Activating Heme Oxygenase-1 Research Articles

High Hemin Concentration Induces Escape from Senescence of Normoxic and Hypoxic Colon Cancer Cells.

Simple SummaryHigh red-meat consumption as well as bleeding or bruising can promote oxidative stress and, in consequence, cancer development. However, the mechanism of that phenomenon is not understood. The induction of therapy-induced senescence (TIS) might also be induced by oxidative stress. Recently, TIS cells, despite their inhibited proliferation potential, have been identified as one of the sources of tumor re-growth. Here, with the use of molecular analyses, we found that oxidative stress, promoted by high doses of hemin or H2O2, can trigger TIS escape and cell re-population. It is closely related to the activity of antioxidative enzymes, especially heme oxygenase-1. Hypoxia might accelerate these effects. Therefore, we propose that the prevention of excessive oxidative stress could be a potential target in senolytic therapies.Hemoglobin from either red meat or bowel bleeding may promote oxidative stress and increase the risk of colorectal cancer (CRC). Additionally, solid cancers or their metastases may be present with localized bruising. Escape from therapy-induced senescence (TIS) might be one of the mechanisms of tumor re-growth. Therefore, we sought to study whether hemin can cause escape from TIS in CRC. To induce senescence, human colon cancer cells were exposed to a chemotherapeutic agent irinotecan (IRINO). Cells treated with IRINO exhibited common hallmarks of TIS. To mimic bleeding, colon cancer cells were additionally treated with hemin. High hemin concentration activated heme oxygenase-1 (HO-1), induced escape from TIS and epithelial-to-mesenchymal transition, and augmented progeny production. The effect was even stronger in hypoxic conditions. Similar results were obtained when TIS cells were treated with another prooxidant agent, H2O2. Silencing of antioxidative enzymes such as catalase (CAT) or glutathione peroxidase-1 (GPx-1) maintained colon cancer cells in a senescent state. Our study demonstrates that a high hemin concentration combined with an increased activity of antioxidative enzymes, especially HO-1, leads to escape from the senescence of colon cancer cells. Therefore, our observations could be used in targeted anti-cancer therapy.

Schisandrin B promotes Foxp3+ regulatory T cell expansion by activating heme oxygenase-1 in dendritic cells and exhibits immunomodulatory effects in Th2-mediated allergic asthma

Allergic asthma is induced by T helper 2 (Th2) responses and allergen-specific immunoglobulin E (IgE). In asthma, regulatory T (Treg) cells play a crucial role in controlling immune homeostasis, and induction of Treg cells is a good strategy to treat Th2-mediated allergic asthma. Schisandrin B (Sch B), the main component isolated from Schisandra chinensis, reportedly possesses various pharmacological properties, but its immunomodulatory mechanism in allergic asthma remains unclear. In the present study, we explored whether Sch B exerts an antiallergic effect through modifying the function of dendritic cells (DCs) to regulate T-cell polarization and further investigated the immunomodulatory effects of Sch B in allergic asthma. Herein, an in vitro study revealed that 20 μM of Sch B-treated bone-marrow-derived DCs exhibited a semi-mature phenotype that secreted low amounts of proinflammatory cytokines including interleukin (IL)-12, IL-1β, IL-6, and tumor necrosis factor (TNF)-α, and expressed decreased levels of surface molecules of cluster of differentiation 80 (CD80) and CD86. Compared to fully mature DCs, these Sch B-treated DCs displayed a regulatory ability to promote CD4+Foxp3+ Treg cell generation via upregulation of heme oxygenase (HO)-1 expression. Of note, in a murine model of ovalbumin (OVA)-induced asthma, levels of Th2-type cytokines such as IL-4, IL-5, and IL-13, and C–C motif chemokine 11 (CCL11) were dampened, whereas numbers of forkhead box P3 (Foxp3)-positive Treg cells were augmented in Sch B-treated mice. Moreover, administration of 5 mg/kg of Sch B alleviated the cardinal features of Th2-mediated allergic asthma, namely, serum OVA-specific IgE production, the development of airway hyperresponsiveness (AHR), and airway inflammation. Collectively, these findings indicate that the effectiveness of Sch B treatment against Th2-mediated allergic asthma was at least partially due to enhancement of DC induction of Treg cells, and Sch B can possibly be developed as an immunomodulatory adjuvant to treat allergic asthma.

Bardoxolone-Methyl Prevents Oxidative Stress-Mediated Apoptosis and Extracellular Matrix Degradation in vitro and Alleviates Osteoarthritis in vivo.

PurposeOxidative stress-induced chondrocyte apoptosis and extracellular matrix (ECM) degradation plays an important role in the progression of osteoarthritis (OA). Bardoxolone methyl (BM), a semisynthetic triterpenoid, exerts strong effect against oxidative stress. The purpose of the present study was to determine the effectiveness of bardoxolone-methyl (BM) in preventing oxidative stress-induced chondrocyte apoptosis and extracellular ECM degradation in vitro and the role of alleviating OA progression in vivo.MethodsOxidative damage was induced by 25 mM tert-butyl hydroperoxide (TBHP) for 24 h in rat chondrocytes. 0.025 and 0.05 µM bardoxolone-methyl (BM) were used in vitro treatment. Ex-vivo cartilage explant model was established to evaluate the effect of BM on oxidative stress-induced ECM degradation. The mouse OA model was induced by surgical destabilization of the medial meniscus.ResultsIn vitro, 0.025 and 0.05 µM BM reduced TBHP-induced excessive ROS generation, improved cell viability, increased malondialdehyde level and decreased superoxide dismutase level. 0.025 and 0.05 µM BM prevented TBHP-induced mitochondrial damage and apoptosis in chondrocytes BM activated heme oxygenase-1 (HO-1)/NADPH quinone oxidoreductase 1 (NOQ1) signaling pathway through targeting nuclear factor erythroid derived-2-related factor 2 (Nrf2). Additionally, BM treatment enhanced the expression levels of aggrecan and collagen II and inhibited the expression levels of matrix metalloproteinase 9 (MMP 9), MMP 13, Bax and cleaved-caspase-3. BM increased proteoglycan staining area and IOD value in ex vivo cultured experiment cartilage explants and improved the OARSI score, stands, max contact mean intensity, print area and duty cycle in mouse OA model.ConclusionBM prevented oxidative stress-induced chondrocyte apoptosis and ECM degradation in vitro and alleviated OA in vivo, suggesting that BM serves as an effective drug for treatment with OA.

Antioxidant Effects and Cytoprotective Potentials of Herbal Tea against H2O2-Induced Oxidative Damage by Activating Heme Oxygenase1 Pathway.

Herbal tea with antioxidant ingredients has gained increasing attention in the field of functional foods due to their amelioration potential in aging-related diseases. Wanglaoji herbal tea (WHT) is a kind of traditional beverage made from herbal materials. This study was performed to investigate its antioxidant activity and identify its protective effect on a H2O2-induced cell damage model. In this study, we identified six kinds of phenolic acids with antioxidant activity in WHT, among which rosmarinic acid had the highest content and the highest contribution ratio to the antioxidant activity of WHT. Moreover, compared with the H2O2-induced damage group, the WHT treatment group can significantly increase the viability of cells and decrease the ratio of senescence-associated β-galactosidase-positive cells, intracellular malondialdehyde levels, and the percentage of G1 phase. Furthermore, enrichment analysis of differentially expressed genes revealed that heme oxygenase1 (HMOX1) was a key gene for protective effect of WHT on oxidative stress-induced cell damage. Thus, WHT exerted protective effects not only by scavenging reactive oxygen species but also by inducing the expression of cytoprotective genes by activating the HMOX1 pathway, which showed that WHT had a potential of promoting health by reducing oxidative stress-induced cell damage.

Dexmedetomidine attenuates oxidative/nitrative stress in lung tissues of septic mice partly via activating heme oxygenase-1.

Excessive reactive oxygen/nitrogen species are considered to be one of the primary events that cause lung injury during sepsis. The present study aimed to determine whether dexmedetomidine (Dex) exhibits antioxidative and antinitrative effects on sepsis-induced lung injury and its effect on heme oxygenase-1 (HO-1) activation. The cecal ligation and puncture (CLP) mouse model was used, where male C57BL/6J mice were randomized into groups: Sham, CLP, Dex and Dex + zinc protoporphyrin (ZnPP). Following CLP or sham operation, intraperitoneal injections of 40 µg/kg Dex or saline were administered in the Dex + ZnPP group, intraperitoneal injections of ZnPP (40 mg/kg) were administered 1 h prior to the CLP operation. Subsequently, histopathological examination of the lungs and measurement of HO-1 activity in the lung, as well as oxidative and nitrative stress were determined 24 h following CLP. Dex significantly decreased the levels of oxidative and nitrative stress, as demonstrated by the decreased levels of malondialdehyde and nitrotyrosine, and the protein expression of inducible nitric oxide synthase, as well as increased superoxide dismutase in lung tissues. Also Dex inhibited the elevation of serum interleukin-6 and tumor necrosis factor-α and increased lung HO-1 activity. Furthermore, the effects of Dex were partially reverted by the HO-1 inhibitor ZnPP. In conclusion, Dex inhibited oxidative/nitrative stress in sepsis and attenuated sepsis-induced acute lung injury partially by increasing HO-1 activity.

Oxidative effects of lungs in Wistar rats caused by long-term exposure to four kinds of China representative chrysotile.

Chrysotile accounts for some 90% to 95% of all the asbestos used worldwide. Scientific evidences have shown that asbestos (including chrysotile) exposure is associated with increased rates of lung cancer, asbestosis, and mesothelioma. However, molecular mechanisms underlying the toxicity effects of chrysotile are not clear. This study evaluated the oxidative stress in chronic lung toxicity caused by the intratracheal instillation (IT) of four kinds China representative chrysotile once a month for 12months in Wistar rats. These results indicated that chrysotile exposure led to an obvious increase in lung mass and slowed the growth of body mass. Inflammation and fibrosis were observed by hematoxylin-eosin (HE) staining. Exposure to chrysotile significantly increased the accumulation of reactive oxygen species (ROS) and the level of lipid peroxidation and decreased antioxidant capacity in lung tissues. Furthermore, 1-6-month chrysotile exposure activated heme oxygenase-1 (HO-1) and heat shock protein 70 (HSP70) expression, whereas 12-month exposure caused significant decreases of two-factor expression levels in XK and MN groups when compared to negative control group. Therefore, our results suggested that chronic chrysotile pulmonary injury in Wistar rats is triggered by oxidative damage. Meanwhile, the oxidative damage of MN and XK was stronger than that of SSX and AKS, and the difference of oxidative damage in four chrysotile could have been brought by its properties, morphology, chemical composition, and particle size. With all the above mentioned in view, we hope that the revealed data in the experiment could contribute to the progress of further researches on the toxicity and mechanism of chrysotile.

Novel neurotoxic peptides from Protopalythoa variabilis virtually interact with voltage-gated sodium channel and display anti-epilepsy and neuroprotective activities in zebrafish

We previously reported a novel toxic peptide identified from the anthozoan Protopalythoa variabilis transcriptome which is homologous to a novel structural type of sodium channel toxin isolated from a parental species (Palythoa caribaeorum). The peptide was named, according to its homologous, as Pp V-shape α-helical peptide (PpVα) in the present study. Through molecular docking and dynamics simulation, linear and hairpin folded PpVα peptides were shown to be potential voltage-gated sodium channel blockers. Nowadays, sodium channel blockers have been the mainstream of the pharmacological management of epileptic seizures. Also, sodium channel blockers could promote neuronal survival by reducing sodium influx and reducing the likelihood of calcium importation resulting in suppressing microglial activation and protecting dopaminergic neurons from degeneration. The folded PpVα peptide could decrease pentylenetetrazol (PTZ)-induced c-fos and npas4a expression level leading to reverse PTZ-induced locomotor hyperactivity in zebrafish model. In vitro, the folded PpVα peptide protected PC12 cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via activating heme oxygenase-1 (HO-1) and attenuating inducible nitric oxide synthase (iNOS) expression. In vivo, PpVα peptide suppressed the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish and, importantly, prevented the 6-OHDA-induced excessive ROS generation and subsequent dopaminergic neurons loss. This study indicates that the single S-S bond folded PpVα peptide arises as a new structural template to develop sodium channel blockers and provides an insight on the peptide discovery from cnidarian transcriptome to potentially manage epilepsy and neurodegenerative disorders.

Berberine ameliorates renal injury in a rat model of D‐galactose-induced aging through a PTEN/Akt-dependent mechanism

This study aimed to investigate the protective effects of berberine (BBR) against D-galactose (D-gal)-induced renal aging in rats, pointing to its ability to modulate phosphatase and tensin homolog deleted on chromosome ten (PTEN)/Akt signalling, and to attenuate oxidative stress, inflammation and apoptosis. Renal aging was induced by subcutaneous injection of D-gal for six consecutive weeks along with simultaneous oral administration of BBR and compared to control rats and rats received individual doses of either drug. BBR treatment significantly reduced the serum levels of urea and creatinine, retrieved the alterations in kidney histopathology, and restored redox balance evidenced by alleviations of the level of malondialdehyde, 8-hydroxy-2′-deoxyguanosine and activating heme oxygenase-1 enzyme. Moreover, it markedly reduced the serum levels of pro-inflammatory mediators, along with down-regulation of PTEN expression, enhanced Akt activity, as well as significantly higher immunostaining of the anti-apoptotic marker (Bcl-2). These findings hold a great promise for the use of BBR as a protecting agent against renal aging.

Acceleration of pancreatic tumorigenesis under immunosuppressive microenvironment induced by Reg3g overexpression

Reg3g is a potential risk for pancreatic ductal adenocarcinoma (PDAC). We previously demonstrated that Reg3g promoted pancreatic carcinogenesis via a STAT3 signaling pathway in a murine model of chronic pancreatitis. Whether the immune response is involved in tumorigenesis induced by Reg3g remains unknown. In this study, Reg3g-regulated tumor immunity was evaluated in tumor-implanted murine models, immune cells, and tumor microenvironment. In mice that had been orthotopically or ectopically implanted with Panc02 cells, Reg3g overexpression increased EGFR and Ki67, diminished MHC-I and caspase-3 expression, and accelerated growth of tumors. By interacting with PD-1/PD-L1, Reg3g also promoted differentiation of Tregs and recruitment of MDSC, retarded maturation of DCs and inactivation of CD8+ T cells, and suppressed cross-priming of CD8+ T-cell responses by DCs in tumor-bearing mice. Knockdown of Reg3g delayed tumor development in normal mice, but not in CD8+ T-cell-deficient mice. In vitro, Reg3g upregulated EGFR in DCs, activated heme oxygenase-1 (Hmox1) involved JAK2/STAT3 signaling, raised levels of Th2 cytokines in and suppressed maturation of DCs, and enhanced tumor cell proliferation. These results reveal a novel role of Reg3g as an immunosuppressive promoter that weakens tumor-specific antigenicity and suppresses antitumor effects of CD8+ T cells in a murine model of pancreatic cancer. Reg3g produces these effects by activating the JAK2/STAT3 signaling pathway in DCs, triggering the generation of an immunosuppressive tumor microenvironment.

Lithium ameliorates rat spinal cord injury by suppressing glycogen synthase kinase-3β and activating heme oxygenase-1.

Glycogen synthase kinase (GSK)-3β and related enzymes are associated with various forms of neuroinflammation, including spinal cord injury (SCI). Our aim was to evaluate whether lithium, a non-selective inhibitor of GSK-3β, ameliorated SCI progression, and also to analyze whether lithium affected the expression levels of two representative GSK-3β–associated molecules, nuclear factor erythroid 2-related factor-2 (Nrf-2) and heme oxygenase-1 (HO-1) (a target gene of Nrf-2). Intraperitoneal lithium chloride (80 mg/kg/day for 3 days) significantly improved locomotor function at 8 days post-injury (DPI); this was maintained until 14 DPI (P<0.05). Western blotting showed significantly increased phosphorylation of GSK-3β (Ser9), Nrf-2, and the Nrf-2 target HO-1 in the spinal cords of lithium-treated animals. Fewer neuropathological changes (e.g., hemorrhage, inflammatory cell infiltration, and tissue loss) were observed in the spinal cords of the lithium-treated group compared with the vehicle-treated group. Microglial activation (evaluated by measuring the immunoreactivity of ionized calcium-binding protein-1) was also significantly reduced in the lithium-treated group. These findings suggest that GSK-3β becomes activated after SCI, and that a non-specific enzyme inhibitor, lithium, ameliorates rat SCI by increasing phosphorylation of GSK-3β and the associated molecules Nrf-2 and HO-1.

A novel nuclear factor erythroid 2-related factor 2 (Nrf2) activator RS9 attenuates brain injury after ischemia reperfusion in mice

Recanalization of occluded vessels leads to ischemia–reperfusion injury (IRI), with oxidative stress as one of the main causes of injury, despite the fact that recanalization therapy is the most effective treatment for ischemic stroke. The nuclear factor erythroid 2-related factor 2 (Nrf2) is one of the transcription factors which has an essential role in protection against oxidative stress. RS9 is a novel Nrf2 activator obtained from bardoxolone methyl (BARD), an Nrf2 activator that has already been tested in a clinical trial, using a biotransformation technique. RS9 has been reported to lead to higher Nrf2 activation and less cytotoxicity than BARD. In this study, we investigated the effects of RS9 on IRI. Mice were intraperitoneally treated immediately after 2h of transient middle cerebral artery occlusion (MCAO) with a vehicle solution or 0.2mg/kg of RS9. Post-onset treatment of RS9 attenuated the infarct volume and improved neurological deficits 22h after reperfusion. RS9 activated Nrf2 2 and 6h after reperfusion and activated heme oxygenase-1 at 6 and 22h after reperfusion. RS9 also attenuated the phosphorylation of NF-κB p65 2 and 6h after reperfusion. Finally, RS9 improved the survival rate and neurological deficits 7days after MCAO. Our results suggest that the activation of Nrf2 by RS9 has a neuroprotective effect, mediated by attenuating both oxidative stress and neuroinflammation, and that RS9 is an effective therapeutic candidate for the treatment of IRI.

Hydrogen-rich saline ameliorates the expressions of cytokines in a rat model of inflammatory pain through activating heme oxygenase-1

Objective To investigate whether hydrogen-rich saline could ameliorate the expressions of cytokines in a rat model of inflammatory pain through activating heme oxygenase-1 (HO-1). Methods Eighty male Sprague-Dawley (SD) rats, weighing 180~220 g, were randomly divided into five groups (n=16 in each group): Control group (Con), inflammation pain group (CFA), inflammation pain+ hydrogen-rich saline group (CFA+ H2), inflammation pain+ HO-1 inhibitor Znpp-IX group (CFA+ Znpp-IX), and inflammation pain+ hydrogen-rich saline+ HO-1inhibitor Znpp-IX group (CFA+ H2+ Znpp-IX). The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were tested on days 1 (T1), 2 (T2), 3 (T3), 5 (T4), 7 (T5), and 14 (T6) after inflammation pain. The expressions of spinal HO-1 mRNA and protein were measured with real-time quantitative polymerase chain reaction (RT-PCR) and Western blot, and spinal inflammatory cytokines were measured with enzyme-linked immunosorbent assay (ELISA) on day 7 after inflammatory pain. Results Compared to Con group, MWT and TWL were significantly reduced; the spinal HO-1 mRNA level, protein expression and activity were increased; and the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-10 in spinal tissues were also increased in CFA group (P<0.05). Compared to CFA group, MWT and TWL were significantly increased; the spinal HO-1 mRNA level, protein expression and activity were further increased; and the levels of TNF-α, IL-1β and IL-6 were decreased, while IL-10 was further increased in CFA+ H2 group (P<0.05). Compared to CFA+ H2 group, MWT and TWL were decreased; the spinal HO-1 mRNA level, protein expression and activity were decreased; and the levels of TNF-α, IL-1β and IL-6 in spinal tissue were significantly increased, while IL-10 was decreased in CFA+ H2+ Znpp-IX group (P<0.05). Conclusions Hydrogen-rich saline can ameliorate the mechanical and thermal allodynia in a rat model of inflammatory pain, and reduce the release of inflammatory cytokines via activating HO-1. Key words: Hydrogen/PD; Heme oxygenase-1/ME; Pain/ME/TH; Tumor necrosis factor-alpha/ME; Interleukin-6/ME; Interleukin-1/ME; Interleukin-10/ME

Tussilagone inhibits dendritic cell functions via induction of heme oxygenase-1.

Sesquiterpenoid tussilagone (TUS) has a variety of pharmacological activities, such as anti-oxidant, anti-cancer, and anti-inflammatory activities. In this study, we investigated the effects of TUS on dendritic cell (DC) functions and the underlying mechanisms. TUS inhibited lipopolysaccharide (LPS)-induced activation of DCs, as shown by decrease in surface molecule expression, cytokine production, cell migration, and allo-T cell activation. In addition, TUS inhibited LPS-induced activation of NF-κB, MAPKs, and IRF-3 signalings in DCs, although it did not directly affect kinase activities of IRAK1/4, TAK1, and IKK, which suggests that TUS might indirectly inhibit TLR signaling in DCs. As a critical mechanism, we showed that TUS activated heme oxygenase-1 (HO-1), which degrades heme to immunosuppressive products, such as carbon monoxide and bilirubin. HO-1 inhibitor reversed the inhibitory activity of TUS in DCs. In conclusion, this study suggests that TUS inhibits DC function through the induction of HO-1.

Tussilagone inhibits dendritic cell functions via up-regulating heme oxygenase-1 expression (THER5P.833)

Abstract Sesquiterpenoid tussilagone (TUS) has a variety of pharmacological activities. In this study, the effects of TUS on dendritic cell (DC) functions and the underlying mechanisms were investigated. TUS inhibited lipopolysaccharide (LPS)-induced activation of DCs, which was proved by the decreases of surface molecule expression, cytokine production, antigen uptake, migration, and allogenic T cell activation. TUS inhibited LPS-induced activation of NF-kB, MAPKs, and IRF-3 signalings. However, TUS did not directly inhibit kinase activities of IRAK4, IRAK1, TAK1 and IKK, suggesting that signaling molecules downstream from toll-like receptor (TLR) 4 might not be the direct targets of TUS. Instead, TUS activated heme oxygenase-1 (HO-1), which was a rate-limiting enzyme involved in heme degradation to immunosuppressive products, such as carbon monoxide and biliverdin. HO-1 antagonist reversed the inhibitory activity of TUS in DCs. Taken together, this study suggests that TUS might be an inducer of HO-1, which indirectly inhibits TLR signalings and functional activation of LPS-treated DCs

Overexpression of Smad7 suppressed ROS/MMP9-dependent collagen synthesis through regulation of heme oxygenase-1

We previously reported that AngiotensinII receptor blocker effectively inhibited TGF-β1-mediated epithelial-to-mesenchymal transition progress through regulating Smad7. However, the underlying mechanism by which Smad7 exerted in regulating MMP9 and fibrogenic response has not been fully elucidated. In the current study, we proved that NADPH p47(phox)-dependent reactive oxygen species (ROS) production contributed to MMP9 activation and collagen expression, which was suppressed by transfecting pcDNA3-Smad7 in cardiac fibroblasts. The effect of Smad7 overexpression on MMP9 activity and collagen expression was further reversed by adding H2O2 (10μmol/L). In contrast, knockdown of Smad7 caused the enhanced collagen synthesis in cardiac fibroblasts, which was also reversed by treating cells with a ROS inhibitor, YCG063 (2μmol/L). Further investigation showed that Smad7 regulated NADPH-mediated ROS production through activating Heme oxygenase-1 (HO-1). Meanwhile, the intercellular level of bilirubin (product of hemin) and nitric oxide (NO) in cell supernatant were not significantly increased in cells treated with AngII or transfected with Smad7. Knockdown of HO-1 in Smad7-overexpressed cardiac fibroblasts or cells pretreated with SnPP IX, a competitive inhibitor of HO-1 activity, resulted in increased productions of ROS and NADPH p47(phox), and abolished the inhibitory effects of Smad7 on MMP9 activity and collagen expression. Our results indicated that HO-1 might be critically involved in Smad7-mediated regulation of MMP9 activity and fibrogenic genes expression via antagonizing the enhanced myocardial oxidative stress.

Ischemic postconditioning downregulates Egr-1 expression and attenuates postischemic pulmonary inflammatory cytokine release and tissue injury in rats

BackgroundThe early growth response-1 (Egr-1) gene is upregulated after an ischemia–reperfusion (IR) challenge and upregulates target genes, such as proinflammatory cytokines. Ischemic postconditioning (IPostC) attenuates lung IR injury and reduces the systemic inflammatory response by activating heme oxygenase-1 (HO-1). However, the role of Egr-1 in IPostC protection against lung IR injury and inflammation and its interplay with HO-1 in IPostC protection is unknown. Materials and methodsSprague-Dawley rats or cultured A549 cells were subjected to IR or hypoxia/reoxygenation with or without IPostC or hypoxic postconditioning in the presence or absence of Egr-1 inhibition using Egr-1 antisense oligodeoxyrinonucleotide or Egr-1 small interfering RNA transfection. Lung injury was assessed by measuring the lung wet/dry weight ratio, histologic change, and malondialdehyde content. The amount of lactate dehydrogenase release in culture medium was detected to evaluate cell injury. The protein expression of Egr-1, interleukin (IL)-1β, and HO-1 was assessed by Western blot. ResultsInhibition of Egr-1 significantly attenuated lung IR injury and the inflammation response caused by IR or hypoxia/reoxygenation, as shown by the alleviated lung pathologic changes, decreased pulmonary malondialdehyde content, wet/dry ratio, reduced release of the cytokines tumor necrosis factor-α, IL-6, and IL-8 in the bronchoalveolar lavage, and reduced Egr-1, IL-1β, and HO-1 protein expression and HO-1 activity. IPostC or hypoxic postconditioning reduced the postischemic Egr-1 expression and conferred similar protection against lung IR injury as Egr-1 inhibition. ConclusionsEgr-1 plays an important role in regulating the HO-1 production induced by IR or hypoxia/reoxygenation. Thus, downregulation of Egr-1 expression might represent one of the major mechanisms whereby IPostC confers protection against pulmonary IR insult.

Heme oxygenase-1 mediates the inhibitory actions of brazilin in RAW264.7 macrophages stimulated with lipopolysaccharide

Brazilin, the main constituent of Caesalpinia sappan L., is a natural red pigment that has been reported to possess anti-inflammatory properties. This study aimed to identify a novel anti-inflammatory mechanism of brazilin. We found that brazilin did not cause cytotoxicity below 300 μM, and activated heme oxygenase-1 (HO-1) protein synthesis in a concentration-dependent manner at 10–300 μM in RAW264.7 macrophages without affecting mRNA transcription of HO-1. Additionally, brazilin increased bilirubin production and HO-1 activity in RAW264.7 macrophages. In lipopolysaccharide (LPS)-stimulated macrophages, brazilin suppressed the release of nitric oxide (NO), prostaglandin E 2 (PGE 2), interleukin (IL)-1β and tumor necrosis factor-α (TNF-α), and reduced the expression of inducible nitric oxide synthase (iNOS). A specific inhibitor of HO-1, Zn(II) protoporphyrin IX, blocked the suppression of NO production, cytokines release and iNOS expression by brazilin. These results suggest that brazilin possesses anti-inflammatory actions in macrophages and works through a novel mechanism involving the action of HO-1.