β-Glucan from Saccharomyces cerevisiae alleviates oxidative stress in LPS-stimulated RAW264.7 cells via Dectin-1/Nrf2/HO-1 signaling pathway

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.     

LL202 ameliorates colitis against oxidative stress of macrophage by activation of the Nrf2/HO-1 pathway

LL202, a newly synthesized flavonoid derivative, has been confirmed to inhibit the mitogen-activated protein kinase pathway and activation protein-1 activation in monocytes; however, the anti-inflammatory mechanism has not been clearly studied. Uncontrolled overproduction of reactive oxygen species (ROS) has involved in oxidative damage of inflammatory bowel disease. In this study, we investigated that LL202 reduced lipopolysaccharide (LPS)-induced ROS production and malondialdehyde levels and increased superoxide dismutase, glutathione, and total antioxidant capacity in RAW264.7 cells. Mechanically, LL202 could upregulate heme oxygenase-1 (HO-1) via promoting nuclear translocation of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) to regulate LPS-induced oxidative stress in macrophages. In vivo, we validated the role of LL202 in dextran sulfate sodium- and TNBS-induced colitis models, respectively. The results showed that LL202 decreased the proinflammatory cytokine expression and regulated colonic oxidative stress by activating the Nrf2/HO-1 pathway. In conclusion, our study showed that LL202 exerts an anti-inflammatory effect by enhancing the antioxidant capacity of the Nrf2/HO-1 pathway to macrophages.     

Anti-oxidizing effect of the dichloromethane and hexane fractions from Orostachys japonicus in LPS-stimulated RAW 264.7 cells via upregulation of Nrf2 expression and activation of MAPK signaling pathway

Orostachys japonicus shows various biological activities. However, the molecular mechanisms remain unknown in LPS-stimulated macrophages. Here, we investigated the anti-oxidizing effect of the dichloromethane (DCM) and hexane fractions from O. japonicus (OJD and OJH) against oxidative stress in RAW 264.7 cells stimulated by LPS. OJD and OJH significantly increased the expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Additionally, it was found that the expression of HO-1 was stimulated by Nrf2 activated via degradation of Keap1. ERK and p38 inhibitors repressed HO-1 induced by OJD and OJH in LPS-stimulated cells, respectively. In conclusion, these results suggest that OJD and OJH may block oxidative damage stimulated by LPS, via increasing the expression of HO-1 and Nrf2, and MAPK signaling pathway. [BMB Reports 2014; 47(2): 98-103]     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

Protective effects of aminoethyl-chitooligosaccharides against oxidative stress in mouse macrophage RAW 264.7 cells

The aim of this study is to investigate the inhibitory effects of aminoethyl-chitooligosaccharides (AE-COS) on oxidative stress in mouse macrophages (RAW 264.7 cells). The inhibitory effects of AE-COS on DNA and protein oxidation were studied in RAW 264.7 cells. Furthermore, free radical scavenging effect of AE-COS were determined in RAW264.7 cells by 2',7'-dichlorofluorescein (DCF) intensity and intracellular glutathione (GSH) level. AE-COS also inhibited myeloperoxidase (MPO) activity in human myeloid cells (HL-60). These results suggest that AE-COS acts as a potential free radical scavenger in RAW 264.7 cells.     

PPARγ stabilizes HO-1 mRNA in monocytes/macrophages which affects IFN-β expression

NADPH oxidase activation in either RAW264.7 cells or peritoneal macrophages (PM) derived from PPARγ wild-type mice increased reactive oxygen species (ROS) formation, caused PPARγ activation, heme oxygenase-1 (HO-1) induction, and concomitant IFN-β expression. In macrophages transduced with a dominant negative (d/n) mutant of PPARγ (RAW264.7 AF2) as well as PPARγ negative PM derived from Mac-PPARγ-KO mice, NADPH oxidase-dependent IFN-β expression was attenuated. As the underlying mechanism, we noted decreased HO-1 mRNA stability in RAW264.7 AF2 cells as well as PPARγ negative PM, compared to either parent RAW264.7 cells or wild-type PM. Assuming mRNA stabilization of HO-1 by PPARγ we transfected macrophages with a HO-1 3′-UTR reporter construct. The PPARγ agonist rosiglitazone significantly up-regulated luciferase expression in RAW264.7 cells, while it remained unaltered in RAW264.7 AF2 macrophages. Deletion of each of two AU-rich elements in the 3′-UTR HO-1 decreased luciferase activity in RAW264.7 cells. Using LPS as a NADPH oxidase activator, PM from Mac-PPARγ-KO mice showed a decreased HO-1 mRNA half-life in vitro and in vivo compared to PPARγ wild-type mice. These data identified a so far unappreciated role of PPARγ in stabilizing HO-1 mRNA, thus, contributing to the expression of the HO-1 target gene IFN-β.     

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress

AimsProtection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms.Main methodsThe cytoprotective effect of chamomile was examined on H₂O₂-induced cellular stress in RAW 264.7 murine macrophages.Key findingsRAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H₂O₂. Treatment with 50 μM H₂O₂ for 6 h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10–20 μg/mL for 16 h followed by H₂O₂ treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus.SignificanceThese molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties.     

CME‐1, a novel polysaccharide,suppresses iNOS expression in lipopolysaccharide‐stimulated macrophages through ceramide‐initiated protein phosphatase 2A activation

CME‐1, a novel water‐soluble polysaccharide purified from Ophiocordyceps sinensis mycelia, has anti‐oxidative, antithrombotic and antitumour properties. In this study, other major attributes of CME‐1, namely anti‐inflammatory and immunomodulatory properties, were investigated. Treating lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells with CME‐1 concentration‐dependently suppressed nitric oxide formation and inducible nitric oxide synthase (iNOS) expression. In the CME‐1‐treated RAW 264.7 cells, LPS‐induced IκBα degradation and the phosphorylation of p65, Akt and mitogen‐activated protein kinases (MAPKs), including extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38, were reduced. Treatment with a protein phosphatase 2A (PP2A)‐specific inhibitor, significantly reversed the CME‐1‐suppressed iNOS expression; IκBα degradation; and p65, Akt and MAPK phosphorylation. PP2A activity up‐regulation and PP2A demethylation reduction were also observed in the cells. Moreover, CME‐1‐induced PP2A activation and its subsequent suppression of LPS‐activated RAW 264.7 cells were diminished by the inhibition of ceramide signals. LPS‐induced reactive oxygen species (ROS) and hydroxyl radical formation were eliminated by treating RAW 264.7 cells with CME‐1. Furthermore, the role of ceramide signalling pathway and anti‐oxidative property were also demonstrated in CME‐1‐mediated inhibition of LPS‐activated primary peritoneal macrophages. In conclusion, CME‐1 suppressed iNOS expression by up‐regulating ceramide‐induced PP2A activation and reducing ROS production in LPS‐stimulated macrophages. CME‐1 is a potential therapeutic agent for treating inflammatory diseases.     

Anti-Inflammatory Effects of New Naphthyridine from Sponge Aaptos suberitoides in LPS-Stimulated RAW 264.7 Macrophages via Regulation of MAPK and Nrf2 Signaling Pathways

Two new naphthyridine compounds, 4-methoxycarbonyl-5-oxo-1,6-naphthyridine ( 1 ) and 5-methoxycarbonyl-4-oxo-1,6-naphthyridine ( 2 ) were obtained from the MeOH extracts of sponge Aaptos suberitoides. Their structures were determined by spectroscopic methods, including HR-ESI-MS, 1D-NMR (¹H-NMR, ¹³C-NMR), 2D-NMR (COSY, HSQC, HMBC). The structure of compound 1 was further confirmed via single crystal X-ray diffraction analysis. Compound 1 was found to reduce NO production in LPS-induced RAW 264.7 macrophages with IC₅₀ value of 0.15 mM. In addition, it decreased the mRNA expression levels of pro-inflammatory mediators, such as the tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) in LPS-induced macrophages. It also decreased the protein expression of iNOS and COX-2 in LPS-induced macrophages. Mechanistic studies further revealed that compound 1 inhibited the mitogen-activated protein kinase (MAPK), and activated the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathways in LPS-induced RAW 264.7 macrophages.     

Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin

The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.     

Macrophage-derived heme-oxygenase-1: expression, regulation, and possible functions in skin repair.

BACKGROUND: Expression and enzymatic activity of heme oxygenase (HO) has been implicated in the development, as well as in the resolution, of inflammatory conditions. Because inflammation is central to tissue repair, we investigated the presence and potential functions of HO in an excisional model of normal and diabetes-impaired wound repair in mice. MATERIALS AND METHODS: Expression of HO-1 during cutaneous healing was analyzed by RNase protection assay, Western blot, and immunohistochemical techniques in a murine model of excisional repair. Furthermore, we determined HO-1-dependent release of proinflammatory cytokines from RAW 264.7 macrophages by enzyme-linked immunosorbent assay (ELISA). RESULTS: Upon injury, we observed a rapid and strong increase in HO-1 mRNA and protein levels at the wound site. By contrast to normal repair, late stages of diabetes-impaired repair were associated with elevated HO-1 expression. Besides a few keratinocytes of the hyperproliferative epithelium, immunohistochemistry revealed infiltrating macrophages as the predominant and major source of HO-1 at the wound site. In vitro studies demonstrated the potency of exogenous and also endogenous nitric oxide (NO) to strongly induce HO-1 expression in RAW 264.7 macrophages. However, L-NIL-mediated enzymatic inhibition of inducible NO-synthase (iNOS) at the wound site in vivo was not paralleled by decreased HO-1 levels. In vitro inhibition of HO-1 enzymatic activity by tin protoporphyrin IX (SnPPIX) in RAW 264.7 macrophages markedly attenuated tumor necrosis factor-alpha (TNF-alpha), but strongly increased interleukin-1beta (IL-1beta) release in RAW 264.7 macrophages in vitro. CONCLUSIONS: The observed injury-mediated increase in HO-1 mRNA and protein at the wound site was due to infiltrating HO-1 expressing monocytic cells. Macrophage-derived HO-1 expression was not under regulatory control by NO in skin repair. We provide evidence that HO-1 might exert a regulatory role in macrophage-derived cytokine release.     

The influence of wine polyphenols on reactive oxygen and nitrogen species production by murine macrophages RAW 264.7

The aim was to study the antioxidant properties of four wine polyphenols (flavonoids catechin, epicatechin, and quercetin, and hydroxystilbene resveratrol). All three flavonoids exerted significant and dose-dependent scavenging effects against peroxyl radical and nitric oxide in chemical systems. The scavenging effect of resveratrol was significantly lower. All polyphenols decreased production of reactive oxygen species (ROS) by RAW264.7 macrophages. Only quercetin quenched ROS produced by lipopolysaccharide-stimulated RAW264.7 macrophages incubated for 24 h with polyphenols. Quercetin and resveratrol decreased the release of nitric oxide by these cells in a dose-dependent manner which corresponded to a decrease in iNOS expression in the case of quercetin. In conclusion, the higher number of hydroxyl substituents is an important structural feature of flavonoids in respect to their scavenging activity against ROS and nitric oxide, while C-2,3 double bond (present in quercetin and resveratrol) might be important for inhibition of ROS and nitric oxide production by RAW 264.7 macrophages.     

Selective activation of adrenergic beta1 receptors induces heme oxygenase 1 production in RAW264.7 cells

We hypothesized that catecholamines through beta-adrenoceptor might modulate macrophage function. We showed that isoproterenol concentration-dependently induced HO-1 production through beta(1)-but not beta(2)-adrenoceptor. Production was increased by forskolin and inhibited by pretreatment with the PKA inhibitor, H-89. Furthermore, induction of HO-1 by isoproterenol effectively protected RAW264.7 cells from effects of glucose oxidase treatment, which was abrogated either by HO-1 inhibitor, ZnPP IX and beta-adenoceptor antagonist, propranolol. Thus, stimulation of HO-1 production through beta(1)-adenoceptors, and via the PKA pathways by isoproterenol, can enable RAW264.7 cells to resist oxidant stress, suggesting that catecholamine hormones may be necessary, at least, to maximize defending role of macrophages.     

Induction of heme oxygenase-1 expression in macrophages by diesel exhaust particle chemicals and quinones via the antioxidant-responsive element

Diesel exhaust particles (DEP) contain organic chemicals that contribute to the adverse health effects of inhaled particulate matter. Because DEP induce oxidative stress in the lung and in macrophages, effective antioxidant defenses are required. One type of defense is through the expression of the antioxidant enzyme, heme oxygenase I (HO-1). HO-1 as well as phase II detoxifying enzymes are induced via antioxidant response elements (ARE) in their promoters of that gene. We show that a crude DEP total extract, aromatic and polar DEP fractions, a benzo(a)pyrene quinone, and a phenolic antioxidant induce HO-1 expression in RAW264.7 cells in an ARE-dependent manner. N-acetyl cysteine and the flavonoid, luteolin, inhibited HO-1 protein expression. We also demonstrate that the same stimuli induce HO-1 mRNA expression in parallel with the activation of the SX2 enhancer of that gene. Mutation of the ARE core, but not the overlapping AP-1 binding sequence, disrupted SX2 activation. Finally, we show that biological agents, such as oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, could also induce HO-1 expression via an ARE-dependent mechanism. Prior induction of HO-1 expression, using cobalt-protoporphyrin, protected RAW264.7 cells against DEP-induced toxicity. Taken together, these data show that HO-1 plays an important role in cytoprotection against redox-active DEP chemicals, including quinones.     

Cytosolic NADP(+)-dependent isocitrate dehydrogenase protects macrophages from LPS-induced nitric oxide and reactive oxygen species

Macrophages activated by microbial lipopolysaccharides (LPS) produce bursts of nitric oxide and reactive oxygen species (ROS). Redox protection systems are essential for the survival of the macrophages since the nitric oxide and ROS can be toxic to them as well as to pathogens. Using suppression subtractive hybridization (SSH) we found that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is strongly upregulated by nitric oxide in macrophages. The levels of IDPc mRNA and of the corresponding enzymatic activity were markedly increased by treatment of RAW264.7 cells or peritoneal macrophages with LPS or SNAP (a nitric oxide donor). Over-expression of IDPc reduced intracellular peroxide levels and enhanced the survival of H2O2- and SNAP-treated RAW264.7 macrophages. IDPc is known to generate NADPH, a cellular reducing agent, via oxidative decarboxylation of isocitrate. The expression of enzymes implicated in redox protection, superoxide dismutase (SOD) and catalase, was relatively unaffected by LPS and SNAP. We propose that the induction of IDPc is one of the main self-protection mechanisms of macrophages against LPS-induced oxidative stress.     

Anti-inflammatory effect of heme oxygenase 1: glycosylation and nitric oxide inhibition in macrophages

Flavonoids including the aglycones, hesperetin (HT; 5,7,3'-trihydroxy-4'-methoxy-flavanone), and naringenin (NE; 5,7,4'-trihydroxy flavanone) and glycones, hesperidin (HD; 5,7,3'-trihydroxy-4'-methoxy-flavanone 7-rhamnoglucoside) and naringin (NI; 5,7,4'-trihydroxy flavanone 7-rhamno glucoside), were used to examine the importance of rutinose at C7 on the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced nitric oxide production in macrophages. Both HT and NE, but not their respective glycosides HD and NI, induced heme oxygenase 1 (HO-1) protein expression in the presence or absence of LPS and showed time and dose-dependent inhibition of LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW264.7, J774A.1, and thioglycolate-elicited peritoneal macrophages. Additive inhibitory effect of an HO-1 inducer hemin and NE or NI on LPS-induced NO production and iNOS expression was identified, and HO enzyme inhibitor tin protoporphyrin (SnPP) attenuated the inhibitory effects of HT, NE, and hemin on LPS-induced NO production. Both NE and HT showed no effect on iNOS mRNA and protein stability in RAW264.7 cells. Removal of rutinose at C7 of HD and NI by enzymatic digestion using hesperidinase (HDase) and naringinase (NIase) produce inhibitory activity on LPS-induced NO production, according to the production of the aglycones, HT and NE, by high-performance liquid chromatography (HPLC) analysis. Furthermore, the amount of NO produced by LPS or lipoteichoic acid (LTA) was significantly reduced in HO-1-overexpressing cells (HO-1/RAW264.7) compared to that in parental cells (RAW264.7). Results of the present study provide scientific evidence to suggest that rutinose at C7 is a negative moiety in flavonoid inhibition of LPS-induced NO production, and that HO-1 is involved in the inhibitory mechanism of flavonoids on LPS-induced iNOS and NO production.