Orazipone Inhibits Activation of Inflammatory Transcription Factors Nuclear Factor-κB and Signal Transducer and Activator of Transcription 1 and Decreases Inducible Nitric-Oxide Synthase Expression and Nitric Oxide Production in Response to Inflammatory Stimuli

Orazipone (OR-1384) and its derivative OR-1958 are novel thiol-modulating compounds that exert their effects most likely by forming reversible conjugates with the thiol groups of glutathione and proteins (Wrobleski et al., 1998; Aho et al., 2001). The reaction with glutathione is readily reversible, which makes orazipone and OR-1958 unique among thiolmodulating compounds. Orazipone has been shown to express anti-inflammatory properties in various models of experimentally induced colitis (Wrobleski et al., 1998; Boerma et al., 2006). In addition, orazipone was shown to inhibit eosinophil accumulation in animal models of asthma (Ruotsalainen et al., 2000) and to possess antieosinophilic activity (Kankaanranta et al., 2006). However, the cellular and molecular mechanisms of the anti-inflammatory actions of orazipone remain mostly unknown.

Increased NO production is associated with inflammatory diseases such as inflammatory bowel disease (Kolios et al., 2004), arthritis (Cuzzocrea, 2006), and asthma (Ricciardolo et al., 2004). NO is a small gaseous molecule synthesized in inflammatory and tissue cells by nitric-oxide synthase enzymes. In mammalian cells, two isoforms of the enzyme are constitutively expressed, but the third isoform, iNOS, is induced in response to bacterial products and proinflammatory cytokines (Alderton et al., 2001; Kleinert et al., 2004; Korhonen et al., 2005). NO production through iNOS pathway is regulated mainly at the level of iNOS expression (Kleinert et al., 2004; Korhonen et al., 2005). In inflammation, NO has regulatory and proinflammatory effects (Coleman, 2001; Korhonen et al., 2005). Compounds that inhibit iNOS expression or iNOS activity possess antiinflammatory properties (Vallance and Leiper, 2002).

Bacterial lipopolysaccharide (LPS) induces the expression of iNOS in various cells, including murine macrophages. LPS activates toll-like receptor (TLR) 4 (Poltorak et al., 1998). TLR4 activation leads to the activation of nuclear factor κB (NF-κB) (Zhang and Ghosh, 2000), which is an important transcription factor for iNOS. Another transcription factor involved in LPS-induced iNOS expression is the signal transducer and activator of transcription (STAT) 1, which is activated through the Janus kinase (JAK)-STAT pathway (Gao et al., 1998; Jacobs and Ignarro, 2001). In addition, post-transcriptional mechanisms are important regulators of iNOS expression (Kleinert et al., 2004; Korhonen et al., 2005).

In the present study, we investigated the anti-inflammatory properties of orazipone by studying its effects and mechanisms of action on iNOS expression and NO production in activated macrophages and epithelial cells.

Materials and Methods

Reagents. Orazipone (OR-1384), its derivative OR-1958, and the nonthiol-modulating control compound OR-2149 were provided by Orion Pharma (Espoo, Finland). OR compounds were dissolved in dimethyl sulfoxide just before the experiments. They were further diluted into culture medium 1:1000 to give the final concentration of 0.1% dimethyl sulfoxide. Rabbit anti-iNOS (sc-650), STAT1α p91 (sc-345), NF-κB p65 (sc-7151), actin (sc-1616-R), lamin A/C (sc-20681), and goat anti-rabbit horseradish peroxidase-conjugated polyclonal (sc-2004) antibodies were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). JAK inhibitor AG-490 (tyrphostin B42) was from Calbiochem (San Diego, CA). All other reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

Cell Culture. J774 macrophages (TIB-67; American Type Culture Collection, Manassas, VA) were cultured at 37°C in 5% CO₂ atmosphere in Dulbecco's modified Eagle's medium with Gluta-MAX-I (Lonza Verviers SPRL, Verviers, Belgium) containing 10% heat-inactivated fetal bovine serum (Lonza Verviers SPRL), 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B (all obtained from Invitrogen, Paisley, UK). Cells were seeded on 24-well plates for nitrite measurement, Western blot analysis, and reverse transcription (RT)-PCR, and on 10-cm dishes for nuclear extract preparation. Cells were grown for 72 h to confluence before experiments.

To study the regulation of human iNOS promoter, pools of stably transfected A549/8 cells containing the full-length (16-kilobase) human iNOS promoter (GenBank accession number AC005697) cloned in front of a luciferase reporter gene (Hausding et al., 2000) were used. These A549/8-pNOS2(16)Luc cells were kindly provided by Prof. Hartmut Kleinert (Johannes Gutenberg University, Mainz, Germany), and they were cultured at 37°C in 5% CO₂ atmosphere in Dulbecco's modified Eagle's medium with 1 mM sodium pyruvate (Lonza Verviers SPRL) containing 5% heat-inactivated fetal bovine serum (Lonza Verviers SPRL), 2.5 μg/ml polymyxin B, and 0.5 mg/ml G-418 disulfate salt (Sigma-Aldrich; for selection of cells stably transfected with the vector containing iNOS promoter construct and neomycin resistance gene). Cells were seeded on 24-well plates for nitrite measurement and RT-PCR. They were then grown for 48 h to confluence before experiments unless otherwise stated.

Cell viability after treatment with combinations of LPS or cytokine mixture and the tested compounds was assessed using modified 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide test (Cell Proliferation Kit II; Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Compounds were considered cytotoxic if the response was affected by more than 20%.

Preparation of Cell Lysates. At indicated time points, cells were rapidly washed with ice-cold phosphate-buffered saline. Cells were solubilized in ice-cold lysis buffer containing 1% Triton X, 50 mM NaCl, 10 mM Tris-base, pH 7.4, 5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 40 μM leupeptin, 50 μg/ml aprotinin, 5 mM NaF, 2 mM sodium pyrophosphate, and 10 μM N-octyl-β-d-glucopyranoside. After incubation for 15 min on ice, lysates were centrifuged at 13,500g for 5 min. The protein content of the supernatants was measured by the Coomassie Blue method (Bradford, 1976). The extracts were boiled in SDS sample buffer, and they were stored at -20°C.

Preparation of Nuclear Extracts. Nuclear proteins were extracted as described previously (Sareila et al., 2006). The protein content of the extracts was measured by the Coomassie Blue method (Bradford, 1976). The extracts were boiled in SDS sample buffer, and they were stored at -20°C.

Western Blotting. Protein (20 μg of lysates and nuclear extracts unless otherwise stated) was analyzed according to standard Western blotting procedure as described previously (Sareila et al., 2006). The membrane was incubated with the primary antibodies anti-iNOS, anti-STAT1α, anti-NF-κB p65, anti-lamin A/C, or anti-actin antibody in the blocking solution at 4°C overnight. The quantitation of the chemiluminescent signal was carried out with the use of FluorChem software version 3.1 (Alpha Innotech, San Leandro, CA).

RNA Extractions and Quantitative Reverse Transcription-PCR. RNA extractions and quantitative RT-PCR were performed as described previously (Sareila et al., 2006). In brief, total RNA was extracted and converted to cDNA. iNOS, luciferase and GAPDH mRNA were measured by quantitative RT-PCR. iNOS and luciferase mRNA levels were normalized to GAPDH, which was considered as a housekeeping gene. Each sample was determined in duplicate.

Luciferase, and human and murine iNOS and GAPDH primer and probe (6-carboxyfluorescein as 5′-reporter dye and 6-carboxytetramethylrhodamine as 3′-quencher) sequences are described in Table 1.

Nitrite Assays. For nitrite assays, J774 cells were treated with the compounds of interest in cell culture media volume of 1 ml and A549/8-pNOS2(16)Luc cells in a volume 0.5 ml. After 24-h incubation, the culture medium was collected for the nitrite measurement, which was used as a measure of NO production. Culture medium (100 μl) was incubated with 100 μl of Griess reagent (0.1% napthalethylenediamine dihydrochloride, 1% sulfanilamine, and 2.4% H₃PO₄), and the absorbance was measured at 540 nm. The concentration of nitrite was calculated with sodium nitrite as a standard (Green et al., 1982).

Statistics. Results are expressed as mean + S.E.M. When indicated, statistical analysis was carried out by analysis of variances followed by Dunnett's multiple comparisons test. Differences were considered significant at P < 0.05.

Results

Effect of Orazipone on LPS-Induced iNOS mRNA Expression in J774 Macrophages. We studied the effects of orazipone and its derivatives (Fig. 1) on LPS-induced iNOS mRNA expression in J774 macrophages. iNOS mRNA was measured by quantitative RT-PCR after 3-h incubation with the combination of LPS and the drugs (Fig. 2). LPS (10 ng/ml) induced iNOS mRNA expression, which was dose-dependently inhibited by orazipone (OR-1384), and a significant reduction was obtained also with its derivative OR-1958 (60 μM). iNOS mRNA expression was not affected by the nonthiol-modulating control compound OR-2149 (60 μM).

Effect of Orazipone on LPS-Induced NF-κB p65 Activation in J774 Macrophages. Because iNOS mRNA expression was inhibited by orazipone, we investigated the effects of orazipone on the activation of NF-κB, which is an important transcription factor in iNOS expression (Xie et al., 1994). NF-κB activation in LPS-stimulated J774 cells was studied by measuring the nuclear translocation of NF-κB subunit p65 by Western blot. LPS caused nuclear translocation of NF-κB p65, which peaked at 30 min. Both orazipone (OR-1384; 60 μM) and its derivative OR-1958 (60 μM) decreased significantly the nuclear levels of NF-κB p65 in LPS-activated macrophages (Fig. 3a). The nonthiol-modulating control compound OR-2149 (60 μM) did not alter LPS-induced NF-κB activation. In a similar manner to orazipone and OR-1958, an NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; 100 μM) inhibited LPS-induced nuclear translocation of NF-κB by approximately 50% (Fig. 3b) and iNOS mRNA expression by approximately 75% (Fig. 3c).

Effect of Orazipone on LPS-Induced STAT1 Activation in J774 Macrophages. In addition to NF-κB, transcription factor STAT1 is involved in LPS-induced iNOS expression (Gao et al., 1998). We studied the effects of orazipone on LPS-induced STAT1 activation in J774 cells by measuring nuclear translocation of STAT1α by Western blot. LPS caused nuclear translocation of STAT1α, which increased up to the 6-h follow-up after addition of LPS. Orazipone (OR-1384; 60 μM) and its derivative OR-1958 (60 μM) inhibited LPS-induced nuclear translocation of STAT1α, whereas the nonthiol-modulating control compound OR-2149 (60 μM) had no effect (Fig. 4a). The effects of orazipone and OR-1958 on LPS-induced STAT1α activation were similar to the inhibitory effect of JAK inhibitor AG-490, which inhibited STAT1α activation (Fig. 4b) and iNOS mRNA expression (Fig. 4c).

Effect of Orazipone on LPS-Induced iNOS mRNA Decay in J774 Macrophages. In addition to transcriptional regulation, iNOS expression is regulated at the level of iNOS mRNA degradation (Kleinert et al., 2004; Korhonen et al., 2005). In J774 macrophages, dexamethasone and SP600125, an inhibitor of c-Jun NH₂-terminal kinase (Bennett et al., 2001), have been shown to reduce LPS-induced iNOS expression by destabilizing the mRNA (Korhonen et al., 2002; Lahti et al., 2003). To study whether orazipone enhances iNOS mRNA degradation, the actinomycin D assay was applied. Cells were stimulated with LPS (10 ng/ml) in the presence and in the absence of orazipone (60 μM). After 6 h, actinomycin D (0.5 μg/ml) was added into the culture medium to stop transcription. The decay of iNOS mRNA was followed by measuring iNOS mRNA levels at 2-h intervals. Orazipone did not affect iNOS mRNA decay (Fig. 5), when measured by quantitative RT-PCR.

Effect of Orazipone on LPS-Induced iNOS Protein Expression in J774 Macrophages. To study whether the reduction in iNOS mRNA levels by orazipone results in decreased iNOS protein expression, we measured iNOS protein levels in J774 cells by Western blot after 24-h LPS treatment. LPS (10 ng/ml) induced iNOS protein expression, which was inhibited by orazipone (OR-1384) in a concentration-dependent manner (Fig. 6). The nonthiol-modulating control compound OR-2149 (60 μM) did not affect LPS-induced iNOS expression. Orazipone derivative OR-1958 (60 μM) reduced iNOS protein levels, but it was somewhat less potent than orazipone.

Effect of Orazipone on LPS-Induced NO Production in J774 Macrophages. Because iNOS protein levels were decreased by the drugs, we investigated the effects of orazipone (OR-1384), its derivative OR-1958, and the nonthiol-modulating control compound OR-2149 on LPS-induced NO production in J774 macrophages. LPS (10 ng/ml) induced NO production, which was inhibited by OR-1384 in a concentration-dependent manner (Fig. 7a). Orazipone was somewhat more potent than its derivative OR-1958, whereas the nonthiol-modulating control compound OR-2149 had no effect.

When orazipone was added to the culture 6 h after LPS, NO production was not altered (Fig. 7b). These data suggest that orazipone does not inhibit iNOS enzyme activity, but the effect is due to its suppressive action on iNOS expression (see above).

Because orazipone has been described to be labile in aqueous solutions (Vendelin et al., 2005), we incubated the cells with LPS and orazipone for only 4 h (which is needed for induction of iNOS), changed fresh medium thereafter, and measured accumulated nitrite after an additional 20-h incubation in medium free of LPS and orazipone. In these conditions, orazipone was more potent as an inhibitor of NO production than when LPS and orazipone were present in the culture medium for the whole 24-h incubation time (Fig. 7, a and c). That result supports the idea that the labile action of orazipone is partly overtaken by LPS in longer incubations.

Cytotoxicity as a contributing factor was ruled out by 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide test. None of the tested compounds at highest concentrations used (60 μM) showed cytotoxicity in combination with LPS in 24-h incubation.

Effect of Orazipone on iNOS mRNA Expression and iNOS Promoter Activity in Human Alveolar Epithelial Cells. The human alveolar epithelial A549/8-pNOS2(16)Luc cells are genetically modified to express luciferase reporter gene under the control of full-length human iNOS promoter (16 kilobases) (Hausding et al., 2000).

To investigate the mechanism how orazipone lowered iNOS expression and NO production, we studied the effects of orazipone on iNOS and luciferase mRNA expression induced by a combination of proinflammatory cytokines [cytokine mixture of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-1β; 10 ng/ml each] in A549/8-pNOS2(16)Luc cells. The combination of proinflammatory cytokines was used to induce NO production in these cells, which produce only low amounts of NO when exposed to LPS. The effects of orazipone were compared with those of PDTC, which prevents the activation of NF-κB, an important transcription factor in human iNOS expression, and thereby inhibits iNOS expression at the level of transcription.

iNOS mRNA and luciferase mRNA were measured by quantitative RT-PCR after 6-h incubation with the combination of cytokine mixture and the drugs. Cytokine mixture induced iNOS mRNA expression, which was inhibited by orazipone (OR-1384; 60 μM) and PDTC (100 μM) (Fig. 8a).

In A549/8-pNOS2(16)Luc cells, luciferase gene expression is controlled by full-length human iNOS promoter. In this experiment, luciferase mRNA was used as a reporter of iNOS promoter activity. Unstimulated cells expressed a basal level of luciferase mRNA as a marker of some iNOS promoter activity in resting cells, which was expected according to previous data (Kleinert et al., 2004). To examine cytokine-induced portion of luciferase mRNA expression, the basal expression was subtracted from the luciferase expression in cells stimulated by cytokine mixture. Cytokine mixture induced luciferase mRNA expression, which was decreased by both orazipone and PDTC (Fig. 8a). The inhibitory effects of orazipone and PDTC on both iNOS mRNA and luciferase mRNA were about equal compared with mRNA expression induced by cytokine mixture. These results suggest that orazipone inhibits iNOS expression mostly at transcriptional level rather than by regulating mRNA expression through 3′-untranslated region of iNOS mRNA or by other post-transcriptional mechanisms.

In A549/8-pNOS2(16)Luc cells, also NO production was inhibited by orazipone (60 μM) and PDTC (100 μM), but the nonthiol-modulating control compound OR-2149 (60 μM) was ineffective (Fig. 8b). The effects of the drug compounds on A549/8-pNOS2(16)Luc cell viability were tested. Orazipone and its derivatives (60 μM) did not affect cell viability in combination with the cytokine mixture in 24-h incubation.

Effect of Orazipone on iNOS mRNA Expression and NO Production in the Presence of N-Acetyl-l-cysteine or Glutathione. To evaluate whether the effects of orazipone are dependent on the thiol-modulating property of orazipone, we tested the effects of orazipone in the presence of exogenously added thiols, i.e., N-acetyl-l-cysteine (NAC) and glutathione (GSH). J774 macrophages were preincubated with NAC or GSH for 1 h before they were stimulated with LPS to induce iNOS expression and NO production. iNOS mRNA levels (Fig. 9a) and NO production (Fig. 9b) were decreased by orazipone, but when NAC or GSH were present, the effects of orazipone were abolished. Likewise, the effect of orazipone on NO production was abolished in the presence of NAC or GSH also in A549/8-pNOS2(16)Luc human epithelial cells stimulated by proinflammatory cytokines (data not shown). These results support the assumption that thiol-modulating properties of orazipone are involved in the down-regulation of LPS or cytokine-induced iNOS expression and NO production by orazipone.

Discussion

Orazipone is a novel anti-inflammatory sulfhydryl-modulating compound that forms reversible conjugates with thiol groups in proteins and glutathione (Wrobleski et al., 1998). In the present study, we showed that the thiol-modulating compounds orazipone and its derivative OR-1958 inhibited activation of transcription factors NF-κB and STAT1, and iNOS expression in response to inflammatory stimuli. The latter effect was related to the ability of orazipone to inhibit activation of iNOS promoter, which may well be explained by its ability to inhibit transcription factors NF-κB and STAT1.

LPS-induced NO production has been shown to be decreased in murine RAW264.7 macrophages in which glutathione was depleted by inhibiting its synthesis (Srisook and Cha, 2005). Because orazipone reacts with thiols in, e.g., glutathione, at least part of its effects on NO production could be mediated through inactivation of glutathione. In the present study, the effects of orazipone were abolished when an excess of glutathione or N-acetyl-l-cysteine was present in the culture, supporting the assumption that the effects of orazipone are mediated through its reactions with glutathione and/or thiol groups in intracellular signal transduction proteins. This idea is further supported by the present results showing that the nonthiol-modulating derivative of orazipone (OR-2149) did not affect NF-κB or STAT1 activation, iNOS expression, or NO production. Recently, protein glutathionylation has become of interest as an important post-translational modification that serves to transduce redox signals (Ghezzi, 2005; Gallogly and Mieyal, 2007). Therefore, we studied the existing literature to find out whether glutathionylation of transcription factors NF-κB or STAT1 has been documented to have functional consequences that could explain the present results. We did not find reports on glutathionylation of STAT1. Glutathionylation of p50 subunit of NF-κB has been reported to inhibit its DNA binding (Pineda-Molina et al., 2001). However, in the present study, we found that orazipone inhibited nuclear translocation of NF-κB. Another explanation is that orazipone targets some of the TLR4-receptor-activated signaling mechanisms upstream of NF-κB and STAT1 either by a mechanism related or unrelated to glutathionylation.

In the present study, we found that orazipone decreased iNOS expression and NO production in a concentration-dependent manner in activated macrophages. LPS-induced NF-κB activation and iNOS mRNA accumulation were inhibited by orazipone, when measured after 30-min and 3-h incubation, respectively. For comparison, effects of an NF-κB inhibitor PDTC were tested, and it was shown to inhibit nuclear translocation of NF-κB, along with its inhibitory effect on iNOS mRNA expression. PDTC has also previously been demonstrated to inhibit LPS-induced NO production in J774 macrophages (Ruetten et al., 1999). In the present study, we found that orazipone inhibited nuclear translocation of NF-κB. In further experiments, we studied the effects of orazipone on iNOS promoter activity in A549/8-pNOS2(16)Luc human epithelial cells. In these cells, orazipone and NF-κB inhibitor PDTC inhibited the expression of a reporter gene that was under the control of human iNOS promoter similarly as the expression of iNOS and the production of NO. NF-κB is known to be an important transcription factor in human and murine iNOS expression (Kleinert et al., 2004; Korhonen et al., 2005), and inhibition of NF-κB by orazipone or PDTC resulted in decreased iNOS mRNA expression along with inhibition of iNOS promoter activity. These data together suggest that orazipone inhibits NF-κB activation and iNOS transcription, which leads to lowered levels of iNOS expression and NO production.

In addition to NF-κB, another important transcription factor in iNOS expression is STAT1 (Kleinert et al., 2004; Korhonen et al., 2005). Orazipone and OR-1958 inhibited STAT1 activation and iNOS expression. Likewise, JAK2 inhibitor AG-490 inhibited both STAT1 activation and iNOS mRNA expression in J774 macrophages. AG-490 has also previously been shown to inhibit LPS-induced NO production in J774 macrophages (Salonen et al., 2006) and other cell lines (Cruz et al., 1999, 2001). JAK2 is regarded to be the kinase primarily responsible for phosphorylation and activation of STAT1 (Leonard and O'Shea, 1998), and JAK2 inhibition leads to lowered STAT1 activity. These results suggest that the decrease in iNOS expression and in NO production caused by orazipone may be partly mediated through the inhibition of STAT1 activation.

The reaction between glutathione and the thiol-modulating compounds orazipone and OR-1958 has been shown to be reversible (Nissinen et al., 1997). In addition, orazipone has been described to be labile in aqueous solutions (Vendelin et al., 2005). Therefore, orazipone and OR-1958 might be partly degraded during prolonged incubation periods (e.g., 12–24 h, which we used in the present study). Orazipone extensively reduced NF-κB activation in J774 macrophages when measured after 30-min incubation and iNOS mRNA expression when measured after 3-h incubation, whereas it was less potent as an inhibitor of iNOS protein expression and NO production, which were measured after 24-h incubation. When the cells were incubated with LPS and orazipone for only 4 h (which is enough to induce iNOS) and then cultured in fresh medium (without LPS and orazipone) for another 20 h, orazipone was more potent as an inhibitor of NO production than in experiments where the cells were incubated with LPS and orazipone for 24 h. Therefore, we concluded that orazipone might have been partly degraded or its effects deteriorated during time in the culture, and while LPS was present its effects overcame those of orazipone. Therefore, it is possible that our experimental conditions (24-h incubations) may underestimate the potency of orazipone. This is in line with the studies in colitis models, where orazipone seemed to be more potent when it was given locally (intracolonic administration) than systemically (Wrobleski et al., 1998).

Orazipone has been tested in models of inflammatory bowel disease, with favorable results (Wrobleski et al., 1998), and it has been reported to ameliorate intestinal radiation injury in a rat model (Boerma et al., 2006). Orazipone also has been shown to have beneficial effects in an animal model of asthma (Ruotsalainen et al., 2000) and to have antieosinophilic activity in human cells (Kankaanranta et al., 2006). Orazipone has been previously shown to inhibit IL-1β, TNF-α, and IL-6 production in human monocytes (Wrobleski et al., 1998) and TNF-α production in human mast cell line HMC-1 (Vendelin et al., 2005). The present study extends the previous data by showing that orazipone inhibits activation of inflammatory transcription factors NF-κB and STAT1, and iNOS expression in activated macrophages and epithelial cells. At least part of the beneficial effects of orazipone in colitis could be explained by lowered iNOS expression and NO production, because selective inhibition of iNOS has been shown to ameliorate inflammation in 2,4,6-trinitrobenzenesulfonic acid-induced model of colitis (Kankuri et al., 2001; Menchén et al., 2001) and in dextran sodium sulfate-induced colitis in rats (Rumi et al., 2004). More importantly, the observed inhibitory effects on the activation of NF-κB and STAT1 are probably involved in the general anti-inflammatory mechanisms of orazipone because those transcription factors regulate the expression of various inflammatory factors in addition to iNOS. Moreover, the anti-inflammatory effects of glucocorticoids are mainly transmitted through suppressed NF-κB-mediated transcription (Clark, 2007).

In conclusion, we have shown that orazipone and its derivative OR-1958 inhibited activation of NF-κB and STAT1, and iNOS expression and NO production in response to inflammatory stimuli. Therefore, they are candidates for the treatment of inflammatory diseases whose pathogenesis is related to the activation of those inflammatory factors.