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AKT pathway and polarizing M1 macrophages to M2-like macrophages
European Journal of Pharmacology 708 (2013) 21–29
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Arctigenin ameliorates inflammation in vitro and in vivo by inhibiting the PI3K/AKT pathway and polarizing M1 macrophages to M2-like macrophages Supriya R. Hyam a, In-Ah Lee a, Wan Gu a, Kyung-Ah Kim a, Jin-Ju Jeong a, Se-Eun Jang b, Myung Joo Han b, Dong-Hyun Kim a,n a b
Department of Life and Nanopharmaceutical Sciences and Department of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea Department of Food and Nutrition, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e i n f o
abstract
Article history: Received 30 August 2012 Received in revised form 18 December 2012 Accepted 14 January 2013 Available online 31 January 2013
Seeds of Arctium lappa, containing arctigenin and its glycoside arctiin as main constituents, have been used as a diuretic, anti-inflammatory and detoxifying agent in Chinese traditional medicine. In our preliminary study, arctigenin inhibited IKKb and NF-kB activation in peptidoglycan (PGN)- or lipopolysaccharide (LPS)-induced peritoneal macrophages. To understand the anti-inflammatory effect of arctigenin, we investigated its anti-inflammatory effect in LPS-stimulated peritoneal macrophages and on LPS-induced systemic inflammation as well as 2,4,6-trinitrobenzene sulfonic acid (TNBS)induced colitis in mice. Arctigenin inhibited LPS-increased IL-1b, IL-6 and TNF-a expression in LPSstimulated peritoneal macrophages, but increased LPS-reduced IL-10 and CD204 expression. Arctigenin inhibited LPS-induced PI3K, AKT and IKKb phosphorylation, but did not suppress LPS-induced IRAK-1 phosphorylation. However, arctigenin did not inhibit NF-kB activation in LPS-stimulated PI3K siRNAtreated peritoneal macrophages. Arctigenin suppressed the binding of p-PI3K antibody and the nucleus translocation of NF-kB p65 in LPS-stimulated peritoneal macrophages. Arctigenin suppressed blood IL-1b and TNF-a level in mice systemically inflamed by intraperitoneal injection of LPS. Arctigenin also inhibited colon shortening, macroscopic scores and myeloperoxidase activity in TNBS-induced colitic mice. Arctigenin inhibited TNBS-induced IL-1b, TNF-a and IL-6 expression, as well as PI3K, AKT and IKKb phosphorylation and NF-kB activation in mice, but increased IL-10 and CD204 expression. However, it did not affect IRAK-1 phosphorylation. Based on these findings, arctigenin may ameliorate inflammatory diseases, such as colitis, by inhibiting PI3K and polarizing M1 macrophages to M2-like macrophages. & 2013 Elsevier B.V. All rights reserved.
Keywords: Arctigenin Inflammation Macrophages PI3K Polarization
1. Introduction Inflammation is the body’s response to injury or infection (Mehta et al., 1998; Baldwin, 1996). It can be classified as both acute and chronic inflammations. Acute inflammation is a normal and helpful component of physiologic response to injury. However, chronic inflammation is persistent inflammation that becomes excessive. This persistent inflammatory response causes progressive damage to the body leading to a variety of diseases, such as colitis, rheumatoid arthritis, atherosclerosis and even cancer. The mechanisms include macrophage and T lymphocyte activation and the release of pro-inflammatory cytokines that induce inflammatory activity (Tousoulis et al., 2005; Lin and Karin, 2007). Inflammation can be mediated by inflammatory mediators, including interleukin (IL)-1b, IL-6, tumor necrosis n
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0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.01.014
factor (TNF)-a, interferon-g, IL-12, IL-18, nitric oxide and prostaglandins (Fairweather and Rose, 2005). Among these inflammatory mediators, pro-inflammatory cytokines such as IL-1b and TNF-a are activated through nuclear factor-kappaB (NF-kB), but they can also activate this transcription factor (Baldwin, 1996; Collins et al., 1995). These mediators arouse the innate immune response but their over-expression causes acute phase endotoxemia leading to organ failure, shock, tissue injury and even death (Astiz and Rackow, 1998). Toll-like receptors (TLRs), a major connection between innate and adaptive mucosal immune responses, act as transmembrane receptors in the cellular response to insults, such as gram-negative bacterial lipopolysaccharides (LPS) (Chow et al., 1999; Ingalls et al., 1999), and grampositive bacterial peptidoglycans (PGN) (Olson and Miller, 2004). Among this family of receptors, TLRs, such as TLR2 and TLR4, recognize pathogen-associated molecular patterns (Akira and Hemmi, 2003; O’Neill and Dinarello, 2000) and interact with myeloid differentiation factor 88 (MyD88). The interaction results
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in the activation of NF-kB and MAP kinases to elicit regulatory responses for the generation of inflammation mediators. TLR4/MyD88 initiates a signaling cascade through the Toll/IL-1R (TIR) domain of its cytoplasmic tail, allowing for subsequent activation of IL-1R-associated kinases (IRAKs). Phosphorylated IRAK-1 activates a multimeric protein complex composed of TRAF6, TAK1, TAB1 and TAB2, leading to NF-kB-mediated pathway as well as the induction of proinflammatory cytokines. Recently, TLR4/MyD88 has been also reported to initiate phosphatidylinositol 3-kinases (PI3K)/AKT/(IkB kinase) IKK signaling pathway. Regulating expression of these different inflammatory mediators is therefore beneficial in decreasing inflammatory diseases. To prevent inflammation, the use of dietary supplements has recently become a topic of interest (Chan et al., 1998; Paradkar et al., 2004; Davis et al., 2006). The seed of Arctium lappa is one of the well-known diuretic, anti-inflammatory and detoxifying agents in Chinese traditional medicine (Park et al., 2007). Arctigenin displays a memoryenhancing effect (Lee et al., 2011) and anti-inflammatory activity via NF-kB or JAK-STAT pathway in LPS-stimulated RAW264.7 cells (Kang et al., 2008; Kou et al., 2011; Zhao et al., 2009), inhibit T lymphocyte proliferation and the gene expression of IL-2, IFN-g and NF-AT (Tsai et al., 2011), as well as anti-tumor activity against PANC-1 cells (Awale et al., 2006). Nevertheless, its antiinflammatory mechanism is not understood thoroughly. Therefore, we isolated arctigenin from the seed of Arctium lappa as an inhibitor for NF-kB activation in LPS- or PGNstimulated peritoneal macrophages, investigated its antiinflammatory mechanism in LPS-stimulated peritoneal macrophages, and evaluated its inhibitory effect against LPS-induced systemic inflammation and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice.
2. Materials and methods 2.1. Materials DMEM, RPMI 1640, TNBS, and LPS purified from Escherichia coli O111:B4 were purchased from Sigma Co. (St. Louis, MO, U.S.A.). Antibodies for IRAK1, AKT, p-AKT, p65 and b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, L.A. U.S.A.). Antibody for p-IRAK1 and p-p65 was purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Enzyme-linked immunosorbent assay (ELISA) kits for cytokines were purchased from R&D Systems (Minneapolis, MN, U.S.A.). Other chemicals used were of the highest grade available. Arctigenin (purity, 495%) was isolated from the seeds of Arctium lappa Linne as previously reported (Lee et al., 2011). 2.2. Animals Male C57BL/6 (18–22 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 507 10% humidity, fed standard laboratory chow (Samyang Co., Seoul, Korea), and allowed water ad libitum. All experiments were performed in accordance with the NIH and Kyung Hee University guidelines for Laboratory Animals Care and Use and approved by the Committee for the Care and Use of Laboratory Animals in the College of Pharmacy, Kyung Hee University. 2.3. Isolation and culture of peritoneal macrophages The mice were intraperitoneally injected with 2 ml of 4% sodium thioglycolate solution. Mice were sacrificed 4 days
after injection and the peritoneal cavities were flushed with 10 ml of RPMI 1640. The peritoneal lavage fluids were centrifuged at 200 g for 10 min and the cells were resuspended with RPMI 1640 and plated. After incubation for 1 h at 37 1C, the cells were washed three times and nonadherent cells were removed by aspiration. Cells were cultured in 24-well plates (0.5 106 cells/ well) at 37 1C in RPMI 1640 plus 10% FBS. The attached cells were used as peritoneal macrophages. To examine anti-inflammatory effect of arctigenin, peritoneal macrophages were incubated in the absence or presence of arctigenin with 50 ng/ml LPS or PGN. 2.4. Immunofluorescent confocal and fluorescent microscopy For the p65 assay, peritoneal macrophages were stimulated with LPS (50 ng/ml) in the presence or absence of arctigenin for 60 min. The cells were then fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100. The cells were stained with goat polyclonal anti-p65 antibody for 2 h at 4 1C and then incubated with secondary antibodies conjugated with Alexa 488 and propidium iodide (10 mg/ml, Calbiochem Co., San Diego, CA, U.S.A.) for 1 h. Images were observed by confocal microscopy. For the p-PI3K assay, peritoneal macrophages were stimulated with LPS (50 ng/ml) in the presence or absence of arctigenin for 30 min. The cells were then fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100. The cells were stained with goat polyclonal anti-p-PI3K antibody for 2 h at 4 1C and then incubated with secondary antibodies conjugated with Alexa 594 for 1 h. The stained macrophages were observed with 5 ml of DAPI mounting (Mounting Medium with DAPI, Abcam, Cambridge, CA, U.S.A.), at 5 min by fluorescent microscope. 2.5. 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 one day prior to the transfection. PI3K small interfering RNA was purchased from Santa Cruz (Santa Cruz, CA, U.S.A.). The cells were transfected with 100 nM PI3K siRNA using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instruction. At 48 h after transfections, the cells were treated with or without arctigenin (10 and 20 mM) and/or LPS (50 ng/ml). 2.6. Assay of serum pro-inflammatory cytokines TNF-a and IL-1b in PGN- or LPS-stimulated mice The mice were intraperitoneally injected with or without arctigenin (5 mg/kg) and/or PGN (4 mg/kg) or LPS (4 mg/kg). Each group consisted of 6 mice. Normal control was treated with vehicle alone instead of LPS, PGN and arctigenin. Mice were sacrificed 4 h after LPS or PGN injection, whole blood was obtained by cardiac puncture and serum was obtained by centrifugation at 12,000g for 20 min. The serum levels of IL-1b and TNF-a were measured using ELISA kit. 2.7. Preparation of experimental colitic mice The mice were randomly divided into 5 groups: normal and TNBS-induced colitic groups treated with or without arctigenin or sulfasalazine. Each group consisted of 7 mice. TNBS-induced colitis was induced by the administration of 2.5% (w/v) TNBS solution (100 ml) in 50% ethanol into the colon of anesthetized mice via a thin round-tip needle equipped with a 1 ml syringe (Joh et al., 2010). The normal group was treated with just the vehicle. The needle was inserted so that the tip was 3.5–4 cm proximal to the anal verge. To distribute the agents within the entire colon and cecum, mice were held in a vertical position for
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Fig. 1. Effect of arctigenin on IKKb and NF-kB phosphorylation in pathogen-stimulated peritoneal macrophages. The peritoneal macrophages isolated from mice were treated with 50 ng/ml LPS or PGN in the absence or presence of arctigenin (10 mM). The phosphorylation of IKKb and NF-kB was detected by immunoblottig. All data are expressed as mean 7 S.D. (n¼ 3 in a single experiment). ]P o0.05, significantly different vs. NOR group. nP o 0.05 vs. group treated with LPS alone.
Fig. 2. Inhibitory effect of arctigenin on the expression of pro-inflammatory and anti-inflammatory cytokines (A) and polarization markers (B) in LPS-stimulated peritoneal macrophages. Peritoneal macrophages (0.5 106 cells) were treated with 50 ng/ml LPS in the absence (LPS) or presence of arctigenin (10 and 20 mM) for 20 h. L (A) Effect on the expression of pro-inflammatory and anti-inflammatory cytokines. Levels of TNF-a, IL-1b, IL-6 and IL-10 in culture supernatants were measured by ELISA. (B) Effect on the expression of macrophage polarization markers. Levels of TNF-a, IL-1b, IL-10 and CD204 mRNA were measured by real time–PCR. All data are expressed as mean 7 S.D. (n¼ 6 in a single experiment). ]P o0.05, significantly different vs. normal control group. nP o 0.05 vs. group treated with LPS alone.
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30 s after the injection. Using this procedure, 495% of the mice retained the TNBS enema. If an animal quickly excreted the TNBS– ethanol solution, it was excluded from the remainder of the study. Arctigenin (30 and 60 mg/kg) or sulfasalazine (50 mg/kg) dissolved in 2% tween 80 were orally administered once a day for 3 days after TNBS administration. The mice were sacrificed on next day after the final administration of test agents. The colon was quickly removed, opened longitudinally, and gently cleared of stool by PBS. Macroscopic assessment of the disease grade was scored according to a previously reported scoring system (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; 5, ulceration extending more than 2 cm, and the colon tissue was then used for immunoblotting and enzyme-linked immunosorbent assay (ELISA) analysis. For the histological exam, the colons were fixed over-night in phosphate buffer (50 mM, pH7.4) containing 4% para-formaldehyde, then immersed in a 30% sucrose solution (in 50 mM phosphate-buffered saline) and stored at 4 1C until sectioning. Frozen colons were sectioned along in the coronal plane (10 mm) using a cryostat (Leica Microsy stems AG, Germany) and stained with hematoxylin-eosin.
2.8. Colon tissue preparation Colon tissues were excised, perfused with ice-cold perfusion solution containing 0.15 M KCl and 2 mM EDTA (pH 7.4), and homogenized in 50 mM Tris–HCl buffer (pH 7.4). The homogenates were centrifuged at 10,000 g at 4 1C for 30 min. The supernatant was used for the estimation of the antioxidant defense system. For immunoblotting and ELISA of colon tissues were carefully homogenized in 1 ml of ice cold RIPA lysis buffer containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail. 2.9. Assay of myeloperoxidase activity in colonic mucosa 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 20,000 g at 4 1C. A 50 ml aliquot of the supernatant was added to the reaction mixture consisting of 1.6 mM tetramethyl benzidine and 0.1 mM H2O2, and incubated at 37 1C. The absorbance was measured at 650 nm spectrophotometrically time-scanned. The myeloperoxidase activity was defined as the quantity of enzyme degrading 1 mmol/ml of
Fig. 3. Effect of arctigenin on IRAK-1 phosphorylation and NF-kB activation in LPS-stimulated peritoneal macrophages. The peritoneal macrophages isolated from mice were treated with 50 ng/ml LPS in the absence or presence of arctigenin (10 or 20 mM). (A) Effect in IRAK-1 and NF-kB phosphorylation. b-Actin was used as a control. (B) Effect on NF-kB nuclear translocation. Peritoneal macrophages were incubated with 50 ng/ml LPS in the absence or presence of arctigenin (10 or 20 mM) for 60 min. NF-kB nuclear translocation was detected by confocal analysis using an antibody for p65 subunit. All data are expressed as mean 7 S.D. (n¼ 3 in a single experiment). ] Po 0.05, significantly different vs. normal control group. nP o 0.05 vs. group treated with LPS alone.
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peroxide at 37 1C, and expressed in unit/mg protein (Mullane et al., 1985). The protein content was assayed by Bradford’s method (Bradford (1996)). 2.10. Cytokine analysis and immunoblotting Proinflammatory cytokines, TNF-a, IL-1b, IL-6 and IL-10, were assessed by ELISA according to the manufacturer’s instructions. The supernatant extracts prepared from colon or peritoneal macrophages were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (PVDF). The membranes were blocked with 5% non-fat dried-milk proteins in PBST, and probed with p-IRAK1, IRAK1, p-PI3K, PI3K, p-AKT, AKT, p65, p-p65 or b-actin antibody. After washing with PBST, proteins were detected with HRP-conjugated secondary antibodies for 1 h. Bands were visualized with enhanced chemiluminescence reagent (Bai et al., 2007).
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utilizing Takara thermal cycler, which used SYBER premix agents, as per the instructions from Takara biology incorporation. Thermal cycling conditions were as follows: activation of DNA polymerase at 95 1C for 10 min, followed by 40 cycles of amplification at 95 1C for 30 s and at 60 1C for 30 s. The normalized expression of the target gene, with respect to b-actin, was computed for all samples by using the Microsoft Excel data spreadsheet. 2.12. Statistical analysis Results are presented as the means7standard deviation of at least four replicates. One-way ANOVA and Student t-test were used. Po0.05 was regarded statistically significant.
3. Results
2.11. Real time–polymerase chain reaction (RT–PCR)
3.1. Anti-inflammatory effect of arctigenin in LPS or PGN-stimulated peritoneal macrophages
Total RNA was extracted by RIPA buffer. Reverse transcription was performed with 2 mg of total RNA. RT–PCR for TNF-a, IL-1b, IL10, CD204 and b-actin was performed as described previously,
We tested the inhibitory effect of arctigenin in PGN- or LPSstimulated peritoneal macrophages (Fig. 1). Stimulation with PGN or LPS markedly induced IKKb phosphorylation and NF-kB
Fig. 4. Effect of arctigenin on activation of PI3K in LPS-stimulated peritoneal macrophages. The peritoneal macrophages isolated from mice were treated with 50 ng/ml LPS in the absence or presence of arctigenin (10 or 20 mM). (A) Effect in PI3K and AKT phosphorylation by immunoblotting. b-Actin was used as a control. (B) Effect in PI3K phosphorylation by a fluorescent microscopy. Peritoneal macrophages were incubated with 50 ng/ml LPS in the absence or presence of arctigenin (10 or 20 mM) for 30 min. PI3K phosphorylation was detected by confocal analysis using an antibody for p-PI3K. All data are expressed as mean 7 S.D. (n¼ 3 in a single experiment). ]Po 0.05, significantly different vs. normal control group. nP o0.05 vs. group treated with LPS alone.
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activation. However, treatment with PGN or LPS in the presence of arctigenin potently suppressed IKKb phosphorylation and NFkB activation. Therefore, to clarify the anti-inflammatory mechanism of arctigenin, we stimulated peritoneal macrophages with LPS in the presence or absence of arctigenin and measured the expression of proinflammatory and inflammatory cytokines (Fig. 2A). Treatment with LPS significantly increased TNF-a, IL-1b and IL-6 expression, but reduced IL-10 expression. However, treatment with LPS in the presence of arctigenin inhibited the expression of proinflammatory cytokines, TNF-a, IL-1b and IL-6, but increased the expression of anti-inflammatory cytokine IL-10. We also examined the ability of arctigenin to polarize LPS-stimulated macrophages to M2 macrophages (Fig. 2B) by real time–PCR. Arctigenin suppressed the LPS-elevated levels of IL-1b and TNF-a and increased the LPS-reduced expression of IL-10 and CD204. Next, we stimulated peritoneal macrophages with LPS in the presence or absence of arctigenin and measured the phosphorylation of IRAK-1 and the activation of NF-kB (Fig. 3A). IRAK-1 phosphorylation and NF-kB activation were significantly elicited by LPS treatment. Although arctigenin inhibited LPS-induced NF-kB activation (p65 phosphorylation), it did not inhibit the phosphorylation of IRAK-1. Thus, arctigenin did not increase IRAK-1 expression reduced by LPS. We also examined the ability of arctigenin to inhibit the nuclear translocation of NF-kB in LPS-stimulated peritoneal macrophages by confocal microscopy (Fig. 3B). Treatment with arctigenin attenuated the LPS-translocated NF-kB into the nucleus and subsequently increased the cytosolic p65 level. 3.2. Effect of arctigenin on the PI3K/AKT pathway in LPS-stimulated peritoneal macrophages We investigated the effect of arctigenin on the phosphorylation PI3K and AKT in LPS-stimulated peritoneal macrophages by immunoblotting and fluorescent microscopy. Single LPS treatment significantly increased the phosphorylation of PI3K and AKT. Arctigenin potently suppressed LPS-induced PI3K and AKT phosphorylation (Fig. 4A). Furthermore, arctigenin inhibited LPSstimulated PI3K phosphorylation by a fluorescent microscopy (Fig. 4B). To confirm the inhibition of PI3K activation by arctigenin, we investigated its effect in PI3K siRNA-transfected peritoneal macrophages (Fig. 5). Transfection of PI3K siRNA significantly inhibited PI3K expression by 72%. LPS stimulation in PI3K siRNA-treated peritoneal macrophages reduced NF-kB activation to 53% of normal peritoneal macrophages with no further reduction after treatment with arctigenin. 3.3. Effect of arctigenin on the expression of serum pro-inflammatory cytokines IL-1b and TNF-a in LPS- or PGN-stimulated mice We prepared systemic inflammation in mice by the intraperitoneal injection of LPS or PGN, and investigated the inhibitory effect of arctigenin in vivo. Treatment with LPS or PGN significantly induced the expression of proinflammatory cytokines IL-1b and TNF-a. Treatment with arctigenin (30 mg/kg) significantly inhibited LPS- and PGN-induced proinflammatory cytokines by 71% and 68% for IL-1b and 81% and 62% for TNF-a, respectively (Fig. 6). 3.4. Inhibitory effect of arctigenin in TNBS-induced colitis We tested the ability of arctigenin to inhibit colitis induced by TNBS in mice. Intrarectal injection of TNBS caused inflammation, manifested by shortened, thickened and erythematous colons in
Fig. 5. Effect of arctigenin on PI3K activation in PI3K siRNA-transfected peritoneal macrophages. Peritoneal macrophages were incubated with PI3K siRNA and/or 50 ng/ml LPS in the absence or presence of arctigenin (10 or 20 mM) for 30 min. PI3K activation was detected by immunoblotting. All data are expressed as mean 7S.D. (n¼ 3 in a single experiment). ]P o0.05, significantly different vs. normal control group. nP o 0.05 vs. group treated with LPS alone.
mice. Treatment with arctigenin (30 and 60 mg/kg) in TNBStreated mice inhibited body weight loss, colon shortening and myeloperoxidase activity (Fig. 7A and B). Histologic examination of TNBS-treated mouse colon showed increased neutrophils, massive bowel edema, dense infiltration of the superficial layers of the mucosa and epithelial cell disruption by large ulcerations (data not shown). We also measured the effect of arctigenin on the phosphorylation of PI3K, AKT and p65 in TNBS-induced colitic mice (Fig. 7C). Arctigenin inhibited the activation of PI3K, AKT and NF-kB. We also tested the inhibitory effect of arctigenin on the expression of pro-inflammatory cytokines, TNF-a, IL-1b and IL-6 in addition to the anti-inflammatory cytokine, IL-10, in the colons of TNBS-induced colitis in mice (Fig. 7D). TNBS increased the expression of TNF-a 5.2-fold, IL-1b 4.4-fold and IL-6 3.8-fold, but reduced IL-10 expression by 74%. Treatment arctigenin inhibited the expression of proinflammatory cytokines, while b-actin expression (a house-keeping protein) was not affected. Treatment with arctigenin at 60 mg/kg inhibited the expression of these cytokines by 53% for IL-1b (Po 0.05), 39% for TNF-a (Po0.05), and 59% for IL-6 (Po0.05), while increasing IL-10 expression by 62% (Po0.05). We measured the effect of arctigenin on the expression of TNF-a, IL1b, IL10 and CD204, markers of M1/M2 macrophages, in TNBS-induced colitic mice by real time–PCR (Fig. 7E). Treatment with TNBS increased the expression of IL-1b and TNF-a, which are markers of M1 macrophages, did not affect CD204 expression, but reduced IL-10 expression. Treatment with arctigenin in TNBS-treated mice reduced the expression of TNF-a and IL-1b, M1 macrophage markers, but increased the expression of IL10 and CD204, M2 macrophage markers.
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Fig. 6. Inhibitory effect of arctigenin on the production of IL-1b and TNF-a in mice intraperitoneally injected with LPS (A) or PGN (B). The mice were intraperitoneally injected with or without arctigenin (5 mg/kg) in the presence or absence of LPS (4 mg/kg) or PGN (4 mg/kg). Normal control was treated with vehicle alone instead of LPS, PGN and arctigenin. Mice were sacrificed 4 h after LPS or PGN injection, whole blood was obtained by cardiac puncture and serum was obtained by centrifugation at 12,000 g for 20 min. The serum levels of IL-1b and TNF-a were measured using ELISA kit. All data shown are the mean 7 S.D. (n¼ 6). ]Po 0.05, significantly different vs. normal control group. nPo 0.05 vs. group treated with LPS or PGN alone.
4. Discussion Inflammation is a highly regulated defensive process characterized by the release of cytokines, chemokines and growth factors with transmigration of inflammatory cells, such as neutrophils, monocytes and lymphocytes, from the blood to the affected tissue (Bradding et al., 1993). Systemic inflammation and colitis caused by pathogens are regulated by inflammatory mediators, including interleukin (IL)1b, IL-6, tumor necrosis factor (TNF)-a, interferon-g, IL-12, IL-18, nitric oxide and prostaglandins (Joh and Kim, 2011). Of various pathogens, LPS containing gram-negative microbes and PGN containing gram-positive microbes potently induce inflammatory reactions. Among the inflammatory mediators, pro-inflammatory cytokines, such as IL-1b and TNF-a, are expressed through NF-kB activation, but they also activate NF-kB (Kumar et al., 2004). Interestingly, IL-1b and/or TNF-a levels are negatively correlated with life span (Fernandes et al., 1997). Thus, while these mediators are an integral part of the immune response, over-expression causes acute phase endotoxemia leading to organ failure, shock, tissue injury and even death (Strieter et al., 2002). This suggests that preventing overexpression of these inflammatory mediators can be beneficial in decreasing chronic inflammatory diseases. The use of the constituents of natural products has recently become a focus of interest (Joh and Kim, 2011; Joh et al., 2011). For example, compound K, lancemaside A and platycodins ameliorate several inflammatory disorders, including endotoxemia, colitis, itching and rheumatoid arthritis by regulating the activation of NF-kB (Joh et al., 2010 and 2011; Ahn et al., 2005). We isolated arctigenin from the seeds of Arctium lappa as an inhibitor
for NF-kB activation in LPS- or PGN-stimulated peritoneal macrophages. Arctigenin potently inhibited the expression of iNOS and COX-2 and NF-kB in LPS-stimulated peritoneal macrophages, like previous reports that arctigenin inhibited the expression of iNOS and proinflammatory cytokines in LPS-stimulated RAW264.7 cells (Kang et al., 2008; Kou et al., 2011; Zhao et al., 2009). Arctigenin also inhibited AKT phosphorylation, similar to a previous report where arctigenin inhibited AKT activation in tumor cells (Awale et al., 2006). Moreover, arctigenin inhibited the activation of PI3K and AKT, similar to previous report where tat-transduced CHME5 cells were killed by inhibiting PI3K and AKT phosphorylation (Kim et al., 2011). However, arctigenin did not inhibit IRAK-1 phosphorylation. Based on these findings, arctigenin may ameliorate inflammation in LPS-stimulated macrophages by inhibiting PI3K phosphorylation rather than IRAK-1 phosphorylation. LPS potently induced NF-kB activation in peritoneal macrophages treated without PI3K siRNA. However, LPS-induced NF-kB activation was significantly reduced in PI3K siRNA treated peritoneal macrophages compared to that in the cells treated without PI3K siRNA, and no further reduction was observed after treatment with arctigenin. Thus, arctigenin inhibited LPS-induced NF-kB activation through PI3K inhibition. These results were in agreement with previous reports that the PI3K/AKT signaling pathway may be involved in the pathogenesis of ulcerative colitis in mice (Huang et al., 2011) and that an PI3K inhibitor might ameliorate dextran sodium sulfate-induced colitis in mice (Peng et al., 2010). Toll-like receptors (TLRs), a major connection between innate and adaptive mucosal immune responses, act as membrane-bound
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Fig. 7. Effect of arctigenin on colon length (A), MPO activity (B), phosphorylation of PI3K, AKT and p65 (C), protein expression of IL-1b, TNF-a, IL-6 and IL-10 (D), and mRNA expression of T IL-1b, TNF-a, IL-10 and CD204 (E) in TNBS-induced colitic mice. TNBS, except in normal control group, was intrarectally administered to mice treated with saline, arctigenin or sulfasalazine. Arctigenin (AG30, 30 mg/kg; AG60, 60 mg/kg), sulfasalazine (S50, 50 mg/kg) or saline was orally administered for 3 days after TNBS treatment. The mice were anesthetized with ether and sacrificed 3 days after TNBS treatment. All values are mean 7 S.D. (n¼7). The levels of PI3K, p-PI3K, AKT, p-AKT, p65 and p-p65 were measured by immunoblotting. The protein expression levels of TNF-a, IL-1b, IL-6 and IL-10, and mRNA levels of TNF-a, IL-1b, IL-10 and CD204, in colons were measured ELISA and RT–PCR, respectively. All data shown are the mean 7 S.D. (n¼ 7). ]Significantly different vs. normal control group (P o0.05); nsignificantly different vs. group treated with TNBS alone (Po 0.05).
receptors in the cellular response to insults, such as PGN and LPS (Takeuchi et al., 1999). Among TLR family, TLR2 and TLR4, which are associated with the activation of NF-kB, may serve as the primary mediators of PGN and LPS cell signaling, respectively. The binding of LPS to TLR4 or of PGN to TLR2 induces the activation of IRAKs and/or PI3K/AKT, which are involved in host defense mechanisms, either by the identification of pathogens or as receptors for pro-inflammatory cytokines (Kim et al., 2011). Of the IRAK members, the phosphorylation of IRAK-1 activates a multimeric protein complex composed of TRAF6, TAK1, TAB1 and TAB2, leading to activation of NF-kB and MAPK via IKKb phosphorylation, as well as induction of pro-inflammatory cytokines. At the same time, the phosphorylated PI3K activates NF-kB via the phosphorylation of PDK-1 and AKT, leading to the induction of proinflammatory cytokines. Various inflammatory diseases involve the over-expression of pro-inflammatory cytokines, such as TNF-a and IL-1b, in addition to inflammatory mediators such as NO and PGE2 via the activation of NF-kB pathway in macrophages. Arctigenin inhibited the expression of serum TNF-a, IL-1b and IL-6 on LPS-induced systemic inflammation and TNBS-induced colitis in mice, as well as the activation of NF-kB in TNBS-induced colitic mice. Arctigenin also inhibited the PI3K/AKT pathway in TNBS-induced colitis in mice. When the effect of arctigenin in macrophage polarization was examined, arctigenin suppressed the
expression of TNF-a and IL-1b in LPS-stimulated macrophages, but increased the expression of IL-10 and CD204. Arctigenin also inhibited TNBS-induced the expression of TNF-a and IL-1b in TNBS-induced colitic mice, but increased the expression of IL-10 and CD204, M2 macrophage markers, although treatment with TNBS alone reduced IL-10 expression and did not alter CD204 expression. These results suggest that arctigenin may polarize LPS-stimulated macrophages to M2-like macrophages in vivo like an in vitro study, although the expression of CD204, a marker of M2 macrophages, due to the difference of microenvironment between TNBS-induced colitis in mice and LPS-treated peritoneal macrophages could be different. Arctigenin was also able to reverse a series of molecular, cellular and immunological responses observed during the inflammation process in vivo. Based on these findings, arctigenin may ameliorate inflammatory diseases, such as colitis, by inhibiting PI3K and polarizing M1 macrophages to M2-like macrophages.
Acknowledgment This study was supported by a grant from World Class University Program through the National Research Foundation
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Joh, E.H., Kim, D.H., 2011. Kalopanaxsaponin A ameliorates experimental colitis in mice by inhibiting IRAK-1 activation in the NF-kB and MAPK pathways. Br. J. Pharmacol. 162, 1731–1742. Joh, E.H., Lee, I.A., Jung, I.H., Kim, D.H., 2011. Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation—the key step of inflammation. Biochem. Pharmacol. 82, 278–286. Kang, H.S., Lee, J.Y., Kim, C.J., 2008. Anti-inflammatory activity of arctigenin from Forsythiae Fructus. J. Ethnopharmacol. 116, 305–312. Kim, Y., Hollenbaugh, J.A., Kim, D.H., Kim, B., 2011. Novel PI3K/Akt inhibitors screened by the cytoprotective function of human immunodeficiency virus type 1 Tat. PLoS One 6, e21781. Kou, X., Qi, S., Dai, W., Luo, L., Yin, Z., 2011. Arctigenin inhibits lipopolysaccharideinduced iNOS expression in RAW264.7 cells through suppressing JAK–STAT signal pathway. Int. Immunopharmacol. 11, 1095–1102. Kumar, A., Takada, Y., Boriek, A.M., Aggarwal, B.B., 2004. Nuclear factor-kappaB: its role in health and disease. J. Mol. Med. (Berl) 82, 434–448. Lee, I.A., Joh, E.H., Kim, D.H., 2011. Arctigenin Isolated from the seeds of Arctium lappa ameliorates memory deficits in mice. Planta Med. 77, 1525–1527. Lin, W.W., Karin, M., 2007. A cytokine-mediated link between innate immunity, inflammation and cancer. J. Clin. Invest. 117, 1175–1183. Mehta, J.L., Saldeen, T.G., Rand, K., 1998. Interactive role of infection, inflammation and traditional risk factors in atherosclerosis and coronary artery disease. J. Am. Coll. Cardiol. 31, 1217–1225. Mullane, K.M., Kraemer, R., Smith, B., 1985. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J. Pharmacol. Methods 14, 157–167. Olson, J.K., Miller, S.D., 2004. Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs. J. Immunol. 173, 3916–3924. O’Neill, L.A., Dinarello, C.A., 2000. The IL-1 receptor/toll-like receptor superfamily: crucial receptors for inflammation and host defense. Immunol. Today 21, 206–209. Paradkar, P.N., Blum, P.S., Berhow, M.A., Baumann, H., Kuo, S.M., 2004. Dietary isoflavones suppress endotoxin-induced inflammatory reaction in liver and intestine. Cancer Lett. 215, 21–28. Park, S.Y., Hong, S.S., Han, X.H., Hwang, J.S., Lee, D., Ro, J.S., Hwang, B.Y., 2007. Lignans from Arctium lappa and their inhibition of LPS-induced nitric oxide production. Chem. Pharm. Bull. (Tokyo) 55, 150–152. Peng, X.N., Wu, X.H., Chen, L.J., Wang, Z.L., Hu, X.H., Song, L.F., He, C.M., Luo, Y.F., Chen, K.J., Lin, H.G., Li, X.I., Wang, Y.S., Wei, Y.Q., 2010. Inhibition of phosphoinositide 3-Kinase ameliorates dextran sodium sulfate-induced colitis in mice. J. Pharmacol. Exp. Ther. 332, 46–56. Strieter, R.M., Belperio, J.A., Keane, M.P., 2002. Cytokines in innate host defense in the lung. J. Clin. Invest. 109, 699–705. Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K., Akira, S., 1999. Differential roles of TLR2 and TLR4 in recognition of gramnegative and gram-positive bacterial cell wall components. Immunity 11, 443–451. Tousoulis, D., Charakida, M., Stefanadis, C., 2005. Endothelial function and inflammation in coronary artery disease. Heart 92, 441–444. Tsai, W.J., Chang, C.T., Wang, G.J., Lee, T.H., Chang, S.F., Lu, S.C., Kuo, Y.C., 2011. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes. Chin. Med. 6, 12. Zhao, F., Wang, L., Liu, K., 2009. In vitro anti-inflammatory effects of arctigenin, a lignan from Arctium lappa L., through inhibition on iNOS pathway. J. Ethnopharmacol. 122, 457–462.
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Arctigenin ameliorates inflammation in vitro and in vivo by inhibiting the PI3K/AKT pathway and polarizing M1 macrophages to M2-like macrophages Supriya R. Hyam a, In-Ah Lee a, Wan Gu a, Kyung-Ah Kim a, Jin-Ju Jeong a, Se-Eun Jang b, Myung Joo Han b, Dong-Hyun Kim a,n a b
Department of Life and Nanopharmaceutical Sciences and Department of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea Department of Food and Nutrition, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e i n f o
abstract
Article history: Received 30 August 2012 Received in revised form 18 December 2012 Accepted 14 January 2013 Available online 31 January 2013
Seeds of Arctium lappa, containing arctigenin and its glycoside arctiin as main constituents, have been used as a diuretic, anti-inflammatory and detoxifying agent in Chinese traditional medicine. In our preliminary study, arctigenin inhibited IKKb and NF-kB activation in peptidoglycan (PGN)- or lipopolysaccharide (LPS)-induced peritoneal macrophages. To understand the anti-inflammatory effect of arctigenin, we investigated its anti-inflammatory effect in LPS-stimulated peritoneal macrophages and on LPS-induced systemic inflammation as well as 2,4,6-trinitrobenzene sulfonic acid (TNBS)induced colitis in mice. Arctigenin inhibited LPS-increased IL-1b, IL-6 and TNF-a expression in LPSstimulated peritoneal macrophages, but increased LPS-reduced IL-10 and CD204 expression. Arctigenin inhibited LPS-induced PI3K, AKT and IKKb phosphorylation, but did not suppress LPS-induced IRAK-1 phosphorylation. However, arctigenin did not inhibit NF-kB activation in LPS-stimulated PI3K siRNAtreated peritoneal macrophages. Arctigenin suppressed the binding of p-PI3K antibody and the nucleus translocation of NF-kB p65 in LPS-stimulated peritoneal macrophages. Arctigenin suppressed blood IL-1b and TNF-a level in mice systemically inflamed by intraperitoneal injection of LPS. Arctigenin also inhibited colon shortening, macroscopic scores and myeloperoxidase activity in TNBS-induced colitic mice. Arctigenin inhibited TNBS-induced IL-1b, TNF-a and IL-6 expression, as well as PI3K, AKT and IKKb phosphorylation and NF-kB activation in mice, but increased IL-10 and CD204 expression. However, it did not affect IRAK-1 phosphorylation. Based on these findings, arctigenin may ameliorate inflammatory diseases, such as colitis, by inhibiting PI3K and polarizing M1 macrophages to M2-like macrophages. & 2013 Elsevier B.V. All rights reserved.
Keywords: Arctigenin Inflammation Macrophages PI3K Polarization
1. Introduction Inflammation is the body’s response to injury or infection (Mehta et al., 1998; Baldwin, 1996). It can be classified as both acute and chronic inflammations. Acute inflammation is a normal and helpful component of physiologic response to injury. However, chronic inflammation is persistent inflammation that becomes excessive. This persistent inflammatory response causes progressive damage to the body leading to a variety of diseases, such as colitis, rheumatoid arthritis, atherosclerosis and even cancer. The mechanisms include macrophage and T lymphocyte activation and the release of pro-inflammatory cytokines that induce inflammatory activity (Tousoulis et al., 2005; Lin and Karin, 2007). Inflammation can be mediated by inflammatory mediators, including interleukin (IL)-1b, IL-6, tumor necrosis n
Corresponding author. Tel.: þ82 2 961 0357; fax: þ82 2 957 5030. E-mail address: [email protected] (D.-H. Kim).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.01.014
factor (TNF)-a, interferon-g, IL-12, IL-18, nitric oxide and prostaglandins (Fairweather and Rose, 2005). Among these inflammatory mediators, pro-inflammatory cytokines such as IL-1b and TNF-a are activated through nuclear factor-kappaB (NF-kB), but they can also activate this transcription factor (Baldwin, 1996; Collins et al., 1995). These mediators arouse the innate immune response but their over-expression causes acute phase endotoxemia leading to organ failure, shock, tissue injury and even death (Astiz and Rackow, 1998). Toll-like receptors (TLRs), a major connection between innate and adaptive mucosal immune responses, act as transmembrane receptors in the cellular response to insults, such as gram-negative bacterial lipopolysaccharides (LPS) (Chow et al., 1999; Ingalls et al., 1999), and grampositive bacterial peptidoglycans (PGN) (Olson and Miller, 2004). Among this family of receptors, TLRs, such as TLR2 and TLR4, recognize pathogen-associated molecular patterns (Akira and Hemmi, 2003; O’Neill and Dinarello, 2000) and interact with myeloid differentiation factor 88 (MyD88). The interaction results
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in the activation of NF-kB and MAP kinases to elicit regulatory responses for the generation of inflammation mediators. TLR4/MyD88 initiates a signaling cascade through the Toll/IL-1R (TIR) domain of its cytoplasmic tail, allowing for subsequent activation of IL-1R-associated kinases (IRAKs). Phosphorylated IRAK-1 activates a multimeric protein complex composed of TRAF6, TAK1, TAB1 and TAB2, leading to NF-kB-mediated pathway as well as the induction of proinflammatory cytokines. Recently, TLR4/MyD88 has been also reported to initiate phosphatidylinositol 3-kinases (PI3K)/AKT/(IkB kinase) IKK signaling pathway. Regulating expression of these different inflammatory mediators is therefore beneficial in decreasing inflammatory diseases. To prevent inflammation, the use of dietary supplements has recently become a topic of interest (Chan et al., 1998; Paradkar et al., 2004; Davis et al., 2006). The seed of Arctium lappa is one of the well-known diuretic, anti-inflammatory and detoxifying agents in Chinese traditional medicine (Park et al., 2007). Arctigenin displays a memoryenhancing effect (Lee et al., 2011) and anti-inflammatory activity via NF-kB or JAK-STAT pathway in LPS-stimulated RAW264.7 cells (Kang et al., 2008; Kou et al., 2011; Zhao et al., 2009), inhibit T lymphocyte proliferation and the gene expression of IL-2, IFN-g and NF-AT (Tsai et al., 2011), as well as anti-tumor activity against PANC-1 cells (Awale et al., 2006). Nevertheless, its antiinflammatory mechanism is not understood thoroughly. Therefore, we isolated arctigenin from the seed of Arctium lappa as an inhibitor for NF-kB activation in LPS- or PGNstimulated peritoneal macrophages, investigated its antiinflammatory mechanism in LPS-stimulated peritoneal macrophages, and evaluated its inhibitory effect against LPS-induced systemic inflammation and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice.
2. Materials and methods 2.1. Materials DMEM, RPMI 1640, TNBS, and LPS purified from Escherichia coli O111:B4 were purchased from Sigma Co. (St. Louis, MO, U.S.A.). Antibodies for IRAK1, AKT, p-AKT, p65 and b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, L.A. U.S.A.). Antibody for p-IRAK1 and p-p65 was purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Enzyme-linked immunosorbent assay (ELISA) kits for cytokines were purchased from R&D Systems (Minneapolis, MN, U.S.A.). Other chemicals used were of the highest grade available. Arctigenin (purity, 495%) was isolated from the seeds of Arctium lappa Linne as previously reported (Lee et al., 2011). 2.2. Animals Male C57BL/6 (18–22 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 507 10% humidity, fed standard laboratory chow (Samyang Co., Seoul, Korea), and allowed water ad libitum. All experiments were performed in accordance with the NIH and Kyung Hee University guidelines for Laboratory Animals Care and Use and approved by the Committee for the Care and Use of Laboratory Animals in the College of Pharmacy, Kyung Hee University. 2.3. Isolation and culture of peritoneal macrophages The mice were intraperitoneally injected with 2 ml of 4% sodium thioglycolate solution. Mice were sacrificed 4 days
after injection and the peritoneal cavities were flushed with 10 ml of RPMI 1640. The peritoneal lavage fluids were centrifuged at 200 g for 10 min and the cells were resuspended with RPMI 1640 and plated. After incubation for 1 h at 37 1C, the cells were washed three times and nonadherent cells were removed by aspiration. Cells were cultured in 24-well plates (0.5 106 cells/ well) at 37 1C in RPMI 1640 plus 10% FBS. The attached cells were used as peritoneal macrophages. To examine anti-inflammatory effect of arctigenin, peritoneal macrophages were incubated in the absence or presence of arctigenin with 50 ng/ml LPS or PGN. 2.4. Immunofluorescent confocal and fluorescent microscopy For the p65 assay, peritoneal macrophages were stimulated with LPS (50 ng/ml) in the presence or absence of arctigenin for 60 min. The cells were then fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100. The cells were stained with goat polyclonal anti-p65 antibody for 2 h at 4 1C and then incubated with secondary antibodies conjugated with Alexa 488 and propidium iodide (10 mg/ml, Calbiochem Co., San Diego, CA, U.S.A.) for 1 h. Images were observed by confocal microscopy. For the p-PI3K assay, peritoneal macrophages were stimulated with LPS (50 ng/ml) in the presence or absence of arctigenin for 30 min. The cells were then fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100. The cells were stained with goat polyclonal anti-p-PI3K antibody for 2 h at 4 1C and then incubated with secondary antibodies conjugated with Alexa 594 for 1 h. The stained macrophages were observed with 5 ml of DAPI mounting (Mounting Medium with DAPI, Abcam, Cambridge, CA, U.S.A.), at 5 min by fluorescent microscope. 2.5. 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 one day prior to the transfection. PI3K small interfering RNA was purchased from Santa Cruz (Santa Cruz, CA, U.S.A.). The cells were transfected with 100 nM PI3K siRNA using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instruction. At 48 h after transfections, the cells were treated with or without arctigenin (10 and 20 mM) and/or LPS (50 ng/ml). 2.6. Assay of serum pro-inflammatory cytokines TNF-a and IL-1b in PGN- or LPS-stimulated mice The mice were intraperitoneally injected with or without arctigenin (5 mg/kg) and/or PGN (4 mg/kg) or LPS (4 mg/kg). Each group consisted of 6 mice. Normal control was treated with vehicle alone instead of LPS, PGN and arctigenin. Mice were sacrificed 4 h after LPS or PGN injection, whole blood was obtained by cardiac puncture and serum was obtained by centrifugation at 12,000g for 20 min. The serum levels of IL-1b and TNF-a were measured using ELISA kit. 2.7. Preparation of experimental colitic mice The mice were randomly divided into 5 groups: normal and TNBS-induced colitic groups treated with or without arctigenin or sulfasalazine. Each group consisted of 7 mice. TNBS-induced colitis was induced by the administration of 2.5% (w/v) TNBS solution (100 ml) in 50% ethanol into the colon of anesthetized mice via a thin round-tip needle equipped with a 1 ml syringe (Joh et al., 2010). The normal group was treated with just the vehicle. The needle was inserted so that the tip was 3.5–4 cm proximal to the anal verge. To distribute the agents within the entire colon and cecum, mice were held in a vertical position for
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Fig. 1. Effect of arctigenin on IKKb and NF-kB phosphorylation in pathogen-stimulated peritoneal macrophages. The peritoneal macrophages isolated from mice were treated with 50 ng/ml LPS or PGN in the absence or presence of arctigenin (10 mM). The phosphorylation of IKKb and NF-kB was detected by immunoblottig. All data are expressed as mean 7 S.D. (n¼ 3 in a single experiment). ]P o0.05, significantly different vs. NOR group. nP o 0.05 vs. group treated with LPS alone.
Fig. 2. Inhibitory effect of arctigenin on the expression of pro-inflammatory and anti-inflammatory cytokines (A) and polarization markers (B) in LPS-stimulated peritoneal macrophages. Peritoneal macrophages (0.5 106 cells) were treated with 50 ng/ml LPS in the absence (LPS) or presence of arctigenin (10 and 20 mM) for 20 h. L (A) Effect on the expression of pro-inflammatory and anti-inflammatory cytokines. Levels of TNF-a, IL-1b, IL-6 and IL-10 in culture supernatants were measured by ELISA. (B) Effect on the expression of macrophage polarization markers. Levels of TNF-a, IL-1b, IL-10 and CD204 mRNA were measured by real time–PCR. All data are expressed as mean 7 S.D. (n¼ 6 in a single experiment). ]P o0.05, significantly different vs. normal control group. nP o 0.05 vs. group treated with LPS alone.
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30 s after the injection. Using this procedure, 495% of the mice retained the TNBS enema. If an animal quickly excreted the TNBS– ethanol solution, it was excluded from the remainder of the study. Arctigenin (30 and 60 mg/kg) or sulfasalazine (50 mg/kg) dissolved in 2% tween 80 were orally administered once a day for 3 days after TNBS administration. The mice were sacrificed on next day after the final administration of test agents. The colon was quickly removed, opened longitudinally, and gently cleared of stool by PBS. Macroscopic assessment of the disease grade was scored according to a previously reported scoring system (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; 5, ulceration extending more than 2 cm, and the colon tissue was then used for immunoblotting and enzyme-linked immunosorbent assay (ELISA) analysis. For the histological exam, the colons were fixed over-night in phosphate buffer (50 mM, pH7.4) containing 4% para-formaldehyde, then immersed in a 30% sucrose solution (in 50 mM phosphate-buffered saline) and stored at 4 1C until sectioning. Frozen colons were sectioned along in the coronal plane (10 mm) using a cryostat (Leica Microsy stems AG, Germany) and stained with hematoxylin-eosin.
2.8. Colon tissue preparation Colon tissues were excised, perfused with ice-cold perfusion solution containing 0.15 M KCl and 2 mM EDTA (pH 7.4), and homogenized in 50 mM Tris–HCl buffer (pH 7.4). The homogenates were centrifuged at 10,000 g at 4 1C for 30 min. The supernatant was used for the estimation of the antioxidant defense system. For immunoblotting and ELISA of colon tissues were carefully homogenized in 1 ml of ice cold RIPA lysis buffer containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail. 2.9. Assay of myeloperoxidase activity in colonic mucosa 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 20,000 g at 4 1C. A 50 ml aliquot of the supernatant was added to the reaction mixture consisting of 1.6 mM tetramethyl benzidine and 0.1 mM H2O2, and incubated at 37 1C. The absorbance was measured at 650 nm spectrophotometrically time-scanned. The myeloperoxidase activity was defined as the quantity of enzyme degrading 1 mmol/ml of
Fig. 3. Effect of arctigenin on IRAK-1 phosphorylation and NF-kB activation in LPS-stimulated peritoneal macrophages. The peritoneal macrophages isolated from mice were treated with 50 ng/ml LPS in the absence or presence of arctigenin (10 or 20 mM). (A) Effect in IRAK-1 and NF-kB phosphorylation. b-Actin was used as a control. (B) Effect on NF-kB nuclear translocation. Peritoneal macrophages were incubated with 50 ng/ml LPS in the absence or presence of arctigenin (10 or 20 mM) for 60 min. NF-kB nuclear translocation was detected by confocal analysis using an antibody for p65 subunit. All data are expressed as mean 7 S.D. (n¼ 3 in a single experiment). ] Po 0.05, significantly different vs. normal control group. nP o 0.05 vs. group treated with LPS alone.
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peroxide at 37 1C, and expressed in unit/mg protein (Mullane et al., 1985). The protein content was assayed by Bradford’s method (Bradford (1996)). 2.10. Cytokine analysis and immunoblotting Proinflammatory cytokines, TNF-a, IL-1b, IL-6 and IL-10, were assessed by ELISA according to the manufacturer’s instructions. The supernatant extracts prepared from colon or peritoneal macrophages were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (PVDF). The membranes were blocked with 5% non-fat dried-milk proteins in PBST, and probed with p-IRAK1, IRAK1, p-PI3K, PI3K, p-AKT, AKT, p65, p-p65 or b-actin antibody. After washing with PBST, proteins were detected with HRP-conjugated secondary antibodies for 1 h. Bands were visualized with enhanced chemiluminescence reagent (Bai et al., 2007).
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utilizing Takara thermal cycler, which used SYBER premix agents, as per the instructions from Takara biology incorporation. Thermal cycling conditions were as follows: activation of DNA polymerase at 95 1C for 10 min, followed by 40 cycles of amplification at 95 1C for 30 s and at 60 1C for 30 s. The normalized expression of the target gene, with respect to b-actin, was computed for all samples by using the Microsoft Excel data spreadsheet. 2.12. Statistical analysis Results are presented as the means7standard deviation of at least four replicates. One-way ANOVA and Student t-test were used. Po0.05 was regarded statistically significant.
3. Results
2.11. Real time–polymerase chain reaction (RT–PCR)
3.1. Anti-inflammatory effect of arctigenin in LPS or PGN-stimulated peritoneal macrophages
Total RNA was extracted by RIPA buffer. Reverse transcription was performed with 2 mg of total RNA. RT–PCR for TNF-a, IL-1b, IL10, CD204 and b-actin was performed as described previously,
We tested the inhibitory effect of arctigenin in PGN- or LPSstimulated peritoneal macrophages (Fig. 1). Stimulation with PGN or LPS markedly induced IKKb phosphorylation and NF-kB
Fig. 4. Effect of arctigenin on activation of PI3K in LPS-stimulated peritoneal macrophages. The peritoneal macrophages isolated from mice were treated with 50 ng/ml LPS in the absence or presence of arctigenin (10 or 20 mM). (A) Effect in PI3K and AKT phosphorylation by immunoblotting. b-Actin was used as a control. (B) Effect in PI3K phosphorylation by a fluorescent microscopy. Peritoneal macrophages were incubated with 50 ng/ml LPS in the absence or presence of arctigenin (10 or 20 mM) for 30 min. PI3K phosphorylation was detected by confocal analysis using an antibody for p-PI3K. All data are expressed as mean 7 S.D. (n¼ 3 in a single experiment). ]Po 0.05, significantly different vs. normal control group. nP o0.05 vs. group treated with LPS alone.
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activation. However, treatment with PGN or LPS in the presence of arctigenin potently suppressed IKKb phosphorylation and NFkB activation. Therefore, to clarify the anti-inflammatory mechanism of arctigenin, we stimulated peritoneal macrophages with LPS in the presence or absence of arctigenin and measured the expression of proinflammatory and inflammatory cytokines (Fig. 2A). Treatment with LPS significantly increased TNF-a, IL-1b and IL-6 expression, but reduced IL-10 expression. However, treatment with LPS in the presence of arctigenin inhibited the expression of proinflammatory cytokines, TNF-a, IL-1b and IL-6, but increased the expression of anti-inflammatory cytokine IL-10. We also examined the ability of arctigenin to polarize LPS-stimulated macrophages to M2 macrophages (Fig. 2B) by real time–PCR. Arctigenin suppressed the LPS-elevated levels of IL-1b and TNF-a and increased the LPS-reduced expression of IL-10 and CD204. Next, we stimulated peritoneal macrophages with LPS in the presence or absence of arctigenin and measured the phosphorylation of IRAK-1 and the activation of NF-kB (Fig. 3A). IRAK-1 phosphorylation and NF-kB activation were significantly elicited by LPS treatment. Although arctigenin inhibited LPS-induced NF-kB activation (p65 phosphorylation), it did not inhibit the phosphorylation of IRAK-1. Thus, arctigenin did not increase IRAK-1 expression reduced by LPS. We also examined the ability of arctigenin to inhibit the nuclear translocation of NF-kB in LPS-stimulated peritoneal macrophages by confocal microscopy (Fig. 3B). Treatment with arctigenin attenuated the LPS-translocated NF-kB into the nucleus and subsequently increased the cytosolic p65 level. 3.2. Effect of arctigenin on the PI3K/AKT pathway in LPS-stimulated peritoneal macrophages We investigated the effect of arctigenin on the phosphorylation PI3K and AKT in LPS-stimulated peritoneal macrophages by immunoblotting and fluorescent microscopy. Single LPS treatment significantly increased the phosphorylation of PI3K and AKT. Arctigenin potently suppressed LPS-induced PI3K and AKT phosphorylation (Fig. 4A). Furthermore, arctigenin inhibited LPSstimulated PI3K phosphorylation by a fluorescent microscopy (Fig. 4B). To confirm the inhibition of PI3K activation by arctigenin, we investigated its effect in PI3K siRNA-transfected peritoneal macrophages (Fig. 5). Transfection of PI3K siRNA significantly inhibited PI3K expression by 72%. LPS stimulation in PI3K siRNA-treated peritoneal macrophages reduced NF-kB activation to 53% of normal peritoneal macrophages with no further reduction after treatment with arctigenin. 3.3. Effect of arctigenin on the expression of serum pro-inflammatory cytokines IL-1b and TNF-a in LPS- or PGN-stimulated mice We prepared systemic inflammation in mice by the intraperitoneal injection of LPS or PGN, and investigated the inhibitory effect of arctigenin in vivo. Treatment with LPS or PGN significantly induced the expression of proinflammatory cytokines IL-1b and TNF-a. Treatment with arctigenin (30 mg/kg) significantly inhibited LPS- and PGN-induced proinflammatory cytokines by 71% and 68% for IL-1b and 81% and 62% for TNF-a, respectively (Fig. 6). 3.4. Inhibitory effect of arctigenin in TNBS-induced colitis We tested the ability of arctigenin to inhibit colitis induced by TNBS in mice. Intrarectal injection of TNBS caused inflammation, manifested by shortened, thickened and erythematous colons in
Fig. 5. Effect of arctigenin on PI3K activation in PI3K siRNA-transfected peritoneal macrophages. Peritoneal macrophages were incubated with PI3K siRNA and/or 50 ng/ml LPS in the absence or presence of arctigenin (10 or 20 mM) for 30 min. PI3K activation was detected by immunoblotting. All data are expressed as mean 7S.D. (n¼ 3 in a single experiment). ]P o0.05, significantly different vs. normal control group. nP o 0.05 vs. group treated with LPS alone.
mice. Treatment with arctigenin (30 and 60 mg/kg) in TNBStreated mice inhibited body weight loss, colon shortening and myeloperoxidase activity (Fig. 7A and B). Histologic examination of TNBS-treated mouse colon showed increased neutrophils, massive bowel edema, dense infiltration of the superficial layers of the mucosa and epithelial cell disruption by large ulcerations (data not shown). We also measured the effect of arctigenin on the phosphorylation of PI3K, AKT and p65 in TNBS-induced colitic mice (Fig. 7C). Arctigenin inhibited the activation of PI3K, AKT and NF-kB. We also tested the inhibitory effect of arctigenin on the expression of pro-inflammatory cytokines, TNF-a, IL-1b and IL-6 in addition to the anti-inflammatory cytokine, IL-10, in the colons of TNBS-induced colitis in mice (Fig. 7D). TNBS increased the expression of TNF-a 5.2-fold, IL-1b 4.4-fold and IL-6 3.8-fold, but reduced IL-10 expression by 74%. Treatment arctigenin inhibited the expression of proinflammatory cytokines, while b-actin expression (a house-keeping protein) was not affected. Treatment with arctigenin at 60 mg/kg inhibited the expression of these cytokines by 53% for IL-1b (Po 0.05), 39% for TNF-a (Po0.05), and 59% for IL-6 (Po0.05), while increasing IL-10 expression by 62% (Po0.05). We measured the effect of arctigenin on the expression of TNF-a, IL1b, IL10 and CD204, markers of M1/M2 macrophages, in TNBS-induced colitic mice by real time–PCR (Fig. 7E). Treatment with TNBS increased the expression of IL-1b and TNF-a, which are markers of M1 macrophages, did not affect CD204 expression, but reduced IL-10 expression. Treatment with arctigenin in TNBS-treated mice reduced the expression of TNF-a and IL-1b, M1 macrophage markers, but increased the expression of IL10 and CD204, M2 macrophage markers.
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Fig. 6. Inhibitory effect of arctigenin on the production of IL-1b and TNF-a in mice intraperitoneally injected with LPS (A) or PGN (B). The mice were intraperitoneally injected with or without arctigenin (5 mg/kg) in the presence or absence of LPS (4 mg/kg) or PGN (4 mg/kg). Normal control was treated with vehicle alone instead of LPS, PGN and arctigenin. Mice were sacrificed 4 h after LPS or PGN injection, whole blood was obtained by cardiac puncture and serum was obtained by centrifugation at 12,000 g for 20 min. The serum levels of IL-1b and TNF-a were measured using ELISA kit. All data shown are the mean 7 S.D. (n¼ 6). ]Po 0.05, significantly different vs. normal control group. nPo 0.05 vs. group treated with LPS or PGN alone.
4. Discussion Inflammation is a highly regulated defensive process characterized by the release of cytokines, chemokines and growth factors with transmigration of inflammatory cells, such as neutrophils, monocytes and lymphocytes, from the blood to the affected tissue (Bradding et al., 1993). Systemic inflammation and colitis caused by pathogens are regulated by inflammatory mediators, including interleukin (IL)1b, IL-6, tumor necrosis factor (TNF)-a, interferon-g, IL-12, IL-18, nitric oxide and prostaglandins (Joh and Kim, 2011). Of various pathogens, LPS containing gram-negative microbes and PGN containing gram-positive microbes potently induce inflammatory reactions. Among the inflammatory mediators, pro-inflammatory cytokines, such as IL-1b and TNF-a, are expressed through NF-kB activation, but they also activate NF-kB (Kumar et al., 2004). Interestingly, IL-1b and/or TNF-a levels are negatively correlated with life span (Fernandes et al., 1997). Thus, while these mediators are an integral part of the immune response, over-expression causes acute phase endotoxemia leading to organ failure, shock, tissue injury and even death (Strieter et al., 2002). This suggests that preventing overexpression of these inflammatory mediators can be beneficial in decreasing chronic inflammatory diseases. The use of the constituents of natural products has recently become a focus of interest (Joh and Kim, 2011; Joh et al., 2011). For example, compound K, lancemaside A and platycodins ameliorate several inflammatory disorders, including endotoxemia, colitis, itching and rheumatoid arthritis by regulating the activation of NF-kB (Joh et al., 2010 and 2011; Ahn et al., 2005). We isolated arctigenin from the seeds of Arctium lappa as an inhibitor
for NF-kB activation in LPS- or PGN-stimulated peritoneal macrophages. Arctigenin potently inhibited the expression of iNOS and COX-2 and NF-kB in LPS-stimulated peritoneal macrophages, like previous reports that arctigenin inhibited the expression of iNOS and proinflammatory cytokines in LPS-stimulated RAW264.7 cells (Kang et al., 2008; Kou et al., 2011; Zhao et al., 2009). Arctigenin also inhibited AKT phosphorylation, similar to a previous report where arctigenin inhibited AKT activation in tumor cells (Awale et al., 2006). Moreover, arctigenin inhibited the activation of PI3K and AKT, similar to previous report where tat-transduced CHME5 cells were killed by inhibiting PI3K and AKT phosphorylation (Kim et al., 2011). However, arctigenin did not inhibit IRAK-1 phosphorylation. Based on these findings, arctigenin may ameliorate inflammation in LPS-stimulated macrophages by inhibiting PI3K phosphorylation rather than IRAK-1 phosphorylation. LPS potently induced NF-kB activation in peritoneal macrophages treated without PI3K siRNA. However, LPS-induced NF-kB activation was significantly reduced in PI3K siRNA treated peritoneal macrophages compared to that in the cells treated without PI3K siRNA, and no further reduction was observed after treatment with arctigenin. Thus, arctigenin inhibited LPS-induced NF-kB activation through PI3K inhibition. These results were in agreement with previous reports that the PI3K/AKT signaling pathway may be involved in the pathogenesis of ulcerative colitis in mice (Huang et al., 2011) and that an PI3K inhibitor might ameliorate dextran sodium sulfate-induced colitis in mice (Peng et al., 2010). Toll-like receptors (TLRs), a major connection between innate and adaptive mucosal immune responses, act as membrane-bound
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Fig. 7. Effect of arctigenin on colon length (A), MPO activity (B), phosphorylation of PI3K, AKT and p65 (C), protein expression of IL-1b, TNF-a, IL-6 and IL-10 (D), and mRNA expression of T IL-1b, TNF-a, IL-10 and CD204 (E) in TNBS-induced colitic mice. TNBS, except in normal control group, was intrarectally administered to mice treated with saline, arctigenin or sulfasalazine. Arctigenin (AG30, 30 mg/kg; AG60, 60 mg/kg), sulfasalazine (S50, 50 mg/kg) or saline was orally administered for 3 days after TNBS treatment. The mice were anesthetized with ether and sacrificed 3 days after TNBS treatment. All values are mean 7 S.D. (n¼7). The levels of PI3K, p-PI3K, AKT, p-AKT, p65 and p-p65 were measured by immunoblotting. The protein expression levels of TNF-a, IL-1b, IL-6 and IL-10, and mRNA levels of TNF-a, IL-1b, IL-10 and CD204, in colons were measured ELISA and RT–PCR, respectively. All data shown are the mean 7 S.D. (n¼ 7). ]Significantly different vs. normal control group (P o0.05); nsignificantly different vs. group treated with TNBS alone (Po 0.05).
receptors in the cellular response to insults, such as PGN and LPS (Takeuchi et al., 1999). Among TLR family, TLR2 and TLR4, which are associated with the activation of NF-kB, may serve as the primary mediators of PGN and LPS cell signaling, respectively. The binding of LPS to TLR4 or of PGN to TLR2 induces the activation of IRAKs and/or PI3K/AKT, which are involved in host defense mechanisms, either by the identification of pathogens or as receptors for pro-inflammatory cytokines (Kim et al., 2011). Of the IRAK members, the phosphorylation of IRAK-1 activates a multimeric protein complex composed of TRAF6, TAK1, TAB1 and TAB2, leading to activation of NF-kB and MAPK via IKKb phosphorylation, as well as induction of pro-inflammatory cytokines. At the same time, the phosphorylated PI3K activates NF-kB via the phosphorylation of PDK-1 and AKT, leading to the induction of proinflammatory cytokines. Various inflammatory diseases involve the over-expression of pro-inflammatory cytokines, such as TNF-a and IL-1b, in addition to inflammatory mediators such as NO and PGE2 via the activation of NF-kB pathway in macrophages. Arctigenin inhibited the expression of serum TNF-a, IL-1b and IL-6 on LPS-induced systemic inflammation and TNBS-induced colitis in mice, as well as the activation of NF-kB in TNBS-induced colitic mice. Arctigenin also inhibited the PI3K/AKT pathway in TNBS-induced colitis in mice. When the effect of arctigenin in macrophage polarization was examined, arctigenin suppressed the
expression of TNF-a and IL-1b in LPS-stimulated macrophages, but increased the expression of IL-10 and CD204. Arctigenin also inhibited TNBS-induced the expression of TNF-a and IL-1b in TNBS-induced colitic mice, but increased the expression of IL-10 and CD204, M2 macrophage markers, although treatment with TNBS alone reduced IL-10 expression and did not alter CD204 expression. These results suggest that arctigenin may polarize LPS-stimulated macrophages to M2-like macrophages in vivo like an in vitro study, although the expression of CD204, a marker of M2 macrophages, due to the difference of microenvironment between TNBS-induced colitis in mice and LPS-treated peritoneal macrophages could be different. Arctigenin was also able to reverse a series of molecular, cellular and immunological responses observed during the inflammation process in vivo. Based on these findings, arctigenin may ameliorate inflammatory diseases, such as colitis, by inhibiting PI3K and polarizing M1 macrophages to M2-like macrophages.
Acknowledgment This study was supported by a grant from World Class University Program through the National Research Foundation
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