Ursolic Acid Isolated from the Seed of Cornus officinalis Ameliorates

Publication Date (Web): September 12, 2014 ... Ursolic acid, which was isolated from an ethanol extract of Cornus officinalis seed, potently inhibited...
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Ursolic Acid Isolated from the Seed of Cornus officinalis Ameliorates Colitis in Mice by Inhibiting the Binding of Lipopolysaccharide to Toll-like Receptor 4 on Macrophages Se-Eun Jang,†,‡ Jin-Ju Jeong,† Supriya R. Hyam,† Myung Joo Han,‡ and Dong-Hyun Kim*,†,§ †

Department of Life and Nanopharmaceutical Sciences, ‡Department of Food and Nutrition, and §Department of Pharmacy, Kyung Hee University, Seoul 130-701, Korea S Supporting Information *

ABSTRACT: Ursolic acid, which was isolated from an ethanol extract of Cornus officinalis seed, potently inhibited nuclear factor κ light-chain enhancer of activated B cells (NF-κB) activation in lipopolysaccharide (LPS)-stimulated peritoneal macrophages. Therefore, we investigated the anti-inflammatory mechanism of ursolic acid in LPS-stimulated macrophages and colitic mice. Ursolic acid inhibited phosphorylation of interleukin 1 receptor-associated kinase (IRAK)1, TAK1, inhibitor of nuclear factor κB kinase subunit β (IKKβ), and IκBα as well as activation of NF-κB and MAPKs in LPS-stimulated macrophages. Ursolic acid suppressed LPS-stimulated interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and inducible NO synthetase (iNOS) expression as well as PGE2 and NO levels. Ursolic acid not only inhibited the Alexa Fluor 488conjugated LPS-mediated shift of macrophages but also reduced the intensity of fluorescent LPS bound to the macrophages transiently transfected with or without MyD88 siRNA. However, ursolic acid did not suppress NF-κB activation in peptidoglycan-stimulated macrophages. Oral administration of ursolic acid significantly inhibited 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colon shortening and myeloperoxidase (MPO) activity in mice. Ursolic acid also suppressed TNBSinduced COX-2 and iNOS expression as well as NF-κB activation in colon tissues. Ursolic acid (20 mg/kg) also inhibited TNBSinduced IL-1β, IL-6, TNF-α by 93, 86, and 85%, respectively (p < 0.05). However, ursolic acid reversed TNBS-mediated downregulation of IL-10 expression to 79% of the normal control group (p < 0.05). On the basis of these findings, ursolic acid may ameliorate colitis by regulating NF-κB and MAPK signaling pathways via the inhibition of LPS binding to TLR4 on immune cells. KEYWORDS: ursolic acid, Cornus off icinalis, macrophage, toll-like receptor 4, colitis

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INTRODUCTION Inflammation is an adaptive response to pathogen infection or injury and is highly regulated by the secretion of cytokines from inflammatory cells, such as neutrophils, monocytes, and lymphocytes.1 Among these cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β are expressed through canonical and non-canonical nuclear factor κ light-chain enhancer of activated B cells (NF-κB) signaling pathways, but they also regulate NF-κB activation.2 The canonical NF-κB signaling pathway is primarily involved in natural immunity and most inflammation processes, while the non-canonical pathway plays a role in B-cell maturation and autoimmune diseases.3 NF-κB transcription factor is essential for proper immune functions, but its excess activation by inflammatory stimuli, including lipopolysaccharide (LPS), peptidoglycan, IL-1β, and TNF-α, often causes inflammation.4,5 These stressors activate inflammatory signaling pathways via toll-like receptors (TLRs) and/or cytokine receptors.3 TLRs act as transmembrane co-receptors with cluster determinant 14 in the cellular response to onslaughts, such as LPS and peptidoglycan.3,6 Of those, TLR2 specifically recognizes components from Gram-positive bacteria, including lipoteicoic acid and macrophage-activating lipopeptide, with the assistance of CD36.7 TLR4 recognizes LPS on Gram-negative bacteria with the assistance of LPS-binding protein (LBP).8 © XXXX American Chemical Society

TLR-2 and TLR4 are associated with the peptidoglycan- and LPS-mediated activation of transcription factor NF-κB. The binding of peptidoglycan or LPS to TLR2 or TLR4, respectively, is initiated through the Toll/IL-1R domain of its cytoplasmic tail, which recruits myeloid differentiation factor 88 (MyD88), resulting in the activation of NF-κB and MAPK pathways via subsequent activation of interleukin 1 receptorassociated kinases (IRAKs), and induces expression of proinflammatory cytokines.6,9 Therefore, regulating inflammatory signaling pathways can be beneficial in curing chronic inflammatory diseases, such as colitis and rheumatism.10,11 The dried fruit of Cornus officinalis (FCO, Cornaceae) has been used as a tonic and anti-inflammatory functional food in China, Japan, and Korea. FCO contains ursolic acid, a wellknown compound distributed in many plants, and is a major constituent in many natural products. FCO has been reported to possess antidiabetic, antioxidative, and antihyperglycemic effects.12,13 However, the anticolitic effect of FCO has not been studied thoroughly. Received: March 29, 2014 Revised: September 4, 2014 Accepted: September 12, 2014

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resuspended with RPMI 1640 and seeded in a 12-well microplate. After incubation for 1 h at 37 °C, the cells were washed 3 times and non-adherent cells were removed by aspiration. Attached cells were used as peritoneal macrophages. The cells (0.5 × 106 cells/well) were cultured in 24-well plates at 37 °C for 3 days in RPMI 1640 plus 10% fetal bovine serum (FBS). To count the number of macrophage cells, the adherent macrophages were then scraped with a rubber policeman, suspended into fresh phosphate-buffered saline (PBS), stained with Giemsa, and counted by a hemocytometer. To examine the antiinflammatory effect of ursolic acid, macrophages were cultured in the absence or presence of ursolic acid (dissolved in 0.1% dimethyl sulfoxide) with 100 ng/mL LPS or peptidoglycan. ELISA and Immunoblotting in Peritoneal Macrophages. For the assay of NF-κB activation-inhibitory activity of FCO ethanol extract, peritoneal macrophages (0.5 × 106 cells) were stimulated with LPS (100 ng/mL) for 90 min in the presence or absence of FCO ethanol extract (10 or 20 μg/mL), then lysed, and centrifuged at 2000g for 10 min.15 The macrophages (0.5 × 106 cells) were also stimulated with LPS (100 ng/mL) or peptidoglycan (100 ng/mL) for 90 min (for the assay of IRAK-4, IRAK-1, p-IKK-β, IκB-α, p65, p-p65, p38, p-p38, ERK, p-ERK, JNK, p-JNK, and β-actin) or 20 h (for the assay of COX2, iNOS, and β-actin) in the presence or absence of ursolic acid (10 and 20 μM) (dissolved in 0.1% dimethyl sulfoxide), then lysed, and centrifuged at 2000g for 10 min. Cell supernatants were used for immunoblot and ELISA analyses. Cytokine levels were assayed using an ELISA kit. Protein expression was analyzed by immunoblotting. Briefly, the supernatants were separated by 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dried-milk proteins in phosphatebuffered saline containing 0.05% Tween 20 (PBST, Biosesang, Inc., Seoul, Korea) and then probed with COX-2, iNOS, IRAK-4, IRAK-1, p-IKK-β, IκB-α, p65, p-p65, p38, p-p38, ERK, p-ERK, JNK, p-JNK, or β-actin antibodies. After washing with PBST, proteins were detected by incubating with horseradish peroxidase (HRP)-conjugated secondary antibodies for 50 min. Bands were visualized using an enhanced chemiluminescence reagent (Millipore Cooperation, Billerica, MA).15 Immunofluorescent Confocal Microscopy. For the assay of LPS/TLR4 complex formation, peritoneal macrophages plated on cover slides were cultured at 37 °C for 20 h. Cells were treated with Alexa Fluor 594-conjugated LPS (100 ng/mL, Invitrogen) for 60 min in the absence or presence of ursolic acid (10 and 20 μM), as previously reported.16 Cells were fixed with 4% formaldehyde and 3% sucrose for 20 min, then treated with rabbit polyclonal anti-TLR4 antibody (Santa Cruz Biotechnology) for 90 min at 4 °C, and incubated with Alexa Fluor 488-conjugated secondary antibodies (Invitrogen) for 1 h. Stained cells were observed with a confocal microscope. Flow Cytometry. Peritoneal macrophages (1 × 105 cells) were incubated with or without Alexa Fluor 448-conjugated LPS (10 μg/ mL) in the absence or presence of ursolic acid (5, 10, and 20 μM) for 30 min. Cells were fixed in PBS containing 3% sucrose and 4% paraformaldehyde for 20 min, as previously reported,16 and then analyzed by flow cytometer (C6 Flow Cytometer System, Ann Arbor, MI). Transient Transfection of Small Interfering RNA (siRNA). Peritoneal macrophages were seeded at 3 × 105 cells/well in 24-well plates and allowed to rest for 2 days prior to transfection. Cells were transfected with 100 nM siRNA for TLR4 (Dharmacon, Chicago, IL) or MyD88 (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the protocol of the manufacturer. At 48 h after transfection, cells were treated with or without ursolic acid (10 and 20 μM) and/or LPS (100 ng/mL). Preparation of Experimental Colitis in Mice. To investigate the curative effect of ursolic acid against colitis in mice, mice were divided into five groups: normal control and TNBS-induced colitis groups treated in the absence or presence of ursolic acid (10 and 20 mg/kg) or mesalazine (10 mg/kg) dissolved in 2% Tween 80.21 Each group consisted of six mice. TNBS-induced colitis was induced by intrarectal

In a preliminary experiment, FCO seed ethanol extract inhibited NF-κB activation in LPS-stimulated peritoneal macrophages. Therefore, we isolated the primary antiinflammatory constituent, ursolic acid, from the seed of FCO and investigated its anti-inflammatory effect in LPS- or peptidoglycan-stimulated peritoneal macrophages and 2,4,6trinitrobenzenesulfonic acid (TNBS)-induced colitis mice.

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MATERIALS AND METHODS

Chemicals. RPMI 1640, peptidoglycan purified from Staphylococcus aureus, LPS purified from Escherichia coli O111:B4, and TNBS were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies for cyclooxygenase (COX)-2, inducible NO synthetase (iNOS), and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for IRAK-4, IRAK-1, p-IRAK-1, IκB-α, p-IKK-β, ERK, p-ERK, JNK, p-JNK p38, p-p38, p65, and p-p65 were purchased from Cell Signaling Technology (Danvers, MA). Enzymelinked immunosorbent assay (ELISA) kits for cytokines and prostaglandin E2 (PGE2) were from R&D Systems (Minneapolis, MN). 3,3′-Diaminobenzidine tetrahydrochloride substrate was purchased from Thermo Scientific (Rockford, IL). Isolation of Ursolic Acid. The dried seed of FCO (500 g) was extracted 3 times with hexane (3 L) at room temperature and 80% ethanol (2.5 L) in boiling water, successively, and evaporated under reduced pressure (35.2 g). The ethanol extract was suspended in distilled water, fractionated with BuOH twice, and evaporated under reduced pressure (21.5 g). The BuOH-soluble fraction was subjected to silica gel column chromatography (40 × 5 cm) with a gradient of CHCl3/MeOH (100:1, 50:1, 25:1, 20:1, 10:1, 5:1, and 0:1, each 1.2 L) to afford seven subfractions. Of them, subfraction E2 (2.5 g) most potently inhibited NF-κB activation in LPS-stimulated peritoneal macrophages. Therefore, it was applied for isolation of the active constituents. Subfraction E2 was subjected to silica gel column chromatography (30 × 5 cm) with a gradient of CHCl3/MeOH (40:1−10:1) to give four subfractions (E2.1−E2.4). Of them, the most potent anti-inflammatory constituent E2.2 (53 mg) was obtained as crystals in MeOH. The isolated compound was identified to be ursolic acid by instrumental analysis [nuclear magnetic resonance (NMR), Varian UNITY INOV-500 spectrophotometer, API-2000 spectrometer] and compared to an authentic standard, as reported in the literature.14 Ursolic Acid (Purity of >95%). Amorphous white powder. IR νmax (cm−1): 1692, 2928, 3421. Electrospray ionization mass spectrometry (ESI−MS) (m/z): [M+] 456. 1H NMR (CDCl3, 500 MHz) δ: 5.52 (1H, t, J = 3.5 Hz, H-12), 3.48 (1H, dd, J = 10.5 and 4.0 Hz, H-3), 2.63 (1H, d, J = 3.5 Hz, H-18), 1.27, 1.26, 1.10, 1.08 (each 3H, s H-23, 27, 26, 24), 1.02 (3H, d, J = 6.5 Hz, H-30), 0.98 (3H, d, J = 6.5 Hz, H29), 0.89 (3H, s, H-25). 13C NMR (CDCl3, 125 MHz) δ: 180.1 (C28), 139.7 (C-13), 126.1 (C-12), 78.6 (C-3), 56.2 (C-18), 53.8 (C-5), 47.1 (C-17), 42.3 (C-14), 39.6 (C-4), 39.4 (C-1), 37.5 (C-22), 34.6 (C-10), 33.6 (C-7), 29.1 (C-19, C-20), 29.0 (C-15), 26.4 (C-21), 24.2 (C-27), 24.1 (C-11), 24.1 (C-2), 24.0 (C-23, C-30), 23.8 (C-16), 21.7 (C-29), 19.1 (C-6), 16.9 (C-24, C-25), 16.0 (C-26). Animals. All animal experiments were approved by the Committee for the Care and Use of Laboratory Animals in the College of Pharmacy, Kyung Hee University (KHP-2012-11-01-R1) and performed according to the Kyung Hee University guides for Laboratory Animals Care and Usage. Male C57BL/6J mice (male, 19−23 g, 6 weeks old) and TLR4deficient C57BL/10ScNJ mice (male, 19−23 g, 6 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME). All animals were fed standard laboratory chow (Samyang Co., Seoul, South Korea), housed in wire cages at 20−22 °C and 50 ± 10% relative humidity, and allowed water ad libitum. Isolation and Culture of Peritoneal Macrophages. The mice were intraperitoneally injected with 4% thioglycolate solution (2 mL) and sacrificed on the fourth day after injection, and their peritoneal cavities were rinsed with RPMI 1640 (10 mL).15 The peritoneal lavage fluid was centrifuged at 200g for 10 min. Collected cells were B

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Figure 1. Effect of ursolic acid (UA) on IRAK1 phosphorylation and NF-κB activation in normal TLR4+/+ and TLR4−/− peritoneal macrophages stimulated with lipopolysaccharide (LPS) or peptidoglycans (PG). (A) Effect in (a) LPS- or (b) PG-stimulated TLR4+/+ peritoneal macrophages. (B) Effect in (a) LPS- or (b) PG-stimulated TLR4−/− peritoneal macrophages. Peritoneal macrophages were isolated from TLR4+/+ and TLR4−/− mice and then incubated with LPS (100 ng/mL) or PG (100 ng/mL) in the absence or presence of UA (5, 10, or 20 μM) for 60 min. Protein expression of IRAK1, p-IRAK1, p65, p-p65, and β-actin was assayed by immunoblotting. (C) Cell viability of UA for (a) TLR4+/+ and (b) TLR4−/− peritoneal macrophages. Cell viability was measured by a tryphan blue staining assay. Ursolic acid was treated for 48 h. All data are expressed as the mean ± SD (n = 4 in a single experiment). injection of 2.5% (w/v) TNBS solution (100 μL) in 50% ethanol into the colon of lightly anesthetized (with ether) mice via a thin round-tip needle equipped with a 1 mL syringe.15 The normal control group was treated with vehicle alone instead of TNBS and test agents. A needle was inserted to 3.5−4 cm proximal to anal verge. To distribute agents within the entire colon, the mice were held in a vertical position for 30 s after injection. Using this procedure, >96% of the mice retained the TNBS enema. If an animal quickly excreted the TNBS−ethanol solution, it was excluded. Ursolic acid (10 and 20 mg/kg) or mesalazine (10 mg/kg) was orally administered once a day for 3 days from the first day after TNBS treatment. Mice were sacrificed 18 h after the final administration of test agents. The colon was removed

quickly, then opened longitudinally, and gently washed by PBS. Macroscopic assessment of the colitis grade was scored: 0, no ulcer and no inflammation; 1, no ulceration and local hyperemia; 2, ulceration without hyperemia; 3, ulceration and inflammation at one site only; 4, two or more sites of ulceration and inflammation; and 5, ulceration extending more than 2 cm, as previously described.21 The colon was then stored at −80 °C for immunoblotting and ELISA. For histological exams, the colon was fixed with 4% paraformaldehyde and then stored in 30% sucrose solution. For histologic exams, colons were fixed in 10% buffered formalin solution, embedded in paraffin, cut into 7 μm sections, stained with hematoxylin−eosin, and then observed under a light microscope. C

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Figure 2. Effect of ursolic acid (UA) on the activation of TLR4/NF-κB and MAPKs in lipopolysaccharide (LPS)-stimulated peritoneal macrophages. (A) Effect on TLR4/NF-κB signaling pathway. (B) Effect on translocation of NF-κB into the nuclei. (C) Effect on the activation of MAPKs. Peritoneal macrophages were incubated with LPS (100 ng/mL) in the absence or presence of UA (10 and 20 μM) for 60 min. Protein expressions were assayed by immunoblotting. Assay of Myeloperoxidase (MPO) Activity. Colons were homogenized in 10 mM potassium phosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethylammonium bromide and then centrifuged at 15000g and 4 °C for 20 min.16 The supernatant (50 μL) was added in a reaction mixture preincubated at 37 °C for 5 min, which contained 0.1 mM H2O2 and 1.6 mM tetramethylbenzidine and scanned at 650 nm at 37 °C for 10 min. MPO activity was defined as the enzyme quantity degrading 1 μmol of hydrogen peroxide/mg of protein. The amount of protein was determined by the Bradford protein assay kit, as previously reported.16 Determination of Cytokines, PGE2, and Nitrite in the Colons of Mice. Cytokine and PGE2 levels were determined using an ELISA kit. Briefly, colons were homogenized in ice-cold RIPA lysis buffer (1 mL) containing 1% phosphatase inhibitor cocktail and 1% protease inhibitor cocktail. The lysate was centrifuged at 15000g and 4 °C for 10 min, and the resulting supernatant was used for immunoblotting and ELISA, as previously described.16 Nitrite was measured using Griess reagent.15 The supernatant (100 μL) was mixed with 100 μL of Griess reagent [the mixture of an equal volume of 1% sulfanilamide in 5% H3PO4 and 0.1% N-(1naphthyl)ethylenediamine dihydrochloride in H2O] in a 96-well plate, and then the absorbance was spectrophotometrically measured at 550 nm. The concentration of nitrite was determined, using a sodium nitrite, as a standard. Statistical Analysis. Data are presented as the mean ± standard deviation (SD) and were statistically analyzed using one-way analysis

of variation (ANOVA) and Turkey’s multiple comparison tests (12.0K for Windows, SPSS, Inc., Chicago, IL). p values of 0.05 or less were considered statistically significant.

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RESULTS Inhibitory Effect of Ursolic Acid on the Expression of Inflammatory Mediators in LPS- or PeptidoglycanStimulated Peritoneal Macrophages. In a preliminary study, FCO seed 80% ethanol extract potently inhibited NFκB activation in LPS-stimulated peritoneal macrophages (see Supplemental Figure S1 of the Supporting Information). Therefore, we isolated ursolic acid, a potent inhibitor for NFκB activation, through activity-guided fractionation and investigated the inhibitory effect of ursolic acid on LPS-linked TLR4/NF-κB activation in TLR4+/+ or TLR4−/− peritoneal macrophages activated with LPS or peptidoglycan. The stimulation of LPS or peptidoglycan increased IRAK1 phosphorylation and NF-κB activation in TLR4+/+ cells (Figure 1A). Treatment with ursolic acid inhibited LPS-induced IRAK1 phosphorylation and NF-κB activation. In contrast, ursolic acid did not inhibit peptidoglycan-induced IRAK1 phosphorylation and NF-κB activation in TLR4+/+ cells. However, the stimulation of LPS failed to significantly activate IRAK1 and NF-κB in TLR4-deficient (TLR−/−) cells, but the stimulation of D

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Figure 3. Effect of ursolic acid (UA) on the expression of inflammatory markers in peritoneal macrophages stimulated with lipopolysaccharide (LPS). (A) Effect on TNF-α, IL-1β, IL-6, and IL-10. (B) Effect on the production of PGE2 and NO. (C) Effect on the expression of iNOS and COX-2. Peritoneal macrophages were incubated with LPS (100 ng/mL) in the absence or presence of UA (10 or 20 μM) for 20 h. Cytokines and PGE2 were assayed by ELISA. iNOS and COX-2 were assayed by immunoblotting. NO was assayed, using Griess reagent. All data are the mean ± SD (n = 4). (#) Significantly different versus the normal group (p < 0.05). (∗) Significantly different versus the control group (p < 0.05).

Phosphorylation of inhibitor of nuclear factor κB kinase subunit β (IKKβ) and IκBα (through ubiquitination and proteolytic degradation) leads to the translocation of NF-κB into the nuclei.17 Therefore, we measured the effect of ursolic acid on phosphorylation of TAK1, IKKβ, and IκBα and degradation of IRAK1 and IRAK4 in LPS-stimulated peritoneal macrophages (Figure 2A). Treatment with LPS significantly induced these processes. Ursolic acid significantly inhibited the phosphorylation of these proteins. To confirm the inhibitory effect of ursolic acid on NF-κB activation, we measured its

peptidoglycan significantly increased IRAK1 phosphorylation and NF-κB activation (Figure 1B). Notably, ursolic acid could not inhibit peptidoglycan-induced IRAK1 and NF-κB activation in the TLR4-deficient cells. To investigate the cytotoxicity of ursolic acid, peritoneal macrophages were treated with ursolic acid (10 and 20 μM) for 48 h. Cells were then trypsinized, stained with Trypan blue solution, and assessed for viability. No cytotoxic effect of ursolic acid was observed under the experimental conditions (Figure 1C). E

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Figure 4. Inhibitory effect of ursolic acid (UA) on the binding of LPS to TLR4 in peritoneal macrophages. Peritoneal macrophages isolated from mice were incubated with Alexa Fluor 488-conjugated LPS for 30 min for a flow cytometry or 60 min for a confocal microscopy in the absence or presence of UA (5, 10, and 20 μM), and the cells were stained with rabbit polyclonal anti-TLR4 antibody for 2 h at 4 °C, incubated with secondary antibodies conjugated with TRITC for 1 h, and then analyzed using a (A) flow cytometer and (B) confocal microscope. The normal group was treated with vehicle alone instead of LPS and UA.

inhibitory effect on the translocation of NF-κB into the nuclei in LPS-stimulated peritoneal macrophages (Figure 2B). LPS

significantly increased the translocation of NF-κB into the nuclei, which was significantly inhibited by ursolic acid. We also F

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Figure 5. Effect of ursolic acid (UA) on the expression of TLR4 and MyD88 and activation of NF-κB in peritoneal macrophages transfected with or without TLR4 siRNA and/or MyD88 siRNA. (A) Peritoneal macrophages isolated from mice were incubated with Alexa Fluor 594-conjugated LPS (100 ng/mL) for 30 min in the absence or presence of UA (10 and 20 μM), and then LPS binding on the surface of TLR4 and/or MyD88 siRNAtransfected peritoneal macrophages was measured using a confocal microscope. (B) Effect on NF-κB activation. TLR4, MyD88, p65, p-p65, and βactin proteins were assayed by immunoblotting.

investigated the effect of ursolic acid on MAPK activation in LPS-stimulated peritoneal macrophages (Figure 2C). LPS stimulation activated ERK, JNK, and p38. However, ursolic acid significantly suppressed their phosphorylation. Next, we measured the effect of ursolic acid on the expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α in LPS-stimulated peritoneal macrophages (Figure 3A). LPS significantly increased the expression of these cytokines. Treatment with ursolic acid inhibited TNF-α, IL-1β, and IL-6 expression. Urosolic acid also decreased LPS-induced PGE2 and NO production (Figure 3B). Further, ursolic acid suppressed LPS-induced COX-2 and iNOS expression (Figure 3C). Ursolic Acid Inhibits the Binding of LPS to TLR4. Treatment with ursolic acid (5, 10, and 20 μM) inhibited the phosphorylation of IRAK1, IκBα, and p65 in LPS-stimulated peritoneal macrophages. Therefore, we examined the ability of ursolic acid to inhibit LPS binding to TLR4 on peritoneal macrophages using a flow cytometer (Figure 4A). Treatment of peritoneal macrophages with Alexa Fluor 488-conjugated LPS induced a significant shift. However, treatment with ursolic acid

remarkably restored the Alexa Fluor 488-conjugated LPSmediated shift of macrophages. Next, we examined the ability of ursolic acid to inhibit LPS binding to TLR4 on peritoneal macrophages using a confocal microscope (Figure 4B). Alexa Fluor 488-conjugated TLR4 antibody was found to bind the macrophages, and this binding was unaffected by ursolic acid treatment. Alexa Fluor 594-conjugated LPS was also found to potently bind to macrophages. However, ursolic acid treatment significantly inhibited the binding of Alexa Fluor 594conjugated LPS to the macrophages. To further confirm whether ursolic acid inhibits LPS binding to TLR4 on peritoneal macrophages, the macrophages were transiently transfected with TLR4 or MyD88 siRNA for 48 h and then protein expression of TLR4 and MyD88 was detected (Figure 5). Knockdown efficiencies of TLR4 and MyD88 were 93 and 89%, respectively, as determined by immunoblotting. When the cells were treated with Alex 594-conjugated LPS, the LPS did not bind to the macrophages transfected with TLR4 siRNA. Ursolic acid did not influence the binding of LPS to TLR4 or MyD88 siRNA-transfected macrophages (data not shown). However, it was observed that LPS was bound to G

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Figure 6. Effect of ursolic acid (UA) on (A) body weight, (B) colon length, (C) macroscopic score, (D) peritoneal MPO activity, and (E) histology in the colon tissues of TNBS-induced colitic mice. TNBS (2.5% TNBS in 50% ethanol) was intrarectally administered in mice. Test agents (TNBS, vehicle alone; UA10, 10 mg/kg of UA; UA20, 20 mg/kg of UA; and MEL, 10 mg/kg of mesalazine) were orally administered. The normal group (NOR) was treated with vehicle alone in normal mice. Test agents (dissolved in 2% Tween 80) were orally administered once daily for 3 days after TNBS treatment. Mice were sacrificed 18 h after the final administration of test agents. Colons were stained with hematoxylin−eosin and then assessed by light microscopy. All data are the mean ± SD (n = 6). (#) Significantly different versus the normal group (p < 0.05). (∗) Significantly different versus the control group (p < 0.05).

ulcerations. Orally administered ursolic acid suppressed edema and epithelial cell disruption. We also measured the levels of proinflammatory cytokines, IL-1β, IL-6, and TNF-α, and an anti-inflammatory cytokine, IL-10, in the colons of TNBS-treated mice (Figure 7A). TNBS significantly increased TNF-α, IL-1β, IL-6, and IL-10 expression. Ursolic acid inhibited the expression of pro-inflammatory cytokines TNFα, IL-1β, and IL-6, although β-actin expression was not affected. Thus, ursolic acid (20 mg/kg) inhibited expression of these cytokines by 93% for IL-1β (p < 0.05), 86% for TNF-α (p < 0.05), and 85% for IL-6 (p < 0.05). However, ursolic acid reversed the TNBS-mediated downregulation of IL-10 expression to 79% of the normal control group (p < 0.05). Treatment with TNBS also increased the expression of COX-2 and iNOS, the degradation of IRAK1 and IRAK4, and the activation of IRAK1, IKKβ, and NF-κB (p-p65) (Figure 7B), all of which were blocked by ursolic acid. Further, ursolic acid inhibited the degradation of IRAK1 and IRAK4.

MyD88 siRNA-transfected cells. Nevertheless, LPS did not activate NF-κB. When ursolic acid (20 μM) was treated prior to stimulation with LPS in MyD88 siRNA-transfected macrophages, ursolic acid potently inhibited the binding of LPS to TLR4 on these cells. Ursolic Acid Inhibits the Expression of Proinflammatory Cytokines and Activation of NF-κB in TNBS-Induced Colitic Mice. We also investigated the inhibitory effect of ursolic acid on TNBS-induced colitis in mice. When TNBS was intrarectally injected in mice, colitis was caused, as observed in previous reports.15 Oral administration of ursolic acid significantly inhibited TNBS-induced colon shortening and increased the macroscopic score (Figure 6). Ursolic acid (20 mg/kg) inhibited the TNBS-induced MPO activity by 92% compared to normal mice treated with TNBS alone (p < 0.05). Histologic examination of the colon in TNBS-induced colitic mice showed edema, dense infiltration of the superficial layers of the mucosa, and epithelial cell disruption by large H

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Figure 7. Effect of ursolic acid (UA) on the expression of inflammatory cytokines IL-1β, IL-6, and TNF-α, iNOS, and COX-2 and the activation of NF-κB in the colon tissues of TNBS-induced colitic mice. TNBS was intrarectally administered in control (2.5% TNBS in 50% ethanol), UA (10 or 20 mg/kg), and mesalazine (MEL, 10 mg/kg) groups; the normal group was treated with vehicle alone. Mice were sacrificed 18 h after the final administration of test agents. (A) Effect on the expression of proinflammatory cytokines. Cytokines were assayed by ELISA. (B) Effect on the expression of TLR4, iNOS, and COX-2 and the activation of NF-κB, as determined by immunoblotting. All data values are the mean ± SD (n = 6). (#) Significantly different versus the normal group (p < 0.05). (∗) Significantly different versus the control group (p < 0.05).

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rats25 as well as TPA/croton oil-induced ear edema.26 Ursolic acid also inhibits LPS-induced cognitive deficits by suppressing p38/NF-κB mediated inflammatory pathways,27 LPS-stimulated lung inflammation via NF-κB activation,28 ovalbumininduced airway inflammation,29 and zymosan-induced acute inflammation in mice.30 Therefore, we investigated the anti-inflammatory effect on TNBS-stimulated colitis in mice. We found that ursolic acid inhibited the expression of inflammatory enzymes, COX-2 and iNOS, as well as activation of their transcription factor NF-κB. Ursolic acid also inhibited the expression of pro-inflammatory cytokines, TNF-α and IL-1β, but increased the expression of anti-inflammatory cytokine, IL-10, which was reduced by TNBS. These results suggest that ursolic acid may inhibit colitis by inhibiting NF-κB activation as well as the activation of Th1 cells, which are activated by pro-inflammatory cytokines TNF-α and IL-1β,31 rather than Th2 cells, which are activated by IL-4 and IL-10.31 This suggestion is supported by previous studies showing that ursolic acid protects CCl4-induced inflammation by inhibiting MAPK, STAT3, and NF-κB signaling pathways.32 Ursolic acid also inhibits Th1 cytokine IL-2 production and induces Th2 cytokine IL-10 production in murine primary splenocytes.33 In the present study, we found that ursolic acid inhibited the expression of inflammatory markers COX-2, iNOS, TNF-α, and IL-1β as well as activation of NF-κB and MAPKs in LPSstimulated peritoneal macrophages, as previously reported in RAW264.7 cells.34,35 Ursolic acid inhibits the expression of proinflammaotry cytokines in murine T cells, B cells, and macrophages as well as activation of NF-κB in T cells.36 Ursolic acid also inhibits TPAinduced COX-2 expression and PGE2 synthesis in human mammary epithelial cells via the protein kinase C/MAPK signaling pathway37 as well as the leucine-stimulated mROC1 signaling pathway in C2C12 myotube cells by suppressing mTOR localization to lysosome.38

DISCUSSION Inflammation is a complex, highly sequential series of events provoked by a variety of stimuli, including pathogens.18 Inflammation is classified as acute or chronic. Acute inflammation is a normal and beneficial response to injury or infection. However, chronic inflammation is excessive and persistent. This chronically inflammatory response causes progressive injuries to the body, resulting in a variety of severe inflammatory diseases, such as colitis and rheumatoid arthritis.19,20 The inflammatory process is usually tightly regulated by inflammatory mediators, such as TNF-α, IL-1β, and IL-6, and immune cells, such as neutrophils, monocytes, and lymphocytes.1 Of these inflammatory mediators, TNF-α and IL-1β are activated through NF-κB, which consists of five members, including RelA, cRel, RelB, p50, and p52.3,21 There are canonical and non-canonical NF-κB signaling pathways. The canonical NF-κB is primarily involved in natural immunity and most inflammation, while non-canonical NF-κB plays a role in B-cell maturation and autoimmune diseases. NF-κB is an essential transcription factor for immune functions. However, excessive NF-κB activation often causes inflammation.4 Bacterial lipopolysaccharides or peptidoglycans increase blood IL-1β and TNF-α levels via canonical and non-canonical TLRlinked NF-κB signaling pathways and cause inflammation, although blood IL-1β and TNF-α levels are hardly detectable in mice without any stimuli or treatment.5,22 Therefore, to regulate the expression of proinflammatory cytokines via the canonical NF-kB signaling pathway in macrophages, many constituents isolated from natural products, including saponins and flavonoids, have recently been applied.23,24 In a preliminary study, FCO seed 80% ethanol extract potently inhibited NF-κB activation in LPS-stimulated peritoneal macrophages. We isolated ursolic acid as a potent inhibitor for NF-κB activation from FCO seed. Ursolic acid, which is distributed in vegetables and functional foods as a main constituent, inhibits carrageenan-induced paw edema in I

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However, its anti-inflammatory mechanism has not been thoroughly studied. To understand the anti-inflammatory mechanism of ursolic acid, we investigated its effect on IRAK phosphorylation and binding of LPS to TLR4 in macrophages. Ursolic acid not only inhibited the binding of LPS to TLR4 on peritoneal macrophages transfected with or without MyD88 siRNA but also suppressed the phosphorylation of IRAK1 and IκBα and the degradation of IRAK1 and IRAK4. Further, ursolic acid inhibited the expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 as well as activation NF-kB and MAPKs. However, ursolic acid did not inhibit IP-10 synthesis in T84 colon carcinoma cells.39 These results suggest that ursolic acid may suppress NF-κB and MAPK signaling pathways by inhibiting the binding of LPS to TLR4 on immune cells, such as macrophages. Additionally, we investigated the effect of ursolic acid on systemic inflammation and septic death in mice peritoneally injected with LPS (see Supplemental Figure S2 of the Supporting Information). Ursolic acid [10 mg/kg, per os (po)] significantly reduced LPS-induced blood proinflammatory cytokines IL-1β and TNF-α levels. Further, Ursolic acid inhibited LPS-induced septic death (systemic inflammation). These results also suggest that ursolic acid may inhibit LPSstimulated inflammatory responses. On the basis of these findings, we conclude that ursolic acid may ameliorate inflammatory diseases, such as colitis, by regulating NF-κB and MAPK signaling pathways via the inhibition of LPS binding to TLR4 on immune cells.

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ASSOCIATED CONTENT

Effect of C. officinalis seed 80% ethanol extract (FCO) on NFκB activation in lipopolysaccharide-stimulated peritoneal macrophages (Supplemental Figure S1) and Inhibitory effect of ursolic acid (UA) on IL-1β and TNF-α production and septic death in mice intraperitoneally injected with LPS (Supplemental Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Telephone: +82-2-961-0357. Fax: +82-2-957-5030. E-mail: [email protected]. Funding

This study was supported by a grant from BK21 Plus Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2013). Notes

The authors declare no competing financial interest.

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S Supporting Information *

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Article

ABBREVIATIONS USED

COX, cyclooxygenase; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; IKK-β, inhibitor of nuclear factor κB kinase subunit β; IL, interleukin; iNOS, inducible NO synthetase; IRAK, interleukin 1 receptor-associated kinase; LPS, lipopolysaccharide; MPO, myeloperoxidase; NF-κB, nuclear factor κ light-chain enhancer of activated B cells; TLR, toll-like receptor; TNBS, 2,4,6-trinitrobenzenesulfonic acid; TNF, tumor necrosis factor J

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