Suppression of Inflammatory Responses by Handelin, a Guaianolide

Apr 1, 2014 - ABSTRACT: The anti-inflammatory activity of handelin (1), a guaianolide dimer from Chrysanthemum boreale flowers, was evaluated in vivo ...
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Suppression of Inflammatory Responses by Handelin, a Guaianolide Dimer from Chrysanthemum boreale, via Downregulation of NF-κB Signaling and Pro-inflammatory Cytokine Production Yuna Pyee, Hwa-Jin Chung, Tae Jun Choi, Hyen Joo Park, Ji-Young Hong, Ju Sun Kim, Sam Sik Kang, and Sang Kook Lee* College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742, Korea ABSTRACT: The anti-inflammatory activity of handelin (1), a guaianolide dimer from Chrysanthemum boreale flowers, was evaluated in vivo, and the effects on mediators nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) and the nuclear factorκB (NF-κB) and ERK/JNK signaling pathways were investigated in vitro. Compound 1 inhibited lipopolysaccharide (LPS)-induced production of NO and PGE2 in cultured mouse macrophage RAW 264.7 cells. The suppression of NO and PGE2 production by 1 was correlated with the downregulation of mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Compound 1 also suppressed the induction of pro-inflammatory cytokines TNF-α and IL-1β in LPS-stimulated RAW 264.7 cells. To further clarify the transcriptional regulatory pathway in the expression of iNOS and COX-2 by 1, the role of NF-κB was determined in RAW 264.7 cells. Compound 1 inhibits the binding activity of NF-κB into the nuclear proteins. The transcriptional activity of NF-κB stimulated with LPS was also suppressed by 1, which coincided with the inhibition of IκB degradation. Compound 1 also suppressed the activation of mitogen-activated protein kinases, including ERK and JNK signaling. In addition, the LPS-stimulated upregulation of miRNA-155 expression was suppressed by 1. The oral administration of 1 inhibited acute inflammation in carrageenan-induced paw and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear edema models. The serum level of IL-1β was also inhibited by 1 in a carrageenan-induced paw edema model. These findings suggest that the suppression of NF-κB activation and pro-inflammatory cytokine production may be a plausible mechanism of action for the antiinflammatory activity of handelin.

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factor-κB (NF-κB) is an essential factor in controlling inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).5 NF-κB is activated by the phosphorylation of IκB, which occurs after the activation of MAPKs. NF-κB regulates the production of pro-inflammatory cytokines in stimulated macrophages.6 Since inflammatory mediators can cause severe damage by contributing to inflammatory diseases, the effective blockade of these inflammatory responses might be an essential target for the development of therapeutic agents.7,8 Chrysanthemum boreale Makino (Asteraceae), a medicinal plant that is widely distributed in Asian countries, such as mainland China and Korea, has been used traditionally for its antipyretic, anticancer, and antiangiogenesis effects. It has also been utilized for the treatment of vertigo and inflamed eyes.9−11 A recent study also showed that the methanol extract of C. boreale possesses anti-inflammatory effects through the induction of HO-1.12 However, no reports have addressed the anti-inflammatory activity of isolated compounds from C.

nflammation is a complex biological response to cell damage and vascularized tissues.1 Depending on the time of onset, inflammation can be classified as either an acute or chronic response. Acute inflammation is the primary cellular response to injurious stimuli and is produced by the local vascular and immune response. In contrast, chronic inflammation is a pathological condition characterized by the progressive destruction and recovery of injured tissue from inflammatory responses.2 Therefore, many pathophysiological disorders, such as rheumatoid arthritis, atherosclerosis, asthma, Alzheimer’s disease, and cancer, are considered to be highly associated with inflammatory responses. Macrophages play a central role in inflammation and host defense mechanisms. The production of inflammatory mediators, such as nitric oxide (NO) and pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interferon-γ (IFNγ), and interleukins, by macrophages in response to various pro-inflammatory stimuli is considered essential for inflammatory response.3 The inflammatory response initially requires signal transduction that is mediated by phosphoinositide 3kinases (PI3K), mitogen-activated protein kinases (MAPKs), and transcription factors.4 The transcription factor nuclear © 2014 American Chemical Society and American Society of Pharmacognosy

Received: November 27, 2013 Published: April 1, 2014 917

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boreale extracts. A previous phytochemical investigation of this species revealed that its constituents include flavonoids, polyacetylenes, sterols, and guaianolide sesquiterpenoids.13−17 Kang et al. have also reported the isolation of the guaianolide dimer handelin from the flowers of C. boreale.18 Cytotoxic activity and antiapoptotic effects induced by etoposide have been reported in guaianolide-type monomers from C. boreale.12,16,17 Handelin (1), a guaianolide dimer, was found to have no growth-inhibitory activity against cultured human cancer cell lines.18 In an ongoing program to search for antiinflammatory agents from natural products and based on the anti-inflammatory activity of the extracts of the plant, studies were extended to evaluate the effect of 1 on inflammatory responses.

treated with compound 1 for 20 h, the PGE2 production induced by LPS was inhibited significantly in a concentrationdependent manner with an IC50 value of 14.3 μM. Celecoxib (20 μM), a positive control of a selective COX-2 inhibitor, resulted in 1.2 ng/mL PGE2 (19% of LPS control) under the same assay conditions. The overproduction of NO and PGE2 is associated with the overexpression of iNOS and COX-2 in activated macrophages. To elucidate the mechanism of action mediated by 1 on the inhibition of NO and PGE2 production, the effects of 1 on iNOS and COX-2 protein and gene expression were determined. RAW 264.7 cells were incubated with LPS in the presence or absence of various concentrations of 1. Compound 1 suppressed the LPS-induced overexpression of iNOS and COX-2 protein levels in a concentration-dependent manner (Figure 1C) in Western blotting. To further investigate the effect of 1 on the LPS-induced enhancement of mRNA expression, steady-state levels of iNOS and COX-2 mRNA were evaluated by RT-PCR analysis. When cells were treated with 1, iNOS and COX-2 mRNA levels were significantly reduced in comparison to LPS stimulation (Figure 1D), suggesting that the inhibition of NO and PGE2 production by 1 might be correlated with the suppression of iNOS and COX-2 expression at the translational and transcriptional levels. No significant effect on cell viability was observed at a test concentration up to 20 μM 1, as determined by an MTT assay (>80% cell survival), indicating that the inhibition of NO and PGE2 production by 1 was not mediated by a cytotoxic effect (Figure 1E). NF-κB is a crucial transcriptional factor in the regulation of iNOS and COX-2 expression.5,6 In general, NF-κB is composed of two subunits (p65 and p50) and is present in the cytosol as inactive heterodimers bound to the inhibitory protein IκB-α.21 Upon stimulation by pro-inflammatory signals, including LPS, IκB-α is phosphorylated by IκB kinase and proteolytically degraded via a 26S proteasome-mediated pathway that facilitates NF-κB translocation into the nucleus and thus regulates target gene transcription.22 To investigate whether NF-κB is an important target of 1, a reporter gene assay for NFκB transcriptional activity (SEAP) was employed. The stimulation of RAW 264.7 cells with LPS for 16 h elicited a 5.1-fold increase in NF-κB transcriptional activity, but concurrent treatment with 1 effectively inhibited the LPSstimulated increase of NF-κB transcriptional activity (Figure 2A). To examine further whether this inhibition of NF-κB transcriptional activity is associated with the level of NF-κB subunits p65 and p50 in the nucleus, cells were treated with various concentrations of 1 and LPS for 30 min, and Western blot analysis was performed. As shown in Figure 2B, the nuclear p65 and p50 levels were increased after LPS stimulation, but the levels of p65 and p50 in the nuclear extracts were downregulated in cells treated with 1. In addition, the degradation of IκB-α in the cytosol by LPS was suppressed by treatment with 1. To determine whether the decrease in iNOS and COX-2 gene expressions by 1 is associated with the inhibition of the binding activity of NF-κB, RAW264.7 cells were treated with 10, 20, or 40 μM 1 for 1.5 h, and then the nuclear extracts were prepared and analyzed by using a TransAM NF-κB binding assay (Active Motif). As shown in Figure 2C, compound 1 downregulated LPS-stimulated NF-κB p50 or p65 binding activity. This result was well correlated with the suppressive effects on the mRNA gene expression of iNOS and COX-2 in 1-treated RAW 264.7 cells. Competition experiments

In the present study, the anti-inflammatory activity of 1 was determined by an animal model, and its possible mechanisms of action were investigated in vitro.

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RESULTS AND DISCUSSION The overproduction of NO and its corresponding enzyme, iNOS, is highly associated with inflammation. The pathway that regulates the production of NO and the expression of iNOS is considered a useful target for the procurement of antiinflammatory agents. To evaluate the inhibitory effect of handelin (1) on the production of nitric oxide, RAW 264.7 cells were stimulated with lipopolysaccharide (LPS), and the amount of nitrite, the stable metabolite of NO, was monitored in the medium. Lipopolysaccharide, a cell component of Gramnegative bacteria, is the most common cause of macrophagy activation. LPS-induced activation of macrophages has been reported to cause an inflammatory response.19,20 As shown in Figure 1A, stimulation with LPS markedly increased the production of NO over the basal level of 5.1 to 43.0 μM after 20 h of incubation. In this assay system, L-NMMA (50 μM), a nonselective inhibitor of NOS used as a positive control, inhibited 74% of NO production. When the cells were pretreated with various concentrations of 1 (0−40 μM) 30 min prior to LPS stimulation, NO production was inhibited significantly in a concentration-dependent manner, with an IC50 value of 6.7 μM. Over 84% inhibition of NO production was shown at 20 μM of 1. While 1 reduced slightly cell viability (83% of control) at 40 μM, the effect was negligible when considering the high inhibition (98%) of NO production. To determine the inhibitory effect of 1 on prostaglandin E2 (PGE2) production, a PGE2 enzyme immunometric assay (EIA) was used in LPS-stimulated RAW 264.7 cells. The stimulation of RAW 264.7 cells with LPS for 20 h increased dramatically the production of PGE2 from endogenous arachidonic acid from the basal level of 0.2 ng/mL without LPS to 6.3 ng/mL (Figure 1B). When RAW 264.7 cells were 918

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Figure 1. Inhibitory effects of 1 on LPS-induced NO (A) and PGE2 (B) production in macrophage cells. RAW 264.7 cells were stimulated with LPS (1 μg/mL) in the presence or absence of 1. After 20 h, cultured media were collected and analyzed for nitrite and PGE2 concentrations. The values are expressed as the mean ± SD of triplicate tests. The modulation of iNOS and COX-2 protein (C) and mRNA (D) expression by 1 was determined. Cells were stimulated with LPS (1 μg/mL) in the presence or absence of 1 for 16 and 5 h, and total RNA and protein were isolated and further analyzed by RT-PCR and Western blotting, respectively, as described in the Experimental Section. (E) Cell viability was measured by MTT assay as described in the Experimental Section. All experiments were performed in triplicate. The data are expressed as mean ± SD of triplicate tests.

with the addition of excess unlabeled wild-type (WT) but not mutant (MT) NF-κB p50 or p65 oligonucleotide were shown to be completely blocked in the signal of NF-κB p50 or p65 binding activity. A competition experiment confirms the specific binding activity of LPS-stimulated NF-κB p50 or p65 DNA binding in the nucleus. These findings indicate that the anti-inflammatory effect of 1 is associated partially with the suppression of NF-κB activation. Further studies were performed to correlate the antiinflammatory effect of 1 with the regulation of the expression of pro-inflammatory cytokines. A body of evidence suggests that pro-inflammatory cytokines, such as TNF-α, IFN-γ, and

interleukins, are upregulated in response to various proinflammatory stimuli.23,24 NF-κB also plays an essential role in the expression of these pro-inflammatory cytokines.25 Therefore, the effect of 1 on the expression of the proinflammatory cytokines TNF-α and IL-1β was investigated in LPS-stimulated macrophage cells. As shown in Figure 3A, LPS stimulation for 4 h increased the expression of TNF-α and IL1β proteins, but 1 effectively suppressed the LPS-induced expression of these proteins in a concentration-dependent manner. Further analysis also revealed that the upregulation of steady-state transcripts of TNF-α and IL-1β by LPS was suppressed significantly by treatment with 1 (Figure 3B). These 919

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Figure 2. Effect of 1 on LPS-induced NF-κB transcriptional activity. (A) SEAP-RAW cells were stimulated with LPS (1 μg/mL) in the presence or absence of 1. After 16 h, the supernatants were analyzed for the determination of SEAP activity. The values are expressed as the mean ± SD of triplicate tests. (B) Effects of 1 on expression of NF-κB and the degradation of IκB-α in macrophage cells. RAW 264.7 cells were treated with various concentrations of 1 for 30 min prior to LPS stimulation. After 30 min, proteins were extracted and analyzed by Western blot analysis. Tests were performed in triplicate, and data are representative of three separate experiments. (C) Effects of 1 on the LPS-induced NF-κB DNA-binding activity. Cells were stimulated with LPS (1 μg/mL) in the presence or absence of 1 for 1 h, and nuclear extracts were isolated and analyzed for NF-κB DNAbinding activity. The values are expressed as the means ± SD of triplicate tests.

pro-inflammatory cytokines.28,29 As shown in Figure 3D, compound 1 suppressed the activation (phosphorylation) of JNK and ERK in LPS-stimulated RAW 264.7 cells, but the activation of p38 was not altered by 1. These data suggest the anti-inflammatory activity of 1 with the suppression of MAPK signaling pathways. To assess the anti-inflammatory effect of 1 in vivo, a carrageenan-induced paw edema model was established in rats. Paw edema was induced by the subplantar injection of carrageenan (0.1 mL in 1% solution), and the volume of paw edema was monitored for 6 h. The volume of paw edema gradually increased and peaked 4 h after treatment with carrageenan. As shown in Figure 4A, oral treatment with 1 reduced the formation of paw edema significantly when compared with the vehicle-treated control groups. The rate of inhibition was 22% and 35% after 4 h treatment with 1 at concentrations of 10 and 20 mg/kg, respectively. Under the same experimental conditions, indomethacin (20 mg/kg) was shown to inhibit edema by 51%. The anti-inflammatory effect

data suggest that 1 suppresses inflammatory responses through a blockade of the expression of pro-inflammatory cytokines, such as TNF-α and IL-1β. Recent studies have demonstrated that miRNA-155 (miR155) plays an important role in the innate immune response and inflammation and that it positively regulates proinflammatory cytokines, such as TNF-α and IL-1β.26,27 Therefore, it was determined as to whether 1 affects the expression of miR-155 in LPS-stimulated macrophage cells. The stimulation of cells with LPS for 8 h resulted in a 20.1-fold increase in the expression of miR-155 over unstimulated cells, as measured by qRT-PCR. The LPS-stimulated upregulation of miR-155 expression was reduced significantly by treatment with 1 in a concentration-dependent manner (Figure 3C). These results suggest that the anti-inflammatory activity of 1 is in part associated with the downregulation of miRNA-155. LPS activates not only NF-κB but also MAPK family members, such as JNK, ERK, and p38. The MAPK signaling pathway also plays a crucial role in mediating the induction of 920

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Figure 3. Effect of 1 on LPS-induced pro-inflammatory cytokine protein (A) and mRNA (B) expression in macrophage cells. Cells were stimulated with LPS (1 μg/mL) in the presence or absence of 1 for 4 h. Total RNA and protein were isolated and further analyzed by RT-PCR and Western blotting, respectively, as described in the Experimental Section. Tests were performed in triplicate, and data are representative of three separate experiments. (C) Effect of 1 on the expression of miRNA-155 in LPS-stimulated RAW 264.7 cells. The cells were stimulated with LPS in the presence or absence of 1 for 8 h. Total RNA was isolated, and the expression of miRNA-155 was determined by qRT-PCR. The values are expressed as the means ± SD of triplicate tests. (D) Effect of 1 on the activation of ERK and JNK in macrophage cells. RAW 264.7 cells were treated with various concentrations of 1 for 30 min prior to LPS stimulation. After stimulation with LPS for an additional 30 min, proteins were extracted and analyzed by Western blot analysis. Tests were performed in triplicate, and data are representative of three separate experiments.

inflammatory biomarkers such as iNOS, COX-2, and IL-1β (Figure 5B). In summary, the present study demonstrates the potent antiinflammatory activity of handelin (1) in both in vitro and in vivo acute inflammatory models. A plausible mechanism of action for the anti-inflammatory activity of 1 has been shown for the first time, and it involves the suppression of proinflammatory cytokine production and suppression of JNK and ERK signaling pathways. These findings suggest that handelin (1) is a promising new chemotherapeutic candidate for the management of inflammatory responses.

of 1 was also observed by the reduced production of proinflammatory mediators in inflamed paw tissue (Figure 4B). When treated with carrageenan for 6 h, the expression levels of iNOS, COX-2, and IL-1β in paw tissues with edema were upregulated when compared to the untreated normal groups. However, compound 1 (20 mg/kg) significantly inhibited the production of these mediators. In addition, the effect of 1 on the production of IL-1β in the serum was measured after the subplantar injection of carrageenan for 6 h. The serum IL-1β level in carrageenan-treated groups was increased significantly from 27.1 to 129.1 pg/mL, when compared to untreated groups (Figure 4C). However, 1 (10 or 20 mg/kg) effectively suppressed the production of serum IL-1β in a dose-dependent manner in rats with carrageenan-induced paw edema. These findings demonstrated that 1 has in vivo anti-inflammatory activity in an acute animal model system. The in vivo efficacy of 1 was also evaluated in a mouse ear edema model, where inflammation was induced by the topical application of TPA. As shown in Figure 5A, ear edema was increased by the application of TPA for 4 h, with a weight increase of approximately 9.3 mg/mouse. However, the oral administration of 1 (10 or 20 mg/kg) significantly inhibited TPA-induced ear edema. Doses of 10 and 20 mg/kg led to a 43% and 56% reduction of edema, respectively. Indomethacin (20 mg/kg), a positive control, exhibited a similar percentage inhibition (53%) to 1. In addition, the analysis of inflamed ear tissues showed that 1 suppresses the protein expression of the

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EXPERIMENTAL SECTION

Chemicals. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), sodium pyruvate, L-glutamine, antibiotics− antimycotics solution, and trypsin-EDTA were purchased from Invitrogen Co. (Grand Island, NY, USA). Lipopolysaccharide (E. coli 0111: B4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and other chemicals were purchased from SigmaAldrich (St. Louis, MO, USA), unless otherwise indicated. Goat antirabbit IgG-HRP, goat anti-mouse IgG-HRP, goat anti-goat IgG-HRP, iNOS, COX-2, IL-1β, ERK1/2, p-ERK1/2, p50, p65, IκB-α, and p-IκBα antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against SAPK/JNK, p-SAPK/JNK, p38, p-p38, and TNF-α were purchased from Cell Signaling Technology (Beverly, MA, USA). Gene-specific primers were synthesized by Bioneer (Daejeon, Korea). AMV reverse transcriptase, dNTP mixture, random primer, RNasin, and Taq polymerase were purchased from Promega (Madison, WI, USA). Enzyme immunoassay 921

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Figure 4. Inhibitory effect of 1 on carrageenan-induced paw edema. Compound 1 was orally administered 30 min prior to carrageenan injection into the right paw of SD rats. The paw volume was measured before (0 h) and at 0.5, 1, 2, 3, 4, 5, and 6 h after carrageenan injection using a plethysmometer. (B) The expression of iNOS, COX-2, and IL-1β was determined in the carrageenan-inflamed paw tissues by Western blot analysis. Tests were performed in triplicate, and data are representative of three separate experiments. (C) IL-1β levels were measured after the in vivo administration of 1. Serum was collected from rats with carrageenan-induced paw edema and analyzed for IL-1β levels using an ELISA kit. Data represent the means ± SD (n = 6) (*p < 0.01 indicates a statistically significant difference from the control group).

Figure 5. Inhibitory effect of 1 on TPA-induced ear edema in mice. (A) Inhibitory effect of 1 on TPA-induced ear edema. Compound 1 was administered orally 30 min prior to TPA (1.0 μg/ear) application in the right ear of ICR mice. The mice were sacrificed 4 h after topical TPA treatment, and ear biopsies were obtained for the determination of edema formation by measurement of the ear weight and analysis of inflammatory proteins by Western blotting. (B) The expression of iNOS, COX-2, and IL-1β was determined using homogenates of ear biopsies and Western blot analysis. Data are representative of three independent experiments. β-Actin was used as an internal standard. 922

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kits used for the measurement of PGE2 and IL-1β were purchased from R&D Systems (Minneapolis, MN, USA). Handelin (1; purity >98% by HPLC analysis) was isolated from a CHCl3-soluble extract of the flowers of Chrysanthemum boreale, as described previously.18 Cell Culture. Mouse macrophage RAW 264.7 cells, obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/ mL amphotericin B. Cells were incubated at 37 °C, with 5% CO2 in a humidified atmosphere. Nitrite Assay. To evaluate the inhibitory activity of the test compound on LPS-induced NO production, RAW 264.7 cells were cultured in 10% FBS−DMEM without phenol red, plated in 24-well plates (3 × 105 cells/mL), and incubated for 24 h. Cells were washed with PBS, fresh medium was added, and the cells were then incubated with 1 μg/mL LPS in the presence or absence of test compounds. After an additional 20 h of incubation, the media were collected and analyzed for nitrite accumulation as an indicator of NO production by the Griess reaction. Briefly, 180 μL of Griess reagent (0.1% N-(1naphthyl)ethylenediamine dihydrochloride in H2O and 1% sulfanilamide in 5% H3PO4) was added to 100 μL of each supernatant from LPS or sample-treated cells in 96-well plates. The absorbance was measured at 540 nm, and nitrite concentration was determined by comparison with a sodium nitrite standard curve. Percent inhibition was expressed as [1 − (NO level of test samples/NO levels of vehicletreated control)] × 100. The IC50 value, the sample concentration resulting in 50% inhibition of NO production, was determined using nonlinear regression analysis (% inhibition vs concentration). MTT Cell Viability Assay. After the Griess reaction, MTT solution (final concentration of 500 μg/mL) was added to each well and further incubated for 4 h at 37 °C. Each medium was discarded, and dimethyl sulfoxide (DMSO) was added to each well to dissolve generated formazan. The absorbance was measured at 570 nm, and percent survival was determined by comparison with a control group. Prostaglandin E2 Assay. The expression level of PGE2 was measured with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) according to the manufacturer’s instructions. Briefly, RAW 264.7 cells (3 × 105 cells/mL) were plated in 24-well plates and pretreated with the indicated concentration of 1 for 1 h prior to stimulation with 1 μg/mL of LPS for 20 h. Culture medium supernatants were collected for the determination of PGE 2 concentration by ELISA. Western Blot Analysis. Cells were incubated with various concentrations of 1 for the indicated times. The proteins from cell lysates were resolved by 6−15% SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with blocking buffer [5% nonfat dry milk in PBS containing 0.1% Tween-20 (PBST)] for 1 h at room temperature. After washing three times with PBST, the membranes were incubated with primary antibodies diluted in 3% nonfat dairy milk in PBST (1:200−1:2000) overnight at 4 °C. The membranes were washed three times with PBST and incubated with corresponding secondary antibodies diluted in 3% nonfat dry milk in PBST (1:1000−1:5000) for 2 h at room temperature. The membranes were washed three times with PBST and visualized with an enhanced chemiluminescence (ECL) detection kit (LabFrontier, Suwon, Korea). Blots were analyzed by LAS 3000 (Fuji Film Corp., Tokyo, Japan). Reverse Transcriptase−Polymerase Chain Reaction (RTPCR). RAW 264.7 cells were stimulated with 1 μg/mL LPS in the presence or absence of 1 for 5 h. Total cellular RNA was extracted with TRI reagent (Sigma). Briefly, 1 μg of total RNA was reverse transcribed using oligo-(dT)15 primers and avian myeloblastosis virus (AMV) reverse transcriptase (Promega). Specific primers were designed using Roche Applied System (Basel, Swiss) and custom synthesized by Bioneer Corporation (Seoul, Korea). The following sequences were used: iNOS F5′-ATGTCCGAAGCAAACATCAC-3′; iNOS R5′-TAATGTCCAGGAAGTAGGTG-3′; COX-2 F5′ GGAGAGACTATCAAGATAGTGATC-3′; COX-2 R5′ATGGTCAGTAGACTTTTACAGCTC-3′; IL-1β F5′TGCAGAGTTCCCCAACTGGTACATC-3′; IL-1β R5′-

GTGCTGCCTAATGTCCCCTTGAATC-3′; TNF-α F5′ATGAGCACAGAAAGCATGAT-3′; TNF-α R5′-TACAGGCTTGTCACTCGAAT-3′; β-actin F5′-TGTGATGGTGGGAATGGGTCAG-3′; β-actin R5′-TTTGATGTCACGCACGATTTCC-3′. PCR was performed in a reaction mixture containing the obtained cDNA, 0.2 mM dNTP mixture (Promega), 10 pmol of target gene-specific primers, and 0.25 unit of Taq DNA polymerase (Promega) using GeneAmp PCR system 2400 (Applied Biosystems, Foster, CA, USA). Each of the PCR steps was performed as follows: an initial denaturation step for 4 min at 94 °C; 25−30 cycles of an amplification step consisting of denaturation for 30 s at 94 °C, annealing for 30 s at 55 °C, and elongation for 30 s at 72 °C; and a final extension step for 5 min at 72 °C. The PCR products were separated on a 2% agarose gel by electrophoresis, and DNA bands were visualized by SYBR-Safe staining. Reporter Gene (SEAP; Secreted Embryonic Alkaline Phosphatase) Assay. To determine the effect of test compound on the activation of NF-κB, a reporter gene assay was performed as described previously, with some modifications.30 The cells were treated with test compound for 2 h and then further stimulated with LPS for an additional 16 h. Cell culture supernatants were heated at 65 °C for 5 min and reacted with SEAP assay buffer [2 M diethanolamine, 1 mM MgCl2, 500 μM 4-methylumbelliferyl phosphate (MUP)] in the dark at 37 °C for 1 h. Fluorescence from the product of the SEAP/MUP reaction was measured in relative fluorescence units using a 96-well plate fluorometer with excitation at 360 nm and emission at 449 nm and normalized by protein concentration. Data are expressed as the proportion to vehicle-treated control cells without LPS. Preparation of Nuclear Extracts. Nuclear extracts were prepared by the method of Beg et al.31 Briefly, cells were washed in phosphatebuffered saline, pelleted, and resuspended in lysis buffer (10 mM TrisHCl, pH 8.0, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 100 μM PMSF, and 0.2% NP-40). After 5 min on ice, the lysates were spun at 2500 rpm in a microcentrifuge at 4 °C for 4 min. The pelleted nuclei were briefly washed in lysis buffer without NP-40. The nuclear pellet was then resuspended in an equal-volume nuclear extract buffer (20 mM Tris-HCl, pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol). After a 10 min incubation at 4 °C, the nuclei were briefly vortexed and spun at 14 000 rpm for 5 min. The supernatant was then removed and used as a nuclear extract. Protein concentrations were determined by the Bradford assay.32 NF-κB DNA-Binding Activity. NF-κB binding activity was measured with the Trans-AM NF-κB transcription factor assay kit (Active Motif Japan, Tokyo, Japan) according to the manufacturer’s instructions. Nuclear extracts (5 μg) were incubated with plate-coated NF-κB consensus oligonucleotide (5′-GGGACTTTCC-3′). Plates were washed, and anti-NF-kB antibody (p50 or p65) was added to the well plates. The binding of each antibody (p50 or p65) was detected with the incubation of an HRP-conjugated secondary antibody and developed with tetramethylbenzidine (TMB) substrate. The reaction intensity was measured by the absorbance at 450 nm. MicroRNA-155 Determination. Quantitative RT-PCR analysis for microRNA-155 was performed using TaqMan miRNA assays (Applied Biosystems). After concentrations were determined with a NanoDrop instrument, 10 ng of RNA per reaction was subjected to an RT reaction. The RT reaction was carried out with a target-specific stem-loop primer under the following conditions: 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. The expression levels of the miRNA were determined by qRT-PCR using TaqMan Expression Assays (Applied Biosystems). Relative miRNA concentrations are given as the ratio between the amount of the target gene and the control β-actin. Animals. Male ICR mice (18−20 g, 5 weeks old) and male Sprague−Dawley (SD) rats (150−170 g, 5 weeks old) were purchased from Central Laboratory Animal, Inc. (Seoul, Korea). The animals were housed under standard laboratory conditions with free access to food and water. The temperature was thermostatically regulated to 22 ± 2 °C, and a 12 h light/dark schedule was maintained. Prior to their use, the mice were allowed to acclimatize for 1 week within the work area environment. All animal experiments were carried out in 923

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accordance with the Institutional Animal Care and Use Committee Guidelines of Seoul National University (permission number: SNU201110-4). Carrageenan-Induced Paw Edema. The carrageenan-induced hind paw edema model in rats was used for the assessment of antiinflammatory activity.33−35 The test compound 1 (10 or 20 mg/kg) was dissolved in 0.5% CMC-Na. Thirty minutes after the administration of 1, vehicle, or indomethacin, paw edema was induced by the subplantar injection of 0.1 mL of 1% freshly prepared carrageenan suspension in normal saline. The left hind paw was injected with 0.1 mL of normal saline. The paw volume was measured before (0 h) and at 0.5, 1, 2, 4, and 6 h after carrageenan injection using a plethysmometer (Ugo Basile, Comerio, Italy). TPA-Induced Ear Edema. 12-O-Tetradecanoylphorbol 13-acetate (TPA)-induced ear edema was established using the method previously reported by Rao et al.36 TPA (1.0 μg) was dissolved in acetone (20 μL) and applied to the inner and outer surfaces of the right ear of ICR mice. Compound 1, vehicle, or indomethacin was administered orally 30 min prior to the TPA application. The animals were sacrificed by cervical dislocation after 4 h, and ear biopsies were obtained with a punch (a diameter of 5 mm) and weighed. An increase in the weight of the right ear punch over the left indicated edema.37 Determination of the Production of Pro-Inflammatory Mediators in Inflamed Tissues. The carrageenan-inflamed left hind paws of rats and the TPA-inflamed left ears of mice were amputated at the end of each indicated time point. The tissues were homogenized using a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Protein levels of pro-inflammatory mediators (iNOS, COX-2, and IL-1β) in the supernatants were determined by Western blotting. Measurement of the Levels of IL-1β in Serum. Blood collection was performed 6 h after carrageenan injection. The serum collected was analyzed for IL-1β using an ELISA assay (R&D Systems), according to the manufacturer’s instructions. Statistical Analysis. All experiments were repeated at least three times. Data are presented as the means ± SD for the indicated number of independently performed experiments. Statistical significance (p < 0.05) was assessed by a one-way analysis of variation (ANOVA) coupled with Dunnett’s t-test.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-2-880-2475. Fax: +82-2-762-8322. E-mail: sklee61@ snu.ac.kr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (MRC No. 2009-0083533).

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