NF-κB In RAW Research Articles

Evaluating the anti-inflammatory potential of Tectaria cicutaria L. rhizome extract in vitro as well as in vivo

Ethnopharmacological relevanceThe rhizome of Tectaria cicutaria has been used in the folklore system of Indian traditional medicine (Ayurveda) for the treatment of various disorders such as rheumatic pain, chest complaints, burns, sprain, poisonous bites, tonsilitis, toothache, gum complaints, cuts and wounds. The present work has for the first time tried to elucidate the anti-inflammatory potential of aqueous extract of Tectaria cicutaria rhizome (TCRaq) in vitro as well as in vivo. Materials and methodsAnti-inflammatory potential of TCRaq was analyzed in vivo in carrageenan induced rat paw edema model. Serum antioxidant status in TCRaq-treated as well as untreated control rodents was measured by oxygen radical absorbance capacity (ORAC) assay. In vitro experiments for analyzing the anti-inflammatory potential of TCRaq were performed on murine macrophage cell line, RAW 264.7. Analysis of nitric oxide release in RAW 264.7 cells was done by Griess reaction. RT-PCR and western blotting experiment was performed to analyze the expression of iNOS. Expression of COX-2 and NFκB proteins was evaluated by western blotting. ResultsTCRaq significantly reduced the paw volume in Sprague-Dawley rats at a dose of 200mg/kg body weight, which was comparable with the standard diclofenac treatment. The rats treated with TCRaq showed a significant increase in the serum antioxidant levels compared to the untreated control animals. TCRaq was able to reduce the nitric oxide (NO) levels in RAW 264.7 cells that had been stimulated with lipopolysaccharide (LPS). This was accompanied by a corresponding decrease in iNOS expression at mRNA and protein level. Interestingly, TCRaq was found to decrease the expression of COX-2 as well as the nuclear translocation of NFκB in RAW 264.7 cells. ConclusionOur study signifies the anti-inflammatory potential of Tectaria cicutaria and scientifically validates its traditional use in inflammatory conditions.

Downregulation of pro-inflammatory mediators by a water extract of Schisandra chinensis (Turcz.) Baill fruit in lipopolysaccharide-stimulated RAW 264.7 macrophage cells

Schisandra chinensis has a long-standing history of medicinal use as a tonic, a sedative, an anti-tussive, and an anti-aging drug. Nevertheless, the antagonistic effects of S. chinensis against lipopolysaccharide (LPS)-stimulated responses have not yet been studied. In this study, we investigated whether water extract of S. chinensis fruit (WESC) has the ability to attenuate the expression of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) in LPS-stimulated RAW 264.7 macrophage cells. WESC inhibited the expression of LPS-induced pro-inflammatory mediators, namely, NO, PGE2, and TNF-α. Furthermore, gene expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α was inhibited both at mRNA and protein synthesis levels, without any cytotoxic effect. Moreover, WESC significantly suppressed LPS-induced DNA-binding activity of NF-κB by inhibiting degradation of IκBα. It was found that pyrrolidine dithiocarbamate (PDTC), a specific NF-κB inhibitor, downregulates the expression of these pro-inflammatory genes to be closely regulated by NF-κB activity. Furthermore, we found that WESC retains dephosphorylation of Akt in response to LPS, and consequently suppressed the DNA-binding activity of NF-κB in RAW 264.7 macrophage cells. LY294002, a specific Akt inhibitor, attenuated LPS-induced pro-inflammatory gene expression via suppression of NF-κB activity. Taken together, our results indicate that WESC downregulates the expression of pro-inflammatory genes involved in the synthesis of NO, PGE2, and TNF-α in LPS-stimulated RAW 264.7 macrophage cells by suppressing Akt-dependent NF-κB activity.

Water Extract of Deer Bones Activates Macrophages and Alleviates Neutropenia

Extracts from deer bones, called nok-gol in Korean, have long been used to invigorate Qi. While neutropenia is not well detected in normal physiological condition, it could be a cause of severe problems to develop diseases such as infectious and cancerous diseases. Thus, a prevention of neutropenia in normal physiology and pathophysiological states is important for maintaining Qi and preventing disease progress. In cell biological aspects, activated macrophages are known to prevent neutropenia. In this study, we demonstrate that water extract of deer bone (herein, NG) prevents neutropenia by activating macrophages. In mouse neutropenia model system in vivo where ICR mice were treated with cyclophosphamide to immunosuppress, an oral administration of NG altered the number of blood cells including lymphocytes, neutrophils, basophils, and eosinophils. This in vivo effect of NG was relevant to that of granulocyte colony stimulating factor (G-CSF) that was known to improve neutropenia. Our in vitro studies further showed that NG treatment increased intracellular reactive oxygen species (ROS) and promoted macrophagic differentiation of mouse monocytic Raw264.7 cells in a dose-dependent manner. In addition, NG enhanced nitric oxide (NO) synthesis and secretions of cytokines including IL-6 and TNF-α. Consistently, NG treatment induced phosphorylation of ERK, JNK, IKK, IκBα, and NF-κB in Raw264.7 cells. Thus, our data suggest that NG is helpful for alleviating neutropenia.

Evaluation of the new anti-inflammatory compound ethyl salicylate 2-O-β-d-glucoside and its possible mechanism of action

Ethyl salicylate 2-O-β-d-glucoside (ESG) is a derivative of natural salicylate isolated from Gaultheria yunnanensis (Franch.) Rehder, it has been used for the treatments of rheumatoid arthritis, swelling and pain. The aim of this study was to evaluate the anti-inflammatory effects of ESG and explore the anti-inflammatory mechanisms. We found that ESG had potent anti-inflammatory effects on the lipopolysaccharide (LPS)-activated murine macrophages RAW264.7. ESG exerted a dose-dependent inhibition of the LPS-stimulated release of the pro-inflammatory cytokines TNF-α and IL-1β. Moreover, it significantly inhibited LPS-stimulated the production of NO and PGE2 by repressing the expression of iNOS and COX protein respectively. Western blot analysis showed that ESG prominently inhibited LPS-induced activation of NF-κB in RAW264.7 cells by blocking phosphorylation of inhibitor IκBα and p65. Consistent with these results, we found that ESG prevented the nuclear translocation of NF-κB induced by LPS. Our study suggests that ESG may be effective in the treatment of inflammatory diseases by inhibiting the pro-inflammatory cytokine production and regulating the NF-κB signal pathway.

Mechanism of anti-inflammatory activity of umbelliferone 6-carboxylic acid isolated from Angelica decursiva

Aims of the studyWe recently reported the potential antioxidant and anti-inflammatory activities of umbelliferone 6-carboxylic acid (UMC) isolated from the whole plants of Angelica decursiva. In this study, we elucidated the anti-inflammatory mechanisms of UMC in vitro and in vivo. MethodsThe inhibitory effects of UMC on the production of nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α), the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the activation of nuclear factor kappa B (NF-κB) were evaluated using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The reactive oxygen species (ROS) generation inhibitory activity of UMC was evaluated using t-butyl hydroperoxide (t-BHP)-induced RAW 264.7 cells. Furthermore, the in vivo anti-inflammatory activity of UMC was evaluated using carrageenan induced mouse paw edema model. ResultsUMC dose-dependently inhibited NO and PGE2 production by down-regulating iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 macrophages. UMC also suppressed the production of the proinflammatory cytokine TNF-α in LPS stimulated RAW 264.7 cells in a concentration dependent manner. In addition, UMC dose-dependently prevented LPS-induced nuclear translocation of NF-κB in RAW 264.7 macrophages. Furthermore, UMC exhibited the inhibitory activity against t-BHP-induced ROS generation in RAW 264.7 cells with an IC50 value of 705.1μg/ml. Moreover, UMC inhibited λ-carrageenan induced mouse paw edema by 70.40 and 60.20% at doses of 50 and 25mg/kg body weight, respectively. ConclusionThe combined results of this study indicate that UMC is an important anti-inflammatory constituent of A. decursiva and its anti-inflammatory effect was due to its ability to inhibit the production of inflammatory mediators via inhibition of NF-κB activation pathway.

Anti-inflammatory activity of edible oyster mushroom is mediated through the inhibition of NF-κB and AP-1 signaling

BackgroundMushrooms are well recognized for their culinary properties as well as for their potency to enhance immune response. In the present study, we evaluated anti-inflammatory properties of an edible oyster mushroom (Pleurotus ostreatus) in vitro and in vivo.MethodsRAW264.7 murine macrophage cell line and murine splenocytes were incubated with the oyster mushroom concentrate (OMC, 0-100 μg/ml) in the absence or presence of lipopolysacharide (LPS) or concanavalin A (ConA), respectively. Cell proliferation was determined by MTT assay. Expression of cytokines and proteins was measured by ELISA assay and Western blot analysis, respectively. DNA-binding activity was assayed by the gel-shift analysis. Inflammation in mice was induced by intraperitoneal injection of LPS.ResultsOMC suppressed LPS-induced secretion of tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6), and IL-12p40 from RAW264.7 macrophages. OMC inhibited LPS-induced production of prostaglandin E2 (PGE2) and nitric oxide (NO) through the down-regulation of expression of COX-2 and iNOS, respectively. OMC also inhibited LPS-dependent DNA-binding activity of AP-1 and NF-κB in RAW264.7 cells. Oral administration of OMC markedly suppressed secretion of TNF-α and IL-6 in mice challenged with LPS in vivo. Anti-inflammatory activity of OMC was confirmed by the inhibition of proliferation and secretion of interferon-γ (IFN-γ), IL-2, and IL-6 from concanavalin A (ConA)-stimulated mouse splenocytes.ConclusionsOur study suggests that oyster mushroom possesses anti-inflammatory activities and could be considered a dietary agent against inflammation. The health benefits of the oyster mushroom warrant further clinical studies.

Pro-inflammatory effects of commercial alpha-lactalbumin on RAW 264.7 macrophages is due to endotoxin contamination

This study investigated the effects of alpha-lactalbumin (α-LA) on cellular signaling molecules associated with inflammatory responses in RAW 264.7 macrophages. The results indicated that commercial α-LA could increase prostaglandin E2 (PGE2) and the expression of COX-2 via increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and jun N-terminal kinase (JNK), and increase nitric oxide (NO) and the expression of iNOS via the activation of ERK1/2 and JNK. Furthermore, commercial α-LA could increase nuclear translocation of p65 nuclear factor-kappa B (p65 NF-κB) through stimulation on inhibitor kappa B-alpha (IκB-α) degradation. Since endotoxin also has these effects, we assayed the content of endotoxin in the commercial α-LA. We found to our surprise that endotoxin was there and that α-LA-induced NO and PGE2 production could be suppressed by polymyxin B, a specific inhibitor of endotoxin. Thus, the pro-inflammatory effects of commercial α-LA might be caused by endotoxin contamination through activation and expression of iNOS and COX-2 which were upregulated by MAPKs or nuclear translocation of p65 NF-κB in RAW 264.7 cells. It is therefore crucial to assess the possibility of endotoxin contamination within any biological product being studied for immune augmenting activities before a meaning result can be obtained.

Sulfuretin isolated from heartwood of Rhus verniciflua inhibits LPS-induced inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokines expression via the down-regulation of NF-κB in RAW 264.7 murine macrophage cells

It has been reported that Rhus verniciflua exhibits anti-inflammatory, anti-oxidant and anti-cancer activities. However, little is known about biological activity of sulfuretin, a flavonoid isolated from R. verniciflua. In the present study, we investigated the anti-inflammatory effect and the underlying molecular mechanisms of sulfuretin in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Sulfuretin dose-dependently reduced the productions of nitric oxide (NO), prostaglandin E 2 (PGE 2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) induced by LPS. Consistent with these findings, sulfuretin significantly suppressed the LPS-induced expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, and IL-1β. In addition, sulfuretin attenuated LPS-induced DNA binding and the transcriptional activities of nuclear factor-kappa B (NF-κB), which was accompanied by a parallel reduction of degradation and phosphorylation of inhibitory kappa B-α (IκB-α) and consequently by decreased nuclear translocation of p65 subunit of NF-κB. Furthermore, pretreatment with sulfuretin significantly inhibited the LPS-stimulated activation of IκB kinase β (IKKβ). Taken together, these results suggest that the anti-inflammatory effect of sulfuretin in LPS-treated RAW 264.7 macrophages is associated with the suppression of NF-κB transcriptional activity via the inhibitory regulation of IKKβ phosphorylation.

Quaternary alkaloid, pseudocoptisine isolated from tubers of Corydalis turtschaninovi inhibits LPS-induced nitric oxide, PGE 2, and pro-inflammatory cytokines production via the down-regulation of NF-κB in RAW 264.7 murine macrophage cells

It is well known that pro-inflammatory mediators like nitric oxide (NO), prostaglandin E 2 (PGE 2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) contribute to the courses of many inflammatory diseases. In the present study, the authors investigated the anti-inflammatory effects of pseudocoptisine, a quaternary alkaloid with a benzylisoquinoline skeleton, which was isolated from the tubers of Corydalis turtschaninovii by examining its inhibitory effects on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. Pseudocoptisine caused dose-dependent reductions in the levels of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) at both protein and mRNA levels and concomitant decreases in PGE 2 and NO production. In addition, it was found that pseudocoptisine suppressed the production and mRNA expressions of inflammatory cytokines, such as, TNF-α and IL-6. Furthermore, molecular data revealed that pseudocoptisine inhibited the LPS-stimulated DNA binding activity and the transcription activity of nuclear factor-kappa B (NF-κB). Moreover, this effect was accompanied by decreases in the phosphorylation of inhibitory κB (IκB)-α and in the subsequent blocking of p65 subunit of NF-κB translocation to the nucleus. In addition, pseudocoptisine dose-dependently inhibited the phosphorylations of ERK and p38. Taken together, these results suggest that pseudocoptisine reduces levels of the pro-inflammatory mediators, such as, iNOS, COX-2, TNF-α, and IL-6 through the inhibition of NF-κB activation via the suppression of ERK and p38 phosphorylation in RAW 264.7 cells. These findings reveal in part the molecular basis for the anti-inflammatory properties of pseudocoptisine.

Muramyl dipeptide potentiates lipoteichoic acid-induced nitric oxide production in macrophage (135.32)

Abstract Pathogen-associated molecular patterns, such as lipoteichoic acid (LTA) and peptidoglycan (PGN) of gram-positive bacteria, often cooperate together to induce immune responses in the host. In this study, we investigated nitric oxide (NO) production by macrophages in response to LTA and muramyl dipeptide (MDP), a minimal structural unit of PGN. MDP alone could not induce NO production in both murine macrophages and a murine macrophage-like cell line, RAW 264.7, whereas LTA induced NO production. However, MDP potentiated LTA-induced NO production under the same condition. This was due to enhanced activation of Toll-like receptor 2 (TLR2) recognizing LTA since MDP increased TLR2 expression on the surface of macrophages and the potentiation effect was abolished in the macrophages derived from TLR2-deficient mice. Furthermore, MyD88 and MAP kinases such as ERK and p38 kinase also appear to be essentially required for such potentiation, which was determined by using MyD88-deficient macrophages and their specific inhibitors, PD98059 and SB203580, respectively. Subsequently, MDP enhanced the LTA-induced activation of NF-κB in RAW 264.7. These findings suggest that MDP potentiates the ability of LTA to stimulate macrophages via upregulating TLR2 activity. Supported by a grant from 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science and Technology (11-2008-15-005-01).

(−)-Epigallocatechin gallate inhibition of osteoclastic differentiation via NF-κB

People who regularly drink tea have been found to have a higher bone mineral density (BMD) and to be at less risk of hip fractures than those who do not drink it. Green tea catechins such as (−)-epigallocatechin gallate (EGCG) have been reported to increase osteogenic functioning in mesenchymal stem cells. However, its effect on osteoclastogenesis remains unclear. In this study, we investigated the effect of EGCG on RANKL-activation osteoclastogenesis and NF-κB in RAW 264.7, a murine preosteoclast cell line. EGCG (10–100 μM) significantly suppressed the RANKL-induced differentiation of osteoclasts and the formation of pits in murine RAW 264.7 cells and bone marrow macrophages (BMMs). EGCG appeared to target osteoclastic differentiation at an early stage but had no cytotoxic effect on osteoclast precursors. In addition, it significantly inhibited RANKL-induced NF-κB transcriptional activity and nuclear translocation. We conclude that EGCG inhibits osteoclastogenesis through its activation of NF-κB.

Anti-inflammatory effects of essential oil isolated from the buds of Cleistocalyx operculatus (Roxb.) Merr and Perry

Cleistocalyx operculatus (Roxb.) Merr and Perry buds (Myrtaceae) are widely used in folk medicine for the treatment of gastric ailments as well as an antiseptic agent in China, Vietnam and some other tropical countries. However, to be clinically useful, more scientific data are needed. In the present study, we investigated the in vitro and in vivo anti-inflammatory activities of the essential oil of the C. operculatus buds (EO–CO). In the applied tests, EO–CO significantly inhibited lipopolysaccharide (LPS)-induced secretion of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in RAW 264.7 cells, a mouse macrophage-like cell line. Also the mRNA expression of TNF-α and IL-1β was suppressed by treatment with EO–CO in LPS-stimulated RAW 264.7 cells. Moreover, reporter gene analysis revealed that the EO–CO significantly blocked LPS-induced transcriptional activation of NF-κB in RAW 264.7 cells. Nuclear translocation of p65 subunit was also suppressed by EO–CO treatment. In addition, EO–CO inhibited phorbol ester-induced increase in ear swelling and skin water content in BALB/c mice. These results suggest that EO–CO might exert an anti-inflammatory effect by suppressing the expression of pro-inflammatory cytokines which is mediated, at least in part, by blocking NF-κB activation.

Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-κB in RAW 264.7 cells

Induction of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production is thought to have beneficial immunomodulatory effects in acute and chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, withaferin A inhibited LPS-induced expression of both iNOS protein and mRNA in a dose-dependent manner. To investigate the mechanism by which withaferin A inhibits iNOS gene expression, we examined activation of mitogen-activated protein kinases (MAPKs) and Akt in Raw 264.7 cells. We did not observe any significant changes in the phosphorylation of p38 MAPK in cells treated with LPS alone or LPS plus withaferin A. However, LPS-induced Akt phosphorylation was markedly inhibited by withaferin A, while the phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs) was slightly inhibited by withaferin A treatment. Withaferin A prevented IκB phosphorylation, blocking the subsequent nuclear translocation of nuclear factor-κB (NF-κB) and inhibiting its DNA binding activity. LPS-induced p65 phosphorylation, which is mediated by extracellular signal-regulated kinase (ERK) and Akt pathways, was attenuated by withaferin A treatment. Moreover, LPS-induced NO production and NF-κB activation were inhibited by SH-6, a specific inhibitor of Akt. Taken together, these results suggest that withaferin A inhibits inflammation through inhibition of NO production and iNOS expression, at least in part, by blocking Akt and subsequently down-regulating NF-κB activity.

Isoliquiritigenin isolated from the roots of Glycyrrhiza uralensis inhibits LPS-induced iNOS and COX-2 expression via the attenuation of NF-κB in RAW 264.7 macrophages

In this study, the anti-inflammatory effects of flavonoids isolated from the roots of Glycyrrhiza uralensis (Leguminosae), namely, isoliquiritin (the glycoside of isoliquirigenin) and isoliquiritigenin (the aglycone of isoliquiritin) were evaluated on lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Isoliquiritigenin (ILG) more potently inhibited LPS-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2) production than isoliquiritin (ILT). Consistent with these findings, ILG reduced the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in a concentration-dependent manner, as determined by Western blotting and RT-PCR, respectively. In addition, the release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and the mRNA expression levels of these cytokines were reduced by ILG in a dose-dependent manner. Moreover, ILG attenuated the LPS-induced DNA binding activity and the transcription activity of nuclear factor-kappa B (NF-κB), and this was associated with a decrease in inhibitory kappa B-α (IκB-α) phosphorylation and in the subsequent blocking of p65 and p50 protein translocations to the nucleus. Furthermore, ILG suppressed the phosphorylations of IκB kinase (IKK), ERK1/2, and p38, whereas the phosphorylation of JNK1/2 was unaffected. These results suggest that the anti-inflammatory properties of ILG are caused by iNOS, COX-2, TNF-α, and IL-6 down-regulation due to NF-κB inhibition via the suppression of IKK, ERK1/2 and p38 phosphorylation in RAW 264.7 cells.

Effect of expression silencing of NF-κB in RAW264.7 macrophages on secretory cytokines in systemic inflammatory response syndrome

Effect of expression silencing of NF-κB in RAW264.7 macrophages on secretory cytokines in systemic inflammatory response syndrome

Increase of intracellular glutathione by low-level NO mediated by transcription factor NF-κB in RAW 264.7 cells

The mechanism underlying the elevation of intracellular glutathione (GSH) in RAW 264.7 cells exposed to low concentrations of sodium nitroprusside (SNP), a well-known nitric oxide (NO) donor, was investigated. The peak of intracellular GSH was reached at 6 h after exposure of the cells to SNP (0.1–0.5 mM), and this was preceded by the induction of mRNA for γ-glutamylcysteine synthetase (γ-GCS; the rate-limiting enzyme of de novo GSH synthesis), which peaked at 3 h. N-α-Tosyl- l-phenylalanine chloromethyl ketone (TPCK) and caffeic acid phenethyl ester (CAPE), specific inhibitors of NF-κB, significantly suppressed the SNP-induced elevation of GSH protein and γ-GCS mRNA, while curcumin, an inhibitor of AP-1, was less effective. Electrophoretic mobility shift assay (EMSA) showed that SNP exposure markedly increased the DNA binding of NF-κB, but not that of AP-1. Deletion or mutagenesis of the NF-κB site in the γ-GCS gene promoter abolished the SNP-induced up-regulation of GSH protein and γ-GCS mRNA. These results suggest that the elevation of intracellular GSH in RAW 264.7 cells exposed to low concentrations of SNP occurs through the operation of the de novo GSH pathway, and is mediated by transcriptional up-regulation of the γ-GCS gene, predominantly at the NF-κB binding site in its promoter.

Ca 2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-κB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells

Ca 2+ and Ca 2+/calmodulin-dependent protein phosphatase calcineurin (CN) have been known to play crucial roles in immune response and inflammation. Using mouse peritoneal macrophages and RAW 264.7 macrophage cells, we demonstrated that LPS mobilized intracellular free Ca 2+ and induced CN phosphatase activity. iNOS expression and NO secretion in response to LPS were suppressed by Ca 2+ antagonists (TMB-8, BAPTA/AM, and nifedipine) and CN inhibitor (cyclosporin A). Transient expression of constitutively active CN in mouse peritoneal macrophages and RAW 264.7 macrophages strongly activated NF-κB, a key mediator of iNOS expression. We also found that CN mediates NF-κB activation via IκB-α hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKα and -β demonstrates that only IKKβ is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-κB in RAW 264.7 and peritoneal macrophages.

IL-1β induces MMP-9 via reactive oxygen species and NF-κB in murine macrophage RAW 264.7 cells

IL-1β increased the production of proenzyme of MMP-9 (pro-MMP-9) in a time- and dose-dependent manner in murine macrophage RAW 264.7 cells. However, the production of MMP-2 was not significantly changed by IL-1β treatment. The intracellular H 2O 2 content, as determined with H 2O 2-sensitive probe 2 ′,7 ′-dichlorodihydrofluorescein, also increased after IL-1β treatment (5 ng/ml). In addition, exogenous H 2O 2 (50 μM) was found to increase the production of pro-MMP-9. Transient transfection study using a MMP-9 promoter–reporter construct showed that IL-1β enhanced the MMP-9 promoter activity. Electrophoretic mobility shift assay and site-directed mutagenesis study on the consensus binding site for NF-κB revealed that the activation of NF-κB is required for the IL-1β-induced activation of MMP-9 promoter. N-acetylcysteine, an antioxidant, could abrogate the production of pro-MMP-9, H 2O 2 generation, and activation of NF-κB and MMP-9 promoter. These results suggest that IL-1β upregulates the MMP-9 expression via production of reactive oxygen species and activation of NF-κB in RAW 264.7 cells.