International Immunopharmacology 23 (2014) 726–734

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Raphanus sativus L. seeds prevent LPS-stimulated inflammatory response through negative regulation of the p38 MAPK-NF-κB pathway Sung-Ho Kook a,b,1, Ki-Choon Choi c,1, Young-Hoon Lee b, Hyoung-Kwon Cho d, Jeong-Chae Lee a,b,⁎ a

Research Center of Bioactive Materials and Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju 561-756, South Korea Institute of Oral Biosciences and School of Dentistry, Chonbuk National University, Jeonju 561-756, South Korea Grassland and Forage Division, National Institute of Animal Science, RDA, Seonghwan-Eup, Cheonan, Chungnam, 330-801, South Korea d Center for Health Care Technology Development, HanPoong Pharmaceutical Co. Ltd., Jeonju 561-201, South Korea b c

a r t i c l e

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Article history: Received 18 September 2014 Received in revised form 29 October 2014 Accepted 3 November 2014 Available online 13 November 2014 Keywords: Raphanus sativus L. (RSL) Inflammatory mediators Lipopolysaccharide Macrophages Sepsis Sinapic acid

a b s t r a c t The seeds of Raphanus sativus L. (RSL) have long been used as anti-inflammatory traditional medicine. However, scientific bases for the purported potential of the medicine and the associated mechanisms were barely defined. This study investigated the effects of RSL seeds on lipopolysaccharide (LPS)-stimulated inflammatory responses in vitro and in vivo. Treatment with 100 μg/ml ethyl acetate fraction (REF), which was isolated from water extract of the seeds, significantly inhibited LPS-stimulated production of nitric oxide (P b 0.05), interleukin-6 (P b 0.001), and tumor necrosis factor (TNF)-α (P b 0.001) in RAW264.7 cells. Oral supplementation with 30 mg/kg REF protected mice by 90% against LPS-induced septic death and prevented the increases of serum TNF-α and interferon-γ levels in LPS-injected mice. When REF was divided into four sub-fractions (REF-F1–F4), REF-F3 showed the greatest activity to suppress LPS-stimulated production of inflammatory mediators. We subsequently isolated an active fraction from the REF-F3 and identified sinapic acid as the main constituent. The addition of 50 μg/ml active fraction markedly inhibited LPS-stimulated production of inflammatory mediators by suppressing p38 MAPK and nuclear factor-κB activation. Furthermore, supplementation with the active fraction (10 mg/ kg) improved the survival rate of LPS-injected mice by 80% of the untreated control. Additional experiments revealed that sinapic acid was the active component responsible for the anti-inflammatory potential of RSL seeds. Collectively, our current results suggest that both RSL seeds and sinapic acid may be attractive materials for treating inflammatory disorders caused by endotoxins. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Macrophages play crucial roles in host defense against bacterial infection via activation of the innate immunity response. For example, lipopolysaccharide (LPS) signals macrophages to produce a variety of inflammatory mediators including nitric oxide (NO), prostaglandins, and pro-inflammatory cytokines such as interferon-γ (IFN-γ), interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) [1,2]. However, prolonged and persistent activation of macrophages causes excessive production of these inflammatory mediators. Various pathological conditions including sepsis, septic shock, rheumatoid arthritis, and autoimmune diabetes involve the aberrant activation of macrophages and the concomitant over-production of such mediators [3–5]. Sepsis is a systemic response to infection and septic shock develops in a few of cases after surgery as a complication accompanied by

⁎ Corresponding author at: Research Center of Bioactive Materials and Institute of Oral Biosciences, Chonbuk National University, Jeonju 561-756, South Korea. Tel.: +82 63 270 4049; fax: +82 63 270 4004. E-mail address: [email protected] (J.-C. Lee). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.intimp.2014.11.001 1567-5769/© 2014 Elsevier B.V. All rights reserved.

exposure to LPS [6,7]. LPS-activated macrophages in sepsis mediate multiple dysfunctions of organs with secretion of inflammatory mediators, eventually leading to death [8]. Approximately 900,000 cases of sepsis per year are reported in the United States, 20% of which result in mortality [9]. With this regard, many investigators have focused their efforts on the development of bioactive materials to combat the acute and severe inflammation that occurs during sepsis [10,11]. Radish (Raphanus sativus L.; RSL) is an edible root vegetable of the Brassicaceae (mustard family) that is consumed throughout the world. Radish has been traditionally used to treat various disorders of the gastrointestinal, biliary, hepatic, urinary, and respiratory systems. Importantly, the seeds of RSL known as Raphani Semen have long been used as anti-cancer and anti-inflammatory agents in Korean traditional medicine. It was reported that the RSL seeds mediate cardiovascular inhibitory effects via muscarinic receptor activation [12] as well as exert chemopreventive effects [13]. Recently, 4-methylthio-butanyl derivatives were isolated from RSL seeds as the main compositions having anti-inflammatory and/or anti-tumor activities [14]. All these reports suggest a pharmaceutical use of RSL seeds, especially as chemopreventive and anti-inflammatory agents. To date, however, the scientific basis by which RSL seeds exert anti-inflammatory potential is not

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completely understood. Likewise, the ability of RSL seeds to protect against bacterial toxin-mediated immune disorders has hardly been investigated. The aims of the present study were focused in examining the antiinflammatory activities of RSL seeds in vitro and in vivo. For this purpose, we used LPS and RAW264.7 macrophages as model systems to induce cellular inflammatory responses and produce a murine sepsis model. We also tried to identify bioactive constituents responsible for the anti-inflammatory potential of RSL seeds and explored the associated mechanisms. Our current findings revealed that the seeds of RSL inhibit inflammatory responses in LPS-stimulated macrophages and improved survival of septic mice. In particular, sinapic acid was identified as one of the major active compounds responsible for the anti-inflammatory and anti-septic activities of RSL seeds.

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2.4. Active compound identification

2. Materials and methods

The active fraction was applied to a reverse-phase HPLC (model 2695 with a 2487 detector, Waters Co., Milford, MA, USA) and Capcell Pak C18 column (5 μm, 4.6 × 250-mm, Shiseido Co. Japan) as described previously [15]. The fraction was also subjected to EI-MS and NMR analyses as described previously [16,17]. In addition, thin-layer chromatography (TLC) analysis was performed with aluminum silica-gel sheets (0.2 mm layer thickness, silica gel 60 F254, Merck, Darmstadt, Germany). Spots were visualized by UV light (λexc = 254 nm) or I2 vapor. The abundance of the active compound (Comp 1) present in the active fraction was determined by LC-MS analysis using the formula: Comp 1 (mg) = standard compound (mg) × AT (peak area corresponding Comp 1 in the extract)/AS (peak area of standard compound) × 1/MT (weight of the extract). The mobile phase consisted of acetonitrile, purified water, and acetic acid (15:110:1 (v/v)) at a flow rate of 1.0 ml/min. Samples were detected at a wavelength of 280 nm.

2.1. Animal use ethics

2.5. Determination of total phenolic compound (TPC) content

This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals as promulgated by the National Institute of Health. The experimental protocol was approved by the Commission of Ethics of the Laboratory Animal Center (Approved No. CBU 20100007), Chonbuk National University (Jeonju, South Korea).

The TPC contents in RSL seed extracts were determined according to the Folin–Ciocalteu reaction using gallic acid as described previously [18]. TPC content was expressed as gallic acid equivalents (GAE) in mg/g extract. 2.6. Measurement of anti-oxidant activities

2.2. Chemicals, laboratory wares, and animals Unless otherwise specified, all chemicals and laboratory wares were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA) and SPL Life Sciences Co. (Pocheon, South Korea), respectively. Female ICR mice (18– 22 g) were obtained from Orient Co. (Seoul, South Korea). Animals were housed under automatically controlled conditions with a 12-h light/ dark cycle at 22 ± 1 °C with 45–55% relative humidity. All animals had free access to standard rodent food pellets and water. All experiments were carried out according to the guidelines of the Animal Care and Use Committee of Chonbuk National University.

The scavenging activity of RSL samples was initially analyzed using DPPH free radicals. Briefly, 200 μg of each extract dissolved in a volume of 50 μl was mixed with 1 ml of 0.1 mM DPPH–ethanol solution and

2.3. Preparation of RSL seed extracts Dried RSL seeds were obtained from a traditional herbal market located in Jeonju (South Korea) and were identified by Dr. H.K. Cho, a director of the Center for Health Care and Technology Development, HanPoong Pharmaceutical Co. Ltd. (Jeonju, South Korea). A voucher specimen (HP-RSLS) was deposited at the Center. Preparation and separation of RSL seed samples were performed as described previously [15] with slight modifications. Briefly, powdered seeds (1 kg) were extracted with distilled water (2000 ml) by shaking at 20 °C for 24 h. The extract was then lyophilized to give a crude water extract (107 g) and stored at − 20 °C before use. The crude water extract (100 g; RWE) was serially extracted with ethyl acetate, n-butanol, and distilled water in a stepwise manner. Each RWE fraction was concentrated and lyophilized to yield 2.84 (REF), 0.87 (RBF), and 91 g (RWF), respectively. Based on the respective anti-inflammatory activities of the various fractions in LPS-stimulated macrophages and murine sepsis model, REF, an ethyl acetate fraction, was resuspended in 200 ml of n-hexane. The hexane-insoluble fraction (2.62 g) was subjected to silica gel column chromatography and diluted successively with CHCl3–MeOH (20:1, 8:1, 1:1, 0:1) to yield four sub-fractions (REF-F1–F4). REF-F3 was further separated into several fractions by the same silica gel column eluted with CHCl3–MeOH (20:1 → 10:1). Based on the inhibitory activity of these sub-fractions on NO production, an active fraction was isolated from REF-F3 and then lyophilized to give 67.5 mg of material. The procedures used to obtain RSL seed samples are shown in Supplemental Fig. 1.

Fig. 1. RSL seed samples reduce NO production and inflammatory cytokine secretion in LPS-stimulated RAW264.7 cells. Cells were stimulated with 1 μg/ml LPS 1 h before the addition of 100 μg/ml of each sample. After treatment for 48 h the levels of NO (A), TNF-α (B), and IL-6 (C) in the conditioned media were measured. ⁎P b 0.05, ⁎⁎P b 0.01 and ⁎⁎⁎P b 0.001 vs. the LPS treatment alone.

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450 μl of 50 mM Tris–HCl buffer (pH 7.4). After incubation for 30-min the reduction of DPPH free radicals was measured by reading the absorbance at 517 nm. The percentage of inhibition was calculated using the following equation: % inhibition = [(absorbance of control − absorbance of test sample)/absorbance of control] × 100. We also measured the activity of RSL seed extracts towards inhibiting thiobarbituric acid-reactive substances (TBARS) formed by Fe2+-dependent lipid peroxidation as described previously [19]. In the TBARS assay, a mixture of 200 μg/ml of each extract and 100 μl of a mitochondrial suspension (3 mg protein/ml) was incubated for 10 min at 37 °C before addition of the reaction solution (10 μl of 2 mM ascorbic acid and 10 μl of 25 μM FeSO4). 2.7. Cell culture and NO assay RAW264.7 macrophage cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 2 mM L-glutamine, and antibiotics. When cells reached 70–80% confluency in 6- or 96-well flat-bottomed plates, they were treated with RSL samples (0–100 μg/ml) for 1 h before exposure to 1 μg/ml LPS. The nitrite concentration in culture medium was used as an indicator of NO produced by LPS as described previously [15]. 2.8. Survival study ICR mice were randomly divided into eight groups (n = 10 per each group) and caged separately. Mice were orally administered either 200 μl of phosphate-buffered saline (PBS) alone (for control or LPS only group) or 200 μl of PBS containing 30 mg/kg body weight (BW) of RSL seed extract (for experiments with RWE, REF, RBF, and RWF) every other day for 10 days. Two groups of mice (n = 15) were also administered 200 μl of PBS containing 10 mg/kg BW of either the active fraction isolated from REF-F3 or commercial sinapic acid under the same conditions. Mice received an intraperitoneal injection of LPS (30 mg/kg BW) 1 day after the last administration of RSL samples or sinapic acid, and survival rates (n = 10/group) were recorded every 6 h after LPS injection for 4 days. In addition, rectal temperature of mice (n = 5/group) was monitored before and every 3 h after LPS injection for 24 h.

Fig. 2. Oral supplementation with RSL seed samples ameliorates septic death. (A) Mice were orally administered with one of the RSL extracts and survival rates were evaluated every 6 h after LPS challenge for four days. (B) Rectal temperatures of mice were checked every 3 h after LPS injection for 24 h. ⁎P b 0.05 vs. the groups supplemented with REF or RBF.

2.10. Measurement of cytokine levels Levels of cytokines IFN-γ, TNF-α, IL-1β, and prostaglandin E2 (PGE2) in serum or conditioned culture supernatants were determined using

2.9. Mouse serum and liver collections Blood was collected from animals supplemented with REF or LPS alone (n = 5/group) at several time points (1–24 h) after LPS injection and used to determine IFN-γ and TNF-α levels. Livers were also collected from animals that received the active fraction, sinapic acid, or PBS 6 h after LPS injection. Liver samples were reperfused with ice-cold PBS and stored at −80 °C prior to analysis for inflammatory cytokine expression and protein phosphorylation by real time RT-PCR and Western blotting, respectively. Table 1 TPC contents and anti-oxidant activities of RSL seed water extract (RWE) and its fractions partitioned with ethyl acetate (REF), butanol (RBF), and water (RWF).a Extracts

TPC (mg GAE/g extract)

Anti-oxidant activity DPPHb

RWE REF RBF RWF

100.6 ± 1.8b 348.4 ± 2.9a 334.2 ± 4.3a 83.1 ± 2.1c

68.4 81.3 82.6 71.4

± ± ± ±

TBARSc 2.3b 1.4a 2.4a 1.5b

48.2 68.4 70.5 38.3

± ± ± ±

1.8b 3.1a 2.2a 2.1c

a Values are the means ± SD of at least three measurements. Means within a column followed by different letters are significantly different (P b 0.05). b Percent (%) inhibition on DPPH radicals by 200 μg of each extract. c Percent (%) inhibition of TBARS formation (nmol/mg protein) by 200 μg/ml of each extract compared with positive control containing ascorbic acid and Fe2+.

Fig. 3. Attenuation of serum cytokine concentrations by oral treatment with RSL seed samples in a murine sepsis model. Blood was collected from mice administered with REF or RBF at various time points (1–24 h) after LPS injection and serum TNF-α (A) and IFN-γ levels (B) were measured by ELISA. Different letters within the same times indicate significant differences between experiments.

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OptEIA™ kits (R&D Systems, Inc., Minneapolis, MN, USA) specific to each of the cytokines and an ELISA reader (Packard Instrument Co., Downers Grove, IL, USA). The levels of cytokines produced were calculated from standard curves generated using known concentrations of recombinant cytokine proteins.

2.11. Western blot analysis Equal protein amounts for each sample were separated by 12% SDSPAGE and blotted onto PVDF membranes. Blots were probed with primary and secondary antibodies and then developed with enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK) prior to exposure to X-ray film (Eastman-Kodak, Rochester, NY, USA). Mono- and poly-clonal antibodies specific for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), p38 mitogenactivated protein kinase (MAPK), p-p38 MAPK, IκB-α, p-IκB-α, p65, pp65, p50, ERK1/2, p-ERK1/2, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Band intensities were calculated using a gel imaging system and software (model F1-F2 Fuses type T2A, BIO-RAD, Segrate, Italy).

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2.12. Luciferase assay RAW264.7 cells were transfected using Superfect reagent (Qiagen, Hilden, Germany) and a pNF-κB-Luc reporter plasmid (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. After 48 h of transfection, cells were treated with the active fraction or commercial sinapic acid for 1 h before stimulation with 1 μg/ml LPS followed by incubation for an additional 2 h. Cells were lysed and then the luciferase activity was determined using the luciferase assay system (Promega, Fitchburg, WI, USA) and a MicroLumat Plus LB 964 (Berthold Technologies, Bad Wildbad, Germany). 2.13. NF-κB DNA-binding activity assay NF-κB binding activity to specific DNA sequences was determined by electrophoretic mobility shift assay (EMSA). In brief, DNA– protein binding reactions were performed for 30 min with 10– 15 μg of nuclear protein in a 20-μl binding buffer containing 30,000 cpm of [α-32P] dCTP-labeled oligonucleotides and the Klenow fragment of DNA polymerase. The samples were separated on 6% polyacrylamide gels and the dried gels were exposed to X-ray film. The

Fig. 4. Anti-inflammatory potential of REF sub-fractions in LPS-stimulated RAW264.7 macrophages. Cells were treated with or without 100 μg/ml of each sub-fraction (REF-F1–F4) 1 h before stimulation with 1 μg/ml LPS. Conditioned media from the culture supernatants were assayed for the production of NO (A), TNF-α (B), IL-1β (C), and PGE2 (D) after 48 h of incubation. Cells were also exposed to 100 μg/ml of each fraction with or without 1 μg/ml LPS. After 24 h incubation, the protein levels of COX-2 (E) and p-p38 MAPK (F) in whole protein lysates were determined by Western blot analysis. The relative band intensities of proteins were measured by densitometric analysis and were normalized to that of β-actin. ⁎P b 0.05, ⁎⁎P b 0.01 and ⁎⁎⁎P b 0.001 vs. the LPS treatment alone. #P b 0.05 vs. the indicated experiment.

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2.14. Real time RT-PCR Total RNA was prepared at various post-irradiation times using the SV Total RNA Isolation System (Promega) and reverse-transcribed using a RNA PCR kit according to the protocol provided (Access RTPCR System, Promega). Real time RT-PCR amplification was performed using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster, CA, USA) as described previously [20]. PCR primer sequences specific for iNOS, IL-1β, TNF-α, and glyceraldehyde-3phosphate dehydrogenase (GAPDH) were described previously [21]. PCR reactions were performed for five different samples and expression levels were normalized to the respective GAPDH signal. 2.15. Statistical analysis Unless otherwise specified, all data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. One-way analysis of variance (ANOVA; SPSS version 12.0 software) followed by Scheffe's test was applied to determine differences between the groups. A value of P b 0.05 was considered significant. 3. Results 3.1. Sinapic acid is the main component of the active fraction isolated from RSL seeds Fig. 5. Identification of an active compound present in RSL seed extract. (A) The active fraction isolated from REF-F3 was applied to HPLC analysis, and a single compound, named Comp 1, was identified. The spectral data from EI-MS and NMR analyses confirmed that Comp 1 was sinapic acid. (B) TLC analysis was also performed using a, commercial sinapic acid; b, original RSL seeds; and c, RSL seed water extract (RWE). Arrows indicate the presence of sinapic acid. (C) The content of sinapic acid in RSL samples was determined by LC-MS analysis.

oligonucleotide primer sequences specific for NF-κB were: 5′-aag gcc tgt gct ccg gga ctt tcc ctg gcc tgg a-3′ and 3′-gga cac gag gcc ctg aaa ggg acc gga cct gga a-5′.

All of the RSL seed extracts at 100 μg/ml attenuated LPS-stimulated NO production, with RBF exhibiting the highest potency (Fig. 1A). Stimulating the cells with 1 μg/ml LPS markedly increased TNF-α and IL-6 levels, whereas this increase was inhibited by RSL seed extracts (Fig. 1B, C). Of the extracts, REF and RBF exhibited greater inhibitory activity towards LPS-stimulated cytokine production than RWE and RWF. RSL extracts themselves at a dose of 100 μg/ml did not reduce cell viability (data not shown). REF and RBF had higher TPC contents and antioxidant potentials compared with the other RSL samples (Table 1). REF improved survival in a sepsis model and decreased inflammatory

Fig. 6. The active fraction isolated from REF-F3 inhibits the production of inflammatory mediators in a dose-dependent manner. RAW264.7 macrophages were stimulated with 1 μg/ml LPS in the presence of various concentrations (0–50 μg/ml) of the active fraction. After co-incubation for 48 h, the levels of NO (A), TNF-α (B), and IL-1β (C) in conditioned culture supernatants were measured by ELISA. (D) Cells were also exposed to the indicated concentrations of the fraction with or without the addition of 1 μg/ml LPS. After incubation for 24 h iNOS and COX-2 protein expression were determined by Western blot analysis. ⁎P b 0.05, ⁎⁎P b 0.01 and ⁎⁎⁎P b 0.001 vs. the LPS treatment alone.

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cytokine production to a greater extent than other extracts. As shown in Fig. 2A, oral administration of RWE, REF, RBF, and RWF improved the 48 h survival rates to 70%, 90%, 50%, and 60%, respectively, after LPS injection. A time-dependent decrease of rectal temperature in mice injected with LPS alone was significantly reduced by oral supplementation with either REF or RBF (P b 0.05) (Fig. 2B). Oral treatment with REF or RBF also prevented LPS-mediated increases in serum TNF-α (Fig. 3A) and IFN-γ levels (Fig. 3B). Especially, the RSL extract-mediated decrease in serum TNF-α level was more dramatic in mice supplemented with REF than RBF. Based on the capacity to inhibit the production of inflammatory mediators and prevent septic death of mice, REF was further separated into four fractions (REF-F1–F4). REF-F1, REF-F2, and REF-F3 decreased NO production in LPS-stimulated cells, whereas REF-F4 did not exhibit any effects (Fig. 4A). REF-F3 produced the most efficient inhibition of TNFα (P b 0.001), IL-1β (P b 0.01), and PGE2 (P b 0.001) in LPS-stimulated macrophages (Fig. 4B, C, D). LPS-stimulated increases in COX-2 and pp38 MAPK levels were also attenuated upon treating the cells with 100 μg/ml of REF-F1, REF-F2, or REF-F3, whereas REF-F2 and REF-F3 reduced these proteins to nearly basal levels (Fig. 4E, F). To identify the active compounds responsible for the observed anti-inflammatory activities, REF-F3 was further separated into 14 sub-fractions using a silica gel column. Based on inhibition of NO production and DPPH radical formation, the 8th fraction was selected and further applied to reversed-phase HPLC analysis. The major compound, named Comp 1, comprised approximately 73% of the fraction and was detected at tR = 14 min (Fig. 5A). Upon comparison with published data [16,17, 22,23], spectral data revealed that Comp 1 was 3,5-dimethoxy-4hydroxycinnamic acid (sinapic acid; MW 224.21). This was further supported by a comparison of the tR of Comp 1 with commercial sinapic

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acid (98% purity) (data not shown). As shown in Fig. 5B, we confirmed the presence of sinapic acid in RSL seeds by comparing commercial sinapic acid (a) with powdered original RSL seeds (b) and RWE (c) through a TLC assay with CHCl3/MeOH/water =4:1:0.1 (v/v), Fr = 0.4 (1.8/4.5) (UV–vis (λexc = 254 nm), I2 vapors). Furthermore, LC-MS data showed that original RSL seeds, RWE, and REF-F3 contained sinapic acid up to approximately 0.11, 0.87, and 148 mg/g, respectively (Fig. 5C). 3.2. Active fraction inhibits the production of inflammatory mediators and NF-κB binding activity in a dose-dependent manner We next explored whether the active fraction (8th fraction from REF-F3) contained a compound responsible for the anti-inflammation properties observed for RSL seeds. To this end, RAW264.7 macrophages were stimulated with 1 μg/ml LPS in the presence of 5, 10, 25, or 50 μg/ml of the fraction, which contained approximately 16, 32, 81, or 162 μM of sinapic acid. As shown in Fig. 6, the active fraction inhibited the production of NO (Fig. 6A), TNF-α (Fig. 6B), and IL-1β (Fig. 6C) as well as the induction of iNOS and COX-2 (Fig. 6D) in a dose-dependent manner. Similarly, the addition of commercial sinapic acid at similar concentrations resulted in a dose-dependent inhibition of inflammatory mediators (data not shown). Both the active fraction and commercial sinapic acid significantly inhibited LPS-induced increase of NF-κB-dependent luciferase enzyme expression in a dose-dependent manner (Fig. 7A). Specifically, the active fraction at 50 μg/ml reduced luciferase activity up to that of untreated control cells, as did sinapic acid at 200 μM. LPS at 1 μg/ml increased NF-κB-DNA binding activity, even at 10 min after stimulation (Fig. 7B). Pretreatment with either the active fraction or commercial sinapic

Fig. 7. Effects of the active fraction on NF-κB activation and IκB-α phosphorylation in LPS-stimulated RAW264.7 cells. (A) Cells were exposed to various concentrations (0–50 μg/ml) of the active fraction or commercial sinapic acid (100–200 μM) in the presence of LPS (1 μg/ml) and luciferase activity was measured 48 h after transfection. Cells were also stimulated with 1 μg/ ml LPS alone for various times (0–60 min) (B) or for 30 min in the presence of either the active fraction or commercial sinapic acid (C). Nuclear proteins collected from these cells were subjected to EMSA (B and C) and Western blotting (D). (E) The levels of IκB-α and p-IκB-α in cytosolic fractions were determined by Western blot analysis. Panels B, C, D, and E show representative data from triplicate experiments. ⁎⁎P b 0.01 and ⁎⁎⁎P b 0.001 vs. the LPS treatment alone. AF, active fraction; SA, sinapic acid.

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acid inhibited the NF-κB-DNA binding in a dose-dependent manner (Fig. 7C). A nearly complete inhibition of binding was observed upon treating cells with 50 μg/ml of the active fraction or 200 μM of sinapic acid. Consistent with these results, Western blot analysis showed that both the active fraction and commercial sinapic acid diminished nuclear levels of the NF-κB sub-proteins p65 and p50, which were increased in LPS-stimulated cells (Fig. 7D). While LPS stimulation resulted in increased p-IκB-α with subsequent degradation of IκB-α, treatment with the active fraction resulted in a dose-dependent inhibition of these changes (Fig. 7E). The active fraction itself at 50 μg/ml had no effect on the levels of p-IκB-α and IκB-α in cells. Similarly, commercial sinapic acid inhibited the LPS-stimulated increase of p-IκB-α in a dose-dependent manner (data not shown).

3.3. Both commercial sinapic acid and active fraction protect mice from septic death by decreasing production of inflammatory cytokines

3.4. Both active fraction and commercial sinapic acid inhibit the expression of iNOS, IL-1β, and TNF-α and phosphorylation of p38 MAPK and p65 in livers injured by LPS Compared with untreated controls, the levels of iNOS, IL-1β, and TNF-α in liver were increased up to 12.8, 9.9, and 6.9-fold, respectively, at 6 h after LPS injection (Fig. 9A). Both the active fraction and commercial sinapic acid significantly diminished the expression of the cytokines increased after LPS challenge. We next determined the phosphorylated levels of ERK, p38 MAPK, and p65 in injured livers 6 h after LPS injection. As shown in Fig. 9B, LPS challenge increased phosphorylation of all the proteins examined, and a more significant increase in p-p38 MAPK and p-p65 compared with that of p-ERK was noted. Treatment with either the active fraction or commercial sinapic acid significantly (P b 0.001) reduced p-p38 and p-p65 levels in livers injured by septic damage (Fig. 9C). 4. Discussion

The survival of mice administered with 10 mg/kg BW of the active fraction was significantly higher than that of mice injected with LPS alone (Fig. 8A). When the serum levels of TNF-α and IFN-γ in mice were measured 1 and 8 h after LPS injection, the LPS-mediated increases of these cytokines were significantly reduced by oral administration with the active fraction (Fig. 8B). These results were in parallel with that of commercial sinapic acid at the same concentration administered orally. Both the active fraction and commercial sinapic acid diminished the levels of TNF-α to a greater extent than that of IFN-γ.

Fig. 8. Oral administration of the active fraction or commercial sinapic acid improves survival in a mouse sepsis model. Mice were pretreated orally with 10 mg/kg BW of the active fraction or commercial sinapic acid before injection with LPS. (A) The survival rate (%) in the sepsis mice cohort was analyzed every 6 h after LPS challenge for 4 days. (B) Blood from mice was collected at 1 and 8 h after LPS injection and used for the analysis of TNF-α and IFN-γ, respectively. ⁎P b 0.05 and ⁎⁎⁎P b 0.001 vs. the untreated controls. # P b 0.05 and ##P b 0.01 vs. the LPS injection alone.

Numerous studies have demonstrated that the beneficial effects of medicinal plants are closely associated with their phenolic compounds content [24,25]. We identified sinapic acid as the active compound responsible for the anti-inflammatory activity of RSL seeds. Sinapic acid is a cinnamic acid derivative possessing 3,5-dimethoxyl and 4hydroxyl substitutions at the phenyl group of cinnamic acid. Sinapic acid is a widely present in various sources such as rye, fruits, and vegetables [26], and is also known as one of the main phenolic compounds in seeds [27]. While many studies have demonstrated the anti-oxidative and anxiolytic-like effects of sinapic acid [28,29], little information is available with regards to its anti-inflammatory potential. Thus, our results provide strong evidence that sinapic acid is closely associated with the anti-inflammatory potential of RSL seeds and thus may be useful as an anti-inflammatory agent. The pathogenic mechanism of sepsis primarily involves intravascular inflammation mediated by various cytokines and chemokines [30, 31]. Therefore, measuring serum levels of pro-inflammatory cytokines

Fig. 9. Both the active fraction and commercial sinapic acid inhibit mRNA expression of inflammatory cytokines and phosphorylation of MAPKs and p65 in the injured liver of septic mice. Mice were pretreated with 10 mg/kg BW of the active fraction or sinapic acid and livers were collected 6 h after LPS challenge followed by reperfusion with PBS. Total RNA and proteins were prepared from the liver samples and analyzed by real time RTPCR (A) and Western blotting (B), respectively. Panel C indicates the relative induction (fold) of p-p38 MAPK and p-p65 proteins from five different samples normalized to βactin expression. ⁎P b 0.05, ⁎⁎P b 0.01, and ⁎⁎⁎P b 0.001 vs. LPS challenge only.

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in animal models of sepsis is considered to be a primary approach to study the mechanisms by which active materials protect from inflammatory damage [11,32,33]. Considerable evidence suggests a negative correlation between survival rate and serum levels of inflammatory cytokines [34]. This observation indicates that the potential of an active material to reduce serum levels of inflammatory cytokines is directly related to its ability to block inflammatory responses following LPS challenge. We showed that serum levels of pro-inflammatory cytokines rose markedly after LPS injection, but could be prevented by treatment with RSL seed extracts. More closely, the potential of RSL samples to inhibit TNF-α production appeared to be related with its ability to improve survival in a mouse model of sepsis. These results support the idea that decreasing serum levels of inflammatory cytokines by biologically active materials may be a beneficial approach in preventing acute inflammatory damage. In addition, the current findings suggest that a selective inhibition of COX-2 is to be an additional approach to alleviate inflammatory disorders, because COX-2 involves inflammatory responses by mediating the production of prostaglandins [35]. Our current findings also provide evidence that in vitro antiinflammatory and anti-oxidant activities are not always consistent outcomes under in vivo conditions. Despite the high TPC content of RBF, its anti-oxidant and anti-inflammatory activities were similar to that of REF in RAW264.7 macrophages, and its ability to suppress septic death was lower than RWE as well as RWF and REF. These inconsistent effects may be correlated with the ability of the extracts to decrease serum levels of inflammatory cytokines, especially TNF-α. Upon further investigation, we found that the ability of REF to reduce serum TNF-α concentrations was significantly higher than that of RBF. We also considered the possibility that uptake of a compound and its metabolism within cells varied according to the experimental systems employed (in vitro and in vivo), although more detailed experiments will be needed in the future to investigate this possibility. In response to endotoxicity, activation of the NF-κB transcription factor is essential for the production of inflammatory mediators [36]. Numerous studies have shown that the suppression of the NF-κBmediated pathway is related to the potential of bioactive substances to inhibit inflammation in LPS-stimulated macrophages [37,38]. In addition, a recent report showed that sinapic acid suppresses iNOS, COX-2, PGE2, and TNF-α through the inactivation of NF-κB [38]. In the present study, both the active fraction and commercial sinapic acid markedly inhibited the expression of iNOS, IL-1β, and TNF-α as well as the phosphorylation of p65, which was increased in livers injured by LPS challenge. Therefore, we considered the possibility that sinapic acid or another molecule in the active fraction containing sinapic acid may protect mice against sepsis by preventing activation of NF-κB signaling. Of the MAPKs, ERK and p38 MAPK are closely related to the transcriptional and DNA binding activity of NF-κB in LPS-stimulated macrophages [15,37,39]. Consistent with this observation, the levels p-ERK and p-p38 MAPK were increased in the injured livers of LPSchallenged septic animals. Interestingly, however, LPS-stimulated phosphorylation of p38 MAPK was reduced to a greater extent by the active fraction and commercial sinapic acid than that of ERK, suggesting that p38 MAPK may be a specific target for NF-κB activation after LPS challenge. A previous study also showed that LPS treatment causes different patterns of MAPK phosphorylation in injured livers of mice in septic shock, where the phosphorylation of p38 MAPK is more correlated with septic damage than ERK or JNK [21]. Collectively, the reduction of p38 MAPK phosphorylation is thought to be a possible mechanism by which the active fraction or sinapic acid ameliorated septic damage through inactivation of NF-κB-mediated signaling. In conclusion, our findings demonstrate that RSL seeds have antiinflammatory potential in LPS-stimulated macrophages and protect mice from septic damage. This study also suggests that sinapic acid is one of the active compounds responsible for the beneficial effects of RSL seeds on LPS-mediated inflammatory responses. In addition, we suggest that the decreased production of inflammatory cytokines

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accompanying inactivation of p38 MAPK-NF-κB-mediated signaling is an important event regulating the anti-inflammatory potential of RSL seeds. Collectively, RSL seeds may be an attractive multifunctional food with anti-oxidant and anti-inflammatory potential. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2014.11.001.

Acknowledgments This work was supported by a grant from the RDA, Ministry of Agriculture and Forestry, Republic of Korea (Grant No. PJ007538).

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