Food and Chemical Toxicology 63 (2014) 53–61
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Chloroform fraction of Solanum tuberosum L. cv Jayoung epidermis suppresses LPS-induced inflammatory responses in macrophages and DSS-induced colitis in mice Seung-Jun Lee a,b,1, Ji-Sun Shin a,c,d,1, Hye-Eun Choi a,b, Kyoung-Goo Lee a,b, Young-Wuk Cho c,d, Hyo-Jin An e, Dae Sik Jang b, Jin-Cheol Jeong f, Oh-Keun Kwon f, Jung-Hwan Nam f, Kyung-Tae Lee a,b,⇑ a
Department of Pharmaceutical Biochemistry, Kyung Hee University, Seoul, Republic of Korea Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul, Republic of Korea c Reactive Oxygen Species Medical Research Center, School of Medicine, Kyung Hee University, Republic of Korea d Department of Physiology, School of Medicine, Kyung Hee University, Republic of Korea e Department of Pharmacology, College of Oriental Medicine, Sangji University, Wonju-si, Gangwon-do 220-702, Republic of Korea f Highland Agriculture Research Center, NICS, RDA, Pyeongchang 232-955, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 4 July 2013 Accepted 22 October 2013 Available online 1 November 2013 Keywords: Color-fleshed potato peel Inflammation NF-jB p38 MAPK MKK3/6 Colitis
a b s t r a c t In this study, the authors investigated the molecular mechanism underlying the antiinflammatory effects of the chloroform fraction of the peel of ‘Jayoung’ (CFPJ), a color-fleshed potato, on lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in mice with dextran sulfate sodium (DSS)-induced colitis. CFPJ inhibited the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the transcription level, and attenuated the transcriptional activity of nuclear factor-jB (NF-jB) by reducing the translocation of NF-jB depending on degradation of inhibitory jB-a (IjB-a). Furthermore, CFPJ attenuated the phosphorylations of mitogen-activated protein kinase kinases3/6 (MKK3/6) and of p38. In colitis model, CFPJ significantly reduced the severity of colitis and the productions and protein levels of pro-inflammatory mediators in colonic tissue. These results suggest that the anti-inflammatory effects of CFPJ are associated with the suppression of NF-jB and p38 activation in macrophages, and support its possible therapeutic role for the treatment of colitis. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Inflammatory bowel diseases (IBDs), like Crohn’s disease and ulcerative colitis, are chronic inflammatory disorders of the intestinal tract that are associated with excessive productions of cytokines, adhesion molecules, and reactive oxygen species (ROS) (Cho et al., 2011). Moreover, the etiologies of IBDs are believed to involve inappropriate host responses to commensal microbial flora in the gut that result in mucosal barrier dysfunctions, such as, a leaky mucus layer, alterations in tight junction protein expression and increased epithelial cell apoptosis (Gitter et al., 2000). Experimentally induced colitis in the mouse by the oral administration of dextran sulfate sodium (DSS) is widely used to study the etiologies of IBDs. A DSS-induced murine colitis model exhibits symptoms comparable to those of human ulcerative colitis, that is, body ⇑ Corresponding author. Address: Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Dongdaemun-Ku, Hoegi-Dong, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 0860; fax: +82 2 966 3885. E-mail address: [email protected] (K.-T. Lee). 1 These authors contributed equally to this work. 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.10.040
weight loss, diarrhea, bloody feces, mucosal ulceration, and colonic shortening (Lee and Lim, 2007; Okayasu et al., 1990; Wirtz and Neurath, 2007). Histologically, colitis induced by DSS is characterized by infiltration of inflammatory cells into the lamina propria, accompanied by lymphoid hyperplasia, focal crypt damage, and epithelial ulceration. Furthermore, DSS destroys the mucosal barrier of epithelium, and thus, allows bacteria to contact lamina propria cells, which results in the activation of intestinal macrophages (Okayasu et al., 1990). Macrophages were activated by recognition the threats via the specific receptors, especially toll-like receptors (TLRs), which are capable of inducing specialized activation programs. Engagement of these receptors results in signals that modify the functional properties of cells by inducing gene expression changes and upregulating effector molecules, such as, cytokines and adhesion molecules (Kim et al., 2010). TLR4 ligation by lipopolysaccharide (LPS) induces the activations of specific intracellular pathways via receptor dimerization and the recruitments of different adaptor molecules, and the subsequent activations of two distinct downstream signaling pathways, that is, the nuclear factor-jB (NF-jB) pathway and the mitogen-activated protein kinase (MAPK)
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Fig. 1. Effects of CFPJ on LPS-induced iNOS and COX-2 mRNA expressions and their promoter activities in RAW 264.7 macrophages. (A and C) Total RNA was prepared from cells pretreated with/without the indicated concentrations of CFPJ and then stimulated with LPS (1 lg/ml) for 6 h. The mRNA levels of iNOS and COX-2 were determined by qRT-PCR, as described in methods. Control values were obtained in the absence of LPS and CFPJ. (B and D) Cells were transiently transfected with a pGL3-iNOS (B) or a pGL3COX-2 (D) promoter vector; phRL-TK vector was used as an internal control. Cells were treated with/without the indicated concentrations of CFPJ for 1 h and then stimulated with LPS (1 lg/ml) for 24 h. Cells were then harvested and luciferase activity levels were determined as described in Methods. The data presented are the means ± SDs of three independent experiments. #p < 0.05 vs. the control group; p < 0.01, p < 0.001 vs. the LPS-treated group.
pathway. These two pathways then induce the expressions of various inflammatory mediators, such as, inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines [tumor necrosis factor-a (TNF-a) and interleukin (IL)-1, IL-6, IL-12 and others], chemokines, and other antimicrobial responses (Janeway and Medzhitov, 2002). Furthermore, these pro-inflammatory mediators play key roles in the pathogeneses of various acute and chronic inflammatory diseases, including IBD. Therefore, pharmacologically reducing the levels of LPS-inducible inflammatory mediators is regarded an effective therapeutic strategy for alleviating a variety of disorders, including the inflammatory conditions caused by macrophage activation. In this context, RAW 264.7, a murine macrophage cell line, provides an excellent means of screening antiinflammatory drugs and of evaluating inhibitors of pathways that lead to the inductions of pro-inflammatory enzymes and cytokines. Potato cultivars (Solanum tuberosum L.) are widely cultivated potato varieties (commonly called colored potatoes), which were originally bred in the Republic of Korea during a joint program between the Highland Agriculture Research Center (HARC), the Korean National Institute of Crop Science, and the Rural Development Administration (RDA). ‘Jayoung’ has dark purple-flesh, and contains substantial amounts of polyphenols, such as, anthocyanin and phenolic acid (Park et al., 2009; Park et al., 2008). Several studies have shown that plant containing polyphenols provide protection against the developments of cancers, cardiovascular diseases, diabetes, osteoporosis, infections, asthma, and neurodegenerative diseases (Pandey and Rizvi, 2009). Similarly, previous researches showed that colored potatoes have anti-oxidant, anti-hypertensive, anti-mutagenic, and cytotoxic properties (Park et al., 2008).
Recently, we reported on the anti-inflammatory activities of the ethanol (EtOH) extracts and solvent fractions from the peel or tubers of colored (Jayoung, Hongyoung: red-fleshed potato) and general potatoes (Superior potato). In particular, we found that the chloroform fraction of peel from ‘Jayoung’ (CFPJ) exerting markedly inhibitory effect against LPS-induced NO and PGE2 production in RAW264.7 macrophages (Nam et al., 2011). In the present study, we investigated the anti-inflammatory and anti-colitis effects of CFPJ and the mechanisms responsible in LPS-induced RAW264.7 macrophages and in mice with DSS-induced colitis.
2. Materials and methods 2.1. Reagents Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (NY, USA). iNOS (dilution ratio = 1:1000), p65 (dilution ratio = 1:1000), inhibitory jB-a (IjB-a) (dilution ratio = 1:1000), p38 (dilution ratio = 1:1000), poly(ADP ribose)polymerase (PARP) (dilution ratio = 1:1000), b-actin (dilution ratio = 1:2000) monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (CA, USA). Antiphospho-p65 (Ser276) (dilution ratio = 1:1000), antiphospho-IjB-a (dilution ratio = 1:1000), antiphospho-p38 (dilution ratio = 1:1000), antiphospho-MAPK kinases 3/6 (MKK3/6) (dilution ratio = 1:500), MKK3/6 (dilution ratio = 1:1000) antibodies were purchased from Cell Signaling Technology, Inc. (MA, USA). And peroxidase-conjugated secondary antibody was purchased from Jackson Laboratory (ME, USA). The enzyme immunoassay (EIA) kits for IL-1b, IL-6, and TNF-a were obtained from R&D Systems (MN, USA). Random oligonucleotide primers and M-MLV reverse transcriptase were purchased from Promega (WI, USA). dNTP Mix, and SYBR green were obtained from TaKaRa Bio Inc.(Shiga, Japan). iNOS, COX-2, IL-1b, TNF-a, IL-6, and b-actin oligonucleotide primers were purchased from Bioneer (Seoul, Korea). Sulfanilamide, aprotinin, leupeptin, PMSF, DL-dithiothreitol (DTT), LPS
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Fig. 2. Effects of CFPJ on LPS-induced IL-1b, IL-6, and TNF-a production and on their mRNA expressions in RAW 264.7 macrophages. (A–C) Cells were pretreated with/without the indicated concentrations of CFPJ for 1 h and then stimulated with LPS (1 lg/ml) for 24 h. The productions of IL-1b, IL-6, and TNF-a were determined using EIA kits. Control values were obtained in the absence of LPS and CFPJ. (D–F) Total RNA was prepared from cells pretreated with/without the indicated concentrations of CFPJ for 1 h followed by stimulation with LPS (1 lg/ml) for 6 h. The mRNA levels of IL-1b, IL-6, and TNF-a were determined by qRT-PCR as described in Methods. Control values were obtained in the absence of LPS and CFPJ. The data presented are the means ± SDs of three independent experiments. #p < 0.05 vs. the control group; p < 0.01, p < 0.001 vs. the LPS-treated group. (Escherichia coli, serotype 0111:B4), 5-aminosalicylic acid (5-ASA), caffeic acid, ferulic acid, chlorogenic acid, and all other chemicals were purchased from Sigma Chemical Co. (MO, USA).
concentrations of 25, 50, or 100 lg/ml, and then induced with LPS (1 lg/ml) for the indicated time. Various concentrations of test compounds dissolved in DMSO were added together with LPS. Cells were treated with 0.05% DMSO as vehicle control.
2.2. Preparation and standardization of chloroform fraction 2.4. Determination of IL-1b, IL-6, and TNF-a production The colored potato cultivar, Jayoung was supplied by Highland Agriculture Research Center (HARC). The potatoes were cut into small pieces (20%), stool blood (0:negative, 1:+, 2:++, 3:+++, 4:++++) and stool consistency (0:normal, 1&2:loose stool, 3&4:diarrhea). Body weight loss was calculated as the percent difference between the original body weight (day 0) and the body weight on any particular day as previously described (Murthy et al., 1993). At the end of the experiment, mice were sacrificed and the colons were separated from the proximal rectum, close to its passage under the pelvisternum. The colon length was measured between the ileo–cecal junction and the proximal rectum. The spleens were also obtained and their weight was measured. Removed colonic tissue were immediately frozen ( 70 °C) for Western blotting. For measurement of NO, PGE2, and cytokines in colonic tissues, tissues were washed with DMEM medium containing 2% FBS and penicillin and streptomycin before cut into smaller pieces. Then 0.5 cm of tissue was placed 1 ml of DMEM medium containing 0.2% FBS, distributed into 24-well plate, incubated 24 h at 37 °C in 5% CO2. The cell-free culture supernatants of the colon tissue were using to measure NO, PGE2, TNF-a, and IL-6. 2.12. Statistical analysis Results are expressed as the mean ± SDs of triplicate experiments. Statistically significant values were compared using ANOVA and Dunnett’s post hoc test, and p-values of less than 0.05 were considered statistically significant.
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Fig. 4. Effects of CFPJ on the LPS-induced phosphorylation of IjB-a and on the activations of p38 MAPK and MKK3/6 in macrophages. (A and B) RAW 264.7 macrophages were pretreated with/without the indicated concentrations of CFPJ for 1 h and then stimulated with LPS (1 lg/ml) for 15 min (p-IjB-a, IjB-a, p-MKK3/6 or MKK3/6) or 30 min (pp38, p-38). Total proteins were analyzed by Western blotting using specific antibodies. Densitometric analysis was performed using Bio-rad Quantity OneÒ Software. Values shown are the means ± SDs of three independent experiments. #p < 0.05 vs. the control group; p < 0.001 vs. the LPS-treated group.
3. Results 3.1. CFPJ suppressed LPS-induced iNOS and COX-2 mRNA expressions and promoter activity in RAW 264.7 macrophages Because CFPJ was found previously to strongly inhibit the LPSinduced productions of NO and PGE2 (Nam et al., 2011), we investigated whether the inhibitory effects of CFPJ on these productions results from the suppression of their enzyme activities and/or their gene expressions. Initially, we investigated whether the enzyme activities of iNOS and COX-2 were affected by CFPJ, but enzymebased iNOS and COX-catalytic activity determinations showed that CFPJ did not reduce the productions of NO or PGE2 (data not shown). Next, qRT-PCR was used to confirm that CFPJ regulated LPS-induced iNOS and COX-2 expression at the transcriptional level, and as shown in Fig. 1A and C, pretreatment with CFPJ significantly reduced LPS-induced iNOS and COX-2 mRNA levels in a concentration-dependent manner. Next, the transcriptional regulations of iNOS and COX-2 by CFPJ were investigated using a promoter activity assay. As shown in Fig. 1B and D, LPS significantly enhanced iNOS and COX-2 promoter activities, and CFPJ concentration-dependently inhibited these increases.
3.2. CFPJ suppressed LPS-induced production and the mRNA expressions of IL-1b, IL-6, and TNF-a in RAW 264.7 macrophages To examine the inhibitory effects of CFPJ on the LPS-induced productions and expressions of pro-inflammatory cytokines in RAW 264.7 macrophages, we investigated its effects on LPS-induced IL-1b, IL-6, and TNF-a production and their mRNA levels using EIAs and qRT-RCR, respectively. Pretreatment with CFPJ was found to reduce the LPS- induced productions of IL-1b, IL-6, and TNF-a (Fig. 2A–C) and their mRNA expressions (Fig. 2D–F)
concentration-dependently, showing that CFPJ suppresses the expressions of these inflammatory genes at the transcriptional level. 3.3. CFPJ inhibited LPS-induced NF-jB activation in RAW 264.7 macrophages Since the activation of NF-jB is critically required for the LPSinduced transcriptional regulation of inflammation (Uwe, 2008), we examined the effect of CFPJ on LPS-induced NF-jB-dependent reporter gene activities. Analysis of reporter gene expressions using pNF-jB-luc demonstrated that CFPJ concentration-dependently inhibited NF-jB-dependent luciferase activity (Fig. 3A). Because transcriptional activities of NF-jB are mediated by nuclear translocation and phosphorylation of NF-jB (Oeckinghaus et al., 2011), we investigated whether CFPJ prevented the nuclear translocation and phosphorylations of the p65 subunit of NF-jB. It was found that pretreatment with CFPJ concentration-dependently attenuated the LPS-induced nuclear translocation of p65 (Fig. 3B). As shown by Fig. 3B, CFPJ was found to inhibit the phosphorylation of p65 at Ser276 in a concentration-dependent manner. However, the total amount of p65 present in whole cellular extract was not changed by treatment with LPS or LPS plus CFPJ. 3.4. CFPJ inhibited the LPS-induced phosphorylations of IjB-a, MKK3/ 6, and p38 MAPK in RAW 264.7 macrophages In resting cells, NF-jB is sequestered in the cytosol by its inhibitor IjB, and when induced by LPS, IjB-a is phosphorylated by IKK (IjB kinase), ubiquitinated, and rapidly degraded, which results in the release of NF-jB and its translocation to the nucleus (Oeckinghaus et al., 2011). As shown by Fig. 4A, the LPS-induced phosphorylation and degradation of IjB-a were found to be significantly
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Fig. 5. CFPJ attenuated the progression of DSS-induced colitis. (A) Mice were administered 5% DSS in drinking water (ad libitum) for 7 days with or without CFPJ (30 or 75 mg/ kg/day p.o.), or CFPJ (75 mg/kg/day p.o.) alone; 5-ASA (75 mg/kg/day p.o.) was used as a positive control. Changes in disease activity index (DAI) levels were evaluated daily throughout the 7-day experimental period. (B and C) On day 7, mice were sacrificed and colons and spleens were removed, the change of colon lengths and spleen weight measured in each mouse. Values shown are the means ± SDs (n = 6–8). #p < 0.05 vs the vehicle-treated control group; p < 0.05, p < 0.01, p < 0.001 vs. the DSS-induced colitis group.
blocked by CFPJ pretreatment. In addition, since it has been reported that the phosphorylation of p65 at Ser276 is associated with p38 MAPK signaling pathways (Reber et al., 2009), we investigated whether the inhibition of inflammatory response by CFPJ is mediated via p38 MAPK pathways. As shown in Fig. 4B, LPS significantly increased the phosphorylation of p38 MAPK, and CFPJ pretreatment suppressed this phosphorylation, but did not affect the phosphorylation of JNK or ERK1/2 (data not shown). MAPKs are initiated by the activations of upstream MAP3Ks, which, in turn, activate MKK3 and MKK6, two closely related kinases that phosphorylate p38 MAPK at its Thr–Gly–Tyr site (Raingeaud et al., 1996). We found that cells pretreated with CFPJ (100 lg/ml) showed significantly suppressed LPS-induced MKK3/6 phosphorylations, but CFPJ did not affect total amounts of MKK3/6 in RAW 264.7 macrophages (Fig. 4B). 3.5. CFPJ attenuated the progression of DSS-induced colitis in mice To determined the effects of CFPJ on colitis, mice were administered 5% DSS in drinking water for 7 days with or without CFPJ (30 or 75 mg/kg/day, p.o.) once a day. Following induction of colitis, a combinatorial DAI, which incorporate body weight loss, stool consistency, and gross bleeding, was significantly increased in DSS-treated mice, but the administration of CFPJ significantly reduced DAI values versus the only DSS-treated group after 3 days post-treatment (Fig. 5A). Furthermore, the therapeutic effects of
CFPJ were similar to those of 5-ASA (75 mg/kg/day, p.o.; a reference drug). It is generally accepted that colon length is inversely associated with the severity of DSS-induced colitis. To determine whether CFPJ had a preventive effect on DSS-induced colonic shortening, we measured and compared the colon lengths of vehicle-treated control mice, vehicle-treated mice with DSS, 5ASA-treated mice with DSS, CFPJ (30 or 75 mg/kg)-treated mice with DSS and CFPJ (75 mg/kg)-treated mice without DSS. Significantly colon shortening was observed in mice treated with DSS than in vehicle-treated control mice (77.99 ± 2.85 mm vs. 46.83 ± 4.11 mm, p < 0.001). However, oral administration of ASA or CFPJ reduced this shortening of colon length; vehicle-treated mice with DSS versus 5-ASA (75 mg/kg)-treated mice with DSS (46.83 ± 4.11 mm vs. 62.75 ± 8.74 mm, p < 0.01), vehicle-treated mice with DSS versus CFPJ (30 mg/kg)-treated mice with DSS (46.83 ± 4.11 mm vs. 63.80 ± 8.36 mm, p < 0.01), and vehicle-treated mice with DSS versus CFPJ (75 mg/kg)-treated mice with DSS (46.83 ± 4.11 mm vs. 65.24 ± 3.78 mm, p < 0.001). Treatment with CFPJ (75 mg/kg) alone had no effect on colon length (Fig. 5B). Furthermore, in the present study, as compared with vehicle-treated control mice, vehicle-treated mice with DSS exhibited splenic enlargement (0.13 ± 0.01 g vs. 0.24 ± 0.05 g, p < 0.001). 5-ASA and CFPJ both reduced spleen sizes in mice with DSS-induced colitis (5-ASA 75 mg/kg:0.24 ± 0.05 g vs. 0.15 ± 0.03 g, p < 0.001, CFPJ 30 mg/kg:0.24 ± 0.05 g vs. 0.14 ± 0.02 g, p < 0.001, CFPJ 75 mg/ kg:0.24 ± 0.05 g vs. 0.17 ± 0.02 g, p < 0.001) (Fig. 5C).
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Fig. 6. Effects of CFPJ on the productions of pro-inflammatory mediators (NO, PGE2, TNF-a, and IL-6) and on the expressions of iNOS and COX-2 in the colonic tissues of mice with DSS-induced colitis. (A, B, D, and E) Small colonic tissues were collected on day 7 from control mice (saline or vehicle), DSS-induced mice (5% DSS, ad libitum), and DSSinduced mice treated with CFPJ (30 or 90 mg/kg/day po) or 5-ASA (75 mg/kg/day po). Cell-free culture supernatants were tested for NO, PGE2, and cytokines levels using the Griess reaction (NO) and EIAs (PGE2 and cytokines) (C) Total proteins from colonic tissues were prepared, and the levels of iNOS and COX-2 were determined using specific monoclonal antibodies. b-actin was used as internal control. Values shown are the means ± SDs (n = 6–8). #p < 0.05 vs. the vehicle-treated control group; p < 0.05, p < 0.01, p < 0.001 vs. the DSS-induced colitis group.
3.6. CFPJ attenuated the productions of pro-inflammatory mediators (NO, PGE2, TNF-a, and IL-6) and the protein expressions of iNOS and COX-2 in the colonic tissues of DSS treated mice We further confirmed the inhibitory effects of CFPJ on proinflammatory mediators in DSS-exposed colonic tissues. Using the Griess reaction and EIAs, we compared the pro-inflammatory mediator productions of CFPJ-treated mice with DSS with those of vehicle-treated mice with DSS. As shown in Fig. 6, DSS was found to induced NO, PGE2, TNF-a, and IL-6 production significantly, and CFPJ reduced these increases in medium harvesting colonic tissues. Moreover, Western blotting of colonic tissue extracts revealed a decrease in iNOS and COX-2 protein expressions of CFPJtreated mice with DSS as compared with those of vehicle-treated mice with DSS (Fig. 6C).
4. Discussion Several studies have demonstrated that purple potatoes have various biological activities, such as, anti-oxidant, anti-hypertensive, and anti-inflammatory properties. In our previous study, we found that CFPJ inhibited the LPS-induced productions of NO and PGE2 in RAW 264.7 macrophages (Nam et al., 2011). In the present study, we demonstrate for the first time the molecular mechanisms underlying the anti-inflammatory effects of CFPJ, which was found to inhibit inducible NF-jB activation and the subsequent inductions of pro-inflammatory mediators and to protect mice from DSS-induced colitis. Our findings show that CFPJ suppressed the inductions of iNOS and COX-2 expressions by downregulating their promoter activities, and thus, inhibited the productions of NO and PGE2. In
addition, CFPJ concentration-dependently inhibited the expressions of IL-1b, IL-6, and TNF-a at the transcription level, and reduced the productions of IL-1b, IL-6, and TNF-a. Because the gene expressions of pro-inflammatory enzymes and cytokines, such as, iNOS, COX-2, IL-1b, IL-6, and TNF-a, are known to be modulated by NF-jB, NF-jB has been described ‘‘a central mediator of immune response’’ (Uwe, 2008). Furthermore, the inhibition of NF-jB has been shown to be effective at controlling inflammatory diseases in several animal models. For example, blocking NF-jB activity by overexpressing IjBa has been reported to inhibit both inflammatory response and tissue destruction in rheumatoid synovium (Li and Verma, 2002), and it was demonstrated that the inhibition of NF-jB by IKK inhibitor ameliorates colonic inflammatory injury by down-regulating pro-inflammatory cytokine mediated by NF-jB in a colitis model (Shibata et al., 2007). Based on these reports, we examined whether CFPJ inhibited NF-jB activity in RAW 264.7 macrophages using reporter gene assays. We found that CFPJ inhibited LPS-induced transcriptional activity of NF-jB in a dose-dependent manner in RAW 264.7 macrophages. To identify the mechanisms involved in the inhibition of NF-jB by CFPJ, we investigated the regulation of NF-jB by CFPJ. It has been suggested that NF-jB activation is mediated by two signaling pathways, namely, the NF-jB translocation-dependent pathway, which involves IKK-dependent phosphorylation and IjB-a degradation (Karin and Ben-Neriah, 2000), and the NF-jB phosphorylation-dependent pathway by MAPKs, such as, ERK, JNK, and p38 MAPK, and a variety of kinases (Oeckinghaus et al., 2011). In this regard, a number of anti-inflammatory drugs target NF-jB and/or MAPKs to control the transcriptions of pro-inflammatory genes (Kaminska, 2005). In the present study, Western blotting revealed that CFPJ inhibited LPS-induced phosphorylation and degradation
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of IjB-a, and reduced the amount of p65 (a subunit of NF-jB) in nuclear fractions. Furthermore, CFPJ was found to inhibit the LPS-induced phosphorylation of p65 at Ser 276. It has been demonstrated that p38 MAPK triggers the activation of NF-jB via the phosphorylation of p65 at Ser 276 after undergoing the activation of mitogen- and stress-activated protein kinases (MSKs) (Reber et al., 2009). Results obtained in our previous study also suggested that p38 MAPKs participate in the regulations of LPS-induced NFjB activation and pro-inflammatory mediator production (Shin et al., 2011). In line with these reports, CFPJ suppressed the LPS-induced phosphorylations of p38 MAPK and MKK3/6, the latter of which is upstream of p38 MAPK. These findings suggest that CFPJ suppresses NF-jB activation by downregulating the translocation-dependent and phosphorylation-dependent pathway in LPS-induced RAW 264.7 macrophages. To elucidate the anti-inflammatory potency of CFPJ in IBD, we used a DSS-induced colitis model. Our findings show that CFPJ suppressed DSS-induced colitis by recovery of body weight loss, stool consistency, bloody feces, and colon length shortening. Spleen enlargement is a valid organ marker of inflammation. Several authors have reported increases in spleen size during colitis, and interventions that reduce colitis have also been found to inhibit spleen enlargement in animals (Akiyama et al., 2012; Inoue et al., 2011). In the present study, CFPJ administration was found to attenuate spleen enlargement induced by DSS. Similar to human IBD, DSS induces intestinal inflammation in mice, which results in the excessive infiltration of immune cells, such as macrophages, within the colonic mucosa and higher concentrations of pro-inflammatory mediators (Beck et al., 2007). Furthermore, many studies have revealed that elevated levels of NO, PGE2, and pro-inflammatory cytokines are observed in the inflamed gut of IBD patients and in animals with DSS-induced colitis (Cho et al., 2011). In agreement with in vitro findings, CFPJ strongly inhibited the productions of pro-inflammatory mediators, such as NO, PGE2, TNF-a, and IL-6, and expressions of iNOS and COX-2 protein in colonic tissue, which suggests that the ameliorative effects of CFPJ on colitis are related to the suppressions of these proinflammatory mediators. In various studies, it was indicated that NF-jB is strongly activated by experimental colitis models, as well as in patients with IBD (Atreya et al., 2008), and the levels of activated NF-jB are correlated with the severity of intestinal inflammation (Jobin and Sartor, 2000; Schottelius and Baldwin, 1999). And the activity of p38 MAPK were higher in IBD patients compared with normal control (Vallabhapurapu and Karin, 2009) and these data were correlated with increased phosphorylation of p38 MAPK in experimental colitis model (Sun et al., 2013). Furthermore, interestingly, immunohistochemical analysis of inflamed mucosal biopsies in IBD patients showed p38 MAPK and NF-jB protein mainly localized in lamina propria macrophages and neutrophils (Luhrs et al., 2002; Waetzig et al., 2002). On the analogy of these finding, the anti-colitic effects of CFPJ might be related to inhibitory activities on macrophages activation via NF-jB and p38 MAPK. A subsequent phytochemical study on CFPJ resulted in the identifications of three phenolic acids, that is, caffeic acid, ferulic acid, and chlorogenic acid and their contents were analyzed in the present study. The presence of free-form phenolic compounds, such as caffeic acid and chlorogenic acid have been previously reported in potatoes (Onyeneho and Hettiarachchy, 1993), and some studies have been conducted on the anti-oxidant activities of potato extracts (Lachman et al., 2005). Ferulic acid was identified as the active radical scavenging compound among phenolic compounds isolated from potato peel (Nara et al., 2006). Furthermore, it has been reported peels from the red potato contains more polyphenols than those from the brown-skinned varieties, and suggested that chlorogenic acid and caffeic acid are mainly responsible for
the strong antioxidant activities of extracts (Onyeneho and Hettiarachchy, 1993). It is also well known that caffeic and ferulic acid exert anti-inflammatory actions via modulating NF-jB activation in vitro and in vivo. In particular, caffeic acid has been reported to protect against DSS-induced colitis (Chen et al., 2010; da Cunha et al., 2004), and chlorogenic acid has been reported to inhibit staphylococcal exotoxin-induced productions of pro-inflammatory cytokines and chemokines, such as, IL-1b, IL-6, TNF-a, IFNc, MCP-1, MIP-1a, and MIP-1b by human peripheral blood mononuclear cells and T-cell proliferation (Krakauer, 2002). Therefore, it appears that caffeic acid, ferulic acid, and chlorogenic acid are at least partially responsible for the anti-inflammatory activities of CFPJ in LPS-induced macrophages and DSS-induced colitis mice. In summary, the present study shows CFPJ markedly suppresses inflammatory mediators, such as, iNOS, COX-2 and pro-inflammatory cytokines (IL-1b, IL-6, and TNF-a) in LPS-induced RAW 264.7 macrophages via NF-jB and p38 MAPK inactivation, and that is prevents DSS-induced colitis development. Accordingly, we suggest that CFPJ deserves careful consideration as a potential therapeutic for the treatment of inflammatory diseases like colitis. 5. Conflict of Interest The authors have declared no conflicts of interest. Acknowledgements This work was carried out with the support of ‘‘Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009146)’’, Rural Development Administration, Republic of Korea. References Akiyama, S., Nesumi, A., Maeda-Yamamoto, M., Uehara, M., Murakami, A., 2012. Effects of anthocyanin-rich tea ‘‘Sunrouge’’ on dextran sodium sulfate-induced colitis in mice. Biofactors 38, 226–233. Atreya, I., Atreya, R., Neurath, M.F., 2008. NF-kappaB in inflammatory bowel disease. J. Intern. Med. 263, 591–596. Bang, M.H., Lee, D.Y., Oh, Y.J., Han, M.W., Yang, H.J., Chung, H.G., Jeong, T.S., Lee, K.T., Choi, M.S., Baek, N.I., 2008. Development of biologically active compounds from edible plant sources XXII. Isolation of indoles from the roots of Brassica campestris ssp rapa and their hACAT inhibitory activity. J. Korean Soc. Appl. Biol. Chem. 51, 59–69. Beck, P.L., Li, Y., Wong, J., Chen, C.W., Keenan, C.M., Sharkey, K.A., McCafferty, D.M., 2007. Inducible nitric oxide synthase from bone marrow-derived cells plays a critical role in regulating colonic inflammation. Gastroenterology 132, 1778– 1790. Chen, M.P., Yang, S.H., Chou, C.H., Yang, K.C., Wu, C.C., Cheng, Y.H., Lin, F.H., 2010. The chondroprotective effects of ferulic acid on hydrogen peroxide-stimulated chondrocytes: inhibition of hydrogen peroxide-induced pro-inflammatory cytokines and metalloproteinase gene expression at the mRNA level. Inflamm. Res. 59, 587–595. Cho, E.J., Shin, J.S., Noh, Y.S., Cho, Y.W., Hong, S.J., Park, J.H., Lee, J.Y., Lee, K.T., 2011. Anti-inflammatory effects of methanol extract of Patrinia scabiosaefolia in mice with ulcerative colitis. J. Ethnopharmacol. 136, 428–435. da Cunha, F.M., Duma, D., Assreuy, J., Buzzi, F.C., Niero, R., Campos, M.M., Calixto, J.B., 2004. Caffeic acid derivatives: in vitro and in vivo anti-inflammatory properties. Free Radic. Res. 38, 1241–1253. Gitter, A.H., Bendfeldt, K., Schulzke, J.D., Fromm, M., 2000. Leaks in the epithelial barrier caused by spontaneous and TNF-alpha-induced single-cell apoptosis. FASEB J. 14, 1749–1753. Inoue, H., Akiyama, S., Maeda-Yamamoto, M., Nesumi, A., Tanaka, T., Murakami, A., 2011. High-dose green tea polyphenols induce nephrotoxicity in dextran sulfate sodium-induced colitis mice by down-regulation of antioxidant enzymes and heat-shock protein expressions. Cell Stress Chaperones 16, 653–662. Janeway Jr., C.A., Medzhitov, R., 2002. Innate immune recognition. Annu. Rev. Immunol. 20, 197–216. Jobin, C., Sartor, R.B., 2000. NF-kappaB signaling proteins as therapeutic targets for inflammatory bowel diseases. Inflamm. Bowel. Dis. 6, 206–213. Kaminska, B., 2005. MAPK signalling pathways as molecular targets for antiinflammatory therapy-from molecular mechanisms to therapeutic benefits. Biochim. Biophys. Acta 1754, 253–262. Karin, M., Ben-Neriah, Y., 2000. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu. Rev. Immunol. 18, 621–663.
S.-J. Lee et al. / Food and Chemical Toxicology 63 (2014) 53–61 Kim, K.N., Heo, S.J., Yoon, W.J., Kang, S.M., Ahn, G., Yi, T.H., Jeon, Y.J., 2010. Fucoxanthin inhibits the inflammatory response by suppressing the activation of NF-kappaB and MAPKs in lipopolysaccharide-induced RAW 264.7 macrophages. Eur. J. Pharmacol. 649, 369–375. Krakauer, T., 2002. The polyphenol chlorogenic acid inhibits staphylococcal exotoxininduced inflammatory cytokines and chemokines. Immunopharmacol. Immunotoxicol. 24, 113–119. Lachman, J., Hamouz, K., 2005. Red and purple coloured potatoes as a significant antioxidant source in human nutrition – a review. Plant. Soil. Environ. 51, 477– 482. Lee, S.J., Lim, K.T., 2007. Glycoprotein isolated from Ulmus davidiana Nakai regulates expression of iNOS and COX-2 in vivo and in vitro. Food Chem. Toxicol. 45, 990–1000. Li, Q., Verma, I.M., 2002. NF-kappaB regulation in the immune system. Nat. Rev. Immunol. 2, 725–734. Luhrs, H., Gerke, T., Muller, J.G., Melcher, R., Schauber, J., Boxberge, F., Scheppach, W., Menzel, T., 2002. Butyrate inhibits NF-kappaB activation in lamina propria macrophages of patients with ulcerative colitis. Scand J. Gastroenterol. 37, 458– 466. Murthy, S.N., Cooper, H.S., Shim, H., Shah, R.S., Ibrahim, S.A., Sedergran, D.J., 1993. Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin. Dig. Dis. Sci. 38, 1722–1734. Nam, J.H., Jeong, J.C., Yoon, Y.H., Hong, S.Y., Kim, S.J., Jin, Y.I., Park, Y.E., An, S.Y., Kim, H.S., Lee, K.T., Park, H.J., 2011. Anti-inflammatory activity of peel extracts in color-fleshed potatoes Korean. J. Hortic. Sci. Technol. 29, 115–116. Nara, K., Miyoshi, T., Honma, T., Koga, H., 2006. Antioxidative activity of bound-form phenolics in potato peel. Biosci. Biotechnol. Biochem. 70, 1489–1491. Oeckinghaus, A., Hayden, M.S., Ghosh, S., 2011. Crosstalk in NF-kappaB signaling pathways. Nat. Immunol. 12, 695–708. Okayasu, I., Hatakeyama, S., Yamada, M., Ohkusa, T., Inagaki, Y., Nakaya, R., 1990. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 98, 694–702. Onyeneho, S.N., Hettiarachchy, N.S., 1993. Antioxidant activity, fatty acids and phenolic acids compositions of potato peels. J. Sci. Food Agric. 62, 345–350. Pandey, K.B., Rizvi, S.I., 2009. Plant polyphenols as dietary antioxidants in human health and disease. Oxid. Med. Cell Longev. 2, 270–278.
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Park, Y.E., Cho, J.H., Cho, H.M., Yi, J.Y., Seo, H.W., Chung, M.G., 2009. A new potato cultivar ‘Jayoung’, with high concentration of anthocyanin. Korean J. Breed Sci. 41, 51–55. Park, Y.E., Jeong, J.C., Cho, H.M., Hwang, Y.S., Lee, H.J., Choi, S.S., Lee, S.J., Park, E.S., Ko, E.A., Kim, N.S., Lim, J.D., Chung, M.G., 2008. Antimutagenic effect and cytotoxicity to human cancer cell lines of colored potato extracts. Korean J. Crop. Sci. 53, 75–84. Raingeaud, J., Whitmarsh, A.J., Barrett, T., Derijard, B., Davis, R.J., 1996. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol. Cell Biol. 16, 1247–1255. Reber, L., Vermeulen, L., Haegeman, G., Frossard, N., 2009. Ser276 phosphorylation of NF-kB p65 by MSK1 controls SCF expression in inflammation. PLoS ONE 4, e4393. Schottelius, A.J., Baldwin Jr., A.S., 1999. A role for transcription factor NF-kappa B in intestinal inflammation. Int. J. Colorectal. Dis. 14, 18–28. Shibata, W., Maeda, S., Hikiba, Y., Yanai, A., Ohmae, T., Sakamoto, K., Nakagawa, H., Ogura, K., Omata, M., 2007. Cutting edge: the IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks inflammatory injury in murine colitis. J. Immunol. 179, 2681–2685. Shin, J.S., Noh, Y.S., Lee, Y.S., Cho, Y.W., Baek, N.I., Choi, M.S., Jeong, T.S., Kang, E., Chung, H.G., Lee, K.T., 2011. Arvelexin from Brassica rapa suppresses NFkappaB-regulated pro-inflammatory gene expression by inhibiting activation of IkappaB kinase. Br. J. Pharmacol. 164, 145–158. Sun, P., Zhou, K., Wang, S., Li, P., Chen, S., Lin, G., Zhao, Y., Wang, T., 2013. Involvement of MAPK/NF-kappaB signaling in the activation of the cholinergic anti-inflammatory pathway in experimental colitis by chronic vagus nerve stimulation. PLoS ONE 8, e69424. Uwe, S., 2008. Anti-inflammatory interventions of NF-kappaB signaling: potential applications and risks. Biochem. Pharmacol. 75, 1567–1579. Vallabhapurapu, S., Karin, M., 2009. Regulation and function of NF-kappaB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693–733. Waetzig, G.H., Seegert, D., Rosenstiel, P., Nikolaus, S., Schreiber, S., 2002. P38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease. J. Immunol. 168, 5342–5351. Wirtz, S., Neurath, M.F., 2007. Mouse models of inflammatory bowel disease. Adv. Drug Deliv. Rev. 59, 1073–1083.
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Chloroform fraction of Solanum tuberosum L. cv Jayoung epidermis suppresses LPS-induced inflammatory responses in macrophages and DSS-induced colitis in mice Seung-Jun Lee a,b,1, Ji-Sun Shin a,c,d,1, Hye-Eun Choi a,b, Kyoung-Goo Lee a,b, Young-Wuk Cho c,d, Hyo-Jin An e, Dae Sik Jang b, Jin-Cheol Jeong f, Oh-Keun Kwon f, Jung-Hwan Nam f, Kyung-Tae Lee a,b,⇑ a
Department of Pharmaceutical Biochemistry, Kyung Hee University, Seoul, Republic of Korea Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul, Republic of Korea c Reactive Oxygen Species Medical Research Center, School of Medicine, Kyung Hee University, Republic of Korea d Department of Physiology, School of Medicine, Kyung Hee University, Republic of Korea e Department of Pharmacology, College of Oriental Medicine, Sangji University, Wonju-si, Gangwon-do 220-702, Republic of Korea f Highland Agriculture Research Center, NICS, RDA, Pyeongchang 232-955, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 4 July 2013 Accepted 22 October 2013 Available online 1 November 2013 Keywords: Color-fleshed potato peel Inflammation NF-jB p38 MAPK MKK3/6 Colitis
a b s t r a c t In this study, the authors investigated the molecular mechanism underlying the antiinflammatory effects of the chloroform fraction of the peel of ‘Jayoung’ (CFPJ), a color-fleshed potato, on lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in mice with dextran sulfate sodium (DSS)-induced colitis. CFPJ inhibited the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the transcription level, and attenuated the transcriptional activity of nuclear factor-jB (NF-jB) by reducing the translocation of NF-jB depending on degradation of inhibitory jB-a (IjB-a). Furthermore, CFPJ attenuated the phosphorylations of mitogen-activated protein kinase kinases3/6 (MKK3/6) and of p38. In colitis model, CFPJ significantly reduced the severity of colitis and the productions and protein levels of pro-inflammatory mediators in colonic tissue. These results suggest that the anti-inflammatory effects of CFPJ are associated with the suppression of NF-jB and p38 activation in macrophages, and support its possible therapeutic role for the treatment of colitis. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Inflammatory bowel diseases (IBDs), like Crohn’s disease and ulcerative colitis, are chronic inflammatory disorders of the intestinal tract that are associated with excessive productions of cytokines, adhesion molecules, and reactive oxygen species (ROS) (Cho et al., 2011). Moreover, the etiologies of IBDs are believed to involve inappropriate host responses to commensal microbial flora in the gut that result in mucosal barrier dysfunctions, such as, a leaky mucus layer, alterations in tight junction protein expression and increased epithelial cell apoptosis (Gitter et al., 2000). Experimentally induced colitis in the mouse by the oral administration of dextran sulfate sodium (DSS) is widely used to study the etiologies of IBDs. A DSS-induced murine colitis model exhibits symptoms comparable to those of human ulcerative colitis, that is, body ⇑ Corresponding author. Address: Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Dongdaemun-Ku, Hoegi-Dong, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 0860; fax: +82 2 966 3885. E-mail address: [email protected] (K.-T. Lee). 1 These authors contributed equally to this work. 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.10.040
weight loss, diarrhea, bloody feces, mucosal ulceration, and colonic shortening (Lee and Lim, 2007; Okayasu et al., 1990; Wirtz and Neurath, 2007). Histologically, colitis induced by DSS is characterized by infiltration of inflammatory cells into the lamina propria, accompanied by lymphoid hyperplasia, focal crypt damage, and epithelial ulceration. Furthermore, DSS destroys the mucosal barrier of epithelium, and thus, allows bacteria to contact lamina propria cells, which results in the activation of intestinal macrophages (Okayasu et al., 1990). Macrophages were activated by recognition the threats via the specific receptors, especially toll-like receptors (TLRs), which are capable of inducing specialized activation programs. Engagement of these receptors results in signals that modify the functional properties of cells by inducing gene expression changes and upregulating effector molecules, such as, cytokines and adhesion molecules (Kim et al., 2010). TLR4 ligation by lipopolysaccharide (LPS) induces the activations of specific intracellular pathways via receptor dimerization and the recruitments of different adaptor molecules, and the subsequent activations of two distinct downstream signaling pathways, that is, the nuclear factor-jB (NF-jB) pathway and the mitogen-activated protein kinase (MAPK)
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Fig. 1. Effects of CFPJ on LPS-induced iNOS and COX-2 mRNA expressions and their promoter activities in RAW 264.7 macrophages. (A and C) Total RNA was prepared from cells pretreated with/without the indicated concentrations of CFPJ and then stimulated with LPS (1 lg/ml) for 6 h. The mRNA levels of iNOS and COX-2 were determined by qRT-PCR, as described in methods. Control values were obtained in the absence of LPS and CFPJ. (B and D) Cells were transiently transfected with a pGL3-iNOS (B) or a pGL3COX-2 (D) promoter vector; phRL-TK vector was used as an internal control. Cells were treated with/without the indicated concentrations of CFPJ for 1 h and then stimulated with LPS (1 lg/ml) for 24 h. Cells were then harvested and luciferase activity levels were determined as described in Methods. The data presented are the means ± SDs of three independent experiments. #p < 0.05 vs. the control group; p < 0.01, p < 0.001 vs. the LPS-treated group.
pathway. These two pathways then induce the expressions of various inflammatory mediators, such as, inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines [tumor necrosis factor-a (TNF-a) and interleukin (IL)-1, IL-6, IL-12 and others], chemokines, and other antimicrobial responses (Janeway and Medzhitov, 2002). Furthermore, these pro-inflammatory mediators play key roles in the pathogeneses of various acute and chronic inflammatory diseases, including IBD. Therefore, pharmacologically reducing the levels of LPS-inducible inflammatory mediators is regarded an effective therapeutic strategy for alleviating a variety of disorders, including the inflammatory conditions caused by macrophage activation. In this context, RAW 264.7, a murine macrophage cell line, provides an excellent means of screening antiinflammatory drugs and of evaluating inhibitors of pathways that lead to the inductions of pro-inflammatory enzymes and cytokines. Potato cultivars (Solanum tuberosum L.) are widely cultivated potato varieties (commonly called colored potatoes), which were originally bred in the Republic of Korea during a joint program between the Highland Agriculture Research Center (HARC), the Korean National Institute of Crop Science, and the Rural Development Administration (RDA). ‘Jayoung’ has dark purple-flesh, and contains substantial amounts of polyphenols, such as, anthocyanin and phenolic acid (Park et al., 2009; Park et al., 2008). Several studies have shown that plant containing polyphenols provide protection against the developments of cancers, cardiovascular diseases, diabetes, osteoporosis, infections, asthma, and neurodegenerative diseases (Pandey and Rizvi, 2009). Similarly, previous researches showed that colored potatoes have anti-oxidant, anti-hypertensive, anti-mutagenic, and cytotoxic properties (Park et al., 2008).
Recently, we reported on the anti-inflammatory activities of the ethanol (EtOH) extracts and solvent fractions from the peel or tubers of colored (Jayoung, Hongyoung: red-fleshed potato) and general potatoes (Superior potato). In particular, we found that the chloroform fraction of peel from ‘Jayoung’ (CFPJ) exerting markedly inhibitory effect against LPS-induced NO and PGE2 production in RAW264.7 macrophages (Nam et al., 2011). In the present study, we investigated the anti-inflammatory and anti-colitis effects of CFPJ and the mechanisms responsible in LPS-induced RAW264.7 macrophages and in mice with DSS-induced colitis.
2. Materials and methods 2.1. Reagents Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (NY, USA). iNOS (dilution ratio = 1:1000), p65 (dilution ratio = 1:1000), inhibitory jB-a (IjB-a) (dilution ratio = 1:1000), p38 (dilution ratio = 1:1000), poly(ADP ribose)polymerase (PARP) (dilution ratio = 1:1000), b-actin (dilution ratio = 1:2000) monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (CA, USA). Antiphospho-p65 (Ser276) (dilution ratio = 1:1000), antiphospho-IjB-a (dilution ratio = 1:1000), antiphospho-p38 (dilution ratio = 1:1000), antiphospho-MAPK kinases 3/6 (MKK3/6) (dilution ratio = 1:500), MKK3/6 (dilution ratio = 1:1000) antibodies were purchased from Cell Signaling Technology, Inc. (MA, USA). And peroxidase-conjugated secondary antibody was purchased from Jackson Laboratory (ME, USA). The enzyme immunoassay (EIA) kits for IL-1b, IL-6, and TNF-a were obtained from R&D Systems (MN, USA). Random oligonucleotide primers and M-MLV reverse transcriptase were purchased from Promega (WI, USA). dNTP Mix, and SYBR green were obtained from TaKaRa Bio Inc.(Shiga, Japan). iNOS, COX-2, IL-1b, TNF-a, IL-6, and b-actin oligonucleotide primers were purchased from Bioneer (Seoul, Korea). Sulfanilamide, aprotinin, leupeptin, PMSF, DL-dithiothreitol (DTT), LPS
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Fig. 2. Effects of CFPJ on LPS-induced IL-1b, IL-6, and TNF-a production and on their mRNA expressions in RAW 264.7 macrophages. (A–C) Cells were pretreated with/without the indicated concentrations of CFPJ for 1 h and then stimulated with LPS (1 lg/ml) for 24 h. The productions of IL-1b, IL-6, and TNF-a were determined using EIA kits. Control values were obtained in the absence of LPS and CFPJ. (D–F) Total RNA was prepared from cells pretreated with/without the indicated concentrations of CFPJ for 1 h followed by stimulation with LPS (1 lg/ml) for 6 h. The mRNA levels of IL-1b, IL-6, and TNF-a were determined by qRT-PCR as described in Methods. Control values were obtained in the absence of LPS and CFPJ. The data presented are the means ± SDs of three independent experiments. #p < 0.05 vs. the control group; p < 0.01, p < 0.001 vs. the LPS-treated group. (Escherichia coli, serotype 0111:B4), 5-aminosalicylic acid (5-ASA), caffeic acid, ferulic acid, chlorogenic acid, and all other chemicals were purchased from Sigma Chemical Co. (MO, USA).
concentrations of 25, 50, or 100 lg/ml, and then induced with LPS (1 lg/ml) for the indicated time. Various concentrations of test compounds dissolved in DMSO were added together with LPS. Cells were treated with 0.05% DMSO as vehicle control.
2.2. Preparation and standardization of chloroform fraction 2.4. Determination of IL-1b, IL-6, and TNF-a production The colored potato cultivar, Jayoung was supplied by Highland Agriculture Research Center (HARC). The potatoes were cut into small pieces (20%), stool blood (0:negative, 1:+, 2:++, 3:+++, 4:++++) and stool consistency (0:normal, 1&2:loose stool, 3&4:diarrhea). Body weight loss was calculated as the percent difference between the original body weight (day 0) and the body weight on any particular day as previously described (Murthy et al., 1993). At the end of the experiment, mice were sacrificed and the colons were separated from the proximal rectum, close to its passage under the pelvisternum. The colon length was measured between the ileo–cecal junction and the proximal rectum. The spleens were also obtained and their weight was measured. Removed colonic tissue were immediately frozen ( 70 °C) for Western blotting. For measurement of NO, PGE2, and cytokines in colonic tissues, tissues were washed with DMEM medium containing 2% FBS and penicillin and streptomycin before cut into smaller pieces. Then 0.5 cm of tissue was placed 1 ml of DMEM medium containing 0.2% FBS, distributed into 24-well plate, incubated 24 h at 37 °C in 5% CO2. The cell-free culture supernatants of the colon tissue were using to measure NO, PGE2, TNF-a, and IL-6. 2.12. Statistical analysis Results are expressed as the mean ± SDs of triplicate experiments. Statistically significant values were compared using ANOVA and Dunnett’s post hoc test, and p-values of less than 0.05 were considered statistically significant.
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Fig. 4. Effects of CFPJ on the LPS-induced phosphorylation of IjB-a and on the activations of p38 MAPK and MKK3/6 in macrophages. (A and B) RAW 264.7 macrophages were pretreated with/without the indicated concentrations of CFPJ for 1 h and then stimulated with LPS (1 lg/ml) for 15 min (p-IjB-a, IjB-a, p-MKK3/6 or MKK3/6) or 30 min (pp38, p-38). Total proteins were analyzed by Western blotting using specific antibodies. Densitometric analysis was performed using Bio-rad Quantity OneÒ Software. Values shown are the means ± SDs of three independent experiments. #p < 0.05 vs. the control group; p < 0.001 vs. the LPS-treated group.
3. Results 3.1. CFPJ suppressed LPS-induced iNOS and COX-2 mRNA expressions and promoter activity in RAW 264.7 macrophages Because CFPJ was found previously to strongly inhibit the LPSinduced productions of NO and PGE2 (Nam et al., 2011), we investigated whether the inhibitory effects of CFPJ on these productions results from the suppression of their enzyme activities and/or their gene expressions. Initially, we investigated whether the enzyme activities of iNOS and COX-2 were affected by CFPJ, but enzymebased iNOS and COX-catalytic activity determinations showed that CFPJ did not reduce the productions of NO or PGE2 (data not shown). Next, qRT-PCR was used to confirm that CFPJ regulated LPS-induced iNOS and COX-2 expression at the transcriptional level, and as shown in Fig. 1A and C, pretreatment with CFPJ significantly reduced LPS-induced iNOS and COX-2 mRNA levels in a concentration-dependent manner. Next, the transcriptional regulations of iNOS and COX-2 by CFPJ were investigated using a promoter activity assay. As shown in Fig. 1B and D, LPS significantly enhanced iNOS and COX-2 promoter activities, and CFPJ concentration-dependently inhibited these increases.
3.2. CFPJ suppressed LPS-induced production and the mRNA expressions of IL-1b, IL-6, and TNF-a in RAW 264.7 macrophages To examine the inhibitory effects of CFPJ on the LPS-induced productions and expressions of pro-inflammatory cytokines in RAW 264.7 macrophages, we investigated its effects on LPS-induced IL-1b, IL-6, and TNF-a production and their mRNA levels using EIAs and qRT-RCR, respectively. Pretreatment with CFPJ was found to reduce the LPS- induced productions of IL-1b, IL-6, and TNF-a (Fig. 2A–C) and their mRNA expressions (Fig. 2D–F)
concentration-dependently, showing that CFPJ suppresses the expressions of these inflammatory genes at the transcriptional level. 3.3. CFPJ inhibited LPS-induced NF-jB activation in RAW 264.7 macrophages Since the activation of NF-jB is critically required for the LPSinduced transcriptional regulation of inflammation (Uwe, 2008), we examined the effect of CFPJ on LPS-induced NF-jB-dependent reporter gene activities. Analysis of reporter gene expressions using pNF-jB-luc demonstrated that CFPJ concentration-dependently inhibited NF-jB-dependent luciferase activity (Fig. 3A). Because transcriptional activities of NF-jB are mediated by nuclear translocation and phosphorylation of NF-jB (Oeckinghaus et al., 2011), we investigated whether CFPJ prevented the nuclear translocation and phosphorylations of the p65 subunit of NF-jB. It was found that pretreatment with CFPJ concentration-dependently attenuated the LPS-induced nuclear translocation of p65 (Fig. 3B). As shown by Fig. 3B, CFPJ was found to inhibit the phosphorylation of p65 at Ser276 in a concentration-dependent manner. However, the total amount of p65 present in whole cellular extract was not changed by treatment with LPS or LPS plus CFPJ. 3.4. CFPJ inhibited the LPS-induced phosphorylations of IjB-a, MKK3/ 6, and p38 MAPK in RAW 264.7 macrophages In resting cells, NF-jB is sequestered in the cytosol by its inhibitor IjB, and when induced by LPS, IjB-a is phosphorylated by IKK (IjB kinase), ubiquitinated, and rapidly degraded, which results in the release of NF-jB and its translocation to the nucleus (Oeckinghaus et al., 2011). As shown by Fig. 4A, the LPS-induced phosphorylation and degradation of IjB-a were found to be significantly
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Fig. 5. CFPJ attenuated the progression of DSS-induced colitis. (A) Mice were administered 5% DSS in drinking water (ad libitum) for 7 days with or without CFPJ (30 or 75 mg/ kg/day p.o.), or CFPJ (75 mg/kg/day p.o.) alone; 5-ASA (75 mg/kg/day p.o.) was used as a positive control. Changes in disease activity index (DAI) levels were evaluated daily throughout the 7-day experimental period. (B and C) On day 7, mice were sacrificed and colons and spleens were removed, the change of colon lengths and spleen weight measured in each mouse. Values shown are the means ± SDs (n = 6–8). #p < 0.05 vs the vehicle-treated control group; p < 0.05, p < 0.01, p < 0.001 vs. the DSS-induced colitis group.
blocked by CFPJ pretreatment. In addition, since it has been reported that the phosphorylation of p65 at Ser276 is associated with p38 MAPK signaling pathways (Reber et al., 2009), we investigated whether the inhibition of inflammatory response by CFPJ is mediated via p38 MAPK pathways. As shown in Fig. 4B, LPS significantly increased the phosphorylation of p38 MAPK, and CFPJ pretreatment suppressed this phosphorylation, but did not affect the phosphorylation of JNK or ERK1/2 (data not shown). MAPKs are initiated by the activations of upstream MAP3Ks, which, in turn, activate MKK3 and MKK6, two closely related kinases that phosphorylate p38 MAPK at its Thr–Gly–Tyr site (Raingeaud et al., 1996). We found that cells pretreated with CFPJ (100 lg/ml) showed significantly suppressed LPS-induced MKK3/6 phosphorylations, but CFPJ did not affect total amounts of MKK3/6 in RAW 264.7 macrophages (Fig. 4B). 3.5. CFPJ attenuated the progression of DSS-induced colitis in mice To determined the effects of CFPJ on colitis, mice were administered 5% DSS in drinking water for 7 days with or without CFPJ (30 or 75 mg/kg/day, p.o.) once a day. Following induction of colitis, a combinatorial DAI, which incorporate body weight loss, stool consistency, and gross bleeding, was significantly increased in DSS-treated mice, but the administration of CFPJ significantly reduced DAI values versus the only DSS-treated group after 3 days post-treatment (Fig. 5A). Furthermore, the therapeutic effects of
CFPJ were similar to those of 5-ASA (75 mg/kg/day, p.o.; a reference drug). It is generally accepted that colon length is inversely associated with the severity of DSS-induced colitis. To determine whether CFPJ had a preventive effect on DSS-induced colonic shortening, we measured and compared the colon lengths of vehicle-treated control mice, vehicle-treated mice with DSS, 5ASA-treated mice with DSS, CFPJ (30 or 75 mg/kg)-treated mice with DSS and CFPJ (75 mg/kg)-treated mice without DSS. Significantly colon shortening was observed in mice treated with DSS than in vehicle-treated control mice (77.99 ± 2.85 mm vs. 46.83 ± 4.11 mm, p < 0.001). However, oral administration of ASA or CFPJ reduced this shortening of colon length; vehicle-treated mice with DSS versus 5-ASA (75 mg/kg)-treated mice with DSS (46.83 ± 4.11 mm vs. 62.75 ± 8.74 mm, p < 0.01), vehicle-treated mice with DSS versus CFPJ (30 mg/kg)-treated mice with DSS (46.83 ± 4.11 mm vs. 63.80 ± 8.36 mm, p < 0.01), and vehicle-treated mice with DSS versus CFPJ (75 mg/kg)-treated mice with DSS (46.83 ± 4.11 mm vs. 65.24 ± 3.78 mm, p < 0.001). Treatment with CFPJ (75 mg/kg) alone had no effect on colon length (Fig. 5B). Furthermore, in the present study, as compared with vehicle-treated control mice, vehicle-treated mice with DSS exhibited splenic enlargement (0.13 ± 0.01 g vs. 0.24 ± 0.05 g, p < 0.001). 5-ASA and CFPJ both reduced spleen sizes in mice with DSS-induced colitis (5-ASA 75 mg/kg:0.24 ± 0.05 g vs. 0.15 ± 0.03 g, p < 0.001, CFPJ 30 mg/kg:0.24 ± 0.05 g vs. 0.14 ± 0.02 g, p < 0.001, CFPJ 75 mg/ kg:0.24 ± 0.05 g vs. 0.17 ± 0.02 g, p < 0.001) (Fig. 5C).
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Fig. 6. Effects of CFPJ on the productions of pro-inflammatory mediators (NO, PGE2, TNF-a, and IL-6) and on the expressions of iNOS and COX-2 in the colonic tissues of mice with DSS-induced colitis. (A, B, D, and E) Small colonic tissues were collected on day 7 from control mice (saline or vehicle), DSS-induced mice (5% DSS, ad libitum), and DSSinduced mice treated with CFPJ (30 or 90 mg/kg/day po) or 5-ASA (75 mg/kg/day po). Cell-free culture supernatants were tested for NO, PGE2, and cytokines levels using the Griess reaction (NO) and EIAs (PGE2 and cytokines) (C) Total proteins from colonic tissues were prepared, and the levels of iNOS and COX-2 were determined using specific monoclonal antibodies. b-actin was used as internal control. Values shown are the means ± SDs (n = 6–8). #p < 0.05 vs. the vehicle-treated control group; p < 0.05, p < 0.01, p < 0.001 vs. the DSS-induced colitis group.
3.6. CFPJ attenuated the productions of pro-inflammatory mediators (NO, PGE2, TNF-a, and IL-6) and the protein expressions of iNOS and COX-2 in the colonic tissues of DSS treated mice We further confirmed the inhibitory effects of CFPJ on proinflammatory mediators in DSS-exposed colonic tissues. Using the Griess reaction and EIAs, we compared the pro-inflammatory mediator productions of CFPJ-treated mice with DSS with those of vehicle-treated mice with DSS. As shown in Fig. 6, DSS was found to induced NO, PGE2, TNF-a, and IL-6 production significantly, and CFPJ reduced these increases in medium harvesting colonic tissues. Moreover, Western blotting of colonic tissue extracts revealed a decrease in iNOS and COX-2 protein expressions of CFPJtreated mice with DSS as compared with those of vehicle-treated mice with DSS (Fig. 6C).
4. Discussion Several studies have demonstrated that purple potatoes have various biological activities, such as, anti-oxidant, anti-hypertensive, and anti-inflammatory properties. In our previous study, we found that CFPJ inhibited the LPS-induced productions of NO and PGE2 in RAW 264.7 macrophages (Nam et al., 2011). In the present study, we demonstrate for the first time the molecular mechanisms underlying the anti-inflammatory effects of CFPJ, which was found to inhibit inducible NF-jB activation and the subsequent inductions of pro-inflammatory mediators and to protect mice from DSS-induced colitis. Our findings show that CFPJ suppressed the inductions of iNOS and COX-2 expressions by downregulating their promoter activities, and thus, inhibited the productions of NO and PGE2. In
addition, CFPJ concentration-dependently inhibited the expressions of IL-1b, IL-6, and TNF-a at the transcription level, and reduced the productions of IL-1b, IL-6, and TNF-a. Because the gene expressions of pro-inflammatory enzymes and cytokines, such as, iNOS, COX-2, IL-1b, IL-6, and TNF-a, are known to be modulated by NF-jB, NF-jB has been described ‘‘a central mediator of immune response’’ (Uwe, 2008). Furthermore, the inhibition of NF-jB has been shown to be effective at controlling inflammatory diseases in several animal models. For example, blocking NF-jB activity by overexpressing IjBa has been reported to inhibit both inflammatory response and tissue destruction in rheumatoid synovium (Li and Verma, 2002), and it was demonstrated that the inhibition of NF-jB by IKK inhibitor ameliorates colonic inflammatory injury by down-regulating pro-inflammatory cytokine mediated by NF-jB in a colitis model (Shibata et al., 2007). Based on these reports, we examined whether CFPJ inhibited NF-jB activity in RAW 264.7 macrophages using reporter gene assays. We found that CFPJ inhibited LPS-induced transcriptional activity of NF-jB in a dose-dependent manner in RAW 264.7 macrophages. To identify the mechanisms involved in the inhibition of NF-jB by CFPJ, we investigated the regulation of NF-jB by CFPJ. It has been suggested that NF-jB activation is mediated by two signaling pathways, namely, the NF-jB translocation-dependent pathway, which involves IKK-dependent phosphorylation and IjB-a degradation (Karin and Ben-Neriah, 2000), and the NF-jB phosphorylation-dependent pathway by MAPKs, such as, ERK, JNK, and p38 MAPK, and a variety of kinases (Oeckinghaus et al., 2011). In this regard, a number of anti-inflammatory drugs target NF-jB and/or MAPKs to control the transcriptions of pro-inflammatory genes (Kaminska, 2005). In the present study, Western blotting revealed that CFPJ inhibited LPS-induced phosphorylation and degradation
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of IjB-a, and reduced the amount of p65 (a subunit of NF-jB) in nuclear fractions. Furthermore, CFPJ was found to inhibit the LPS-induced phosphorylation of p65 at Ser 276. It has been demonstrated that p38 MAPK triggers the activation of NF-jB via the phosphorylation of p65 at Ser 276 after undergoing the activation of mitogen- and stress-activated protein kinases (MSKs) (Reber et al., 2009). Results obtained in our previous study also suggested that p38 MAPKs participate in the regulations of LPS-induced NFjB activation and pro-inflammatory mediator production (Shin et al., 2011). In line with these reports, CFPJ suppressed the LPS-induced phosphorylations of p38 MAPK and MKK3/6, the latter of which is upstream of p38 MAPK. These findings suggest that CFPJ suppresses NF-jB activation by downregulating the translocation-dependent and phosphorylation-dependent pathway in LPS-induced RAW 264.7 macrophages. To elucidate the anti-inflammatory potency of CFPJ in IBD, we used a DSS-induced colitis model. Our findings show that CFPJ suppressed DSS-induced colitis by recovery of body weight loss, stool consistency, bloody feces, and colon length shortening. Spleen enlargement is a valid organ marker of inflammation. Several authors have reported increases in spleen size during colitis, and interventions that reduce colitis have also been found to inhibit spleen enlargement in animals (Akiyama et al., 2012; Inoue et al., 2011). In the present study, CFPJ administration was found to attenuate spleen enlargement induced by DSS. Similar to human IBD, DSS induces intestinal inflammation in mice, which results in the excessive infiltration of immune cells, such as macrophages, within the colonic mucosa and higher concentrations of pro-inflammatory mediators (Beck et al., 2007). Furthermore, many studies have revealed that elevated levels of NO, PGE2, and pro-inflammatory cytokines are observed in the inflamed gut of IBD patients and in animals with DSS-induced colitis (Cho et al., 2011). In agreement with in vitro findings, CFPJ strongly inhibited the productions of pro-inflammatory mediators, such as NO, PGE2, TNF-a, and IL-6, and expressions of iNOS and COX-2 protein in colonic tissue, which suggests that the ameliorative effects of CFPJ on colitis are related to the suppressions of these proinflammatory mediators. In various studies, it was indicated that NF-jB is strongly activated by experimental colitis models, as well as in patients with IBD (Atreya et al., 2008), and the levels of activated NF-jB are correlated with the severity of intestinal inflammation (Jobin and Sartor, 2000; Schottelius and Baldwin, 1999). And the activity of p38 MAPK were higher in IBD patients compared with normal control (Vallabhapurapu and Karin, 2009) and these data were correlated with increased phosphorylation of p38 MAPK in experimental colitis model (Sun et al., 2013). Furthermore, interestingly, immunohistochemical analysis of inflamed mucosal biopsies in IBD patients showed p38 MAPK and NF-jB protein mainly localized in lamina propria macrophages and neutrophils (Luhrs et al., 2002; Waetzig et al., 2002). On the analogy of these finding, the anti-colitic effects of CFPJ might be related to inhibitory activities on macrophages activation via NF-jB and p38 MAPK. A subsequent phytochemical study on CFPJ resulted in the identifications of three phenolic acids, that is, caffeic acid, ferulic acid, and chlorogenic acid and their contents were analyzed in the present study. The presence of free-form phenolic compounds, such as caffeic acid and chlorogenic acid have been previously reported in potatoes (Onyeneho and Hettiarachchy, 1993), and some studies have been conducted on the anti-oxidant activities of potato extracts (Lachman et al., 2005). Ferulic acid was identified as the active radical scavenging compound among phenolic compounds isolated from potato peel (Nara et al., 2006). Furthermore, it has been reported peels from the red potato contains more polyphenols than those from the brown-skinned varieties, and suggested that chlorogenic acid and caffeic acid are mainly responsible for
the strong antioxidant activities of extracts (Onyeneho and Hettiarachchy, 1993). It is also well known that caffeic and ferulic acid exert anti-inflammatory actions via modulating NF-jB activation in vitro and in vivo. In particular, caffeic acid has been reported to protect against DSS-induced colitis (Chen et al., 2010; da Cunha et al., 2004), and chlorogenic acid has been reported to inhibit staphylococcal exotoxin-induced productions of pro-inflammatory cytokines and chemokines, such as, IL-1b, IL-6, TNF-a, IFNc, MCP-1, MIP-1a, and MIP-1b by human peripheral blood mononuclear cells and T-cell proliferation (Krakauer, 2002). Therefore, it appears that caffeic acid, ferulic acid, and chlorogenic acid are at least partially responsible for the anti-inflammatory activities of CFPJ in LPS-induced macrophages and DSS-induced colitis mice. In summary, the present study shows CFPJ markedly suppresses inflammatory mediators, such as, iNOS, COX-2 and pro-inflammatory cytokines (IL-1b, IL-6, and TNF-a) in LPS-induced RAW 264.7 macrophages via NF-jB and p38 MAPK inactivation, and that is prevents DSS-induced colitis development. Accordingly, we suggest that CFPJ deserves careful consideration as a potential therapeutic for the treatment of inflammatory diseases like colitis. 5. Conflict of Interest The authors have declared no conflicts of interest. Acknowledgements This work was carried out with the support of ‘‘Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009146)’’, Rural Development Administration, Republic of Korea. References Akiyama, S., Nesumi, A., Maeda-Yamamoto, M., Uehara, M., Murakami, A., 2012. Effects of anthocyanin-rich tea ‘‘Sunrouge’’ on dextran sodium sulfate-induced colitis in mice. Biofactors 38, 226–233. Atreya, I., Atreya, R., Neurath, M.F., 2008. NF-kappaB in inflammatory bowel disease. J. Intern. Med. 263, 591–596. Bang, M.H., Lee, D.Y., Oh, Y.J., Han, M.W., Yang, H.J., Chung, H.G., Jeong, T.S., Lee, K.T., Choi, M.S., Baek, N.I., 2008. Development of biologically active compounds from edible plant sources XXII. Isolation of indoles from the roots of Brassica campestris ssp rapa and their hACAT inhibitory activity. J. Korean Soc. Appl. Biol. Chem. 51, 59–69. Beck, P.L., Li, Y., Wong, J., Chen, C.W., Keenan, C.M., Sharkey, K.A., McCafferty, D.M., 2007. Inducible nitric oxide synthase from bone marrow-derived cells plays a critical role in regulating colonic inflammation. Gastroenterology 132, 1778– 1790. Chen, M.P., Yang, S.H., Chou, C.H., Yang, K.C., Wu, C.C., Cheng, Y.H., Lin, F.H., 2010. The chondroprotective effects of ferulic acid on hydrogen peroxide-stimulated chondrocytes: inhibition of hydrogen peroxide-induced pro-inflammatory cytokines and metalloproteinase gene expression at the mRNA level. Inflamm. Res. 59, 587–595. Cho, E.J., Shin, J.S., Noh, Y.S., Cho, Y.W., Hong, S.J., Park, J.H., Lee, J.Y., Lee, K.T., 2011. Anti-inflammatory effects of methanol extract of Patrinia scabiosaefolia in mice with ulcerative colitis. J. Ethnopharmacol. 136, 428–435. da Cunha, F.M., Duma, D., Assreuy, J., Buzzi, F.C., Niero, R., Campos, M.M., Calixto, J.B., 2004. Caffeic acid derivatives: in vitro and in vivo anti-inflammatory properties. Free Radic. Res. 38, 1241–1253. Gitter, A.H., Bendfeldt, K., Schulzke, J.D., Fromm, M., 2000. Leaks in the epithelial barrier caused by spontaneous and TNF-alpha-induced single-cell apoptosis. FASEB J. 14, 1749–1753. Inoue, H., Akiyama, S., Maeda-Yamamoto, M., Nesumi, A., Tanaka, T., Murakami, A., 2011. High-dose green tea polyphenols induce nephrotoxicity in dextran sulfate sodium-induced colitis mice by down-regulation of antioxidant enzymes and heat-shock protein expressions. Cell Stress Chaperones 16, 653–662. Janeway Jr., C.A., Medzhitov, R., 2002. Innate immune recognition. Annu. Rev. Immunol. 20, 197–216. Jobin, C., Sartor, R.B., 2000. NF-kappaB signaling proteins as therapeutic targets for inflammatory bowel diseases. Inflamm. Bowel. Dis. 6, 206–213. Kaminska, B., 2005. MAPK signalling pathways as molecular targets for antiinflammatory therapy-from molecular mechanisms to therapeutic benefits. Biochim. Biophys. Acta 1754, 253–262. Karin, M., Ben-Neriah, Y., 2000. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu. Rev. Immunol. 18, 621–663.
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