Etomidate Attenuates the Ferroptosis in Myocardial Ischemia/Reperfusion Rat Model via Nrf2/HO-1 Pathway.

BACKGROUND: Ferroptosis has been found to play an important role in myocardial ischemia reperfusion (MIR) injury (MIRI). This study aimed to explore whether the improvement effect of Etomidate (Eto) on MIRI was related to ferroptosis. METHODS: The MIRI rats were constructed using left anterior descending artery occlusion for 30 min followed by reperfusion for 3 h. The Eto post-conditioning was performed by Eto administration at the beginning of the reperfusion. For rescue experiments, MIRI rats were pretreated with ferroptosis inducer erastin or Nrf2 inhibitor ML385 intraperitoneally 1 h prior to MIR surgery. RESULTS: Eto mitigated cardiac dysfunction and myocardium damage, as well as the release of creatine kinase and lactate dehydrogenase caused by ischemia/reperfusion (IR). Additionally, Eto reduced the expression of myocardial fibrosis-related proteins (collagen II and α-smooth muscle actin) and the secretion of inflammatory factors (IL-6, IL-1ß, and TNF-α) in MIRI rats. Also, Eto inhibited IR-induced ferroptosis in myocardium, including reducing superoxide dismutase content, glutathione activity, and glutathione peroxidase 4 expression, while increasing the levels of malondialdehyde and iron and Acyl-CoA synthetase long-chain family member 4. Moreover, the inhibition of Eto on IR-induced myocardial fibrosis and inflammation could be eliminated by erastin. The up-regulation of Nrf2 and HO-1 protein expression, and the nuclear translocation of Nrf2 induced by Eto in the myocardial tissues of MIRI rats, could be prevented by erastin. Besides, ML385 eliminated the inhibition of Eto on ferroptosis induced by MIR. CONCLUSIONS: Eto attenuated the myocardial injury by inhibiting IR-induced ferroptosis via Nrf2 pathway, which may provide a new idea for clinical reperfusion therapy.

Crocin ameliorates arsenic trioxide­induced cardiotoxicity via Keap1-Nrf2/HO-1 pathway: Reducing oxidative stress, inflammation, and apoptosis.

Arsenic trioxide (ATO) is an excellent therapy for acute promyelocytic leukemia; however, its use is limited due to its cardiotoxicity. Crocin (CRO) possesses abundant pharmacological and biological properties, including antioxidant, anti-inflammatory, and anti-apoptotic. This study examined the cardioprotective effects of crocin and explored their mechanistic involvement in ATO-induced cardiotoxicity. Forty-eight male rats were treated with ATO to induce cardiotoxicity. In combination with ATO, CRO were given to evaluate its cardioprotection. The results demonstrated that CRO administration not only diminished QTc prolongation, myocardial enzymes and Troponin T levels but also improved histopathological results. CRO administration reduced reactive oxygen species generation. However, the CRO administration caused an increase in glutathione, superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and total sulphydryl levels and a decrease in malondialdehyde content, gamma glutamyl transferase and lipid hydroperoxides levels and proinflammatory cytokines. Importantly, immunohistochemical analysis, real time PCR and western blotting showed a reduction in Caspase-3 and Bcl-2-associated X protein expressions and enhancement of B cell lymphoma-2 expression. Real time PCR and western blotting showed a reduction in proinflammatory cytokines. Moreover, CRO caused an activation in nuclear factor erythroid-2 related factor 2, leading to enhanced Kelch-like ECH-associated protein 1, heme oxygenase-1 and nicotinamide adenine dinucleotide quinone dehydrogenase 1 expressions involved in Nrf2 signaling during ATO-induced cardiotoxicity. CRO was shown to ameliorate ATO-induced cardiotoxicity. The mechanisms for CRO amelioration of cardiotoxicity due to inflammation, oxidative damage, and apoptosis may occur via an up-regulated Keap1-Nrf2/HO-1 signaling pathway.

The Effects of Exercise Training and High Triglyceride Diet in an Estrogen Depleted Rat Model: The Role of the Heme Oxygenase System and Inflammatory Processes in Cardiovascular Risk.

Cardiovascular morbidity and mortality of premenopausal women are significantly lower compared to men of similar age. However, this protective effect evidently decreases after the onset of menopause. We hypothesized that physical exercise could be a potential therapeutic strategy to improve inflammatory processes and cardiovascular antioxidant homeostasis, which can be affected by the loss of estrogen and the adverse environmental factors, such as overnutrition. Ovariectomized (OVX, n= 40) and sham-operated (SO, n= 40) female Wistar rats were randomized to exercising (R) and non-exercising (NR) groups. Feeding parameters were chosen to make a standard chow (CTRL) or a high triglyceride diet (HT) for 12 weeks. Aortic and cardiac heme oxygenase (HO) activity and HO-1 concentrations significantly decreased in all of the NR OVX and SO HT groups. However, the 12-week physical exercise was found to improve HO-1 values. Plasma IL-6 concentrations were higher in the NR OVX animals and rats fed HT diet compared to SO CTRL rats. TNF-α concentrations were significantly higher in the NR OVX groups. 12 weeks of exercise significantly reduced the concentrations of both TNF-α and IL-6 compared to the NR counterparts. The activity of myeloperoxidase enzyme (MPO) was significantly increased as a result of OVX and HT diet, however voluntary wheel-running exercise restored the elevated values. Our results show that estrogen deficiency and HT diet caused a significant decrease in the activity and concentration of HO enzyme, as well as the concentrations of TNF-α, IL-6, and the activity of MPO. However, 12 weeks of voluntary wheel-running exercise is a potential non-pharmacological therapy to ameliorate these disturbances, which determine the life expectancy of postmenopausal women.

Inhaled Carbon Monoxide: From Toxin to Therapy.

Carbon monoxide (CO) is usually recognized as a toxic gas that can be used to assess lung function in the pulmonary function laboratory. The toxicity of CO relates to its high affinity for hemoglobin and other heme molecules, producing carboxyhemoglobin (HbCO). Despite that blood HbCO levels are commonly measured in patients with CO poisoning, the clinical presentation often does not correlate with the HbCO level, and clinical improvement in the patient's condition does not correlate with HbCO clearance. In patients with CO poisoning, administration of 100% O2 is standard practice. If available, hyperbaric O2 can be used, although this is controversial. Measurement of exhaled CO might be useful to estimate HbCO, such as in smoking cessation programs, but assessment of HbCO using pulse oximetry can be misleading. Endogenous CO is generated as the result of heme oxygenase activity. It is becoming increasingly recognized that the results of heme oxygenase activity, specifically CO production, might have important physiologic functions. These include effects on vascular function, inflammation, apoptosis, cell proliferation, and signaling pathways. Given the abundance of basic science supporting a therapeutic role for CO, clinical trials are exploring this potential.

Heme oxygenase-1 protects spinal cord neurons from hydrogen peroxide-induced apoptosis via suppression of Cdc42/MLK3/MKK7/JNK3 signaling.

The mechanisms by which oxidative stress induces spinal cord neuron death has not been completely understood. Investigation on the molecular signal pathways involved in oxidative stress-mediated neuronal death is important for development of new therapeutics for oxidative stress-associated spinal cord disorders. In current study we examined the role of heme oxygenase-1 (HO-1) in the modulation of MLK3/MKK7/JNK3 signaling, which is a pro-apoptotic pathway, after treating primary spinal cord neurons with H2O2. We found that MLK3/MKK7/JNK3 signaling was substantially activated by H2O2 in a time-dependent manner, demonstrated by increase of activating phosphorylation of MLK3, MKK7 and JNK3. H2O2 also induced expression of HO-1. Transduction of neurons with HO-1-expressing adeno-associated virus before H2O2 treatment introduced expression of exogenous HO-1 in neurons. Exogenous HO-1 reduced phosphorylation of MLK3, MKK7 and JNK3. Consistent with its inhibitory effect on MLK3/MKK7/JNK3 signaling, exogenous HO-1 decreased H2O2-induced neuronal apoptosis and necrosis. Furthermore, we found that exogenous HO-1 inhibited expression of Cdc42, which is crucial for MLK3 activation. In addition, HO-1-induced down-regulation of MLK3/MKK7/JNK3 signaling might be related to up-regulation of microRNA-137 (mir-137). A mir-137 inhibitor alleviated the inhibitory effect of HO-1 on JNK3 activation. This inhibitor also increased neuronal death even when exogenous HO-1 was expressed. Therefore, our study suggests a novel mechanism by which HO-1 exerted its neuroprotective efficacy on oxidative stress.

Reciprocal regulation of carbon monoxide metabolism and the circadian clock.

Circadian clocks are cell-autonomous oscillators regulating daily rhythms in a wide range of physiological, metabolic and behavioral processes. Feedback of metabolic signals, such as redox state, NAD+/NADH and AMP/ADP ratios, or heme, modulate circadian rhythms and thereby optimize energy utilization across the 24-h cycle. We show that rhythmic heme degradation, which generates the signaling molecule carbon monoxide (CO), is required for normal circadian rhythms as well as circadian metabolic outputs. CO suppresses circadian transcription by attenuating CLOCK-BMAL1 binding to target promoters. Pharmacological inhibition or genetic depletion of CO-producing heme oxygenases abrogates normal daily cycles in mammalian cells and Drosophila. In mouse hepatocytes, suppression of CO production leads to a global upregulation of CLOCK-BMAL1-dependent circadian gene expression and dysregulated glucose metabolism. Together, our findings show that CO metabolism is an important link between the basic circadian-clock machinery, metabolism and behavior.

Adipose-Derived Stem Cell Modulation of T-Cell Regulation Correlates with Heme Oxgenase-1 Pathway Changes.

BACKGROUND: The authors' previous proteome study revealed that haptoglobin was involved in adipose-derived stem cell modulation of allotransplant survival and T-cell regulation to induce immune tolerance. This study investigated whether adipose-derived stem cells could modulate T-cell regulation through haptoglobin and the downstream heme oxgenase-1 pathway in vitro. METHODS: Splenocytes were isolated from Lewis rat spleens and then CD3 T cells were purified using anti-CD3 beads. Adipose-derived stem cells were harvested from Lewis rats and co-cultured with the T cells. After Transwell co-culture at different periods, the authors analyzed cell proliferation with a bromodeoxyuridine assay. Cell extractions and culture supernatants were collected for further analysis. Heme oxgenase-1 and related protein expression levels from the adipose-derived stem cells and T cells were detected using Western blotting. The related cytokine expression levels were analyzed with enzyme-linked immunosorbent assay kits. Flow cytometry was used to detect the regulatory T-cell proportion. RESULTS: The adipose-derived stem cells significantly suppressed T-cell proliferation. The regulatory T-cell percentages were significantly increased in the adipose-derived stem cells that were co-cultured with T cells compared with T cells alone without adipose-derived stem cell co-culture. Heme oxgenase-1 expression in concanavalin A-stimulated T cells that were co-cultured with adipose-derived stem cells revealed a significant increase compared with concanavalin A-stimulated T cells alone. Cytokine assays of the culture supernatants revealed that transforming growth factor-ß and interleukin-10 were significantly increased and interferon-γ was statistically decreased in the adipose-derived stem cell-co-cultured T-cell group compared with other groups; however, blockade with a heme oxgenase-1 inhibitor (zinc protoporphyrin IX) protected against these changes. CONCLUSION: Adipose-derived stem cells modulate T-cell proliferation and enhance regulatory T-cell expression, and this correlated with heme oxgenase-1 expression and related cytokine pathway changes.

Resveratrol Protects Hippocampal Astrocytes Against LPS-Induced Neurotoxicity Through HO-1, p38 and ERK Pathways.

Resveratrol, a phytoalexin found in grapes and wine, exhibits antioxidant, anti-inflammatory, anti-aging and antitumor activities. Resveratrol also protects neurons and astrocytes in several neurological disease models. Astrocytes are responsible for modulating neurotransmitter systems, synaptic information, ionic homeostasis, energy metabolism, antioxidant defense and inflammatory response. In previous work, we showed that resveratrol modulates important glial functions, including glutamate uptake, glutamine synthetase activity, glutathione (GSH) levels and inflammatory response. Furthermore, astrocytes express toll-like receptors that specifically recognize lipopolysaccharide (LPS), which has been widely used to study experimentally inflammatory response. In this sense, LPS may stimulate pro-inflammatory cytokines release and oxidative stress. Moreover, there is interplay between these signals through signaling pathways such as NFκB, HO-1 and MAPK. Thus, here, we evaluated the effects of resveratrol on LPS-stimulated inflammatory response in hippocampal primary astrocyte cultures and the putative role of HO-1, p38 and ERK pathways in the protective effect of resveratrol. LPS increased the levels of TNF-α, IL-1ß, IL-6 and IL-18 and resveratrol prevented these effects. Resveratrol also prevented the oxidative and nitrosative stress induced by LPS as well as the decrease in GSH content. Additionally, we demonstrated the involvement of NFκB, HO-1, p38 and ERK signaling pathways in the protective effect of resveratrol, providing the first mechanistic explanation for these effects in hippocampal astrocytes. Our findings reinforce the neuroprotective effects of resveratrol, which are mainly associated with anti-inflammatory and antioxidant activities.

[Roles of heme oxygenase-1 promoting regeneration of peribiliary vascular plexus in bile duct ischemia/reperfusion injury].

OBJECTIVE: To study the effect of heme oxygenase-1 (HO-1) on peribiliary vascular plexus (PVP) in rat bile duct ischemia/reperfusion injury. METHODS: Total 128 male SD rats were randomly divided into saline group (Saline), empty virus group (Adv), induced group (Adv-HO-1) and suppressed group (HO-1 siRNA), and there were 32 rats in each group. Rats were injected using 0.5 ml of saline, empty adenovirus, HO-1 adenovirus and siRNA adenovirus (2×10(9) TU/rat) via the dorsal penile vein 24 hours before surgery. Liver function was analyzed at 1 hour and 1, 7, 14 days after reperfusion. HO-1, hypoxiainducible factor-1α (HIF-1α), stromal cell derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) protein content was analyzed by Western blot. The endothelial progenitor cells (EPCs) ratio in the liver and peripheral blood was detected by flow cytometry. Small vascular around the bile duct was observed by α-smooth muscle actin and von Willebrand factor double immunofluorescence staining. RESULTS: Reduced liver injury and higher expression of HIF-1α, SDF-1α and VEGF in the induced group after surgery (q = 5.68-7.52, P < 0.01). EPCs ratio in the liver and peripheral blood was significantly higher in the induced group than saline group (q = 12.14 and 15.26, P < 0.01), and the suppressed group at 7 days after surgery were less than saline group significantly (q = 4.83 and 5.07, P < 0.01). In comparison to the suppressed group, higher density of small vascular around the bile duct was seen in the liver tissue of induced group. CONCLUSIONS: HO-1 can induce the expression of HIF-1α, SDF-1α and VEGF, and mobilize the release of EPCs to the peripheral from the bone marrow. EPCs migrate to the liver and promote damaged PVP repair and regeneration.

Guanosine protects C6 astroglial cells against azide-induced oxidative damage: a putative role of heme oxygenase 1.

Guanosine, a guanine-based purine, is an extracellular signaling molecule that is released from astrocytes and shows neuroprotective effects in several in vivo and in vitro studies. Our group recently showed that guanosine presents antioxidant properties in C6 astroglial cells. The heme oxygenase 1 signaling pathway is associated with protection against oxidative stress. Azide, an inhibitor of the respiratory chain, is frequently used in experimental models to induce oxidative and nitrosative stress. Thus, the goal of this study was to investigate the effect of guanosine on azide-induced oxidative damage in C6 astroglial cells. Azide treatment of these cells resulted in several detrimental effects, including induction of cytotoxicity and mitochondrial dysfunction, increased levels of reactive oxygen/nitrogen species, inducible nitric oxide synthase expression and NADPH oxidase, decreased glutamate uptake and EAAC1 glutamate transporter expression, decreased glutathione (GSH) levels, and decreased activities of glutamine synthetase (GS), superoxide dismutase and catalase (CAT). The treatment also increased nuclear factor-κB activation and the release of proinflammatory cytokines tumor necrosis factor α and IL-1ß. Guanosine strongly prevented these effects, protecting glial cells against azide-induced cytotoxicity and modulating glial, oxidative and inflammatory responses through the activation of the heme oxygenase 1 pathway. These observations reinforce and support the role of guanosine as an antioxidant molecule against oxidative damage. Guanosine protects against azide-induced oxidative damage in C6 astroglial cells. Azide-induced mitochondrial dysfunction (1); increased reactive oxygen species/reactive nitrogen species levels (2); decreased glutamate uptake (3), GS activity (4), GSH levels (5), and SOD (6) and CAT (7) activities; increased glutathione peroxidase (GPx) (8) and NADPH oxidase (9) activities and cellular superoxide levels (10); increased NF-κB activation (11), TNF-α and IL-1ß levels (12); and induced iNOS expression (13). Guanosine prevented these effects through the HO1 signaling pathway, thus our findings support the antioxidant effects of guanosine.

Signaling function of heme oxygenase proteins.

SIGNIFICANCE: Many reports have underscored the importance of the heme degradation pathway that is regulated by heme oxygenase (HO). This reaction releases bile pigments and carbon monoxide (CO), which are important antioxidant and signaling molecules. Thus, the reaction of HO-1 would have significant cytoprotective effects. Nevertheless, the importance of this protein goes beyond its enzymatic action. New evidence outlines significant effects of inactive forms of the HO-1 protein. RECENT ADVANCES: In fact, the role of the HO protein in cellular signaling, including transcription factor activation, binding to proteins, phosphorylation, and modulation of protein function, among others, has started being elucidated. The mechanism by which the inducible form of HO-1, in particular, can migrate to various cellular compartments to mediate important signaling or how and why it binds to key transcription factors and other proteins that are important in DNA repair is also described in several physiologic systems. CRITICAL ISSUES: The signaling functions of HO-1 may have particular relevance in clinical circumstances, including cancer, as redistribution of HO-1 into the nuclear compartment is observed with cancer progression and metastasis. In addition, along with oxidative stress, the pleiotropic functions of HO-1 modulate antioxidant defense. In organ transplantation, HO and its byproducts suppress rejection at multiple levels and in sepsis-induced pulmonary dysfunction, inhaled CO or modulation of HO activity can change the course of the disease in animals. FUTURE DIRECTIONS: It is hoped that a more detailed understanding of the various signaling functions of HO will guide therapeutic approaches for complex diseases.

Anti-inflammatory potential of curcumin and quercetin in rats: role of oxidative stress, heme oxygenase-1 and TNF-α.

Flavonoids are group of compounds that have been shown to possess potent anti-inflammatory effects in both cellular and animal models of inflammation. In the current study, the single and combined effects of the two flavonoids, curcumin and quercetin, against carrageenan-induced acute inflammation in rats were evaluated with emphasis on the role of oxidative stress, anti-inflammatory enzyme, heme oxygenase-1 (HO-1) and proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α). Curcumin (50 mg/kg), quercetin (50 mg/kg) and a combination of both were orally administered for 14 days before carrageenan injection in rats and compared with the reference nonsteroidal anti-inflammatory drug, indomethacin (10 mg/kg). The percentage increase in paw thickness was calculated. Frozen hind paws were used for the estimation of lipid peroxides (malondialdehyde, MDA), nitric oxide (NO), reduced glutathione (GSH), TNF-α level and HO-1 messenger RNA (mRNA) expression. Formalin-fixed hind paws were used for histopathological examination. Results showed that both curcumin and quercetin caused reduction in carrageenin-induced edema and lymphocytes infiltration along with the decrease is being even higher in case of their combination. Additionally, both flavonoids reduced MDA and NO formation, and restored GSH contents in the paw. Furthermore, both flavonoids increased HO-1 mRNA expression and decreased the elevated TNF-α level. Results showed that both flavonoids moderately lowered inflammation, while their combination was more effective. Accordingly, this study suggests that the reduction in oxidative stress and modulation of HO-1 mRNA expression and TNF-α release by curcumin and quercetin may contribute to the synergistic anti-inflammatory effects of these two flavonoids upon combination.

Nicotine paradoxically affects the facilitatory effect of ovarian hormones on the adenosine receptor-mediated renal vasodilation.

We tested the hypothesis that nitric oxide synthase (NOS) and/or heme oxygenase (HO) modulate the hormonally-dependent nicotine interaction with adenosine receptor-mediated renal vasodilations. Vasodilations caused by 5'-N-ethylcarboxamidoadenosine (NECA) in phenylephrine-preconstricted perfused kidneys were reduced in ovariectomized (OVX) rats and restored to sham levels after treatment with estrogen (E2), medroxyprogesterone acetate (MPA) or their combination. The facilitatory action of E2 or MPA was abolished after blockade of their respective receptors by ICI-182780 and mifepristone, or inhibition of NOS (N(ω)-nitro-L-arginine-methyl ester, L-NAME) but not HO (zinc protoporphyrin, ZnPP). NECA vasodilations were (i) decreased and increased by nicotine (1 mg/kg/day, 2 weeks) in OVX/MPA and OVX/E2 kidneys, respectively, and (ii) resistant to nicotine in females with deficient (OVX) or balanced (sham or E2/MPA-replaced OVX) hormonal milieu. The attenuation of NECA responses by nicotine in OVX/MPA kidneys disappeared after treatment with hemin (HO inducer) but not L-arginine (NOS substrate). Alternatively, nicotine enhancement of NECA responses in OVX/E2 kidneys was abolished following treatment with ICI 182780 or L-NAME. Overall, the NOS-coupled E2/progesterone receptor contributes to the enhancement of NECA vasodilations by ovarian hormones. Further, HO inhibition and NOS facilitation mediate the directionally opposite effects of nicotine on NECA responses in MPA- or E2-replaced OVX rats, respectively.

The heme oxygenase system selectively enhances the anti-inflammatory macrophage-M2 phenotype, reduces pericardial adiposity, and ameliorated cardiac injury in diabetic cardiomyopathy in Zucker diabetic fatty rats.

Cardiac function is adversely affected by pericardial adiposity. We investigated the effects of the heme oxygenase (HO) inducer, hemin on pericardial adiposity, macrophage polarization, and diabetic cardiopathy in Zucker diabetic fatty rats (ZDFs) with use of echocardiographic, quantitative real-time polymerase chain reaction, Western immunoblotting, enzyme immunoassay, and spectrophotometric analysis. In ZDFs, hemin administration increased HO activity; normalized glycemia; potentiated insulin signaling by enhancing insulin receptor substrate 1(IRS-1), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (PKB)/Akt; suppressed pericardial adiposity, cardiac hypertrophy, and left ventricular longitudinal muscle fiber thickness, a pathophysiological feature of cardiomyocyte hypertrophy; and correspondingly reduced systolic blood pressure, total peripheral resistance, and pro-inflammatory/oxidative mediators, including nuclear factor κB (NF-κB), cJNK, c-Jun-N-terminal kinase (cJNK), endothelin (ET-1), tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1ß, activating protein 1 (AP-1), and 8-isoprostane, whereas the HO inhibitor, stannous mesoporphyrin, nullified the effects. Furthermore, hemin reduced the pro-inflammatory macrophage M1 phenotype, but enhanced the M2 phenotype that dampens inflammation. Because NF-κB activates TNFα, IL-6, and IL-1ß and TNF-α, cJNK, and AP-1 impair insulin signaling, the high levels of these cytokines in obesity/diabetes would create a vicious cycle that, together with 8-isoprostane and ET-1, exacerbates cardiac injury, compromising cardiac function. Therefore, the concomitant reduction of pro-inflammatory cytokines and macrophage infiltration coupled to increased expressions of IRS-1, PI3K, and PKB may account for enhanced glucose metabolism and amelioration of cardiac injury and function in diabetic cardiomyopathy. The hemin-induced preferential polarization of macrophages toward anti-inflammatory macrophage M2 phenotype in cardiac tissue with concomitant suppression of pericardial adiposity in ZDFs are novel findings. These data unveil the benefits of hemin against pericardial adiposity, impaired insulin signaling, and diabetic cardiomyopathy and suggest that its multifaceted protective mechanisms include the suppression of inflammatory/oxidative mediators.

Diazoxide attenuates ischemia/reperfusion injury via upregulation of heme oxygenase-1 after liver transplantation in rats.

AIM: To evaluate the effects of diazoxide on ischemia/reperfusion (I/R)-injured hepatocytes and further elucidate its underlying mechanisms. METHODS: Male Sprague-Dawley rats were randomized (8 for donor and recipient per group) into five groups: I/R group (4 h of liver cold ischemia followed by 6 h of reperfusion); heme oxygenase-1 (HO-1) small interfering RNA (siRNA) group (injection of siRNA via donor portal vein 48 h prior to harvest); diazoxide (DZ) group (injection of DZ via donor portal vein 10 min prior to harvest); HO-1 siRNA + DZ group; and siRNA control group. Blood and liver samples were collected at 6 h after reperfusion. The mRNA expressions and protein levels of HO-1 were determined by reverse transcription polymerase chain reaction and Western blotting, and tissue morphology was examined by light and transmission electron microscopy. Serum transaminases level and cytokines concentration were also measured. RESULTS: We observed that a significant reduction of HO-1 mRNA and protein levels in HO-1 siRNA and HO-1 siRNA + DZ group when compared with I/R group, while the increases were prominent in the DZ group. Light and transmission electron microscopy indicated severe disruption of tissue with lobular distortion and mitochondrial cristae damage in the HO-1 siRNA and HO-1 siRNA + DZ groups compared with DZ group. Serum alanine aminotransferase, aspartate transaminase, tumor necrosis factor-α and interleukin-6 levels increased in the HO-1 siRNA and HO-1 siRNA + DZ groups, and decreased in the DZ group. CONCLUSION: The protective effect of DZ may be induced by upregulation of HO-1. By inhibiting expression of HO-1, this protection pretreated with DZ was abolished.

Kidney injury accelerates cystogenesis via pathways modulated by heme oxygenase and complement.

AKI accelerates cystogenesis. Because cystogenic mutations induce strong transcriptional responses similar to those seen after AKI, these responses may accelerate the progression of cystic renal disease. Here, we modulated the severity of the AKI-like response in Cys1(cpk/cpk) mice, a model that mimics autosomal recessive polycystic kidney disease. Specifically, we induced or inhibited activity of the renoprotective enzyme heme oxygenase (HO) and determined the effects on renal cystogenesis. We found that induction of HO attenuated both renal injury and the rate of cystogenesis, whereas inhibition of HO promoted cystogenesis. HO activity mediated the response of NFκB, which is a hallmark transcriptional feature common to both cystogenesis and AKI. Among the HO-modulated effects we measured, expression of complement component 3 (C3) strongly correlated with cystogenesis, a functionally relevant association as suggested by Cys1(cpk/cpk) mice with genetically induced C3 deficiency. Because both C3 deficiency and HO induction reduce cyst number and cyst areas, these two factors define an injury-stimulated cystogenic pathway that may provide therapeutic targets to slow the formation of new renal cysts and the growth of existing cysts.

Haem oxygenase-1 counteracts the effects of interleukin-1ß on inflammatory and senescence markers in cartilage-subchondral bone explants from osteoarthritic patients.

IL (interleukin)-1ß plays an important role in cartilage extracellular matrix degradation and bone resorption in OA (osteoarthritis) through the induction of degradative enzymes and pro-inflammatory mediators. In the present study, we have determined the consequences of HO-1 (haem oxygenase-1) induction on markers of inflammation and senescence in the functional unit cartilage-subchondral bone stimulated with IL-1ß. Cartilage-subchondral bone specimens were obtained from the knees of osteoarthritic patients. Treatment with the HO-1 inducer CoPP (cobalt protoporphyrin IX) counteracted the stimulatory effects of IL-1ß on IL-6, nitrite, PGE2 (prostaglandin E2), TGF (transforming growth factor) ß2, TGFß3 and osteocalcin. Immunohistochemical analyses indicated that CoPP treatment of explants down-regulated iNOS (inducible nitric oxide synthase), COX-2 (cyclooxygenase-2) and mPGES-1 (microsomal prostaglandin E synthase-1) induced by IL-1ß. In contrast, the expression of HMGB1 (high-mobility group box 1) was not significantly modified. In addition, CoPP decreased the expression of iNOS and mPGES-1 in cells isolated from the explants and stimulated with IL-1ß, which was counteracted by an siRNA (small interfering RNA) specific for human HO-1. In isolated primary chondrocytes, we determined senescence-associated ß-galactosidase activity and the expression of senescence markers by real-time PCR. We have found that HO-1 induction could regulate senescence markers in the presence of IL-1ß and significantly affected telomerase expression, as well as ß-galactosidase activity and hTERT (human telomerase reverse transcriptase) and p21 expression in chondrocytes. The findings of the present study support the view that HO-1 induction results in the down-regulation of inflammatory and senescence responses in OA articular tissues.

[Expression of heme oxygenase-1 and inducible nitric oxide synthase in the lungs of hyperoxia-exposed preterm rats].

OBJECTIVE: To study the expression and the role of heme oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) in preterm rats with hyperoxia-induced lung injuries. METHODS: Sixty-four three-day-old preterm Sprague-Dawley rats were randomly assigned to a hyperoxia group (90% oxygen exposure) and a control group (room air exposure), with 32 rats in each group. After 3 days or 7 days of exposure, the lung activity of HO-1 and nitric oxide (NO) contents in bronchoalveolar lavage fluid (BALF), pulmonary histopathologic changes, and the cellular distribution and expression of HO-1 and iNOS in the lungs were measured. RESULTS: After 3 days and 7 days of exposure, the hyperoxia group showed acute lung injuries characterized by the presence of hyperaemia, red cell extravasation and inflammatory infiltration. The NO contents in BALF and the iNOS expression in the lungs increased significantly in the hyperoxia group compared with those in the control group 3 and 7 days after exposure. The expression of HO-1 in macrophages in the lungs increased significantly in the hyperoxia group compared with that in the control group 3 and 7 days after exposure. The NO contents in BALF and the iNOS and HO-1 expression in the lungs increased significantly 7 days after hyperoxia exposure compared with 3 days after hyperoxia exposure. CONCLUSIONS: iNOS and HO-1 levels in the lungs increase in preterm rats with hyperoxia-induced lung injuries, suggesting that iNOS and HO-1 may play roles in hyperoxia-induced pulmonary injuries.

Hypoxic pulmonary vasoconstriction: mechanisms of oxygen-sensing.

PURPOSE OF REVIEW: Hypoxic pulmonary vasoconstriction (HPV) is driven by the intrinsic response to hypoxia of pulmonary arterial smooth muscle and endothelial cells. These are representatives of a group of specialized O2-sensing cells, defined by their acute sensitivity to relatively small changes in pO2, which have evolved to modulate respiratory and circulatory function in order to maintain O2 supply within physiological limits. The aim of this article is to discuss recent investigations into the mechanism(s) of hypoxia-response coupling and, in light of these, provide a critical assessment of current working hypotheses. RECENT FINDINGS: Upon exposure to hypoxia state-of-the-art technologies have now confirmed that mitochondrial oxidative phosphorylation is inhibited in all O2-sensing cells, including pulmonary arterial smooth muscle cells. Thereafter, evidence has been presented to indicate a role as principal effector for the 'gasotransmitters' carbon monoxide and hydrogen sulphide, reactive oxygen species or, in marked contrast, reduced cellular redox couples. Considering recent evidence in favour and against these proposals we suggest that an alternative mechanism may be key, namely the activation of adenosine monophosphate-activated protein kinase consequent to inhibition of mitochondrial oxidative phosphorylation. SUMMARY: HPV supports ventilation-perfusion matching in the lung by diverting blood flow away from oxygen-deprived areas towards regions rich in O2. However, in diseases such as emphysema and cystic fibrosis, widespread HPV leads to hypoxic pulmonary hypertension and ultimately right heart failure. Determining the precise mechanism(s) that underpins hypoxia-response coupling will therefore advance understanding of the fundamental processes contributing to related pathophysiology and provide for improved therapeutics.

Hypoxia induces late preconditioning in the rat heart in vivo.

BACKGROUND: Although hypoxic late preconditioning (LPC) limits ischemia-reperfusion injury in vitro, its cardioprotective effect is not established in vivo. METHODS: In part 1, rats were exposed to 4 h of hypoxia (16%, 12%, 8% oxygen) before 24 h of reoxygenation. In part 2, normoxic rats received early preconditioning with sevoflurane (1 minimum alveolar concentration [MAC] for 3 × 5 min), continuous administration of 1 MAC sevoflurane, or 11 mg · kg · h propofol. Thereafter, all rats underwent 25 min of regional myocardial ischemia and 120 min of reperfusion. After reperfusion, hearts were excised for infarct staining. The expression of protein kinase C (PKC)α and PKCε was assessed by Western blot analysis and the expression of heme oxygenase-1 and vascular endothelial growth factor by reverse transcriptase polymerase chain reaction. RESULTS: In normoxic control rats, infarct size was 62 ± 6% of the area at risk. Hypoxic LPC reduced infarct size (LPC16: 36 ± 11%, LPC12: 38 ± 10%, LPC8: 39 ± 11%; each P < 0.001) to approximately the same magnitude as sevoflurane-preconditioning (40 ± 8%; P < 0.001). Combined LPC16 and sevoflurane preconditioning was not superior to either substance alone. Continuous sevoflurane or propofol was not protective. The PKC inhibitor calphostin C abolished the cardioprotective effects of LPC16. PKCε, but not PKCα, expression was increased 6 and 28 h after hypoxic LPC. Heme oxygenase-1 and vascular endothelial growth factor were transiently up-regulated after 6 h. CONCLUSION: Hypoxic LPC at 8%, 12%, and 16% oxygen reduces infarct size in the rat heart in vivo. This effect is as powerful as sevoflurane-preconditioning. PKCε is a key player in mediating hypoxic LPC.