4-methoxycinnamyl p-coumarate reduces neuroinflammation by blocking NF-κB, MAPK, and Akt/GSK-3ß pathways and enhancing Nrf2/HO-1 signaling cascade in microglial cells.

The active compound, 4-methoxycinnamyl p-coumarate (MCC), derived from the rhizome of Etlingera pavieana (Pierre ex Gagnep) R.M.Sm., has been shown to exert anti-inflammatory effects in several inflammatory models. However, its effects on microglial cells remain elusive. In the current study, we aimed to investigate the anti-neuroinflammatory activities of MCC and determine the potential mechanisms underlying its action on lipopolysaccharide (LPS)-induced BV2 microglial cells. Our results revealed that MCC significantly reduced the secretion of nitric oxide (NO) and prostaglandin E2, concomitantly inhibiting the expression levels of inducible NO synthase and cyclooxygenase-2 mRNA and proteins. Additionally, MCC effectively decreased the production of reactive oxygen species in LPS-induced BV2 microglial cells. MCC also attenuates the activation of NF-κB by suppressing the phosphorylation of IκBα and NF-κB p65 subunits and by blocking the nuclear translocation of NF-κB p65 subunits. Furthermore, MCC significantly reduced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK-3ß). In addition, MCC markedly increased the expression of heme oxygenase-1 (HO-1) by upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Collectively, our findings suggest that the anti-inflammatory activities of MCC could be attributed to its ability to suppress the activation of NF-κB, MAPK, and Akt/GSK-3ß while enhancing that of Nrf2-mediated HO-1. Accordingly, MCC has promising therapeutic potential to treat neuroinflammation-related diseases.

Acute and subchronic oral toxicity assessment of extract from Etlingera pavieana rhizomes.

In Southeast Asia, the rhizome of Etlingera pavieana is commonly consumed and parts of the rhizomes have been used as a medicine for the treatment of several disorders. Its pharmacological effects have previously been reported. However, its potential toxicity has not been described. This study aimed to evaluate in vivo toxicity of E. pavieana rhizome extract (EPE) in Sprague Dawley rats. Acute toxicity testing of EPE at a single dose of 2,000 mg/kg produced no toxic effects in female rats after 14 days of treatment. Subchronic toxicity testing showed that all doses of EPE (500, 1,000, and 2,000 mg/kg/day) produced no sign of toxicity during 90 days of treatment. All biochemical and hematological values were within normal ranges. There were no significant histopathological differences in the internal organs among the tested groups. Therefore, the no-observed-adverse-effect level of EPE was 2,000 mg/kg/day in both male and female rats, thereby confirming the safety of EPE for use in traditional medicines.

Enhanced production and immunomodulatory activity of levan from the acetic acid bacterium, Tanticharoenia sakaeratensis.

Levan is a fructose polymer with ß-(2 â†’ 6) glycosidic linkages. It is produced by several microorganisms, and due to its potential biotechnological and industrial applications, various levan-producing bacteria with different levels of production efficiencies have been reported. We investigated the levan-producing ability of the acetic acid bacterium, Tanticharoenia sakaeratensis. The exopolysaccharides produced by the bacterium under a sucrose environment were characterized as levan by FT-IR, and 1H and 13C NMR. The molecular weight of levan thus produced range from 1.0 × 105-6.8 × 105 Da. The maximum yield of levan from T. sakaeratensis is 24.7 g·L-1 in a liquid medium containing 20% (w/v) sucrose and incubated at 37 °C, 250 RPM for 35 h. The levan produced by T. sakaeratensis can promote nitric oxide production in RAW264.7 macrophage cells in a concentration-dependent manner, suggesting it has immunomodulatory effects. Our study reveals that T. sakaeratensis can be potentially employed as a new source of levan for industrial applications.

Etlingera pavieana extract attenuates TNF-α induced vascular adhesion molecule expression in human endothelial cells through NF-κB and Akt/JNK pathways.

The aim of this study was to determine whether ethanol extracts of Etlingera pavieana rhizomes (EPE) can inhibit the expression of ICAM-1 and VCAM-1 in TNF-α-stimulated human vascular endothelial cells. EPE significantly reduced ICAM-1 and VCAM-1 expression in a concentration-dependent manner. EPE also suppressed phospho-IκB level and nuclear translocation of NF-κB. EPE significantly inhibited phosphorylation of JNK and c-Jun, a major component of AP-1, but had no effects on ERK and p38 MAPK pathways. Akt phosphorylation was increased in the presence of EPE, and wortmannin and SP600125 reversed the inhibitory effects of EPE on ICAM-1 and VCAM-1 expression. Furthermore, the active EPE constituents 4-methoxycinnamyl p-coumarate and trans-4-methoxycinnamaldehyde attenuated TNF-α-induced expression of ICAM-1 and VCAM-1. Taken together, our data indicate that EPE protects against vascular inflammation in endothelial cells, in part via NF-κB and Akt/JNK signalings. In future studies, E. pavieana may be developed as a therapeutic agent or dietary supplement for treating and preventing inflammatory diseases.

Anti-inflammatory effect of trans-4-methoxycinnamaldehyde from Etlingera pavieana in LPS-stimulated macrophages mediated through inactivation of NF-κB and JNK/c-Jun signaling pathways and in rat models of acute inflammation.

Trans-4-methoxycinnamaldehyde (MCD) was isolated from the rhizomes of Etlingera pavieana (Pierre ex Gagnep.) R.M.Sm. MCD shows anti-inflammatory effects. However, the molecular mechanism underlying its anti-inflammatory action has not been described. In this study, we investigated this mechanism in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and found MCD significantly inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production in a concentration-dependent manner. MCD could decrease LPS- and Pam3CSK4- induced the expressions of both iNOS and COX-2. The phosphorylation of inhibitory κB (IκB) and translocation of nuclear factor-κB (NF-κB) p65 subunit into the nucleus were also inhibited by MCD. Moreover, MCD suppressed LPS-induced phosphorylation of JNK except for ERK and p38 mitogen-activated protein kinases (MAPKs). Moreover, MCD significantly reduced ethyl phenylpropiolate-induced ear edema and carrageenan-induced paw edema in rat models. These findings indicated MCD has anti-inflammatory activity by inhibiting the production of NO and PGE2 by blocking NF-κB and JNK/c-Jun signaling pathways. Collectively, these data suggest that MCD could be developed as a novel therapeutic agent for inflammatory disorders.

4-methoxycinnamyl p-coumarate isolated from Etlingera pavieana rhizomes inhibits inflammatory response via suppression of NF-κB, Akt and AP-1 signaling in LPS-stimulated RAW 264.7 macrophages.

BACKGROUND: 4-methoxycinnamyl p-coumarate (MCC) was isolated from rhizomes of Etlingera pavieana by bioactivity-guided isolation, however, the molecular mechanism underlying its anti-inflammatory activity remains inadequately understood. PURPOSE: In this study, we elucidated the suppressive effect of MCC on LPS-induced expression of inflammatory mediators and the molecular mechanisms responsible for anti-inflammatory activities in RAW 264.7 macrophages. METHODS: Cell viability of MCC-treated RAW 264.7 macrophage was measured by MTT assay. Anti-inflammatory activity was evaluated by measurement of NO, PGE2, and cytokine production in LPS-stimulated cells. qRT-PCR and Western blotting analysis were used to investigate mRNA and protein levels of inflammatory responsive genes. NF-κB activation and transactivation activity were determined by immunofluorescence and reporter gene assay, respectively. RESULTS: MCC considerably suppressed both the production of NO, PGE2, IL-1ß as well as TNF-α and their expression. MCC inactivated NF-κB by reducing phosphorylation of IκBα and inhibiting NF-κB p65 nuclear translocation. Also, MCC significantly inhibited NF-κB transactivation activity. However, the inhibitory effect of MCC was independent of the MAPK signaling pathway. Furthermore, MCC significantly decreased phosphorylation of Akt and c-Jun, a main component of AP-1. CONCLUSION: These findings suggest that the anti-inflammatory effect of MCC could be mediated by the inhibition of LPS-induced expression of inflammatory mediators by down-regulation of the NF-κB, Akt and AP-1 signaling pathways in murine macrophages.

Biocompatible zwitterionic copolymer-stabilized magnetite nanoparticles: a simple one-pot synthesis, antifouling properties and biomagnetic separation.

A simple one-pot synthesis of biocompatible and antifouling magnetite nanoparticles (Fe3O4NPs) was developed. The process involves co-precipitation and in situ coating of zwitterionic copolymer poly[(methacrylic acid)-co-(2-methacryloyloxyethyl phosphorylcholine)] (PMAMPC). The influence of one-step and two-step coating methods on the performance of modified Fe3O4NP was investigated. The PMAMPC-Fe3O4NP with a narrow particle size distribution obtained from the two-step approach were highly stable in aqueous media within a wide range of pH. The particles exhibited superparamagnetic behavior with high saturation magnetization values so that they could be easily separated from solution by a magnet. Their antifouling characteristics against 2 selected proteins, lysozyme (LYZ) and bovine serum albumin (BSA), as a function of copolymer molecular weight and composition were also evaluated. Moreover, taking advantage of having carboxyl groups in the coated copolymer, the PMAMPC-Fe3O4NP were conjugated with a model biomolecular probe, biotin. The biotin-immobilized PMAMPC-Fe3O4NP were then tested for their specific capturing of a target molecule, streptavidin. The results have demonstrated the potential of PMAMPC-Fe3O4NP prepared by the two-step in situ coating method for probe immobilization and subsequent biomagnetic separation of target molecules. The fact that the developed functionalizable magnetite nanoparticles are biocompatible and antifouling also opens up the possibility of their use in other biomedical-relevant applications.

Non-cytotoxic hybrid bioscaffolds of chitosan-silica: Sol-gel synthesis, characterization and proposed application.

Biohybrid chitosan-silica scaffolds were synthesized through the sol-gel and the freeze drying processes. Hydrolysis and condensation of chitosan with tetraethylorthosilicate (TEOS) in the presence of 3-isocyanatopropyl triethoxysilane (ICPTES) were successfully carried out. Results obtained from FTIR, swelling test and pyrolysis confirmed that the hybrid scaffolds containing covalent coupling between the organic and inorganic networks were formed with high crosslink density of SiOSi bridging and could be classified as the class II material. The hybridization also resulted in improvements on mechanical strength and stability comparing to the pure chitosan. In vitro investigations on the guided bone regeneration and the cytotoxicity were also performed. SEM-EDS was used to examine the proliferation of calcium phosphate mineral at the scaffold surface after an immersion in simulated body fluid. The results revealed that the hybrid scaffolds exhibited a rapid induction of calcium phosphate mineral without cytotoxicity effect, reflecting an excellent in vitro bone bioactivity which was superior to the pure chitosan scaffold.

Anti-inflammatory Effect of Etlingera pavieana (Pierre ex Gagnep.) R.M.Sm. Rhizomal Extract and Its Phenolic Compounds in Lipopolysaccharide-Stimulated Macrophages.

BACKGROUND: In our continuing search for anti-inflammatory agents from Thai herbs, Etlingera pavieana (Pierre ex Gagnep.) R.M.Sm. showed potent inhibition on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages. However, the mechanism behind its inhibitory effect has not been yet explored, and little is known regarding its bioactive compounds responsible for the anti-inflammatory effect. OBJECTIVE: In the present study, anti-inflammatory effect of hexane, ethyl acetate, and water fractions of rhizomal ethanol extracts of E. pavieana was evaluated for their inhibition on NO production and mechanism in LPS-stimulated macrophages. Active compounds responsible for such anti-inflammatory activity were identified. MATERIALS AND METHODS: Inhibitory activities on NO production were performed in LPS-stimulated RAW264.7 macrophage. Cytotoxicity of plant extracts was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, mRNA and protein expressions by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Anti-inflammatory compounds were isolated by activity-guided isolation technique using column chromatography. RESULTS: Ethyl acetate fraction of E. pavieana (EPE) showed the most potent inhibitory effect on NO production in macrophages. EPE significantly decreased NO production and inhibited inducible nitric oxide synthase (iNOS) protein and mRNA expression in a dose-dependent manner. Furthermore, the level of nuclear factor-kappa B p65 subunit was markedly reduced in activated cells treated with EPE. Four phenolic compounds, 4-methoxycinnamyl alcohol (1), trans-4-methoxycinnamaldehyde (2), 4-methoxycinnamyl p-coumarate (3), and p-coumaric acid (4), were obtained from bioactivity-guided isolation technique. CONCLUSIONS: The anti-inflammatory property contained in E. pavieana rhizome extract and conferred through inhibition of iNOS expression, and NO formation provides scientific evidence and support for the development of new anti-inflammatory agents based on extracts from this plant. SUMMARY: Ethyl acetate fraction (EPE) of Etlingera pavieana showed the most potent inhibitory effect on NO production in LPS-induced macrophagesFour phenolic compounds, 4-methoxycinnamyl alcohol (1), trans-4-methoxycinnamaldehyde (2), 4-methoxycinnamyl p-coumarate (3) and p-coumaric acid (4), responsible for the anti-inflammatory effect of EPE were isolated. Abbreviations used: EPE: Ethyl acetate fraction of Etlingera pavieana; EPH: Hexane fraction of Etlingera pavieana; EPW: Water fraction of Etlingera pavieana; NO: Nitric oxide (NO); LPS: Lipopolysaccharide; iNOS: Inducible nitric oxide synthase (iNOS); MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB: Nuclear factor-kappa B; DMSO: Dimethyl sulfoxide; EtOAc: Ethylacetate; MeOH: Methanol; AG: Aminoguanidine; DCM: Dichloromethane; MCA: 4-methoxycinnamyl alcohol; MCD: trans-4-methoxycinnamaldehyde; MCC: 4-methoxycinnamyl p-coumarate; CM: p-coumaric acid.

Involvement of p38 MAPK and ATF-2 signaling pathway in anti-inflammatory effect of a novel compound bis[(5-methyl)2-furyl](4-nitrophenyl)methane on lipopolysaccharide-stimulated macrophages.

Activated macrophages produce various pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) and cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) during inflammatory response. However, overproduction of NO and PGE2 appears to be involved in pathogenesis of various inflammatory diseases. Therefore, inhibition of NO and PGE2 production might be useful for the treatment of inflammatory-related diseases. In this study, the bis[(5-methyl)2-furyl](4-nitrophenyl)methane or BFNM was evaluated for the anti-inflammatory activity and mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage. BFNM inhibited NO and PGE2 production in a concentration-dependent manner and down-regulated the expression of iNOS and COX-2 at mRNA and protein levels. BFNM suppressed nuclear translocation of NF-κB p65 subunit only very slightly, and failed to decrease NF-κB DNA binding activity. In contrast, the compound significantly reduced phosphorylation of p38 MAPK and ATF-2, a component of AP-1 known to be involved in the transcriptional regulation of iNOS and COX-2, in a dose-dependent manner in LPS-induced cells. Collectively, these results suggest that BFNM has an anti-inflammatory effect in RAW 264.7 macrophages, at least in part, by suppression of NO and PGE2 production. The inhibitory effect of BFNM is mediated mainly via the p38 MAPK/ATF-2 signaling pathway. Thus, BFNM would be a lead compound for the development of novel anti-inflammatory agents.

Bioassay-guided isolation and mechanistic action of anti-inflammatory agents from Clerodendrum inerme leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Clerodendrum inerme (L.) Gaertn. have commonly been used in Thai traditional medicine for treatment of inflammatory diseases. However, the bioactive compounds responsible for the anti-inflammatory effect of leaves have not been yet determined. The objective of the present study was to isolate these bioactive compounds by bioassay-guided isolation technique and to determine the mode of action of isolated compounds in LPS-induced macrophages. MATERIALS AND METHODS: Anti-inflammatory effect of various fractions (hexane, ethyl acetate and water) of ethanol extract of C. inerme leaves was determined from the production of nitric oxide (NO) in RAW 264.7 macrophage stimulated with LPS. The mRNA and protein levels were determined also by real-time reverse transcription-polymerase chain reaction and western blot analysis, respectively. Leaf bioactive compounds were isolated by bioassay-guided fractionation technique using column chromatography. RESULTS: The ethyl acetate fraction (EA) among solvent extracts provided the most potent inhibitory activity on NO production. Also, EA reduced the mRNA and protein expressions of inducible nitric oxide synthase (iNOS) in LPS-stimulated macrophages. Three known flavones, acacetin (1), hispidulin (2) and diosmetin (3), were isolated based on inhibition of NO production. Furthermore, hispidulin also inhibited PGE2 production as well as iNOS and cyclooxygenase-2 expressions via the blockade of NF-κB DNA-binding activity and JNKway. CONCLUSIONS: Our results found acacetin (1), hispidulin (2) and diosmetin (3), were responsible for the anti-inflammatory properties of C. inerme leaves. We provide scientific evidence to support the usefulness of C. inerme leaves in traditional medicine for the treatment of inflammation-related diseases.

Green synthesis and anti-inflammatory studies of a series of 1,1-bis(heteroaryl)alkane derivatives.

Molecular iodine has been used as an efficient catalyst for a double Friedel-Crafts reaction of various heteroarenes, i.e. 2-methylfuran, 2-ethylfuran, 2-methylthiophene, pyrrole, N-methylpyrrole and indole, using aldehydes as alkylating agents under "open-flask" conditions with toluene or water as the reaction media. In the presence of 10 mol% iodine in toluene at room temperature, both aliphatic and aromatic aldehydes reacted smoothly to give the corresponding bis(heteroaryl)alkanes in good to excellent yields. Interestingly, with water as the solvent, the bis(heteroaryl)alkane adducts were obtained in moderate to good yields. The use of mild reaction conditions, low catalyst loadings, and eco-friendly reagents in a single step synthesis are the advantages of the present procedure. In an effort to discover novel non-steroidal anti-inflammatory agents, the synthesized bis(heteroaryl)alkanes were evaluated for the anti-inflammatory activity in the lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage model. These compounds (50 µM) significantly inhibited NO production and did not exhibit significant cytotoxic effects on macrophage cells. Among them, bis[(5-methyl)2-furyl](4-nitrophenyl) methane exhibited the most potent inhibition of NO with IC50 value of 42.4 ± 1.9, which is similar to that of the positive control, aminoguanidine (43.3 ± 2.5 µM). Thus, the bis[(5-methyl)2-furyl](4-nitrophenyl) methane could be considered a lead compound for the development of novel anti-inflammatory agents.

Anti-inflammatory effect of ethyl acetate extract from Cissus quadrangularis Linn may be involved with induction of heme oxygenase-1 and suppression of NF-κB activation.

AIM OF THE STUDY: Cissus quadrangularis (family: Vitaceae) has been widely used in traditional herbal medicine for the treatment of hemorrhoids, gastric ulcers and bone healing. In the present study, we determined the anti-inflammatory activity and the molecular mechanism of the ethyl acetate extract of Cissus quadrangularis stem (CQE) in LPS-stimulated RAW 264.7 macrophage cells. MATERIALS AND METHODS: The inhibitory effect of CQE on LPS-induced nitric oxide (NO) production was evaluated in conditioned media. Cell viability was monitored by MTT assay. Protein and mRNA expressions were determined by RT-PCR and Western blotting analysis, respectively. RESULTS: CQE potently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells in a dose-dependent manner. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS) were suppressed also by CQE as was p65 NF-κB nuclear translocation. Further study demonstrated that CQE by itself induced heme oxygenase-1 (HO-1) gene expression at the protein and mRNA levels in dose- and time-dependent manner. In addition, the inhibitory effects of CQE on NO production were abrogated by a HO-1 inhibitor, zinc protoporphyrin IX (ZnPP). CONCLUSIONS: Collectively, these results suggest that CQE exerts an anti-inflammatory effect in macrophages, at least in part, through the induction of HO-1 expression. These findings provide the scientific rationale for anti-inflammatory therapeutic use of Cissus quadrangularis stem.

CO from enhanced HO activity or from CORM-2 inhibits both O2- and NO production and downregulates HO-1 expression in LPS-stimulated macrophages.

Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by the stress-inducible heme oxygenase-1 (HO-1), has recently been demonstrated to provide cytoprotection against cell death in macrophages stimulated with bacterial lipopolysaccharide (LPS). In the present study, we determined the effects of CO on the production of reactive oxygen species (ROS) and nitric oxide (NO) by the LPS-stimulated RAW 264.7 macrophages. In addition, effect of CO-exposure on the production of superoxide (O(2)(-)) in the phorbol myristate acetate (PMA)-stimulated PLB-985 neutrophils was determined. Production of ROS by the LPS-stimulated macrophages pretreated with 50microM [Ru(CO)(3)Cl(2)](2), a CO-releasing molecule (CORM-2), was abolished and the production of O(2)(-) by the PMA-stimulated neutrophils pretreated with the CORM-2 was decreased markedly. The CORM-2 (50microM) was not cytotoxic to both the unstimulated and LPS-stimulated macrophages when determined by employing mitochondrial reductase function test (MTT assay). In macrophages pretreated with increasing doses of CORM-2, both the LPS-derived upregulations of iNOS (NO production) and HO-1 expression (CO production) were suppressed in a dose-dependent manner. Alternatively, when the macrophages were treated with LPS and CO-donor together, the LPS-derived increase in NO production was decreased. Conversely, when the control and LPS-stimulated macrophages were treated with zinc protoporphyrin IX (ZnPP) to inhibit the HO activity blocking endogenous production of CO (basal and enhanced), macrophages died extensively. Interestingly, production of NO in the LPS-stimulated macrophages increased significantly following the ZnPP treatment. Addition of CORM-2 to the LPS-treated cells that were being treated additionally with ZnPP did not prevent the cell death. However, endogenous overproduction of CO by super-induction of HO-1 (obtained by pretreatment of macrophages with either buthionine sulfoximine or hemin) decreased the LPS-derived iNOS expression without affecting cell survival. Combined, these results indicated that enhanced HO activity is essential for the survival of LPS-stimulated macrophages. Thus, upregulation of HO-1 and overproduction of CO may allow the survival of LPS-stimulated macrophages; first, by eliminating the free heme to prevent Fenton reaction, second, by limiting the availability of free heme required for induction of NO-producing heme enzyme (i.e., iNOS), third, by limiting additional production of O(2)(-) and NO via CO-derived inhibition on the activities of heme enzymes like NADPH oxidase and iNOS, respectively. CO may allow the LPS-activated macrophages to return back to the normal quiet state insensitive to additional stimuli causing oxidative stress.

Molecular mechanisms involved in enhancing HO-1 expression: de-repression by heme and activation by Nrf2, the "one-two" punch.

Heme oxygenase (HO)-1 is a stress response protein, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. Molecular mechanisms involved in the inducible transcription of ho-1 occurring in response to numerous and diverse stressful conditions have remained elusive. Since the discovery of E1 and E2, the two upstream enhancers regulating induction of ho-1 transcription in 1989, there have been many studies dealing with molecular mechanisms involved in enhancing HO-1 expression. In this commentary, recent advances in our understanding of the mechanisms involved in the induction of HO-1 expression in mammalian cells are summarized with some supportive results reported by others. Currently available data indicate that activation of ho-1 transcription involves both the heme (native substrate)-dependent selective alleviation of repressor and the oxidative stress-dependent activation of transcriptional activator. The stress-released free-heme (HO-1 substrate) from hemoproteins involved in causing oxidative stress itself appears to act as a molecular switch controlling the repressor- activator antagonism on the enhancer sequences of ho-1. Thus, induction of HO-1 appears to operate in a manner like a simple feedback loop. dox Signal. 7, 1674-1687.

Role of NO in enhancing the expression of HO-1 in LPS-stimulated macrophages.

Macrophages serve as the first-line defense against invading pathogens by (a) overproducing O2- via activation of NADPH-oxidase localized in its plasma membrane, (b) inducing the expression of inducible nitric oxide synthase (iNOS) and overproducing NO, and (c) generating highly toxic peroxynitrite (ONOO-) to kill the invading pathogens without killing the macrophages themselves. Results show that this was due at least in part to the NO-derived induction of heme oxygenase-1 (HO-1) expression. The NO-derived induction of HO-1 caused (a) rapid elimination of toxic heme to inhibit lipid peroxidation and to prevent further induction of iNOS, (b) rapid production of bile pigment antioxidants to scavenge reactive oxygen (O2-) and nitrogen (NO) metabolites, and (c) rapid production of carbon monoxide (CO) to inhibit further production of O2- and NO by blocking the activities of NADPH-oxidase and iNOS, respectively. Thus, the NO overproduced by the O2- -dependent induction of iNOS expression can scavenge O2- to produce ONOO-, first to kill the invading pathogens and second to enhance the HO-1 expression in macrophages. This allows the survival of host tissues from the injuries caused by inflammatory oxidative stress.

Cytotoxic and cytoprotective actions of O2- and NO (ONOO-) are determined both by cellular GSH level and HO activity in macrophages.

Survival of macrophages, which serve as the first-line defense against invading pathogens by invoking the overproduction of highly toxic peroxynitrite (ONOO-), depends on their ability to maintain the intracellular GSH level and to induce the expression of heme oxygenase-1 (HO-1). The ONOO- is produced by macrophages stimulated by pathogens and is a powerful oxidant reacting directly with cellular GSH and proteins, killing both invading pathogens and macrophages themselves. However, macrophages can survive the toxicity of ONOO- by replenishing the depleted GSH level and by inducing HO-1 expression. In macrophages exposed to conditions overproducing O2-, NO, or ONOO-, the cellular level of GSH decreased rapidly, and when excessive, cells died. However, in cells surviving the toxicity caused by lower doses of O2-, NO, or ONOO-, the depleted intracellular GSH level was replenished, and HO-1 expression was increased, but not when they were coexposed to an inhibitor of HO-1 activity. Cells exposed to an inhibitor of GSH synthesis had greater induction of HO-1 expression and survived. However, cells exposed to an inhibitor of HO-1 activity died extensively and could not be revived by addition of N-acetylcysteine (NAC), a precursor of GSH synthesis. Thus, the dichotomous cytotoxic or cytoprotective effects of O2-, NO, or ONOO- in macrophages are determined both by cellular GSH level and by HO-1 activity.

Super-induction of HO-1 in macrophages stimulated with lipopolysaccharide by prior depletion of glutathione decreases iNOS expression and NO production.

In the LPS-stimulated macrophages undergoing oxidative burst, intracellular storage of glutathione (GSH) is depleted, expression of iNOS is enhanced, and NO is overproduced. In response to the depletion of GSH, expression of HO-1 is induced and HO activity is elevated. Thus, in macrophages treated with LPS, productions of NO and CO, catalyzed, respectively, by accumulated iNOS and HO-1, are increased in sequence [Biochem. Pharmacol. 68 (2004) 1709]. In support of this, HO-1 is induced in macrophages treated only with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis depleting the GSH level. Alternatively, when the macrophages were exposed to spermine NONOate, an exogenous NO-donor, HO-1, was induced also. When the GSH-depleted or BSO-pretreated macrophages were exposed to NO, delivered either exogenously from spermine NONOate or endogenously from LPS-derived elevation of iNOS, super-induction of HO-1 was observed. Moreover, both the BSO and LPS treatments increased the accumulation of HO-1 inducing redox-sensitive transcription factor Nrf2 in the nuclear protein fraction. Thus, when the depletion of GSH is combined with NO delivery, expression of HO-1 is enhanced to a greater extent than that enhanced either by GSH depletion or by NO delivery. In these macrophages with super-induced HO-1 and elevated HO activity, LPS-derived increase in iNOS expression was down-regulated and NO production was suppressed. This indicated that induction of HO-1 caused by the NO overproduced from up-regulated iNOS, in turn, produces a causative inhibition on iNOS expression and NO production. Thus, it appears that there is a reciprocal cross-talk between inductions of HO-1 and iNOS in macrophages stimulated with LPS leading to their survival.

Heme oxygenase-1-mediated partial cytoprotective effect by NO on cadmium-induced cytotoxicity in C6 rat glioma cells.

Heme oxygenase-1 (HO-1) is a 32-kDa stress induced enzyme that degrades heme to carbon monoxide (CO) and biliverdin. By employing RT-PCR and Western blotting techniques, we have examined the HO-1 induction in C6 glioma cells that were treated with cadmium chloride (CdCl(2)) or spermine NONOate (SPER/NO). By employing a cell viability assay, we have also examined the cytoprotective effect of HO-1 induction against the cytotoxicity caused by toxic dose of CdCl(2). In C6 glioma cells exposed to CdCl(2), expression of HO-1 (mRNA and protein) was increased in a dose- and time-dependent manner. Nitric oxide (NO) generated from SPER/NO very rapidly increased HO-1 mRNA expression in the C6 glioma cells. The induction of HO-1 by SPER/NO protected the cells from toxic dose of CdCl(2). The up-regulation of HO-1 mRNA expression by CdCl(2) was inhibited by a pre-incubation of the cells with actinomycin D, a potent inhibitor of mRNA transcription. Upon the inhibition of elevated HO-1 mRNA expression by the use of zinc protoporphyrin IX (ZnPP), an inhibitor of HO activity, the change of HO-1 mRNA expression by ZnPP was not observed. Thus, the glial cell may respond to CdCl(2) toxicity by enhancing the HO-1 expression in its effort to minimize the CdCl(2)-derived oxidative damage, and to survive. In the glioma cells, when the HO-1 expression was elevated by a prior incubation with SPER/NO, the cell viability against the cytotoxicity of CdCl(2) was significantly increased. When the results of our experiment are taken together, we discovered that NO provided a rapid enhancement of HO-1 expression, and it provided a protective effect against CdCl(2)-derived oxidative injury in the C6 rat glioma cells.

Biphasic induction of heme oxygenase-1 expression in macrophages stimulated with lipopolysaccharide.

Time course relationship between inductions of iNOS and HO-1 was evaluated in RAW264.7 macrophages stimulated with LPS. Expression of HO-1 mRNA increased in a biphasic pattern, but that of xCT (cystine transporter) and iNOS mRNA increased in a monophasic manner. HO-1 protein level increased also in a biphasic manner, at 1-2 h and again between 8 and 24 h. However, iNOS protein began to increase at 4 h, quickly reaching a high level in a monophasic induction pattern. Production of NO* began to occur at 6 h and nitrite continued to accumulate in the culture medium. Total GSH level decreased markedly (50% of control) by 2 h, began to recover at 4 h, returned to control level by 6 h and increased above the control level during 10-24 h. Collectively, these results indicated that overproduced O2*- depletes GSH and triggers induction of xCT, HO-1, iNOS and HO-1 expression in sequence. Most notably, the second-phase induction of HO-1 was caused by overproduced NO*, resulting from LPS-derived iNOS induction. When this iNOS-derived delivery of NO* was combined with prior depletion of GSH using buthioninesulfoximine, an inhibitor of GSH biosynthesis, induction of HO-1 was potentiated. Furthermore, upon such super-induction of HO-1, NO* production was inhibited along with suppression of iNOS expression. Collectively, these results suggested that HO-1 is induced in a biphasic manner, sequentially by the overproduced O*2- and NO*, and the elevated HO-1 suppresses the production of these radicals in an auto-regulatory manner. This may allow the macrophages to survive from injuries that can be caused by concomitant oxidative and nitrosative stresses initiated by the LPS-driven oxidative burst.