European Journal of Pharmacology 740 (2014) 652–661

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Immunopharmacology and inflammation

Mangiferin ameliorates colitis by inhibiting IRAK1 phosphorylation in NF-κB and MAPK pathways Jin-Ju Jeong a, Se-Eun Jang a,b, Supriya R. Hyam a, Myung Joo Han b, Dong-Hyun Kim a,n a Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, 1, Hoegi, Dongdaemun-gu, Seoul 130-701, Republic of Korea b Department of Food and Nutrition, Kyung Hee University, 1, Hoegi, Dongdaemun-gu, Seoul 130-701, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 25 March 2014 Received in revised form 30 May 2014 Accepted 2 June 2014 Available online 24 June 2014

Mangiferin, a main constituent of the root of Anemarrhena asphodeloides and the leaves of Mangifera indica, inhibits NF-κB activation in macrophages. Therefore, we investigated effect of mangiferin on 2,3,4-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice and its anti-inflammatory mechanism in lipolysaccharide (LPS)- or peptidoglycan-stimulated mouse peritoneal macrophages. Mangiferin inhibited phosphorylation of nuclear factor-kappaB (NF-κB), interleukin-1 receptor-associated kinase 1 (IRAK1), and mitogen-activated protein kinases (MAPK) in peptidoglycan- or LPS-stimulated peritoneal macrophages. Mangiferin in the presence of SN50 inhibited LPS-stimulated NF-κB activation more potently than mangiferin alone. Mangiferin inhibited interaction of fluorescent p-IRAK1 antibody to LPSstimulated peritoneal macrophages, but increased binding of fluorescent IRAK1 antibody. Mangiferin did not influence interaction of fluorescent LPS to toll-like receptor-4 on the macrophages. Molecular peak of mangiferin bound to IRAK1 was detected in the macrophages by mass analysis. Mangiferin (10 μM) inhibited LPS-stimulated expression of TNF-α, IL-1β and IL-6 by 81.0%, 89.5% and 88.3%, respectively, whereas it increased IL-10 expression by 131.8% compared to LPS-nontreated group. Mangiferin furthermore inhibited colon shortening, macroscopic score, and colonic myeloperoxidase activity in TNBS-induced colitic mice. Mangiferin inhibited TNBS-induced IRAK1 phosphorylation and NF-κB activation. Mangiferin suppressed TNBS-induced up-regulation of cyclooxygenase-2 and inducible NO synthase. Furthermore, mangiferin (20 mg/kg) significantly inhibited TNF-α by 78%, IL-1β by 82%, and IL-6 expressions by 88% (P o0.05), but induced IL-10 expression to 79% of the normal control group (Po 0.05). Based on these findings, mangiferin may ameliorate inflammatory diseases such as colitis by regulating NF-κB and MAPK signaling pathways through the inhibition of IRAK1 phosphorylation. & 2014 Elsevier B.V. All rights reserved.

Keywords: Mangiferin Colitis Macrophage IRAK1 NF-κB MAP kinase

1. Introduction Acute and chronic inflammations are the body's response to injury or infection (Mehta et al., 1998; Bistrian, 2007). Acute inflammation is a normal and helpful response to injury. However, chronic inflammation is persistent and excessive. This inflammatory response causes progressive damage to the body, leading to a variety of diseases, such as colitis, rheumatoid arthritis and even cancer. The inflammatory reactions can be mediated by

Abbreviations: COX, cyclooxygenase; ERK, extracellular signal-regulated kinase; IKK, IκB kinase; IL, interleukin; IRAK, interleukin-1 receptor-associated kinase; JNK, c-Jun NH2-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-kappa B; PGE2, prostaglandin E2; TLR, Tolllike receptor; TIR, Toll/IL-1R; TNF, tumor necrosis factor; TNBS, 2,3,4-trinitrobenzene sulfonic acid. n Corresponding author. Tel.: þ 82 2 961 0357; fax: þ 82 2 957 5030. E-mail address: [email protected] (D.-H. Kim). http://dx.doi.org/10.1016/j.ejphar.2014.06.013 0014-2999/& 2014 Elsevier B.V. All rights reserved.

inflammatory mediators, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, interferon-γ, nitric oxide and prostaglandins in immune cells (Fairweather and Rose, 2005; Baldwinm, 1996). Of these inflammatory mediators, pro-inflammatory cytokines IL-1β and TNF-α are activated through nuclear factor-kappaB (NF-κB), but are inhibited by anti-inflammatory cytokine IL-10. NF-κB is a heterodimer consisting of five members, including RelA (p65), cRel, RelB, NF-κB1 p50, and NF-κB2 p52 (Perkins and Gilmore, 2006; Sören and Sten, 2004). There are canonical and noncanonical NF-κB signaling pathways; canonical NF-κB consists of mainly p65/p50 and noncanonical of RelB/p52. The former is mainly involved in natural immunity and mostly inflammation, while the latter in B-cell maturation and autoimmune diseases. NF-κB is an essential transcription factor for proper immune functions, but if excessively activated it often causes inflammation (Li and Verma, 2002). Bacterial lipopolysaccharide (LPS) or peptidoglycan increases blood IL-1β and TNF-α levels via canonical and noncanonical toll-like receptors (TLRs)-NF-κB signaling pathway

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and causes inflammation, although blood IL-1β and TNF-α levels are barely detectable in mice without any stimuli or treatment (Blanqué et al., 1996; Chow et al., 1999; Fabre et al., 2012; Olson and Miller, 2004). Among TLRs, TLR4 recognizes LPS in the canonical NF-κB signaling pathway and initiates a signaling cascade through the Toll/IL-1R (TIR) domain of its cytoplasmic tail and MyD88, allowing for subsequent activation of IL-1R-associated kinases (IRAKs) (O’Neill, 2003; Paradkar et al., 2004). All interleukin-1 receptor-associated kinase (IRAK) members form multimeric receptor complexes. Phosphorylated IRAK1 activates a multimeric protein complex (TRAF6/TAK1/TAB1/TAB2), leading to the activation of NF-κB as well as the expression of proinflammatory cytokines. The use of natural products inhibiting the expression of these inflammatory mediators has recently become a topic of interest in the regulation of inflammatory diseases (Paradkar et al., 2004; Joh et al., 2011). Mangiferin (C2-β-D-glycopyranosyl-1,3,6,7-tetrahydroxyxanthone) is widely distributed in higher plants, such as Mangifera indica L. (Anacardiaceae family), Anemarrhena asphodeloides (Liliaceae family), and Cyclopia intermedia (Fabaceae family) (Jung et al., 2009; Garrido et al., 2004; Mckay and Blumberg, 2007). Mangiferin showed central nervous system-stimulating, anti-oxidant, analgesic, anti-inflammatory, and anti-diabetic activities (Jung et al., 2009; Garrido et al., 2004; Mckay and Blumberg, 2007; Pinto et al., 2005; Márquez et al., 2012). Indeed, mangiferin inhibits oxidative damage and inflammation in immobilized stress-stimulated rats, LPS-induced bronchitis and systemic inflammation and ovalbumin-induced asthma (Márquez et al., 2012). However, the detailed molecular mechanisms underlying the anti-inflammatory effects of mangiferin are not understood thoroughly. Therefore, to clarify anti-inflammatory mechanism of mangiferin, we investigated its anti-inflammatory effect in LPS-induced peritoneal macrophages, and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice.

2. Materials and methods 2.1. Materials Peptidoglycan purified from Staphylococcus aureus cell wall component, LPS purified from Escherichia coli O111:B4, TNBS, DMEM and RPMI 1640 were purchased from Sigma (St. Louis, MO, U.S.A.). Antibodies for TLR4, MyD88, IRAK1, IRAK2, IRAK4, p-IKKβ, p-IκBα, IκBα, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Antibodies for p-TAK1, TAK1, p-IRAK1, JNK, p-JNK, p38, p-p38, ERK, p-ERK, p-PI3K, PI3K, p65, and p-p65 were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, U.S.A.). 2.2. Isolation of mangiferin Mangiferin was isolated according to the method of Jung et al. (2009). Briefly, the dried root of Anemarrhena asphodeloides (1 kg) was extracted five times with MeOH in a boiling water bath, evaporated to dryness under reduced pressure (yield 225 g). This extract was partitioned with n-hexane and H2O. The water fraction was extracted in a stepwise manner with CHCl3 (12 g), ethyl acetate (3 g), and n-butanol (35 g). The butanol extract was separated by column chromatography on silica gel, and eluted with a gradient of MeOH in CH2Cl2. Six fractions (FB1-FB6) were collected on the basis of their thin layer chromatography (TLC) profiles. FB5 was separated by silica gel column chromatography and eluted with 7:2:0.5 (CH2Cl2:MeOH:H2O) to generate 5 subfractions. FB5-5 was further subjected to semi-preparative HPLC

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(35% CH3CN in 50% MeOH at a flow rate of 7 ml/min over 60 min, GS-320 column, 30–500 mm, Japan Analytical Instrument) to produce mangiferin (1.2 g); Mangiferin (purity, 4 95%) colorless amorphous solid, ESI(-)-MS/MS 421, 301 [M–Na]  . 2.3. 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 in accordance with the Kyung Hee University guidelines for Laboratory Animals Care and Usage. Male C57BL/6 (19–23 g, 6 weeks) were supplied from the Orient Animal Breeding Center (Sungnam, Korea). All animals were housed in wire cages at 20–22 1C and 50 710% humidity, fed standard laboratory chow and water ad libitum. 2.4. Isolation and culture of peritoneal macrophages The mice were injected intraperitoneally with 4% sodium thioglycolate solution and killed 4 days after the injection. The peritoneal cavities were washed with RPMI 1640 (10 ml), and centrifuged (200g;10 min) . The cells were resuspended with RPMI 1640. The peritoneal macrophages were isolated by using a biotinlabeled anti-mouse F4/80 antibody (Invitrogen, Carlsbad, CA, U.S.A.) and streptoviridin magnetic beads (Invitrogen). The peritoneal macrophages (0.5  106 cells/well) were cultured in 24-well plates at 37 1C for 3 days in RPMI 1640 plus 10% FBS. To examine the anti-inflammatory effect of mangiferin, peritoneal macrophages were incubated for 15 to 120 min (for the assay of IκB and NF-κB), 90 min (for the assay of NF-κB signaling molecules) or 20 h (for the assay of cytokines, iNOS and COX-2), in the absence or presence of mangiferin (0, 5, 10, and 20 μM) with 100 ng/ml LPS or peptidoglycan. 2.5. Immunofluorescent confocal and fluorescent microscopy The peritoneal macrophages were stimulated with LPS (100 ng/ml) in the absence or presence of mangiferin for 90 min. The cells were fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. For the p65 assay, the cells were stained with goat polyclonal anti-p65 antibody at 4 1C for 2 h and then incubated with Alexa 488-conjgated secondary antibodies and propidium iodide (10 mg/ml, Calbiochem Co., San Diego, CA, U.S.A.) for 1 h. For the LPS–TLR4 complex assay, the cells were stimulated with Alexa Fluor 594-conjugated LPS (10 μg/ml) for 20 min, in the absence or presence of mangiferin. The cells were fixed with 4% paraformaldehyde and 3% sucrose for 20 min. The cells were stained with rabbit polyclonal anti-TLR4 antibody for 2 h at 4 1C, and incubated with Alexa Fluor 488-conjugated secondary antibodies for 1 h. For the IRAK1 and p-IRAK1 analysis, the peritoneal macrophages were incubated with LPS (100 ng/ml) in the absence or presence of mangiferin for 30 min. The cells were fixed, permeabilized, and stained with IRAK1 or p-IRAK antibody for 2 h at 4 1C. Then the cells were incubated with Alexa Fluor 488-conjugated secondary antibodies 1 h. These stained cells were monitored by confocal microscopy. 2.6. Flow cytometry The peritoneal macrophages were treated with Alexa Fluor 488-conjugated LPS (10 μg/ml) for 10 min. The cells were fixed in PBS containing 3% sucrose and 4% paraformaldehyde for 20 min and washed with PBS. The cells were incubated with propidium iodide (10 mg/ml) for 10 min and analyzed by a flow cytometer (C6 Flow Cytometers System).

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2.7. Preparation of experimental colitic mice The mice were randomly divided into 5 groups: normal control, TNBS-induced colitic control groups treated with vehicle, mangiferin (10 or 20 mg/kg) or mesalazine (10 mg/kg). Each group consisted of 6 mice. The colitis was induced by the intrarectal injection of 2.5% (w/v) TNBS solution (100 μl, dissolved in 50% ethanol) into the colon of anesthetized mice using a thin round-tip needle equipped with a syringe (Joh and Kim, 2011). The needle was inserted to 3.5–4 cm proximal to the anus. The normal group was treated with vehicle alone instead of TNBS and test agents. To distribute TNBS within the entire colon, the mice were held in a vertical position for 30 s after the TNBS injection. If the injected TNBS solution was excreted, the mouse was excluded from the experiment. Using this method, 495% of mice caused the TNBS enema. Mangiferin (10 or 20 mg/kg) or mesalazine (10 mg/kg) dissolved in 2% tween 80 were orally administered once a day for 3 days after treatment with TNBS. The mice were killed 18 h after the final administration of test agents. The colon was quickly taken out, opened longitudinally, and gently washed by PBS. Macroscopic evaluation of the colitis grade was scored (0, no ulcer and no inflammation; 1, no ulceration and local hyperemia; 2, ulceration with hyperemia; 3, ulceration and inflammation at one site only; 4, two or more sites of ulceration and inflammation; 5, ulceration extending more than 2 cm). And then the colons were stored at  80 1C until usage for immunoblotting and ELISA.

2.10. Analysis of mangiferin bound to IRAK1 in peritoneal macrophages by mass spectrometry The peritoneal macrophages transfected with or without IRAK siRNA were cultured in 6-well plates (1.5  106 cells/well) at 37 1C in RPMI 1640 plus 10% FBS. To detect IRAK1-bound mangiferin, the macrophages were incubated with or without mangiferin in the presence of 100 ng/ml LPS for 30 min. The cells were lysed with 0.3 ml of lysis buffer per 100-mm culture dish and supplemented with 0.01 ml of NET buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.05% NP-40] and incubated overnight at 4 1C with 0.01 ml of IRAK1 antibody (200 ng/ml). Then the lysates were incubated with 0.05 ml of protein A/G PLUS-Agarose (Santa Cruz, L.A., U.S.A.) at 4 1C for 1 h. The bead-bound complexes were washed thrice with ice-cold NET buffer, denatured for 1 min at 100 1C , centrifuged and analyzed using MS/MS system. Electrospray ionization mass spectrometry (ESI-MS) and tandem MS/MS analyses were carried out by a LCQ DECA XP MS (Thermo Finnigan, CA, USA) equipped with an electrospray ion source. In mass experiments, the spray voltage was 4.5 kV in positive mode and 4 kV in negative mode under N2 sheath gas flow at 50 arbitrary units. The capillary temperature was maintained at 275 1C . Two microliters of samples were injected into the column. Total ion chromatograms from m/z 150 to 2000 in ESI negative mode were obtained. 2.11. Statistical analysis

2.8. Assay of myeloperoxidase activity The colons isolated from the mice were homogenized in a solution containing 0.5% hexadecyl trimethyl ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7.0), and then centrifuged for 30 min at 20,000g at 4 1C. A 50 μL aliquot of the supernatant was added to the reaction mixture consisting of 1.6 mM tetramethyl benzidine and 0.1 mM H2O2, incubated at 37 1C for 3 min, and the absorbance at 650 nm was measured. The myeloperoxidase activity was calculated as the quantity of enzyme degrading 1 μmol/ml of peroxide at 37 1C, and expressed in unit/mg protein (Joh and Kim, 2011). The protein content was determined by the method of Bradford (1976).

2.9. ELISA and immunoblotting The colon tissues were homogenized in 1 ml of RIPA lysis buffer (4 1C) containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (RPP). The lysates or the cultured cells were centrifuged at 15,000g and 4 1C for 15 min. For the cytokine assay, colons and cell-cultured supernatants were transferred to a 96-well microplate. The levels of IL-1β, IL-6, IL-10 and TNF-α were determined using ELISA kits (Pierce Biotechnology, Inc., Rockford, IL, U.S.A.) (Joh and Kim, 2011). For the immunoblot analyses of p-IRAK1, IRAK1, p-TAK1, TAK1, p-IKKβ, p-p65, MAPKs and p-MAPKs and β-actin, the supernatant was subjected to electrophoresis on 8–10% sodium dodecyl sulfate–polyacrylamide gel, and transferred to nitrocellulose membrane. The membrane was blocked with 5% non-fat dried-milk proteins in PBST, probed with p-IRAK1, IRAK1, p-TAK1, TAK1, p-IKKβ, p-p65, MAPKs, p-MAPKs or β-actin antibody and washed with phosphate-buffered saline with tween 20. These proteins were detected with horseradish peroxidase-conjugated secondary antibodies. The protein bands were visualized with an enhanced chemiluminescence detection kit.

All data are indicated as the mean 7standard deviation (S.D.), with statistical significance analyzed using one-way ANOVA followed by a Student-Newman–Keuls test (P o0.05).

3. Results 3.1. Inhibitory effect of mangiferin on IRAK1 phosphorylation and NF-κB activation in LPS- or peptidoglycan-stimulated macrophages First, we tested the ability of mangiferin to inhibit NF-κB activation in LPS or peptidoglycan-induced peritoneal macrophages (Fig. 1). Exposure to LPS increased IRAK1 degradation and phosphorylation, as well as NF-κB activation, as previously reported (Joh and Kim, 2011). Treatment with mangiferin suppressed LPS-induced IRAK1 phosphorylation and NF-κB activation. Furthermore, peptidoglycan, which is produced by gram-positive bacteria, also potently induced degradation and phosphorylation of IRAK1 and activation of NF-κB. Mangiferin also suppressed peptidoglycan-induced IRAK1 phosphorylation and NF-κB activation. To determine whether mangiferin was cytotoxic, peritoneal macrophages were treated with mangiferin (5, 10, 20 μM) for 72 h. The cells were then trypsinized, stained with Trypan blue solution, and assessed for viability. No cytotoxic effect of mangiferin was observed under the experimental conditions (Fig. 1C). 3.2. Inhibitory effect of mangiferin on NF-κB and MAPK signal pathways in LPS-stimulated macrophages Next, we investigated effect of mangiferin on phosphorylation of IKKβ and IκBα and degradation of IκBα in LPS-stimulated peritoneal macrophages (Fig. 2A). Treatment with LPS significantly induced phosphorylation of TAK1, IKKβ and IκBα and degradation of TAK1 and IκBα. However, treatment with mangiferin significantly inhibited LPS-induced IKKβ and IκBα phosphorylation and IκBα degradation. We also investigated effect of mangiferin on degradation of IRAK1, 2 and 4 and phosphorylation of IRAK1 in LPS-stimulated peritoneal macrophages (Fig. 2A). Treatment with

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Fig. 1. Effect of mangiferin on IRAK1 phosphorylation and NF-κB activation in LPS- or peptidoglycan-stimulated peritoneal macrophages. (A) Effect in LPS-stimulated peritoneal macrophages. (B) Effect in peptidoglycan-stimulated peritoneal macrophages. Peritoneal macrophages (0.5  106 cells) were treated with 100 ng/ml of LPS or peptidoglycan in the absence or presence of mangiferin (MF: 5, 10 and 20 mM) for 90 min. IRAK1, p-IRAK1, p65, p-p65 and β-actin were measured by immunoblotting. (C) Cell viability, measured by tryphan blue staining assay. MF was treated for 3 days. All data are expressed as mean 7 S.D. (n¼ 4 in a single experiment).

LPS significantly induced phosphorylation of IRAK1 and degradation of IRAK1, 2 and 4. However, treatment with mangiferin significantly inhibited LPS-induced IRAK1 phosphorylation and IRAK1 degradation, although it did not suppress IRAK2 and IRAK4 degradation. Furthermore, mangiferin inhibited LPS-induced translocation of p65 into the nucleus, as well as NF-κB activation (Fig. 2C). Mangiferin also inhibited phosphorylation of MAPKs p38, ERK and JNK in LPS-induced macrophages, similar to a previous report (Wei et al., 2001) (Fig. 2B). 3.3. Inhibitory effect of mangiferin on inflammatory markers in LPS-stimulated peritoneal macrophages To clarify anti-inflammatory effect of mangiferin, we measured the effect of mangiferin on the production of inflammatory markers in LPS-stimulated peritoneal macrophages (Fig. 3). LPS significantly induced production of PGE2 and NO and expression of TNF-α, IL-1β, IL-6, IL-10, COX-2, and iNOS. Mangiferin significantly reduced LPS-induced production of PGE2 and NO and expression of proinflammatory cytokines TNF-α, IL-1β and IL-6 dose-dependently, but increased IL-10 expression, an anti-inflammatory cytokine. Mangiferin at a concentration of 10 μM inhibited LPSstimulated expression of TNF-α, IL-1β and IL-6 and production of PGE2, and NO by 81.0%, 89.5%, 88.3%, 57.1% and 73.7%, respectively, whereas it increased IL-10 expression by 131.8% compared to LPSnontreated normal control group. Mangiferin also suppressed LPS-induced IκBα and NK-κB activation and COX-2 and iNOS expression (Fig. 3C). The suppression of mangiferin against COX-2 and iNOS expression and IκBα and NF-kB activation was

increased by the addition of SN50, which is a NF-κB inhibitor (Sun et al., 2013). 3.4. Inhibitory effect of mangiferin on IRAK1 phosphorylation in LPS-stimulated macrophages We also examined the ability of mangiferin to inhibit interaction of LPS to TLR4 in peritoneal macrophages using flow cytometry and confocal microscopy analyses (Fig. 4). When peritoneal macrophages were treated with Alexa Fluor 488-conjugated LPS alone, LPS-bound macrophages were significantly shifted by flow cytometry analysis. Treatment with mangiferin did not inhibit shift of macrophages by Alexa Fluor 488-conjugated LPS. Furthermore, mangiferin did not inhibit binding of Alexa Fluor 488conjugated LPS to the macrophages by confocal microscopy. Mangiferin inhibited IRAK1 degradation and phosphorylation in LPS-stimulated macrophages, although it did not suppress LPSinduced IRAK2 and IRAK4 degradation (Fig. 2A). Therefore, we investigated effect of mangiferin on IRAK1 phosphorylation in LPSstimulated peritoneal macrophages transfected with or without IRAK1 siRNA by immunoblotting (Fig. 5A). LPS potently increased IRAK1 phosphorylation in the macrophages transfected without IRAK1 siRNA. Mangiferin inhibited the IRAK1 phosphorylation in the macrophages transfected without IRAK1 siRNA, but not in macrophages with IRAK1 siRNA. On confocal microscopy, LPS increased phosphorylated IRAK1 in macrophage without IAK1 siRNA (fluorescent tagged p-IRAK1 antibody bound to the macrophages), but reduced IRAK expression in macrophages (fluorescent tagged IRAK1 antibody bound to the macrophages) (Fig. 5B and C).

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Fig. 2. Inhibitory effect of mangiferin on NF-κB and MAPK signal pathways in LPS-stimulated macrophages. Peritoneal macrophages were treated with 100 ng/ml LPS in the absence or presence of mangiferin (MF: 5, 10 and 20 mM). (A) Effect on IκB-α phosphorylation and NR-κB activation. The cells were treated with LPS in the absence or presence of MF (20 μM) for 0, 15, 30, 60, 90, and 120 min and then analyzed by immunoblotting. (B) Effect in IRAK1/NF-κB pathway. (C) Effect in MAPKs pathway. (D) Effect in NF-κB nuclear translocation, which was detected by confocal analysis using an antibody for p65 subunit. NF-κB and MAPKs phosphorylation and the nuclear translocation of NF-κB in LPS-stimulated peritoneal macrophages were determined 90 min after treatment with LPS by immunoblotting and confocal microscope, respectively. Nor, vehicle alone in the absence of LPS; LPS, LPS alone; MF10, 10 μM MF with LPS; MF20, 20 μM MF with LPS.

Mangiferin inhibited LPS-induced phosphorylated IRAK1 (fluorescent tagged p-IAK1 antibody bound to the macrophages) in the macrophages transfected without IRAK1 siRNA. However,

treatment with LPS did not induce p-IRAK1 or IRAk1 (fluorescent tagged p-IRAK1 antibody or IRAK1 antibody bound to the macrophages) in the macrophages transfected with IRAK1 siRNA. Despite

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Fig. 3. Inhibitory effect of mangiferin on inflammatory markers in LPS-stimulated peritoneal macrophages. Peritoneal macrophages (0.5  106 cells) were treated with 100 ng/ml LPS in the absence or presence of mangiferin (MF: 5, 10 and 20 mM) and/or SN50 (50 μM) for 90 min or 20 h. (A) Effect on the expression of TNF-α (a), IL-1β (b) and IL-6 (c), and IL-10 (d). (B) Effect on the production of PGE2 (a) and NO (b). (C) Effect on IκBα and NK-κB activation and iNOS and COX-2 expression. The cytokines, PGE2, and NO levels were measured in culture supernatant by ELISA. IkBa, p-IκBa, p65, p-65, and β-actin were measured in the cell lysate at 90 min after LPS treatment by immunoblotting. IκBα, p-IκBα, p65 and p-p65 were in the cell lysate at 90 min after LPS treatment by immunoblotting. COX-2, iNOS and β-actin were in the cell lysate at 20 h after LPS treatment by immunoblotting. All data are expressed as mean 7S.D. (n¼ 4 in a single experiment). #P o 0.05, significantly different vs. control group. nP o 0.05, vs. LPS control.

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Fig. 4. Effect of mangiferin on the binding of LPS to TLR-4 on peritoneal macrophages. Peritoneal macrophages isolated from mice were incubated with Alexa Fluor 488conjugated LPS for 20 min in the absence or presence of mangiferin (MF: 5, 10 and 20 mM) and then analyzed by FACS (A) and confocal microscopy (B).

Fig. 5. Inhibitory effect of mangiferin on IRAK1 and NF-κB activation in LPS-stimulated peritoneal macrophages transfected with or without IRAK1 siRNA. (A) Effect on IRAK1/NF-κB pathway. IRAK1, p-IRAK1, p65 and p-p65 were analyzed by immunoblotting. (B) Effect on IRAK1 degradation. (C) Effect on IRAK1 phosphorylation. The peritoneal macrophages isolated from mice were transfected with or without IRAK1 siRNA, treated with 100 ng/ml LPS in the absence or presence of mangiferin (MF, 20 mM) for 90 min, incubated with Alexa Fluor 488-conjugated IRAK1 or p-IRAK1 antibody, and analyzed by a confocal microscopy. (D) Electrospray ionization mass spectrometry analysis of mangiferin bound to IRAK1. The peritoneal macrophages were treated with 50 ng/ml LPS in the absence or presence of mangiferin (MF, 50 mM) for 90 min. (a), vehicle alone; (b), LPS alone; (c), mangiferin with LPS.

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Fig. 6. Effect of mangiferin on body weight (A), macroscopic disease (B), colon length (C), colonic myeloperoxidase (MPO) activity (D), and histological exam (E) in TNBSinduced colitic mice. TNBS, except in the control group, was intrarectally administered to mice treated with saline, mangiferin or mesalazine. Mangiferin (MF10, 10 mg/kg; MF20, 20 mg/kg), mesalazine (MS10, 10 mg/kg) or saline was orally administered for 3 days after TNBS treatment. The mice were killed 3 days after TNBS treatment. All values are mean 7S.D. (n¼ 7). #Po 0.05, significantly different vs. control group; nPo 0.05, significantly different vs. TNBS group.

mangiferin treatment of LPS-stimulated macrophages transfected with IRAK1 siRNA, IRAK1 phosphorylation was not changed. To confirm whether mangiferin was bound to IRAK1, we incubated LPS-stimulated peritoneal macrophages with mangiferin, immunoprecipitated with IRAK1 antibody and measured mangiferin bound to IRAK1 by mass analysis (Fig. 5D). We detected the molecular peak of mangiferin [Mþ H] þ in peritoneal macrophages treated with mangiferin plus LPS, but not in IRAK1 siRNA-transfected peritoneal macrophages treated with mangiferin plus LPS. 3.5. Inhibitory effect of mangiferin on TNBS-induced colitis in mice Next, we measured the anti-inflammatory effect of mangiferin in mice with TNBS-induced colitis. TNBS caused colon shortening and increased myeloperoxidase activity (Fig. 6). Treatment with mangiferin inhibited TNBS-induced colon shortening and myeloperoxidase activity. Mangiferin (20 mg/kg) also inhibited the

myeloperoxidase activity by 84%, compared with the TNBStreated group (P o0.05). TNBS induced IRAK1 phosphorylation and NF-κB activation (Fig. 7). However, mangiferin inhibited TNBSinduced phosphorylation of IRAK1 and IKKβ, as well as activation of NF-κB. Mangiferin suppressed TNBS-induced up-regulation of COX-2, iNOS and proinflammatory cytokines TNF-α, IL-1β and IL-6. However, IL-10 expression was down-regulated. The anti-colitic effect of mangiferin was comparable to that of mesalazine. Furthermore, mangiferin (20 mg/kg) also inhibited TNF-α by 78%, IL-1β by 82%, and IL-6 expressions by 88%, in TNBS-induced colitic mice. However, IL-10 expression was induced to 79% of normal control group.

4. Discussion Stimulation of LPS or peptidoglycan increases plasma TNF-α and IL-1β levels in mice and causes inflammation (Ingalls et al., 1999;

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Fig. 7. Effect of mangiferin on the phosphorylation of IRAK1 and p65 and the expression of iNOS and COX-2 (A) and proinflammatory cytokines (B) in TNBS-induced colitic mice. TNBS, except in the control group, was intrarectally administered to mice treated with saline, mangiferin or mesalazine. Mangiferin (MF, 10 and 20 mg/kg), mesalazine (MS, 10 mg/kg) or saline was administered orally for 3 days after TNBS treatment. The mice were killed 3 days after TNBS treatment. All values are mean 7S.D. (n ¼7). # P o0.05, significantly different vs. control group; nPo 0.05, significantly different vs. TNBS group.

Olson and Miller, 2004; Blanqué et al., 1996). The suppression of IL-1β and TNF-α expression by natural product constituents, such as luteolin, ginsenosides and berberine ameliorates several chronic inflammatory diseases, including colitis and rheumatoid arthritis (Joh et al., 2011; Kotanidou et al., 2002; Joh and Kim, 2011). Inhibition of NF-κB signaling pathway is the possible mechanism of action. Therefore, the application of natural product constituents has recently been used to regulate inflammatory diseases. A number of in vitro and in vivo studies have shown that mangiferin may suppress inflammation, asthma, arthritis, bronchitis and stress via the NF-kB signaling pathway (Pinto et al., 2005; Márquez et al., 2012; Rivera et al., 2011; García-Rivera et al., 2011; Leiro et al., 2004; Kumar et al., 2003). However, the antiinflammatory mechanism of mangiferin is not clear. In the present study, mangiferin exhibited anti-inflammatory effect in LPS- or peptidoglycan-induced peritoneal macrophages. Mangiferin also inhibited expression of COX-2 and iNOS, as well as their products, PGE2 and NO2, in LPS-stimulated peritoneal macrophages. Therefore, we investigated the effect of mangiferin in canonical NF-kB signaling pathway. Mangiferin potently inhibited LPS-induced IRAK1 degradation, IKKβ phosphorylation and IκB-α degradation, as well as the translocation of NF-κB p65 subunit into the nuclei. Furthermore, mangiferin inhibited peptidoglycan -induced IRAK1 degradation in peritoneal macrophages. Mangiferin inhibited phosphorylation of MAPKs JNK, ERK and p38. However, mangiferin did not inhibit the interaction between LPS and TLR4 on the peritoneal macrophages. Mangiferin significantly inhibited p-IRAK1 in LPS-stimulated peritoneal macrophages, but increased IRAK1 (fluorescent p-IRAK and IRAK1 antibodies binding to macrophages, respectively). However, mangiferin did not influence IRAK1 phosphorylation in LPS-stimulated peritoneal

macrophages transfected with IRAK1 siRNA. When peritoneal macrophages were stimulated by LPS in the absence or presence of mangiferin and immunoprecipitated with IRAK antibody, mangiferin [Mþ H] þ was detected in the precipitate in peritoneal macrophages treated with mangiferin plus LPS, but was not in IRAK1 siRNA-transfected peritoneal macrophages treated with LPS plus mangiferin. These results suggested that mangiferin may inhibit the transfer of LPS-or peptidoglycan -stimulated TLR4/ NF-κB and TLR2/NF-κB signal pathways by regulating IRAK1 phosphorylation. TLR2 and TLR4 are pattern recognition receptor molecules that respond to peptidoglycan and LPS, respectively. The stimulation of LPS or peptidoglycan activates the secretion of pro-inflammatory mediators from monocytes, macrophages and dendritic cells (Kawai and Akira, 2007). These TLRs activate NF-κB and MAPKs signal pathways via MyD88/IRAKs. Thus, the binding of LPS or peptidoglycan to TLRs induces phosphorylation of IRAK1 and/or MAPKs by pathogens or pro-inflammatory cytokines (Li et al., 2010). Various inflammatory diseases up-regulates pro-inflammatory cytokines, such as TNF-α and IL-1β, and inflammatory mediators, such as NO and PGE2, via NF-κB and MAPKs signal pathways in macrophages (Moynagh, 2005; Tak and Firestein, 2001). Mangiferin inhibited MAPKs and IRAK/ NF-κB signal pathways, like kalopanaxsaponin A, compound K and echinocystic acid (Joh et al., 2011, 2012; Joh and Kim, 2011). Furthermore, mangiferin inhibited proinflammatory cytokine expression. These findings suggested that mangiferin can regulate NF-κB and MAPKs signal pathways by inhibiting LPS or peptidoglycan -stimulated IRAK1 phosphorylation in macrophages. Intestinal bowel disease (IBD), including ulcerative colitis and Crohn's disease, might be attributed to mucosal immune

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responses to resident gut bacteria (Jung et al., 1995; Duchmann et al., 1995). Gut microbial antigens stimulates the innate immune system through pattern recognition receptors (Ingalls et al., 1999). Of them, TLR2 and TLR4 are pattern recognition molecules receptors for peptidoglycans and LPS, respectively (Chow et al., 1999; Ingalls et al., 1999; Maldonado-Bernal et al., 2005). The stimulation of these TLRs activates the secretion of pro-inflammatory cytokines from monocytes, macrophages, and dendritic cells, thereby leading to inflammation. Particularly, TLR4 is potently expressed in the colons of patients with IBD (Cario and Podolsky, 2000) and significantly up-regulated in colitic mice (Pasternak et al., 2010). However, in the present study, mangiferin inhibited TNBS-induced colon shortening, myeloperoxidase increase and NF-κB activation in mice. Mangiferin inhibited expression of COX-2 and iNOS, as well as proinflamamtory cytokines TNF-α and IL-1β and IL-6. These results suggested that mangiferin may inhibit colitis by inhibiting IRAK1 phosphorylation. It was supported that PGG inhibited LPS-induced septic death (systemic inflammation), as well as expression of proinflammatory cytokines in mice (Supplementary Fig. 1). Based on these findings, mangiferin may ameliorate inflammatory diseases such as septic shock and colitis by inhibiting IRAK1 phosphorylation in macrophages. Acknowledgments This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C1020). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2014.06.013. References Baldwinm Jr., A.S., 1996. The NF-κB and IκB proteins: new discoveries and insights. Annu. Rev. Immunol. 14, 649–681. Bistrian, B., 2007. Systemic response to inflammation. Nutr. Rev. 65, S170–172. Blanqué, R., Meakin, C., Millet, S., Gardner, C.R., 1996. Hypothermia as an indicator of the acute effects of lipopolysaccharides: comparison with serum levels of IL1 beta, IL6 and TNF alpha. Gen. Pharmacol. 27, 973–977. Bradford, M.M., 1976. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cario, E., Podolsky, D.K., 2000. Differential alteration in intestinal epithelial cell expression of Toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect. Immun. 68, 7010–7017. Chow, J.C., Young, D.W., Golenbock, D.T., Christ, W.J., Gusovsky, F., 1999. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J. Biol. Chem. 274, 10689–10692. Duchmann, R., Kaiser, I., Hermann, E., Mayet, W., Ewe, K., Meyer zum Büschenfelde, K.H., 1995. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease. Clin. Exp. Immunol. 102, 448–455. Fabre, C., Mimura, N., Bobb, K., Kong, S.Y., Gorgun, G., Cirstea, D., Hu, Y., Minami, J., Ohguchi, H., Zhang, J., Meshulam, J., Carrasco, R.D., Tai, Y.T., Richardson, P.G., Hideshima, T., Anderson, K.C., 2012. Dual inhibition of canonical and noncanonical NF-κB pathways demonstrates significant antitumor activities in multiple myeloma. Clin. Cancer Res. 18, 4669–4681. Fairweather, D., Rose, N.R., 2005. Inflammatory heart disease: a role for cytokines. Lupus 14, 646–651. García-Rivera, D., Delgado, R., Bougarne, N., Haegeman, G., Berghe, W.V., 2011. Gallic acid indanone and mangiferin xanthone are strong determinants of immunosuppressive anti-tumour effects of Mangifera indica L. bark in MDAMB231 breast cancer cells. Cancer Lett. 305, 21–31. Garrido, G., Delgado, R., Lemus, Y., Rodriguez, J., García, D., Núñez-Sellés, A.J., 2004. Protection against septic shock and suppression of tumor necrosis factor alpha and nitric oxide production on macrophages and microglia by a standard

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