemin from culture medium, inhibited cell proliferation and increased HO-1 expression in VSMCs were returned to control level within 1 wk. Transfection with HO-1 small interfering RNA significantly knocked down HO-1 expression and decreased HO activity, but had no effect on HO-2 expression, in cells treated with or without hemin for 7 days. The inhibitory effect of hemin on cell proliferation was abolished in HO-1 silenced cells. It is concluded that induction of HO-1 and, consequently, increased HO activity are responsible for the chronic inhibitory effect of hemin on VSMC proliferation. Changes in the levels of p21 and ROS might also participate in the cellular effects of hemin.

Sasim attenuates LPS-induced TNF-α production through the induction of HO-1 in THP-1 differentiated macrophage-like cells

Aim of the study Sasim, a traditional prescription composed of seven herbal mixtures, has been widely used as an oriental medicine for the treatment of cerebral infarction in Korea. However, the regulatory mechanisms by which the formula affects immune processing in cerebral infarction patients remain unknown. Materials and methods The levels of secretory protein of tumor necrosis factor (TNF)-α were determined in both THP-1 differentiated macrophage-like (THP-1/M) cells and Peripheral blood mononuclear cells (PBMCs) from cerebral infarction patients. Also, the levels of protein and mRNA of TNF-α and heme oxygenase-1 (HO-1) were detected in THP-1/M cells under our experimental condition. Results Sasim markedly suppressed lipopolysaccharide (LPS)-induced TNF-α at the levels of secretory protein and mRNA in both PBMCs from cerebral infarction patients and THP-1/M cells. Interestingly, Sasim strongly induced HO-1, the rate-limiting enzyme of heme catabolism, at both the protein and mRNA levels in THP-1/M cells. Treatment with tin protoporphyrin IX (SnPP), an inhibitor of the catalytic activity of HO, significantly abolished the suppressive effect of Sasim on LPS-induced TNF-a production in THP-1/M cells. Conclusions These data indicate that Sasim may be beneficial in the cessation of inflammatory processes associated with cerebral infarction through the induction of HO-1 expression.

HO-1 induction ameliorates experimental murine membranous nephropathy: anti-oxidative, anti-apoptotic and immunomodulatory effects

Therapeutic agents for membranous nephropathy (MN) remain ill-defined. Haeme oxygenase (HO)-1 is considered to play a protective role in various disorders. Here, we assessed the efficacy of HO-1 induction therapy for MN. MN was induced in BALB/c mice with intravenous injections of cationic bovine serum albumin. Three groups of mice were administered 100 micromol/kg Cobalt protoporphyrin (CoPP, a potent HO-1 inducer), Tin protoporphyrin (SnPP, a potent HO-1 inhibitor) or phosphate-buffered saline via intra-peritoneal injections once a week starting from the induction of MN. Disease severity was verified by serum and urine metabolic profiles and by renal histopathology. Cytokine profiles, immunoglobulin production, the expression of oxidative stress markers (thiobarbituric acid reactive substances, TBARS) and apoptosis, as measured by TUNEL, were also determined. Mice treated with CoPP displayed a significant reduction in proteinuria and a marked amelioration of glomerular lesions, accompanied by attenuated immune-complex deposition. The production of immunoglobulins in MN mice treated with CoPP was significantly reduced compared with that of mice in the other two groups. TBARS in the serum and kidneys, as well as apoptosis, were also significantly reduced in CoPP-treated mice. Cytokine mRNA expression in the renal cortex indicated that CoPP not only decreased the expression of proinflammatory cytokines, but also increased the expression of anti-inflammatory cytokines (interleukin-10). HO-1 induction therapy may ameliorate experimental MN via multiple pathways, including anti-oxidative, anti-apoptotic and immunomodulatory effects. HO-1 inducing regimens should be considered as a potential therapeutic intervention in MN in the future.

Cytoprotection of human endothelial cells from menadione cytotoxicity by caffeic acid phenethyl ester: The role of heme oxygenase-1

Caffeic acid phenethyl ester (CAPE), derived from various plant sources, has been shown to ameliorate ischemia/reperfusion injury in vivo, and this has been attributed to its ability to reduce oxidative stress. Here we investigated the cytoprotection of CAPE against menadione-induced oxidative stress in human umbilical vein endothelial cells (HUVEC) to evaluate potential gene expression involvement. CAPE exhibited dose-dependent cytoprotection of HUVEC. A gene screen with microarrays was performed to identify the potential cytoprotective gene(s) induced by CAPE. Heme oxygenase-1 (HO-1) was highly upregulated by CAPE and this was confirmed with reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. Inhibition of HO-1 activity using the HO-1 inhibitor tin protoporphyrin IX (SnPPIX), resulted in loss of cytoprotection. Carbon monoxide, one of HO-1 catabolic products appeared to play a small role in CAPE protection. Caffeic acid, a potential metabolite of CAPE with similar free radical scavenging ability, however, didn't show any cytoprotective effect nor induce HO-1. These findings suggest an important role of HO-1 induction in CAPE cytoprotection against oxidant stress, which may not relate to CAPE structural antioxidant activity nor to its traditional enzymatic activity in decomposing heme but to a yet to be determined activity.

Isoliquiritigenin Inhibits Cell Proliferation by a Heme Oxygenase-Dependent Pathway in Rat Hepatic Stellate Cells

We evaluated whether the antiproliferative effects of isoliquiritigenin (ISL) on rat hepatic stellate cells (HSCs) are related to the induction of heme oxygenase 1 (HO-1) expression. ISL significantly inhibited serum- or growth factor-induced HSC proliferation. The inhibition of platelet-derived growth factor (PDGF)-induced proliferation by ISL was associated with the mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt-p70 (S6K) pathways. ISL induced the expression of HO-1 in HSCs. Using the chemical inhibitor tin protoporphyrin, we also found that the inhibitory action of ISL on PDGF-induced proliferation is mediated by HO-1. These data suggest that HO-1 expression is responsible for the antiproliferative effect of ISL on HSCs.

Non-invasive measurements of exhaled NO and CO associated with methacholine responses in mice

BackgroundNitric oxide (NO) and carbon monoxide (CO) in exhaled breath are considered obtainable biomarkers of physiologic mechanisms. Therefore, obtaining their measures simply, non-invasively, and repeatedly, is of interest, and was the purpose of the current study.MethodsExpired NO (ENO) and CO (ECO) were measured non-invasively using a gas micro-analyzer on several strains of mice (C57Bl6, IL-10-/-, A/J, MKK3-/-, JNK1-/-, NOS-2-/- and NOS-3-/-) with and without allergic airway inflammation (AI) induced by ovalbumin systemic sensitization and aerosol challenge, compared using independent-sample t-tests between groups, and repeated measures analysis of variance (ANOVA) within groups over time of inflammation induction. ENO and ECO were also measured in C57Bl6 and IL-10-/- mice, ages 8–58 weeks old, the relationship of which was determined by regression analysis. S-methionyl-L-thiocitrulline (SMTC), and tin protoporphyrin (SnPP) were used to inhibit neuronal/constitutive NOS-1 and heme-oxygenase, respectively, and alter NO and CO production, respectively, as assessed by paired t-tests. Methacholine-associated airway responses (AR) were measured by the enhanced pause method, with comparisons by repeated measures ANOVA and post-hoc testing.ResultsENO was significantly elevated in naïve IL-10-/- (9–14 ppb) and NOS-2-/- (16 ppb) mice as compared to others (average: 5–8 ppb), whereas ECO was significantly higher in naïve A/J, NOS-3-/- (3–4 ppm), and MKK3-/- (4–5 ppm) mice, as compared to others (average: 2.5 ppm). As compared to C57Bl6 mice, AR of IL-10-/-, JNK1-/-, NOS-2-/-, and NOS-3-/- mice were decreased, whereas they were greater for A/J and MKK3-/- mice. SMTC significantly decreased ENO by ~30%, but did not change AR in NOS-2-/- mice. SnPP reduced ECO in C57Bl6 and IL-10-/- mice, and increased AR in NOS-2-/- mice. ENO decreased as a function of age in IL-10-/- mice, remaining unchanged in C57Bl6 mice.ConclusionThese results are consistent with the ideas that: 1) ENO is associated with mouse strain and knockout differences in NO production and AR, 2) alterations of ENO and ECO can be measured non-invasively with induction of allergic AI or inhibition of key gas-producing enzymes, and 3) alterations in AR may be dependent on the relative balance of NO and CO in the airway.

Role of heme oxygenase-2 in pial arteriolar response to acetylcholine in mice with and without transfusion of cell-free hemoglobin polymers

Carbon monoxide derived from heme oxygenase (HO) may participate in cerebrovascular regulation under specific circumstances. Previous work has shown that HO contributes to feline pial arteriolar dilation to acetylcholine after transfusion of a cell-free polymeric hemoglobin oxygen carrier. The role of constitutive HO2 in the pial arteriolar dilatory response to acetylcholine was determined by using 1) HO2-null mice (HO2-/-), 2) the HO inhibitor tin protoporphyrin IX (SnPPIX), and 3) 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor of casein kinase-2 (CK2)-dependent phosphorylation of HO2. In anesthetized mice, superfusion of a cranial window with SnPPIX decreased arteriolar dilation produced by 10 microM acetylcholine by 51%. After partial polymeric hemoglobin exchange transfusion, the acetylcholine response was normal but was reduced 72% by SnPPIX and 95% by TBB. In HO2-/- mice, the acetylcholine response was modestly reduced by 14% compared with control mice and was unaffected by SnPPIX. After hemoglobin transfusion in HO2-/- mice, acetylcholine responses were also unaffected by SnPPIX and TBB. In contrast, nitric oxide synthase inhibition completely blocked the acetylcholine responses in hemoglobin-transfused HO2-/- mice. We conclude 1) that HO2 activity partially contributes to acetylcholine-induced pial arteriolar dilation in mice, 2) that this contribution is augmented in the presence of a plasma-based hemoglobin polymer and appears to depend on a CK2 kinase mechanism, 3) that nitric oxide synthase activity rather than HO1 activity contributes to the acetylcholine reactivity in HO2-/- mice, and 4) that plasma-based polymeric hemoglobin does not scavenge all of the nitric oxide generated by cerebrovascular acetylcholine stimulation.

Protective Role of Heme Oxygenase-1 in Pancreatic Microcirculatory Dysfunction After Ischemia/Reperfusion in Rats

Microcirculatory derangements caused by ischemia and reperfusion (I/R) play a pivotal role in acute and graft pancreatitis. The inducible enzyme heme oxygenase 1 (HO-1) has been shown to decrease I/R injury by modulation of capillary perfusion in other organs. It was the aim of this study to evaluate the effect of HO-1 induction on pancreatic microcirculation after I/R. Rats were randomized into 4 groups: (1) sham controls; (2) 1-hour ischemia and 2-hour reperfusion (I/R); (3) I/R + cobalt protoporphyrin (CoPP), an HO-1 inducer; and (4) I/R + CoPP + tin protoporphyrin, an HO inhibitor. Functional capillary density (FCD) and leukocyte endothelium interaction were analyzed using intravital microscopy during reperfusion. Expression of HO-1 mRNA, HO-1 protein, and HO activity were assessed by Northern blot, Western blot, and an HO activity assay. Functional capillary density decreased significantly in the I/R group as compared with sham controls. Cobalt protoporphyrin treatment increased FCD to control values. In contrast, HO inhibition in CoPP-pretreated animals lowered FCD and increased leukocyte endothelium interaction significantly. Cobalt protoporphyrin administration increased HO-1 mRNA, protein, and HO activity, whereas activity of the enzyme was reduced after injection of tin protoporphyrin. Heme oxygenase 1 plays a beneficial role in pancreatic microcirculatory derangements after I/R. This could be of therapeutic relevance after pancreas transplantation and other forms of postischemic pancreatitis.

Mycobacterium tuberculosis Senses Host-Derived Carbon Monoxide during Macrophage Infection

Mycobacterium tuberculosis (MTB) expresses a set of genes known as the dormancy regulon in vivo. These genes are expressed in vitro in response to nitric oxide (NO) or hypoxia, conditions used to model MTB persistence in latent infection. Although NO, a macrophage product that inhibits respiration, and hypoxia are likely triggers in vivo, additional cues could activate the dormancy regulon during infection. Here, we show that MTB infection stimulates expression of heme oxygenase (HO-1) by macrophages and that the gaseous product of this enzyme, carbon monoxide (CO), activates expression of the dormancy regulon. Deletion of macrophage HO-1 reduced expression of the dormancy regulon. Furthermore, we show that the MTB DosS/DosT/DosR two-component sensory relay system is required for the response to CO. Together, these findings demonstrate that MTB senses CO during macrophage infection. CO may represent a general cue used by pathogens to sense and adapt to the host environment.

Heme oxygenase-1 inhibits breast cancer invasion via suppressing the expression of matrix metalloproteinase-9

In the present study, we investigated the antitumor effects of the invasiveness and migration of heme oxygenase 1 (HO-1) in human breast carcinoma cells. 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase-9 (MMP-9) enzyme activity and gene expression at both protein and mRNA levels were examined in human breast carcinoma cells (MCF-7 and MDA-MB-231), and the addition of the MMP-9 inhibitor, SB3CT, significantly suppressed TPA-induced invasion and migration according to the in vitro Transwell assay. Elevation of HO-1 gene expression by ferric protoporphyrin IX inhibited TPA-induced invasion of MCF-7 cells, which was blocked by adding the heme oxygenase inhibitor, tin protoporphyrin IX, or transfection of cells with HO-1 short hairpin RNA. MCF-7 cells overexpressing HO-1 (MCF-7/HO-1) were established in the present study, and TPA-induced MMP-9 gene expression, tumor invasion, and colony formation were significantly reduced in MCF-7/HO-1 cells, compared with those in Neo-transfected cells. Activation of protein kinase Calpha/extracellular signal-regulated kinases/AP-1 with stimulation of reactive oxygen species production was involved in TPA-induced invasion of MCF-7 cells, which was attenuated by HO-1 protein induced by ferric protoporphyrin IX or transfection of HO-1 expression vectors. Additionally, the addition of carbon monoxide, but not ferric ions, biliverdin, or bilirubin, inhibited TPA-induced invasion through suppressing MMP-9, extracellular signal-regulated kinases, and AP-1 activation stimulated by TPA. The beneficial role of HO-1 in blocking tumor invasion was first identified in this study.

Hemin Induced Heme Oxygenase-1 Over Expression Involves Nuclear Factor-E2 Related Factor-2, Nuclear Factor-κB and Extracellular Regulated Kinase: An Experimental Study in a Testicular Torsion-Detorsion Rodent Model

Nrf2 (nuclear factor-E2 related factor-2), nuclear factor-kappaB and mitogen-activated protein kinase, including extracellular regulated kinase, c-jun N-terminal kinase and p38 mitogen-activated protein kinase, are crucial for signal transduction. We previously reported that heme oxygenase-1 over expression induced by the heme oxygenase-1 inducer hemin protected testes from torsion-detorsion injury. In this study we elucidated which of these enzymes is involved in hemin induced heme oxygenase-1 over expression in testicular tissues after torsion-detorsion injury. Adult male Sprague-Dawley rats (National Science Council, Taipei, Taiwan, Republic of China) were allocated to undergo testicular torsion-detorsion immediately followed by injection of normal saline, hemin or hemin plus tin protoporphyrin, a heme oxygenase-1 inhibitor. Another set of rats that underwent sham operation were used as controls. Testes were harvested 0 and 30 minutes after detorsion and enzyme expression was analyzed. Hemin alone caused no significant effects on the expression of Nrf2, nuclear factor-kappaB, extracellular regulated kinase and c-jun N-terminal kinase. Similarly testicular torsion-detorsion injury caused no significant effects on Nrf2 expression. In the presence of torsion-detorsion injury hemin significantly up-regulated Nrf2 expression. Moreover, this effect was not affected by tin protoporphyrin. Unlike Nrf2, the expression of nuclear factor-kappaB, extracellular regulated kinase and c-jun N-terminal kinase was significantly up-regulated by testicular torsion-detorsion. Hemin significantly attenuated the testicular torsion-detorsion induced up-regulation of nuclear factor-kappaB and extracellular regulated kinase but not c-jun N-terminal kinase. The effects of hemin on nuclear factor-kappaB and extracellular regulated kinase were significantly reversed by tin protoporphyrin. However, the expression of p38 mitogen-activated protein kinase in rodent testes was not affected by these interventions. Hemin induced heme oxygenase-1 over expression in rodent testes after torsion-detorsion injury involves Nrf2, nuclear factor-kappaB and extracellular regulated kinase.

Tranilast, an orally active anti-allergic drug, up-regulates the anti-inflammatory heme oxygenase-1 expression but down-regulates the pro-inflammatory cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW264.7 macrophages

Tranilast ( N-[3′,4′-dimethoxycinnamonyl] anthranilic acid), an orally active anti-allergic drug, is reported to exert the anti-inflammatory effects, but the underlying mechanisms that could explain the anti-inflammatory actions of tranilast remain largely unknown. Here, we found that tranilast induces heme oxygenase-1 (HO-1) expression through the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway in RAW264.7 macrophages. Tranilast suppressed cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS) expression, and thereby reduced COX-2-derived prostaglandin E 2 (PGE 2) and iNOS-derived NO production in lipopolysaccharide (LPS)-stimulated macrophages. Similarly, tranilast diminished tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production. Interestingly, the effects of tranilast on LPS-induced PGE 2, NO, TNF-α, and IL-1β production were partially reversed by the HO-1 inhibitor tin protoporphyrin, suggesting that tranilast-induced HO-1 expression is at least partly responsible for the resulting anti-inflammatory effects of the drug. Thus, HO-1 expression via ERK1/2 activation may be at least one of the possible mechanisms explaining the anti-inflammatory actions of tranilast.

AICAR preconditioning prevents postischemic leukocyte rolling and adhesion:Role of KATP channels and heme oxygenase

We have shown that eNOS activation plays a critical role as a trigger for the anti‐inflammatory state that is induced by activation of 5′‐adenosine monophosphate‐activated protein kinase (AMPK) with AICAR (AICAR‐PC), such that postcapillary venules fail to support increased leukocyte‐endothelial cell adhesive interactions (LEI) on exposure to ischemia/reperfusion (I/R) 24 hrs later. The aim of the present study was to determine if ATP‐sensitive potassium channels (KATP) and heme oxygenase (HO) are effectors of AICAR‐PC. We also examined the role of KATP as triggers of AICAR‐PC. To address these issues, C57BL/6J mice were treated with either 5‐hydroxydecanoate (5‐HD, mitochondrial KATP (mKATP) inhibitor) or tin protoporphyrin (SnPP, HO inhibitor) during I/R 24 hrs after AICAR‐PC. I/R induced a marked increase in LEI relative to sham control mice, responses that were prevented by AICAR‐PC 24 hrs prior to I/R. The anti‐adhesive effects of AICAR‐PC were abolished by 5‐HD or SnPP but not an inactive PP treatment during I/R. The HO inducer hemin mimicked the anti‐inflammatory actions of AICAR‐PC. Interestingly, AICAR‐PC in the presence of KATP inhibition failed to prevent its anti‐adhesive actions. Our results indicate that AMPK agonists produce an anti‐inflammatory phenotype in postcapillary venules that is mediated by mKATP‐ and HO‐dependent mechanisms during I/R. Supported by AA14945, DK 43785, and HL73414.

Protective effect of p53 in vascular smooth muscle cells against nitric oxide-induced apoptosis is mediated by up-regulation of heme oxygenase-2

The tumor suppressor gene p53 regulates apoptotic cell death and the cell cycle. In this study, we investigated the role of p53 in nitric oxide (NO)-induced apoptosis in vascular smooth muscle cells (VSMCs). We found that the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased apoptotic cell death in p53-deficient VSMCs compared with wild-type cells. The heme oxygenase (HO) inhibitor tin protoporphyrin IX reduced the resistance of wild-type VSMCs to SNAP-induced cell death. SNAP promoted HO-1 expression in both cell types. HO-2 protein was increased only in wild-type VSMCs following SNAP treatment; however, similar levels of HO-2 mRNA were detected in both cell types. SNAP significantly increased the levels of non-heme-iron and dinitrosyl iron-sulfur clusters in wild-type VSMCs compared with p53-deficient VSMCs. Moreover, pretreatment with FeSO4 and the carbon monoxide donor CORM-2, but not biliverdin, significantly protected p53-deficient cells from SNAP-induced cell death compared with normal cells. These results suggest that wild-type VSMCs are more resistant to NO-mediated apoptosis than p53-deficient VSMCs through p53-dependent up-regulation of HO-2.