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Background/aims Heme oxygenase-1 (HO-1) plays a central role in cellular defense against inflammatory insults, and its induction in macrophages potentiates their efferocytic activity. In this study, we explored the potential role of macrophage HO-1 in the resolution of experimentally induced colitis. Methods To induce colitis, male C57BL/6 mice were treated with 2% dextran sulfate sodium (DSS) in the drinking water for 7 days. To investigate efferocytosis, apoptotic colon epithelial CCD 841 CoN cells were coincubated with bone marrow-derived macrophages (BMDMs). Results Administration of the HO-1 inhibitor zinc protoporphyrin IX (ZnPP) blunted the resolution of DSS-induced intestinal inflammation and expression of the proresolving M2 macrophage marker CD206. BMDMs treated with apoptotic colonic epithelial cells showed significantly elevated expression of HO-1 and its regulator Nrf2. Under the same experimental conditions, the proportion of CD206-expressing macrophages was also enhanced. ZnPP treatment abrogated the upregulation of CD206 expression in BMDMs engulfing apoptotic colonic epithelial cells. This result was verified with BMDMs isolated from HO-1-knockout mice. BMDMs, when stimulated with lipopolysaccharide, exhibited increased expression of CD86, a marker of M1 macrophages. Coculture of lipopolysaccharide-stimulated BMDMs with apoptotic colonic epithelial cell debris dampened the expression of CD86 as well as the pro-inflammatory cytokines in an HO-1-dependent manner. Genetic ablation as well as pharmacologic inhibition of HO-1 significantly reduced the proportion of efferocytic BMDMs expressing the scavenger receptor CD36. Conclusions HO-1 plays a key role in the resolution of experimentally induced colitis by modulating the polarization of macrophages.

BACKGROUND: Many women enter pregnancy with iron stores that are insufficient to maintain maternal iron balance and support fetal development and, consequently, often require iron supplements. However, the side effects associated with many currently available iron supplements can limit compliance. OBJECTIVE: This study aimed to test the safety and efficacy of a novel nanoparticulate iron supplement, a dietary ferritin analogue termed iron hydroxide adipate tartrate (IHAT), in pregnant mice. METHODS: Female C57BL/6 mice were maintained on either an iron deficient or a control diet for two weeks prior to timed mating to develop iron deficient or iron sufficient pregnancy models respectively. Mice from each model were then gavaged daily with 10 mg iron/kg body weight as either IHAT or ferrous sulfate, or with water only, beginning on embryonic day (E) 4.5. Mice were euthanized on E18.5 and maternal iron and hematological parameters were measured. The expression of genes encoding iron transporters and oxidative stress markers in the duodenum and placenta were determined, along with hepatic expression of the gene encoding the iron regulatory hormone hepcidin and fetal iron. RESULTS: Oral IHAT and ferrous sulfate were equally effective at increasing maternal hemoglobin (20.2% and 16.9% respectively) and hepatic iron (30.2% and 29.3% respectively), as well as total fetal iron (99.7% and 83.8% respectively), in iron deficient pregnant mice compared to those gavaged with water only, with no change in oxidative stress markers seen with either treatment. However, there was a significant increase in the placental expression of the oxidative stress marker heme oxygenase 1 in iron replete pregnant mice treated with ferrous sulfate when compared to iron replete pregnant mice gavaged with IHAT (96.9%, P

Objectives Early acute kidney injury (AKI) after burn contributes to disastrous prognoses for severely burned patients. Burn-induced renal oxidative stress and secondary proinflammatory mediator release contribute to early AKI development, and Toll-like receptor (TLR) 4 regulates inflammation. Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that plays a vital role in protecting against ischemia-induced organ injury via its antioxidant properties and regulation of inflammation. We investigated the potential effect of HO-1 induction in preventing burn-induced early AKI and its related mechanism. Methods A classic major-burn rat model was established using a 100 °C water bath, and hemin was injected intraperitoneally immediately after the injury to induce HO-1. Histological staining and blood tests were used to assess AKI progression based on structural changes and function. Renal levels of HO-1, oxidative stress, proinflammatory mediators and TLR4-related signals were detected using ELISA, immunostaining, qRT-PCR, and western blotting. The selective TLR4 inhibitor TAK242 and TLR4 inducer LPS were introduced to determine the roles of HO-1 in burn-related renal inflammation and the TLR4 pathway. Results Hemin improved burn-induced renal histological damage and dysfunction, and this beneficial effect was related to reduced renal oxidative stress and the release of proinflammatory mediators, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6 and intracellular adhesion molecule-1 (ICAM-1). Hemin downregulated the expression of TLR4 and the subsequent phosphorylation of IKKα/β, IκBα, and NF-κB p65;. TAK242 exerted an effect similar to but weaker than hemin; and LPS reversed the antiinflammatory effect of hemin and the regulation of TLR4 signals. These results suggested that the TLR4 signaling pathway mediated the HO-1-facilitated regulation of renal inflammation after burn. Conclusion The present study demonstrated that HO-1 induction prevented burn-induced early AKI by targeting renal inflammation, which was mediated via regulation of the TLR4/NF-κB signaling pathway.

Heme is an essential biomolecule and cofactor involved in a myriad of biological processes. In this review, we focus on how heme binding to heme regulatory motifs (HRMs), catalytic sites, and gas signaling molecules as well as how changes in the heme redox state regulate protein structure, function, and degradation. We also relate these heme-dependent changes to the affected metabolic processes. We center our discussion on two HRM-containing proteins: human heme oxygenase-2, a protein that binds and degrades heme (releasing Fe(2+) and CO) in its catalytic core and binds Fe(3+)-heme at HRMs located within an unstructured region of the enzyme, and the transcriptional regulator Rev-erbβ, a protein that binds Fe(3+)-heme at an HRM and is involved in CO sensing. We will discuss these and other proteins as they relate to cellular heme composition, homeostasis, and trafficking. In addition, we will discuss the HRM-containing family of proteins and how the stability and activity of these proteins are regulated in a dependent manner through the HRMs. Then, after reviewing CO-mediated protein regulation of heme proteins, we turn our attention to the involvement of heme, HRMs, and CO in circadian rhythms. In sum, we stress the importance of understanding the various roles of heme and the distribution of the different heme pools as they relate to the heme redox state, CO, and heme binding affinities.

The cytoplasmic heme binding protein from Pseudomonas aeruginosa, PhuS, plays two essential roles in regulating heme uptake and iron homeostasis. First, PhuS shuttles exogenous heme to heme oxygenase (HemO) for degradation and iron release. Second, PhuS binds DNA and modulates the transcription of the prrF/H small RNAs (sRNAs) involved in the iron-sparing response. Heme binding to PhuS regulates this dual function, as the unliganded form binds DNA, whereas the heme-bound form binds HemO. Crystallographic studies revealed nearly identical structures for apo- and holo-PhuS, and yet numerous solution-based measurements indicate that heme binding is accompanied by large conformational rearrangements. In particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of apo- versus holo-PhuS revealed large differences in deuterium uptake, notably in α-helices 6, 7, and 8 (α6,7,8), which contribute to the heme binding pocket. These helices were mostly labile in apo-PhuS but largely protected in holo-PhuS. In contrast, in silico-predicted deuterium uptake levels of α6,7,8 from molecular dynamics (MD) simulations of the apo- and holo-PhuS structures are highly similar, consistent only with the holo-PhuS HDX-MS data. To rationalize this discrepancy between crystal structures, simulations, and observed HDX-MS, we exploit a recently developed computational approach (HDXer) that fits the relative weights of conformational populations within an ensemble of structures to conform to a target set of HDX-MS data. Here, a combination of enhanced sampling MD, HDXer, and dimensionality reduction analysis reveals an apo-PhuS conformational landscape in which α6, 7, and 8 are significantly rearranged compared to the crystal structure, including a loss of secondary structure in α6 and the displacement of α7 toward the HemO binding interface. Circular dichroism analysis confirms the loss of secondary structure, and the extracted ensembles of apo-PhuS and of heme-transfer-impaired H212R mutant, are consistent with known heme binding and transfer properties. The proposed conformational landscape provides structural insights into the modulation by heme of the dual function of PhuS.

Purpose: To investigate the effect of forsythiaside A on heart failure.Methods: An in vitro cell model of myocardial injury was established by incubating H9c2 primary cardiomyocytes with hydrogen peroxide (H2O2). Apoptosis was measured by flow cytometry. Expression of inflammatory factors, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzymelinkedimmunosorbent assay (ELISA). Oxidative stress was evaluated by measuring malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels by ELISA.Results: Incubation with H2O2 increased H9c2 cell apoptosis (p < 0.001). Treatment with forsythiaside A reduced Bax expression and enhanced Bcl-2 expression which suppressed apoptosis of H2O2- induced H9c2 cells. Forsythiaside A also attenuated the H2O2-induced increase in TNF-α and IL-6expressions in H9c2 cells (p < 0.001). The H2O2-induced increase in MDA and decrease in SOD and GSH-Px in H9c2 cells were reversed by treatment with forsythiaside A. IκBα protein expression was downregulated, whereas p65 phosphorylation (p-p65), p-IκBα, nuclear factor erythropoietin-2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) were upregulated in H2O2-induced H9c2 cells. Forsythiaside A increased IκBα, Nrf2, and HO-1 expression and decreased p-p65 and p-IκBα expression in H2O2-induced H9c2 cells.Conclusion: Forsythiaside A exerts anti-inflammatory, anti-oxidant, and anti-apoptotic effects against H2O2-induced H9c2 cells through inactivation of NF-κB pathway and activation of Nrf2/HO-1 pathway. These results support the potential clinical application of forsythiaside A for the treatment of heart failure.

HO-1 is the inducible form of the enzyme heme-oxygenase. HO-1 catalyzes heme breakdown, reducing the levels of this important oxidant molecule and generating antioxidant, anti-inflammatory, and anti-apoptotic byproducts. Thus, HO-1 has been described as an important stress response mechanism during both physiologic and pathological processes. Interestingly, some findings are demonstrating that uncontrolled levels of HO-1 byproducts can be associated with cell death and tissue destruction as well. Furthermore, HO-1 can be located in the nucleus, influencing gene transcription, cellular proliferation, and DNA repair. Here, we will discuss several studies that approach HO-1 effects as a protective or detrimental mechanism in different pathological conditions. In this sense, as the major organs of vertebrates will deal specifically with distinct types of stresses, we discuss the HO-1 role in each of them, exposing the contradictions associated with HO-1 expression after different insults and circumstances.

Objectives This study investigated the involvement of heme oxygenase-1 (HO-1) in the antidepressant-like effects of ursolic acid (UA), a plant-derived compound with neuroprotective and antidepressant-like properties. Methods Mice received intracerebroventricular injections of zinc protoporphyrin IX (ZnPP) or cobalt protoporphyrin IX (CoPP) to inhibit or induce HO-1, respectively, together with effective (0.1 mg/kg, p.o.) or sub-effective (0.01 mg/kg, p.o.) doses of UA or fluoxetine (10 mg/kg, p.o.). Immobility time was assessed using the tail suspension test (TST) and the ambulatory behaviour with the open field test. HO-1 immunocontent was evaluated in mice hippocampus and prefrontal cortex. Key findings ZnPP prevented the anti-immobility effects of UA and fluoxetine. Combined treatment with a sub-effective dose of CoPP and UA synergistically exerted antidepressant-like effects in the TST. Acute administration of UA or CoPP, but not fluoxetine, increased the HO-1 immunocontent in the hippocampus. None of the treatments altered the HO-1 immunocontent in the prefrontal cortex. Conclusions In conclusion, this work shows that increased hippocampal HO-1 content and activity mediate the antidepressant-like effect of UA in the TST.

Ganoderic triterpenoids (GTs) are the primary bioactive constituents of the Basidiomycotina fungus, Ganoderma lucidum. These compounds exhibit antitumor, anti-hyperlipidemic, and immune-modulatory pharmacological activities. This study focused on GT accumulation in mycelia of G. lucidum mediated by the heme oxygenase-1 (HO-1)/carbon monoxide (CO) signaling. Compared with the control, hemin (10 μmol/L) induced an increase of 60.1% in GT content and 57.1% in HO-1 activity. Moreover, carbon monoxide-releasing molecule-2 (CORM-2), CO donor, increased GT content by 56.0% and HO-1 activity by 18.1%. Zn protoporphyrin IX (ZnPPIX), a specific HO-1 inhibitor, significantly reduced GT content by 26.0% and HO-1 activity by 15.8%, while hemin supplementation reversed these effects. Transcriptome sequencing showed that HO-1/CO could function directly as a regulator involved in promoting GT accumulation by regulating gene expression in the mevalonate pathway, and modulating the reactive oxygen species (ROS) and Ca2+ pathways. The results of this study may help enhance large-scale GT production and support further exploration of GT metabolic networks and relevant signaling cross-talk.

Doxorubicin (DOX) is an effective anthracycline chemotherapeutic drug. Nevertheless, the cardiotoxicity adverse effect restricts its clinical benefit. Allyl isothiocyanate (AITC) is a natural antioxidant and anti-inflammatory agent. In the present study, we investigated the effect of AITC on cardiotoxicity of DOX. Thirty-two adult male albino rats were divided into four groups; control, AITC, DOX, and AITC + DOX. AITC was administrated orally (25 mg/kg/day) for 7 days, and DOX was given as a single i.p. injection (15 mg/kg) on the third day. Mortality rate was observed during the experiment. Cardiac toxicity markers (lactate dehydrogenase (LDH), creatine kinase (CK-MB), and cardiac Troponin I (cTn-I)) were evaluated in serum samples obtained from all groups after 48 hours of DOX injection. DOX-treated group showed 40% mortality and a significant increase in cardiac enzymes. This increase was accompanied by degenerated cardiomyocytes, and inflammatory cells infiltrates. Interestingly, AITC administration alleviated myocardial oxidative stress induced by DOX as attenuated the increase in malondialdehyde (MDA), and nitric oxide (NO) while resulted in elevations of the antioxidant reduced glutathione (GSH) level as well as superoxide dismutase (SOD) activity. Furthermore, the inflammatory cytokine, TNF-α, was reduced upon administration of AITC with DOX. The cardio-protection of AITC is attributed to increase the expression of cytoprotective nuclear factor erythroid 2-related factor 2 (Nrf2). Subsequently, heme oxygenase 1 (HO-1) level was elevated by AITC to correct the oxidative stress induced by DOX in the heart. Accordingly, AITC ameliorated acute cardiotoxicity associated with DOX treatment via attenuation of oxidative stress and the induced-tissue inflammatory injury. Abbreviations: DOX: doxrubicin; Nrf2: nuclear factor erythroid 2-related factor 2; HO-1: heme oxygenase 1; AITC: ally isothiocyanate; MDA: malondialdehyde; SOD: superoxide dismutase; GSH: reduced glutathione; TNF-α: tumor necrosis factor alpha.

Bisphenol A (BPA) is an estrogen-like compound, and an environmental hormone, that is commonly used in daily life. Therefore, it may enter the human body through food or direct contact, causing BPA residues in blood and urine. Because most studies focused on the analysis of BPA in reproductive cells or tissues, regarding evidence the effect of BPA on human retinal pigment epithelium (ARPE-19) cells unavailable. Accordingly, the present study explored the cytotoxicity of BPA on ARPE-19 cells. After BPA treatment, the expression of Bcl-XL an antiapoptotic protein, in the mitochondria decreased, and the expression of Bax, a proapoptotic protein increased. Then the mitochondrial membrane potential was affected. BPA changed in mitochondrial membrane potential led to the release of cytochrome C, which activated caspase-9 to promote downstream caspase-3 leading to cytotoxicity. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase 1 (HO-1) pathway play a major role in age-related macular degeneration. Our results showed that expression of HO-1 and Nrf2 suppressed by BPA. Superoxide dismutase and catalase, which Nrf2 downstream antioxidants, were degraded by BPA. AMP-activated kinase (AMPK), which can regulate the phosphorylation of Nrf2, and the phosphorylation of AMPK expression was reduced by BPA. Finally, BPA-induced ROS generation and cytotoxicity were reduced by N-acetyl-l-cysteine. Taken together, these results suggest that BPA induced ARPE-19 cells via oxidative stress, which was associated with down regulated Nrf2/HO-1 pathway, and the mitochondria dependent apoptotic signaling pathway.

Ketamine is clinically used as a narcotic. However, ketamine has certain deficits and produces toxicity to neurons. As a member of the NR4A receptor subfamily, Nur77 decreases neurodegenerative disorders. The study aims to investigate the effects of upregulated Nur77 on ketamine-induced rat hippocampal neurons damage and the active mechanism. Neurons were obtained from rat hippocampal and identified by immunofluorescence assays. The treatment groups contained ketamine group, Nur77 group, ketamine + Nur77 group and ketamine + L-cam group. Neurons apoptosis and reactive oxygen species (ROS) were determined by a related kit using flow cytometry. Enzyme NAD(P)H quinone oxidoreductase 1 (NQO1), enzyme heme oxygenase 1 (HO1), Nur77, the expression of Bax, Bcl-2 and cleaved-caspase-3 and inflammatory cytokines were measured using western blot assays and reverse transcription-quantitative PCR (RT-qPCR) assays. Ketamine-induced neurons apoptosis; however, Nur77 decreased ketamine-induced neurons apoptosis. A low level of ROS was observed in two combination groups. Neurons treated by ketamine only had the lowest levels of Nur77, NQO1 and HO1, compared with other treatment groups. The levels of Bax and cleaved-caspase-3 in two combination groups were lower than those in the ketamine group. Furthermore, the ketamine group had higher levels of tumor necrosis factor alpha, IL-1β and IL-6 but the lowest level of IL-4. Upregulated Nur77 reduced the ketamine-induced toxicity in neurons. The mechanism of Nur77 involved antioxidation, apoptosis signaling pathway and inflammation signaling pathway. Our study provides a novel therapy that could attenuate ketamine-induced toxicity.

Chitosan oligosaccharide (COS), hydrolyzed and deacetylated from chitosan, has been reported to possess varieties of biological activities. Alzheimer’s disease (AD) is a multifactorial progressive neurodegenerative disorder characterized by cognitive decline and memory loss, where oxidative stress was reported to be an overwhelming cause of the occurrence of AD. We have previously reported that COS could significantly decrease cell death, ROS generation, and lipid peroxidation, though the potential mechanism was yet to be determined. This study was designed to investigate the neuroprotective effect of COS against hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in neuronal SH-SY5Y cells. Our results indicated that COS could dose-dependently scavenge H2O2 in the cell-free systems. Accordingly, COS markedly decreased H2O2-induced cell apoptosis and intracellular ROS generation, while increased antioxidant capacity in SH-SY5Y cells. Further, COS significantly reduced the expression of Bax and upregulated Bcl-2. The mRNA and protein expression levels of nuclear Nrf2, heme oxygenase 1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) were significantly increased upon COS treatment. Moreover, Nrf2-siRNA evidently reversed the promotive effect of COS on expression levels of HO-1 and NQO1, and ARE-driven transcriptional activity as determined by double-luciferase reporter gene assay. Besides, COS reversed H2O2-mediated increased phosphorylation of ERK1/2 and p38 MAPK. In conclusion, our findings indicate that COS could protect SH-SY5Y cells from oxidative damage and apoptosis via regulating Nrf2/ARE signaling pathway, which may provide new applications for the prevention and treatment of AD.

The present study aimed to evaluate the protective effects of selenium (Sel) administration against tacrolimus (Tac) – induced lung toxicity and to assess the relation between heme oxygenase 1 (HO-1) and these effects. The study was conducted on 36 Wistar male albino rats equally divided into four groups: (i) normal control; (ii) Sel (0.1 mg/kg per day p.o. for four weeks); (iii) TAC 3 mg/mL as single oral dose on 27th day; and (iv) Tac + Sel. Lung tissues, lung homogenate, and bronchoalveolar lavage of the sacrificed animals were investigated biochemically and histopathologically, by immunohistochemistry or by PCR. The Tac group showed significantly lower expression of HO-1. Administration of Sel was associated with increased HO-1 expression. Oxidative (malondialdehyde, reduced glutathione, superoxide dismutase, myeloperoxidase, and glutathione peroxidase activity) and nitrosative stress (nitric oxide) markers and markers of inflammation (interleukin 1β (IL-1β), IL-6, and IL-10) showed changes corresponding to HO-1 levels in rat groups. Tac group showed the highest expression of caspase-3. Sel exerted a protective role against Tac-induced lung toxicity.

We previously reported that cytoprotective Heme oxygenase-1, HO-1 (HMOX1) gene-modified human placenta-derived mesenchymal stem cell (HO-1-PMSC) improved placental vascularization in vitro. In the current study, we explored the protective benefit of HO-1-PMSC transplantation in a preeclampsia (PE)-like rat model. A model of PE was successfully constructed by intraperitoneal injection of N-nitro-L-arginine methyl ester (L-NAME). Blood pressure and urinary protein levels were measured. Doppler ultrasound was examined to understand uteroplacental perfusion. ELISA was used to examine the serum levels of VEGF, PlGF, sFlt-1, and sEng. The placentas and fetuses were weighed to verify the improvement in pregnancy outcome. Immunohistochemical and H&E staining was used to detect microvessel density (MVD) in placental tissues and kidney pathology, respectively. The distribution of GFP-labeled PMSC in the placenta were observed under fluorescence microscopy. Blood pressure and proteinuria were reduced and kidney damage was improved. PE rat models treated with PMSC and HO-1-PMSC exhibited an increase in the quality of fetuses and placentas, MVD, VEGF, and PlGF expression, but substantially decreased expression of sFlt-1 and sEng. Doppler ultrasound showed that the placental perfusion was improved. Green fluorescent tracing experiments verified that the cells were successfully transplanted into the placenta and distributed in the blood vessels, indicating that the cells might participate in the process of angiogenesis. These results indicate that therapy with HO-1-PMSC could improve placental vascular dysplasia, increase placental perfusion, control PE symptoms, and promote pregnancy outcome by regulating the balance of angiogenic and antiangiogenic factors or directly participating in the repair of placental vessels in a PE-like rat model.

Oxidative stress is associated with the progression of Alzheimer’s disease (AD). Reactive oxygen species can modify lipids, DNA, RNA, and proteins in the brain. The products of their peroxidation and oxidation are readily detectable at incipient stages of disease. Based on these oxidation products, various biomarker-based strategies have been developed to identify oxidative stress levels in AD. Known oxidative stress-related biomarkers include lipid peroxidation products F2-isoprostanes, as well as malondialdehyde and 4-hydroxynonenal which both conjugate to specific amino acids to modify proteins, and DNA or RNA oxidation products 8-hydroxy-2’-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG), respectively. The inducible enzyme heme oxygenase type 1 (HO-1) is found to be upregulated in response to oxidative stress-related events in the AD brain. While these global biomarkers for oxidative stress are associated with early-stage AD, they generally poorly differentiate from other neurodegenerative disorders that also coincide with oxidative stress. Redox proteomics approaches provided specificity of oxidative stress-associated biomarkers to AD pathology by the identification of oxidatively damaged pathology-specific proteins. In this review, we discuss the potential combined diagnostic value of these reported biomarkers in the context of AD and discuss eight oxidative stress-related mRNA biomarkers in AD that we newly identified using a transcriptomics approach. We review these genes in the context of their reported involvement in oxidative stress regulation and specificity for AD. Further research is warranted to establish the protein levels and their functionalities as well as the molecular mechanisms by which these potential biomarkers are involved in regulation of oxidative stress levels and their potential for determination of oxidative stress and disease status of AD patients.

Significance: As the central metabolic organ, the liver is exposed to a variety of potentially cytotoxic, proinflammatory, profibrotic, and carcinogenic stimuli. To protect the organism from these ...

In systemic hemolysis and in hematuric forms of kidney injury, the major heme scavenging protein, hemopexin (HPX), becomes depleted, and the glomerular microvasculature (glomeruli) is exposed to high concentrations of unbound heme, which, in addition to causing oxidative injury, can activate complement cascades, thus, compounding extent of injury. It is unknown whether unbound heme can also activate specific complement regulatory proteins that could defend against complement-dependent injury. Isolated rat glomeruli were incubated in media supplemented with HPX-deficient (HPX−) or HPX-containing (HPX+) sera as a means of achieving different degrees of heme partitioning between incubation media and glomerular cells. Expression of heme oxygenase (HO)-1 and of the complement activation inhibitors, decay-accelerating factor (DAF), CD59, and complement receptor-related gene Y (Crry), was assessed by western blot analysis. Expression of HO-1 and of the GPI-anchored DAF and CD59 proteins increased in isolated glomeruli incubated with HPX− sera with no effect on Crry expression. Exogenous heme (hemin) did not further induce DAF but increased Crry expression. HPX modulates heme-mediated induction of complement activation controllers in glomeruli. This effect could be of translational relevance in glomerular injury associated with hematuria.

Heme oxygenase-1 (HO-1) promotes heme catabolism exercising cytoprotective roles in normal and cancer cells. Herein, we report the design, synthesis, molecular modeling, and biological evaluation of novel HO-1 inhibitors. Specifically, an amide linker in the central spacer and an imidazole were fixed, and the hydrophobic moiety required by the pharmacophore was largely modified. In many tumors, overexpression of HO-1 correlates with poor prognosis and chemoresistance, suggesting the inhibition of HO-1 as a possible antitumor strategy. Accordingly, compounds 7i and 7l-p emerged for their potency against HO-1 and were investigated for their anticancer activity against prostate (DU145), lung (A549), and glioblastoma (U87MG, A172) cancer cells. The selected compounds showed the best activity toward U87MG cells. Compound 7l was further investigated for its in-cell enzymatic HO-1 activity, expression levels, and effects on cell invasion and vascular endothelial growth factor (VEGF) extracellular release. The obtained data suggest that 7l can reduce cell invasivity acting through modulation of HO-1 expression.

In cancer, antigen-presenting cells (APC), including dendritic cells (DCs), take up and process proteins to mount adaptive antitumor immune responses. This often happens in the context of inflamed cancer, where reactive oxygen species (ROS) are ubiquitous to modify proteins. However, the inflammatory consequences of oxidized protein uptake in DCs are understudied. To this end, we investigated human monocyte-derived cell surface marker expression and cytokine release profiles when exposed to oxidized and native proteins. Seventeen proteins were analyzed, including viral proteins (e.g., CMV and HBV), inflammation-related proteins (e.g., HO1 and HMGB1), matrix proteins (e.g., Vim and Coll), and vastly in the laboratory used proteins (e.g., BSA and Ova). The multifaceted nature of inflammation-associated ROS was mimicked using gas plasma technology, generating reactive species cocktails for protein oxidation. Fourteen oxidized proteins led to elevated surface marker expression levels of CD25, CD40, CD80, CD86, and MHC-II as well as strongly modified release of IL6, IL8, IL10, IL12, IL23, MCP-1, and TNFα compared to their native counterparts. Especially IL8, heme oxygenase 2, and vimentin oxidation gave pronounced effects. Furthermore, protein kinase phospho-array studies in monocyte-derived cells pulsed with native vs. oxidized IL8 and insulin showed enhanced AKT and RSK2 phosphorylation. In summary, our data provide for the first time an overview of the functional consequences of oxidized protein uptake by human monocyte-derived cells and could therefore be a starting point for exploiting such principle in anticancer therapy in the future.

Patients with sickle cell disease (SCD) display priapism. Clinical studies have shown a strong positive correlation between priapism and high levels of intravascular hemolysis in men with SCD. However, there are no experimental studies that show that intravascular hemolysis promotes alterations in erectile function. Therefore, we aimed to evaluate the corpus cavernosum smooth muscle relaxant function in a murine model that displays intravascular hemolysis induced by phenylhydrazine (PHZ), as well as the role of intravascular hemolysis in increasing the stress oxidative in the penis. Corpus cavernosum strips were dissected free and placed in organ baths. Acetylcholine and electrical field stimulation (EFS)-induced corpus cavernosum relaxations in vitro were obtained. Increased corpus cavernosum relaxant responses to acetylcholine and EFS were observed in the PHZ group. Protein expression of heme oxygenase-1 increased in the corpus cavernosum of the PHZ group, but PDE5 protein expression was not modified. Preincubation with the heme oxygenase inhibitor 1 J completely reversed the increased relaxant responses to acetylcholine and EFS in PHZ mice. Protein expression of NADPH oxidase subunit gp91phox, 3-nitrotyrosine, and 4-hydroxynonenal increased in the corpus cavernosum of the PHZ group, suggesting a state of oxidative stress. Basal cGMP production was lower in the PHZ group. Our results show that intravascular hemolysis promotes increased corpus cavernosum smooth muscle relaxation associated with increased HO-1 expression, as well as increased oxidative stress associated with upregulation of gp91phox expression. Moreover, our study supports clinical studies that point to a strong positive correlation between priapism and high levels of intravascular hemolysis in men with SCD.

This study demonstrates the effect of a single high-intensity interval training (HIIT) session on the redox status of rat ovaries with excess adiposity. Forty Wistar female rats (mean (±s.e.m.) weight 94.40 ± 13.40 g) were divided into two groups and fed either a standard diet (SD) or a high-fat diet (HFD) for 62 days. At the end of this period, the rats were subjected to a single HIIT session and were killed 24 h after exercise. Both groups subjected to exercise (SDex and HFDex) generated a significantly higher antioxidant environment by presenting a higher thiol content, which represents a lower oxidation rate of GSH than their respective controls (SD and HFD). The percentage of morphologically normal primary follicles decreased, whereas that of antral follicles increased, in the SDex group. In addition, the HFD group had a higher percentage of degenerated antral follicles than the SD and SDex groups. Cells immunoreactive for α-smooth muscle actin were seen in the cortical stroma and thecal layer enclosing late secondary and tertiary follicles in all groups. Moreover, heme oxygenase and cytochrome P450 family 19 subfamily A member 1 (Cyp19A1) labelling was seen in all antral follicles. Progesterone concentrations were significantly higher in the HFDex than SDex group. In conclusion, this study indicates that a single session of HIIT may result in an improvement in ovary redox status because of metabolic muscle activity by inducing physiological adaptation after exercise in a paracrine manner.

Significance:Iron is an essential element required for growth and proper functioning of the body. However, an excess of labile ferrous iron increases the risk of oxidative stress-induced injury due to the high reactivity of the unpaired reactive electrons of both ferrous iron and oxygen. This high reactivity can be exemplified in the outside world by one of its consequences, rust formation. In cells, this redox-active iron is involved in the formation of lipid radicals. Recent Advances:Defect or insufficient membrane-protective mechanisms can result in iron-catalyzed excessive lipid peroxidation and subsequent cell death, now conceptualized as ferroptosis. Growing reports propose the detrimental role of iron and ferroptosis in many experimental disease models such as ischemia-reperfusion, acute and chronic organ injuries. Critical Issues:This review first provides a snapshot of iron metabolism, followed by a brief introduction of the molecular mechanisms of ferroptosis, as an iron-dependent lipid peroxidation-driven mode of cell death. Upon describing how iron dysbiosis affects ferroptosis induction, we elaborate on the detrimental role of the iron-ferroptosis axis in several diseases. Future Directions:Despite compelling findings suggesting a role of ferroptosis in experimental animal models, the exact contribution of ferroptosis in human contexts still needs further investigation. Development of reliable ferroptosis biomarkers will be an important step in characterizing ferroptosis in human disease. This can provide therapeutic opportunities aiming at targeting ferroptosis in human diseases.

This study aimed to evaluate the anti-resorptive activity of a semi-synthetic coumarin derivative from Platymiscium floribundum, named 6,7-dimethoxy-3-nitrocoumarin. Molecular docking studies were performed to test the binding performance of the derivative against targets associated with alveolar bone loss (TNF-α, IL-1β, and catalase) and a target considered an antioxidant defense (HO-1) during periodontitis. Periodontitis was induced by placing a nylon ligature around the second molars. The rats received for 11 days 6,7-dimethoxy-3-nitrocoumarin (0.01, 0.1, or 1 mg/kg) or vehicle. We investigated by RT-qPCR analysis (TNF-α, IL-1β, and HO-1 mRNA expression levels) and by colorimetric assay (catalase activity) the mechanism of action mediated by 6,7-dimethoxy-3-nitrocoumarin. The in vivo toxicity of 6,7-dimethoxy-3-nitrocoumarin was evaluated. 6,7-Dimethoxy-3-nitrocoumarin (0.1 or 1 mg/kg) reduced alveolar bone loss (1.05 ± 0.24), when compared to vehicle-treated group (3.05 ± 0.30). The interactions of 6,7-dimethoxy-3-nitrocoumarin and the four targets (TNF-α, IL-1β, catalase, and HO-1) showed firm bonds above 6.0 kcal/mol. 6,7-dimethoxy-3-nitrocoumarin (1 mg/kg) lowered mRNA expression levels of TNF-α (2.33 ± 0.56) and IL-1β (19.87 ± 2.9), while it increased both the mRNA expression levels of HO-1 (43.40 ± 1.05) and the catalase activity (46.42 ± 4.59), when compared to vehicle-treated group (46.29 ± 8.43; 37.83 ± 4.38; 1.58 ± 0.11; 8.93 ± 1.86, respectively). The animals did not show any signs of toxicity. 6,7-Dimethoxy-3-nitrocoumarin decreased inflammatory bone loss in the ligature-induced periodontitis in rats, and the activation of the HO-1 pathway may contribute, at least partially, to its protective effects by reducing TNF-α and IL-1β mRNA levels and increasing catalase activity. 6,7-Dimethoxy-3-nitrocumarin could be used as an adjunct to subgingival instrumentation during active and supportive periodontal treatment.

Heme oxygenase-1 (HO-1) is an inducible stress protein that degrades heme to carbon monoxide, iron and biliverdin, which subsequently reduces to bilirubin. Many parameters of verdoheme–rat heme oxygenase complex structure and their role and function on heme degradation were unknown. In this work the structure of iron verdoheme in complex with rat heme oxygenase was studied by density functional theory based B3LYP method and 6-31G basis set. The main goal is to obtain structural and energetic information for various transition states and intermediates on reaction pathways. The charge of verdoheme and iron as the central metal, electron distribution, spin multiplicity of the molecule and proximal substituents effect on the verdoheme ring stabilization and their arrangement are discussed. Gas phase computation has shown that the central metal of the five coordinated rat-verdohemeas ferrous (Fe[Formula: see text] (from 1a-1i) and ferric (Fe[Formula: see text] (from 1j–1q). The Mulliken and NBO charge and spin calculation show that iron is considered as ferrous in all of the optimized structures. Assessment results can gain valuable chemical insight into the electronic reorganization during the reactions.

BACKGROUND: Chronic exposure to environmental endocrine disruptors like nonylphenol (NP), has been shown in previous studies to predispose to non-alcoholic fatty liver disease. METHODS: In this work, forty adult male albino rats were divided into four groups, a high sucrose-high-fat diet (HSHFD) group, a group receiving 20 μg/kg/day of NP, an NP + HSHFD group, and a control group. The rats were sacrificed on day 60 after anesthetization. RESULTS: Biochemical tests indicated that serum transaminases (alanine aminotransferase, aspartate aminotransferase) were significantly increased in the NP + HSHFD group. Lipid metabolism was most disrupted in the NP + HSHFD with a highly significant increase (p < 0.001) of serum cholesterol, triglyceride, and low-density lipoprotein cholesterol compared to other groups. Heme oxygenase 1 showed the highest expression in the NP + HSHFD group, with a highly significant difference in comparison with the other groups (p < 0.001). Histopathological studies revealed fatty changes and dilatation in the central vein in the HSHFD group. Lymphoid cell aggregates were detected in the NP group. Massive inflammation and degeneration were revealed in the NP + HSHFD group. There was also marked expression of the apoptotic protein caspase-3 in the NP + HSHFD group. CONCLUSION: In conclusion, exposure to a 20 μg/kg/day of NP induced oxidative stress leading to non-alcoholic steatohepatitis.

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality worldwide. Inflammation and oxidative stress play critical roles in progression of various types of CVD. Broad pharmacological properties of ginger (the rhizome of Zingiber officinale) and its bioactive components have been reported, suggesting that they can be a therapeutic choice for clinical use. Consistent with its rich phenolic content, the anti-inflammatory and antioxidant properties of ginger have been confirmed in many studies. Ginger modifies many cellular processes and in particular was shown to have potent inhibitory effects against nuclear factor kappa B (NF-κB); signal transducer and activator of transcription; NOD-, LRR-, and pyrin domain-containing proteins; toll-like receptors; mitogen-activated protein kinase; and mammalian target of rapamycin signaling pathways. Ginger also blocks pro-inflammatory cytokines and the activation of the immune system. Ginger suppresses the activity of oxidative molecules such as reactive oxygen species, inducible nitric oxide synthase, superoxide dismutase, glutathione, heme oxygenase, and GSH-Px. In this report, we summarize the biochemical pathologies underpinning a variety of CVDs and the effects of ginger and its bioactive components, including 6-shogaol, 6-gingerol, and 10-dehydrogingerdione. The properties of ginger and its phenolic components, mechanism of action, biological functions, side effects, and methods for enhanced cell delivery are also discussed. Together with preclinical and clinical studies, the positive biological effects of ginger and its bioactive components in CVD support the undertaking of further in vivo and especially clinical studies.

Vascular calcification is very commonly observed in patients with chronic kidney disease (CKD), but there is no efficient therapy available. Oxidative stress plays critical roles in the progression of vascular calcification. Celastrol (Cel), a natural constituent derived from Chinese herbals, exhibits anti-oxidative stress activity. Here, we investigated the effect of celastrol on vascular calcification using vascular smooth muscle cells (VSMCs), arterial rings and CKD rats. Alizarin red staining and gene expression analysis showed that Cel dose-dependently inhibited rat VSMC calcification and osteogenic differentiation. Similarly, ex vivo study revealed that Cel inhibited calcification of rat and human arterial rings. In addition, micro-computed tomography, alizarin red staining and calcium content analysis confirmed that Cel inhibited aortic calcification in CKD rats. Interestingly, Cel treatment increased the mRNA and protein levels of heme oxygenase-1 (HMOX-1), and reduced the levels of reactive oxygen species (ROS) in VSMCs. Furthermore, both pharmacological inhibition of HMOX-1 and knockdown of HMOX-1 by siRNA independently counteracted the inhibitory effect of Cel on vascular calcification. Moreover, knockdown of HMOX-1 prevented Cel treatment-mediated reduction in ROS levels. Finally, Cel treatment reduced Vitamin D3-induced aortic calcification in mice and this effect was blocked by HMOX-1 inhibitor ZnPP9. Collectively, our results suggest that up-regulation of HMOX-1 is required for the inhibitory effect of Cel on vascular calcification. Modulation of HMOX-1 may provide a novel strategy for the treatment of vascular calcification in CKD.

CYJ-27, a synthetic analog of decursin, prevents the generation of proinflammatory cytokines and oxidative stress. In this study, the effects of CYJ-27 on the regulation of inducible nitric oxide synthase (iNOS), heme oxygenase (HO)-1, and cyclooxygenase (COX-)2 were characterized in lipopolysaccharide (LPS)-treated human umbilical vein endothelial cells (HUVECs). In addition, the effects of CYJ-27 on the production of iNOS and representative proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, were tested in the lung tissues of LPS-treated mice. CYJ-27 promoted the expression of HO-1, suppressed NF-κB-luciferase activity, and reduced COX-2/PGE2 and iNOS/NO, resulting in a diminution in phosphorylated-STAT-1. Furthermore, CYJ-27 promoted the nuclear translocation of Nrf2, enhanced the combination of Nrf2 to antioxidant response elements, and diminished IL-1β production in LPS-activated HUVECs. CYJ-27-downregulated iNOS/NO expression was rescued after the RNAi suppression of HO-1. In LPS-treated mice, CYJ-27 significantly diminished iNOS production in the lung tissues and TNF-α expression in the bronchoalveolar lavage fluid. These findings indicate that CYJ-27 exerts anti-inflammatory activities by regulating iNOS through downregulation of both NF-κB activation and phosphorylated-STAT-1. Hence, it can act as a template for the development of novel substances to treat inflammatory diseases.

Background: Toxic particles within tobacco smoke are responsible for several respiratory system problems. Among these toxic particles is Carbon Monoxide (CO), produced from environment and Heme Oxygenase induction. Expiratory CO levels can be measured using CO analyzer. CC16 is a pneumoprotein produced by club cells in distal respiratory tract. In acute condition, CC16 level will increase to maintain homeostasis and anti-inflammation. In chronic condition, i.e. in smokers, CC16 will decrease, following destruction of Club cell. This study aims to determine exhaled CO (eCO) levels and serum CC16 levels in active smokers. Methods: This cross-sectional analytical study design has 40 samples of healthy smokers in Brawijaya University who consents to the research from October 2019 until June 2020. The minimum consumption amount is 1 cigarette per day for at least 1 year. eCO levels are measured using CO analyzer (Smokelyzer), while ELISA is used to measure serum CC16 levels. Results: Among 40 subjects, mean eCO level is 10.18 ± 7.42 ppm. Mean serum CC16 level is 3.17 ± 1.78 ng/mL, lower than normal value of 6.4 ng/mL (Lomas et al., 2008). Conclusion: eCO levels increases and serum CC16 level decreases in active smokers, who smokes at least 1 cigarette/day for at least 1 year. This indicates that CO from tobacco smoke could irritate and damage the Club cells in the respiratory system.

Hemin, a substrate of heme oxygenase (HO)-1, induces HO-1 expression on a variety of cells to exert anti-oxidant and anti-inflammatory roles. However, the role of HO-1 in allergic diseases for dendritic cells (DCs) is not fully understood. Here, we report that HO-1 modulates asthmatic airway inflammation by hemin-treated DC-released extracellular vesicles (DCEVs). Following induction of bone marrow-derived DCs by hemin and then by house dust mite (HDM) in vitro, mouse CD4+ naïve T cells were cocultured with DCEVs to determine T helper (h) cell differentiation. C57BL/6 mice were sensitized by different stimuli-induced DCEVs and challenged with HDM to analyze the changes of inflammatory cells and cytokines in the lung and bronchoalveolar lavage fluid. The results showed that hemin-treated DCEVs (hemin-DCEVs) express phosphatidylserine (PS), CD81, heat shock protein 70, and HO-1, which facilitates regulatory T (Treg) cells differentiation in vitro and in vivo. In HDM-induced asthmatic mouse model, hemin-DCEVs inhalation reduced eosinophils infiltration and mucus secretion in the airway, decreased the levels of IL-4, IL-5, and IL-13 in the lung and the number of Th2 cells in mediastinal lymph nodes (MLNs), and increased the number of Treg cells in MLNs. Thus, our study demonstrated, for the first time, that EVs from HO-1-overexpressing DCs alleviate allergic airway inflammation of eosinophilic asthma by potentiating Treg cells differentiation and limiting proinflammatory cytokine secretion, which expands our understanding of HO-1 function, opening the door for HO-1 inducer-like hemin as a novel therapeutic strategy for asthma or other allergic diseases.

Background: The COVID-19 pandemic is the greatest life-threatening disaster currently facing the worldwide population. COVID-19 patients with concomitant diseases such as chronic obstructive pulmonary disease and cardiovascular problems quickly develop severe pneumonia with low arterial oxygen saturation and multiorgan failure, resulting in sudden death. These symptoms are caused by deadly inflammation that occurs in various organs. Objective: Various types of inflammation caused by RNA virus infection have been known to be manageable by the induction of heme oxygenase-1 (HO-1) in local tissues. HO-1 is also known to be a key enzyme for the suppression of RNA viral replication. Therefore, in addition to standard medical care for pneumonic viral infection, we have attempted to treat COVID-19 patients with a highly effective HO-1 inducer, 5-aminolevulinic acid phosphate, in combination with ferrous sodium citrate (5-ALA with SFC). Methods: 5-ALA with SFC is a supplement formulation registered in Japan as food with functional claims. Six patients with typical symptoms of COVID-19 and some suspected COPD associated with heavy smoking were given oral administration of multiple doses of 5-ALA with SFC at the Maximum Tolerated Dose (MTD) for 3 to 7 days, followed by treatment with a lower amount of 5-ALA with SFC for 2 to 3 weeks. Results and Conclusion: Each patient's recovery time was considerably shorter than reported for patients who received only standard care for SARS-CoV-2 infection. The results confirm the safety, tolerability, and efficacy of 5-ALA with SFC as a therapeutic supplement for patients with acute-phase COVID-19.

Appropriately increasing intramuscular fat content can help improve meat quality, so it is necessary to explore the internal molecular mechanism of preadipocyte differentiation. The role of heme oxygenase 1 (HO1) in cell oxidative stress, energy metabolism, cell proliferation, and differentiation has gradually been revealed. Here, we used 3'RACE to identify the full-length 3' untranslated region (3'UTR) of HO1 and found that a very short 3'UTR variant was produced by alternative polyadenylation (APA). HO1 with a long 3'UTR variant was identified as a direct target of miR155-5P and miR377-3P. Our experimental results verified the inhibitory effect of HO1 on preadipocyte differentiation. In addition, our research confirms that by escaping microRNA inhibitory effects, the HO1 3'UTR short variant produced by APA has a higher level of expression. Thus, the HO1 3'UTR short variant has a stronger inhibitory effect on the preadipocyte differentiation than the HO1 3'UTR long variants in 3T3-L1.

Stroke is a lethal event with a high incidence in Egypt. Quick early intervention can be lifesaving. Transient global ischemia (TGI), a type of ischemic stroke, is mainly instigated by cardiac arrest. Ischemia followed by reperfusion causes further neuronal cell damage. In this study, we aimed to evaluate the potential apoptotic, anti-inflammatory, and neuroprotective effects of green (GCBE) and roasted (RCBE) coffee bean water extract against transient global ischemia-induced via a bilateral common carotid artery occlusion (CAO) in rats. Before CAO, 1.5 ml/kg body weight/day of GCBE or RCBE was administered for 14 days by oral gavage. Ischemia/reperfusion (I/R) and sham groups were treated with a vehicle. Oxidative stress biomarkers and antioxidant enzyme activities, such as MDA, NO, GSH, SOD, CAT, GR, GPx, inflammatory markers TNF-α, IL-1β, and NF-κB, and BDNF were investigated. Quantitative real-time PCR analysis of mitogen-activated protein kinase pathways, in addition to heme oxygenase 1, and nuclear factor erythroid 2–related factor 2 were determined. Apoptotic markers, including Bcl-2, Bax, and caspase 3, in addition to the vascular endothelial growth factor-a, were investigated, followed by an examination of hippocampal histopathology. Pre-administration of GCBE and RCBE improved neurological function and neuronal survival, suppressed the spread of oxidative stress, inflammation, and apoptosis, and reversed most of the pathological changes. However, green coffee bean extract was more effective than roasted coffee bean extract, perhaps due to the roasting process, which may affect active compounds. In conclusion, GCBE and RCBE represent a potential clinical strategy for pre-ischemic conditioning.

Hemin can improve the stress resistance of plants through the heme oxygenase system. Additionally, substances contained in plants, such as secondary metabolites, can improve stress resistance. However, few studies have explored the effects of hemin on secondary metabolite content. Therefore, the effects of hemin on saponin synthesis and the mechanism of plant injury relief by hemin in Conyza blinii were investigated in this study. Hemin treatment promoted plant growth and increased the antioxidant enzyme activity and saponin content of C. blinii under osmotic stress and cold stress. Further study showed that hemin could provide sufficient precursors for saponin synthesis by improving the photosynthetic capacity of C. blinii and increasing the gene expression of key enzymes in the saponin synthesis pathway, thus increasing the saponin content. Moreover, the promotion effect of hemin on saponin synthesis is dependent on heme oxygenase-1 and can be reversed by the inhibitor Zn-protoporphyrin-IX (ZnPPIX). This study revealed that hemin can increase the saponin content of C. blinii and alleviate the damage caused by abiotic stress, and it also broadened the understanding of the relationship between hemin and secondary metabolites in plant abiotic stress relief.

Carbon monoxide releasing molecule-3 (CORM-3) has been shown to protect inflammatory diseases via the upregulation of heme oxygenases-1 (HO-1). However, in rat brain astrocytes (RBA-1), the mechanisms underlying CORM-3-induced HO-1 remain poorly defined. This study used western blot, real-time PCR, and promoter activity assays to determine the levels of HO-1 expression and 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE) to measure reactive oxygen species (ROS). We found that CORM-3-induced HO-1 expression was mediated through ROS generation by Nox or mitochondria. The signaling components were differentiated by pharmacological inhibitors and small interfering RNA (siRNA). Subcellular fractions, immunofluorescent staining, and chromatin immunoprecipitation assay were used to evaluate the nuclear translocation and promoter binding activity of Nrf2 induced by CORM-3. The roles of mTOR and FoxO1 in CORM-3-stimulated responses are still unknown in RBA-1 cells. Our results demonstrated that transfection with siRNAs or pretreatment with pharmacological inhibitors attenuated the levels of HO-1 and phosphorylation of signaling components including Akt, mTOR, FoxO1, and Nrf2 stimulated by CORM-3. Moreover, pretreatment with N-acetyl-L-cysteine, diphenyleneiodonium chloride, apocynin, or rotenone blocked nuclear translocation and promoter binding activity of Nrf2 induced by CORM-3. The present study concluded that in RBA-1 cells, CORM-3-induced HO-1 expression is, at least partially, mediated through Nox and mitochondria/ROS-dependent PI3K/Akt/mTOR cascade to activate FoxO1 or ROS leading to activation of Nrf2 activity.

Background and purpose: Low-dose of carbon monoxide delivered by CO-releasing molecule-2 (CORM-2) had been confirmed having anti-inflammatory efficacy in some inflammatory diseases. Herein, we assessed the usefulness of CORM-2 in correcting intracerebral hemorrhage (ICH)-mediated inflammation.Methods: Healthy male Sprague Dawley (SD) rats randomly entered into four groups: sham-ICH, ICH, ICH+CORM-2, and ICH+ inactive carbon monoxide releasing molecule 2 (iCORM-2). ICH was induced by 50 μl of autologous arterial blood injected in situ in the rat brain. Neuro-functions of the ICH rats were evaluated with Garcia 18 scores at the 6th, 24th , 48th hou, and the fifthh day post-ICH. And brain tissues surrounding the hematoma area were collected from all ICH rats and assayed with Western blot and immunofluoresence analysis.Results: Neuro-dysfunctions in ICH rats were very severe than those in ICH +CORM-2 rats. Compared to sham group, the levels of HO-1, IKKβ, NF-κB, and TNF-α in ICH group began to elevate at the 6th hour, and reached to peak at the 48th hour post-ICH, all p < 0.05. While in ICH +CORM-2 group, the expressions of IKKβ, NF-κB, and TNF-α were very weaker than that in ICH group at every time points mentioned above; however, this phenomenon was not reproduced in ICH + iCORM-2 group. HO-1 in ICH+CORM-2 group highlighted in perihematomal area with many activated microglia (Iba-1-positive cells) and co-expressed with TNF-α, all of which were diminished at the fifth day post-ICH.Conclusion: CORM-2 may attenuate ICH-mediated inflammation by inhibiting microglial activation, which may involve the IKK/NF-κB pathway.AbbreviationsICH: intracerebral hemorrhage; CO: carbon monoxide; CORM-2: carbon monoxide releasing molecule-2; iCORM-2: inactive carbon monoxide releasing molecule-2; HO-1: heme oxygenase 1; IKKβ: inhibitor of IκB kinases β; NF-κB: nuclear factor-κB; TNF-α: tumor necrosis factor-α; Iba-1: ionized calcium binding adaptor molecule-1; IκB: inhibitor of NF-κB; iNOS: inducible nitric oxide synthase; Keap1: Kelch-like ECH-associated protein 1; Nrf2: NF-E2-related factor 2; DMSO: dimethylsulfoxide.

Dasatinib, a tyrosine kinase inhibitor, has a protective effect on experimental acute respiratory distress syndrome (ARDS). This study investigated the effect and mechanism of dasatinib in ARDS. C57BL/6 mice were administered with dasatinib (1 and 10 mg/kg) after lipopolysaccharide (LPS) treatment to evaluate the effect of dasatinib on white blood cells (WBC), neutrophils, lymphocytes and macrophages in bronchoalveolar lavage fluid (BALF). The levels and mRNA expressions of inflammation-related cytokines in lung tissues and RAW 264.7 cells were detected by enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. The protein expressions of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO1) were determined by Western blot. MTT assay was performed to detect the viability of RAW 264.7 cell. Rescue experiments were used to assess the effect of Nrf2 silencing on the LPS- and dasatinib-treated mice. Under LPS treatment, levels of the WBC, neutrophils, lymphocytes and macrophages in BALF and mRNA expressions of IL-6, TNF-α and IL-10 as well as expression of iNOS were increased, but the expression of arginase-1 was inhibited, while no obvious changes of the protein expressions of Nrf2 and HO1 were observed. Dasatinib partially reversed the effects of LPS above, and further promoted the mRNA expression of IL-10 and the protein expressions of Nrf2 and HO1, while Nrf2 silencing counteracted the effect of dasatinib. Dasatinib induced the polarization of M2 subtype of macrophages and alleviated LPS-induced ARDS through activating Nrf2 signaling pathway, which may provide a new strategy for the treatment of ARDS.

Objective: Evidence indicates that bone marrow progenitor cells (BMPC) are a major contributor to neointima formation in transplant arteriosclerosis. HO-1 (heme oxygenase-1, Hmox1 ) and carbon monoxide (CO), a product of heme degradation by HO-1, ameliorate neointima formation by inhibiting proliferation of smooth muscle cells. We investigated the mechanism whereby HO-1 and CO modulate BMPC and mitigates neointima formation in transplant arteriosclerosis. Approach and Results: Using a murine model of aortic transplantation, bone marrow chimeric mice, and in vitro experiments, we report that CO does not inhibit mobilization of BMPC into the circulation or their homing to the vessel adventitia, but instead suppresses differentiation of BMPC into smooth muscle cells after they arrive in the adventitia. Specifically, the effect of CO on differentiation of BMPC into smooth muscle cell is mediated in part, by limiting PDGFR-β (platelet derived growth factor receptor-β) signaling. Hmox1 −/− BMPC exhibit a greater propensity to differentiate into smooth muscle cell in vitro, in part by regulating PDGFR-β + expression. Furthermore, wild-type mice transplanted with Hmox1 −/− bone marrow cells show augmented neointima formation after allografting versus control. CO exposure significantly ameliorated neointima formation, which remains more severe with Hmox1 −/− bone marrow cell versus air-treated mice receiving HO-1-expressing bone marrow cell, highlighting the importance of endogenous HO-1 in neointima formation. Conclusions: Host BMPC contribute to neointima formation in transplant arteriosclerosis and the protective effect afforded by HO-1/CO against neointima formation is mediated in part through the regulation of PDGFR-β expression. We propose that suppressing differentiation of BMPC is a major mechanism by which HO-1 and CO prevent neointima expansion after transplant.

OBJECTIVE: To examine early pregnancy levels of serum heme oxygenase-1, soluble vascular endothelial growth factor receptor-1, B-cell lymphoma/leukemia-2 in relation to unfavorable pregnancy outcomes, including preeclampsia, fetal growth restriction, spontaneous preterm birth, gestational diabetes mellitus and fetal macrosomia. STUDY DESIGN: A total of randomly selected 140 pregnancies were included in this prospective study. Peripheral blood samples were obtained between 110/7 and 136/7 gestational weeks. All pregnancies were followed up until the outcomes were obtained and classified as preeclampsia, fetal growth restriction, spontaneous preterm birth, gestational diabetes mellitus, fetal macrosomia, and uncomplicated ones. RESULTS: Significantly high levels of early serum heme oxygenase-1 were found in the cases who subsequently developed preeclampsia, spontaneous preterm birth, and fetal macrosomia (p

Spinal cord injury (SCI) is a devastating event that often leads to permanent neurological deficits. Evidence from emerging studies has implicated oxygen-derived free radicals and high-energy oxidants as mediators of secondary SCI. Therefore, targeting these mediators using antioxidants could be beneficial for the disease. Several signaling pathways, such as the nuclear factor erythroid-2-related factor 2/heme oxygenase 1 (Nrf2/HO-1), have been associated with the regulation of some pathophysiological features of SCI. Curcumin is a plant medicinal agent whose diverse pharmacological properties have been extensively investigated and reported, notably its ability to curtail inflammatory damage by inhibiting the nuclear factor-κ-light-chain-enhancer of activated B cells. In this review, we analyze the role of curcumin in activating Nrf2/HO-1 and scavenging free radicals to repair SCI. With its minimal side effects, curcumin could be a potential therapy for SCI treatment.

Objective: Macrophage-mediated inflammatory response is closely associated with the neovascularization process following hindlimb ischemia. We previously demonstrated that HO-1 (heme oxygenase-1) in macrophages evoked proinflammatory reactions and tissue damage. Here, we evaluated the role played by macrophage-derived HO-1 and elucidated its underlying molecular mechanisms in perfusion recovery after hindlimb ischemia. Approach and Results: We found significant upregulation of HO-1 in mouse ischemic muscles after hindlimb ischemia surgery and with most of this expression occurring in infiltrated macrophages. Myeloid conditional HO-1-deficient mice exhibited higher perfusion recovery, evidenced by restored blood flow, motor function and attenuated tissue damage as well as increased capillary density in the gastrocnemius muscles after hindlimb ischemia, relative to littermate controls. This protective effect was accompanied by reduced NLRP3 (Nod-like receptor family pyrin domain containing 3) inflammasome activation in the infiltrated macrophages without the alteration of macrophage infiltration and polarization. Moreover, suppressing inflammasome activation with NLRP3 inhibitor MCC950 improved blood flow and capillary density in wild-type mice compared with untreated mice. Mechanistically, suppressing HO-1 abolished TNF (tumor necrosis factor)-α-induced NLRP3 protein rather than mRNA expression in bone marrow–derived macrophages, indicating that HO-1 mediated post-transcriptional regulation of NLRP3. Furthermore, HO-1 inhibition promoted autolysosome-dependent degradation of NLRP3 in bone marrow–derived macrophages. Matrigel tube formation assay revealed that HO-1 deletion abrogated the antiangiogenic effect of inflammasome-activated macrophages. Conclusions: Taken together, these findings indicate that macrophage HO-1 deficiency promotes perfusion recovery after hindlimb ischemia by accelerating autolysosomal degradation of NLRP3. The underlying mechanism of action is a potential target for therapeutic angiogenesis in ischemic diseases.

β-Glucan from Saccharomyces cerevisiae has been described to be effective antioxidants, but the specific antioxidation mechanism of β-glucan is unclear. The objectives of this research were to determine whether the β-glucan from Saccharomyces cerevisiae could regulate oxidative stress through the Dectin-1/Nrf2/HO-1 signaling pathway in lipopolysaccharides (LPS)-stimulated RAW264.7 cells. In this study, we examined the effects of β-glucan on the enzyme activity or production of oxidative stress indicators in LPS-stimulated RAW264.7 cells by biochemical analysis and the protein expression of key factors of Dectin-1/Nrf2/HO-1 signaling pathway by immunofluorescence and western blot. The biochemical analysis results showed that β-glucan increased the LPS-induced downregulation of enzyme activity of intracellular heme oxygenase (HO), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) while decreasing the production of reactive oxygen species (ROS) and malondialdehyde (MDA). Furthermore, immunofluorescence results showed that β-glucan can activate the nuclear factor erythroid 2-related factor 2 (Nrf2). The antioxidant mechanism study indicated that β-glucan activated dendritic-cell-associated C-type lectin 1 (Dectin-1) receptors mediated Nrf2/HO-1 signaling pathway, thereby downregulating the production of ROS and thus produced the antioxidant effects in LPS-stimulated RAW 264.7 cells. In conclusion, these results indicate that β-glucan potently alleviated oxidative stress via Dectin-1/Nrf2/HO-1 in LPS-stimulated RAW 264.7 cells.

Heme oxygenase-1 (HO-1) is a vital enzyme in humans that primarily regulates free heme concentrations. The overexpression of HO-1 is commonly associated with cardiovascular and neurodegenerative diseases including atherosclerosis and ischemic stroke. Currently, there are no known chemical probes to detect HO-1 activity, limiting its potential as an early diagnostic/prognostic marker in these serious diseases. Reported here are the design, synthesis, and photophysical and biological characterization of a coumarin–porphyrin FRET break-apart probe to detect HO-1 activity, Fe–L1. We designed Fe–L1 to “break-apart” upon HO-1-catalyzed porphyrin degradation, perturbing the efficient FRET mechanism from a coumarin donor to a porphyrin acceptor fluorophore. Analysis of HO-1 activity using Escherichia coli lysates overexpressing hHO-1 found that a 6-fold increase in emission intensity at 383 nm was observed following incubation with NADPH. The identities of the degradation products following catabolism were confirmed by MALDI-MS and LC–MS, showing that porphyrin catabolism was regioselective at the α-position. Finally, through the analysis of Fe–L2, we have shown that close structural analogues of heme are required to maintain HO-1 activity. It is anticipated that this work will act as a foundation to design and develop new probes for HO-1 activity in the future, moving toward applications of live fluorescent imaging.

Background. The recombinant protein diannexin can inhibit platelet-mediated events, which contribute to acute respiratory distress syndrome (ARDS). Here, we investigated the effect of diannexin and its effect on heme oxygenase-1 (HO-1) in ARDS. Methods. A total of 32 rats were randomized into sham, ARDS, diannexin (D), and diannexin+HO-1 inhibitor (DH) groups. Alveolar-capillary permeability was evaluated by testing the partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) ratio, lung wet/dry weight ratio, and protein levels in the lung. Inflammation was assessed by measuring cytokine levels in the bronchial alveolar lavage fluid (BALF) and serum and nuclear factor-κB (NF-κB) in the lung tissue. Inducible nitric oxide synthase (iNOS), malondialdehyde (MDA), and myeloperoxidase (MPO) were measured to evaluate the oxidative stress response. Lung tissue pathology and apoptosis were also evaluated. We measured HO-1 expression in the lung tissue to investigate the effect of diannexin on HO-1 in ARDS. Results. Compared with the ARDS group, diannexin improved PaO2/FiO2, lung wet/dry weight ratio, and protein levels in the BALF and decreased levels of cytokines and NF-κB in the lung and serum. Diannexin inhibited the oxidative stress response and significantly ameliorated pathological lung injury and apoptosis. The partial reversal of diannexin effects by a HO-1 inhibitor suggests that diannexin may promote HO-1 expression to ameliorate ARDS. Conclusions. We showed that diannexin can improve alveolar-capillary permeability, inhibit the oxidative stress response and inflammation, and protect against ARDS-induced lung injury and apoptosis.

Background Doxorubicin (DOX) is a widely used cancer chemotherapeutic drug with cardiotoxicity effect limiting its clinical use. DOX induced cardiotoxicity is mediated by oxidative stress and mitochondrial damage. Kininogen-1(KNG1) is an important pro-inflammatory and pro-oxidant factor, and studies have found that it can aggravate lung and brain damage. However, it has not been known in terms of cardiotoxicity. Therefore, the purpose of this study is to understand the mechanism of KNG1 in DOX-induced heart injury. Methods C57 mice were selected for intraperitoneal injection of DOX. The model was successfully established, and fresh ventricular tissues were isolated from the ctrl group and the DOX group for mass spectrometry analysis to screen for differentially expressed proteins. Nuclear Factor-Like 2 (Nrf2), Heme Oxygenase 1 (HO-1), 4-Hydroxynonenal (4-HNE) were used to evaluate oxidative stress level, Cytochrome C Oxidase Subunit 4 (COX4) was used to evaluate mitochondria function. Mitochondrial inner membrane potential (ΔΨm) was monitored with JC-1 fluorescence. Results KNG1 was identified as a core gene which was highly expressed in the DOX myocardial injury model. Following this, an overexpression adenovirus was constructed, and KNG1 was overexpressed in vivo (mice) and in vitro (neonatal mouse cardiomyocytes (NMCMs)). It was found that overexpression of KNG1 can aggravate heart oxidative stress and mitochondrial damage. Besides, a knockdown KNG1 model was constructed, and the low expression of KNG1 was performed in cytology. It was found that knockdown of KNG1 can improve cardiomyocyte oxidative stress and mitochondrial damage caused by DOX. Nrf2 is an important antioxidant factor. Further, following KNG1 knock down, Nrf2 was also knocked down, and found that its cardiomyocyte protective effect was weakened. Conclusion The overexpression of KNG1 aggravates the oxidative stress and mitochondrial damage of the heart in vivo and in vitro, which might play a role by regulating Nrf2, providing a therapeutic target for DOX-induced cardiotoxicity.

The incidence of myocardial dysfunction caused by sepsis is high, and the mortality of patients with sepsis can be significantly increased. During sepsis, oxidative stress and inflammation can lead to severe organ dysfunction. Flavone chrysin is one of the indispensable biological active ingredients for different fruits and vegetables and has antioxidant and anti-inflammatory properties. However, it is not clear whether chrysin is an effective treatment for heart dysfunction caused by sepsis. We found that it had protective effects against the harmful effects caused by LPS, manifested in improved survival, normalized cardiac function, improved partial pathological scores of myocardial tissue, and remission of apoptosis, as well as reduced oxidative stress and inflammation. Mechanism studies have found that chrysin is an important antioxidant protein, a key regulator of heme oxygenase 1 (HO-1). We found that HO-1 levels were increased after LPS intervention, and chrysin further increased HO-1 levels, along with the addition of Nrf2, a regulator of antioxidant proteins. Pretreatment with PD98059, an extracellular signal-regulated kinase-specific inhibitor, blocked chrysin-mediated phosphorylation of Nrf2 and the nuclear translocation of Nrf2. The protective effect of chrysin on sepsis-induced cardiac dysfunction was blocked by ZnPP, which is a HO-1 blocker. Chrysin increased antioxidant activity and reduced markers of oxidative stress (SOD and MDA) and inflammation (MPO and IL-1β), all of which were blocked by ZnPP. This indicates that HO-1 is the upstream molecule regulating the protective effect of chrysin. Thus, by upregulation of HO-1, chrysin protects against LPS-induced cardiac dysfunction and inflammation by inhibiting oxidative stress.

Exposure to chronic stress negatively affects the development of cognition, characterized by learning and memory decline. Ginsenoside Re (GRe), an active compound derived from Panax ginseng, exhibited neuroprotective activity in various neurological diseases. In this study, the protective effect of GRe on chronic restraint stress (CRS)-induced memory deficit was investigated. The mice were experienced 35 days of the CRS induction. The GRe was administered daily orally (10, 20, or 40 mg/kg) during the next 3 weeks stress session and the behavior test period. The CRS-induced memory impairment mice were subjected to behavioral tasks, such as the Y-maze, novel objects recognition, and step-through passive avoidance tests. Nissl staining was used to examine the neuron numbers. The levels of antioxidant enzymes, malondialdehyde, and proinflammatory factor were determined by kits and ELISA assays. The expressions of brain-derived neurotrophic factor (BDNF), NOD-like receptor protein 3 (NLRP3), nuclear factor erythroid-2 related factor 2 (Nrf2) and synapse-associated proteins (synaptophysin, SYP, and postsynaptic density 95, PSD95) were measured by Western blotting. Behavioral assessments indicated that GRe could ameliorate the cognitive impairment of CRS-induced mice, as indicated by increased responses in Y-maze (p < .05), novel objects recognition (p < .01), and step-through passive avoidance tests (p < .01). In addition, GRe treatment significantly decreased the neuronal loss in CRS mice in histological examination. Moreover, chronic GRe treatment significantly ameliorated the down-regulated the expressions of BDNF, Nrf2, heme oxygenase (HO)-1, SYP, and PSD95, as well as up-regulated NLRP3, the adaptor protein ASC, and Caspase-1 protein expression in the hippocampus of CRS-treated mice. Taken together, these findings suggest that GRe has a potential therapeutic effect on memory impairment in C57BL/6J mice exposed to CRS paradigm.