Anti-inflammatory effect of ethanolic extract from Myagropsis myagroides on murine macrophages and mouse ear edema

Background This study aims to investigate anti-inflammatory effect of ethanolic extract of Myagropsis myagroides (EMM) in the lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and the phorbol 12-myristate 13-acetate (PMA)-induced ear edema in mice, and to clarify its underlying molecular mechanisms. Methods The levels of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines were measured by Griess assay and enzyme linked immunosorbent assay. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-activated protein kinases (MAPKs), and Akt were measured using Western blotting. Nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) were determined by immunocytochemistry and reporter gene assay, respectively. PMA-induced mouse ear edema was used as the animal model of inflammation. Anti-inflammatory compounds in EMM were isolated using high-performance liquid chromatography and identified by nuclear magnetic resonance. Results EMM significantly inhibited the production of NO, PGE2, and pro-inflammatory cytokines in a dose-dependent manner and suppressed the expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. EMM strongly suppressed nuclear translocation of NF-κB by preventing degradation of inhibitor of κB-α as well as by inhibiting phosphorylation of Akt and MAPKs. EMM reduced ear edema in PMA-induced mice. One of the anti-inflammatory compounds in EMM was identified as 6,6’-bieckol. Conclusions These results suggest that the anti-inflammatory properties of EMM are associated with the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines through the inhibition of NF-κB pathway in LPS-stimulated macrophages.


Background
Inflammation is a complex response of host defense against microbial infection, endotoxin exposure, or cell injury, and ultimately leads to the restoration of normal cell structure and function. Macrophages are known to play a key role in host defense mechanism, and are activated by exposure to interferon-γ, pro-inflammatory cytokines, and bacterial lipopolysaccharides (LPSs) [1,2]. Activated macrophages play a pivotal role in inflammatory diseases by excessive production of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and inflammatory mediators such as nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) [3,4]. Expressions of these cytokines and mediators are regulated by the activation of nuclear factor-kappaB (NF-κB), which is critically involved in the pathogenesis of rheumatism and other chronic inflammatory diseases [5]. In the unstimulated condition, NF-κB is located in the cytoplasm as an inactive complex bound to inhibitor of kappaB and (IκB)-α. Treatment of LPS activates the IκB-α kinase (IKK) complex, resulting in the phosphorylation, ubiquitination, and degradation of IκB-α, inducing the translocation of NF-κB into the nucleus for transcriptional activation. The activation of NF-κB is also regulated by cellular kinases such as mitogen-activated protein kinases (MAPKs) and Akt [6,7]. Hence, substances which inhibit the activation of NF-κB are considered as potential anti-inflammatory agents.
Myagropsis myagroides is one of the most abundant brown algae, which has been used as a part of the diet and folk medicines in Korea, Japan, and China for centuries. This species contains high level of phlorotannins and pigments. Recently, phlorotannins have been reported to have several biological activities, such as antioxidation [8], antidiabetic complications [9], antiamnesia [10], anti-inflammation [11], and hepatoprotection [12]. As a biological activity of M. myagroides, previous study revealed a cytoprotective effect on carbon tetrachloride-induced hepatotoxicity by its antioxidant properties [13]. Recently, fucoxanthin isolated from M. myagroides inhibited the production of inflammatory mediators and pro-inflammatory cytokines via inhibiting NF-κB activation in RAW 264.7 cells [14]. However, we found an anti-inflammatory activity in the ethanolic extract of sun-dried M. myagroides (EMM) in LPSstimulated RAW 264.7 cells. Since fucoxanthin prone to rapidly oxidized during sun-drying due to instability under oxygen and sun light exposure, we hypothesized that other anti-inflammatory compounds might be exist in EMM. Furthermore, we confirmed that EMM does not contain any detectable amount of fucoxanthin by HPLC chromatographic analysis at 410 nm. To our knowledge, no previous study has been reported on the anti-inflammatory activity of M. myagroides except fucoxanthin. Therefore, we investigated the anti-inflammatory and anti-edema properties of EMM and its underlying mechanisms using cultured RAW 264.7 cells and identified antiinflammatory compound in EMM.

Preparation of EMM
M. myagroides was harvested from the coast of Busan, South Korea, in April 2010. Taxonomic identification was confirmed by an agal taxonomist (C.K Choi), at the Department of Ecological Engineering, Pukyong National University, Korea. The seaweed was rinsed in tap water to remove salt, and air-dried under sunshine for 3 days. A dried sample was ground with a hammer mill and the powder was stored at −20°C until used. The dried powder (500 g) of M. myagroides was extracted three times with 96% ethanol (3 L/each) for 3 h at 70°C. The combined extracts were filtered and concentrated using rotary vacuum evaporator (Tokyo Eyela, Tokyo, Japan) at 40°C and freeze-dried EMM (89.7 g) was stored in freezer (−70°C) until used.
Measurement of NO, PGE 2 , TNF-α, IL-1α and IL-6 RAW 264.7 cells were plated in a 12-well plate at a density of 1 × 10 6 cells/well and incubated for 24 h. Cultured cells were treated with various concentrations of EMM for 1 h, and then stimulated with 1 μg/mL LPS for 24 h. Cultured media were collected after centrifugation at 2,000g for 10 min and stored at −70°C until tested. The nitrite concentration in the cultured media was measured as an indicator of NO production, according to the Griess reaction [15]. Levels of PGE 2 , IL-1β, IL-6, and TNF-α in cultured media were quantitated by enzyme-linked immunosorbent assay (ELISA, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Transient transfection and luciferase assay
Murine NF-κB promoter/luciferase DNA (1 μg) along with 20 ng control pRL-TK DNA was transiently transfected into 2 × 10 5 RAW 264.7 cells/well in a 24-well plate using Lipofectamine/Plus reagents for 40 h. Cells pretreated with 0-100 μg/mL EMM for 1 h were stimulated with LPS (1 μg/mL) for 6 h. Each well was washed twice with cold phosphate-buffered saline (PBS), harvested in 100 μL of lysis buffer (0.5 mM HEPES, pH 7.8, 1% Triton N-101, 1 mM CaCl 2 , and 1 mM MgCl 2 ) and used for the measurement of luciferase activity using a luciferase assay kit. Luminescence was measured on a TopCount microplate scintillation and luminescence counter (PerkinElmer, Santa Clara, CA, USA) in singlephoton counting mode for 0.1 min/well, following a 5 min adaptation in the dark. Luciferase activity was normalized to the expression of control pRL-TK.
Western immunoblot analysis RAW 264.7 cells were incubated with various concentrations of EMM for 1 h and stimulated with LPS (1 μg/mL) for 30 min. RAW 264.7 cells were washed twice with cold PBS and lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% IGEPAL CA-630, 1% Tween 20, 0.1% SDS, 1 mM Na 3 VO 4 , 10 μg/mL leupeptin, 50 mM NaF, and 1 mM PMSF) on ice for 1 h. After centrifugation at 18,000g for 10 min, the protein concentrations in the supernatants were determined, and aliquots of the protein (40 μg) were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in Trisbuffered saline with 0.1% Tween 20 (TBST) for 1 h, followed by the incubation for 2 h with primary antibody in TBST containing 5% nonfat dry milk. The blots were treated with horseradish peroxidase-conjugated secondary antibody in TBST containing 5% nonfat dry milk for 1 h, and immune complexes were detected using an ECL detection kit. Densitometric analysis of the data obtained from at least three independent experiments was performed using cooled CCD camera system EZ-Capture II and CS analyzer ver. 3.00 software.
Immunofluorescence analysis RAW 264.7 cells were maintained on glass coverslips (SPL Lifesciences Co., Gyeonggi-do, Korea) to analyze nuclear localization of NF-κB in 24-well plates for 24 h. Cells treated with EMM for 1 h were incubated with LPS (1 μg/mL) for 30 min as described in Lee et al. [16]. Cells were fixed in 4.0% paraformaldehyde in PBS for 15 min at room temperature, and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. Cells were washed with PBS and blocked with 3% BSA/PBS for 30 min. Thereafter, cells were incubated with an anti-NF-κB polyclonal antibody diluted in 3% BSA/PBS for 2 h, and incubated with Alexa Fluor W 488-conjugated secondary antibody diluted in 3% BSA/PBS for 1 h. Cells were stained with 2 μg/mL DAPI and images were captured using an LSM700 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).

Mouse model and PMA-induced ear edema
Animal studies were conducted after the experimental protocols and procedures were approved by the Animal Ethics Committee of the Pukyong National University. ICR mice (male, 25-30 g) were purchased from the Samtako Bio Korea Co. (Gyeonggido, Korea) and permitted free access to a standard chow diet and tap water. All mice were acclimatized for 1 week prior to the experiments and maintained at 22 ± 2°C with a relative humidity of 50 ± 5% and 12 h light-dark cycle. Ear edema was induced on the right ear of mouse with PMA according to Garrido et al. [16]. Briefly, the control group received normal saline, and the other three groups include a PMA alone, PMA + Indo (a positive control), and EMM administered groups (PMA + EMM). The left ear (reference) received the vehicle (30 μL acetone). PMA (6 μg/ear in 30 μL acetone) was applied to the inner surface of the right ear of ICR mouse. EMM (90 μg per ear in 30 μL acetone) or Indo (1 mg/ear in 30 μL acetone) was topically administered 1 h before PMA application. Six hours after PMA application, mice were killed by cervical dislocation and a 6 mm diameter disc from each ear was removed with a metal punch and weighed. Ear edema weight was calculated by subtracting the weight of the left ear from the right ear (treatment). Inhibition percentage (IP) was expressed as a reduction in weight compared to the PMA-treated group.

Isolation of phlorotannins from EMM
Aliquots of EMM were separated by Shimadzu highperformance liquid chromatography (HPLC) system with Luna RP-18 [Luna C18(2), 5 μm, 250 × 10 mm, Phenomenex, Torrence, CA, USA]. The separation of EMM was conducted using the mobile phase of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The elution profile consisted of a linear gradient from 20 to 100% solvent B for 110 min and hold for 15 min and then re-equilibration of the column with 20% solvent B for 15 min. The flow rate was 0.34 mL/min at 35°C oven temperature and detection was performed at 270 nm. The purity was determined by HPLC (purity: >99%) and the chemical structure of the isolated compounds were identified as eckol [17] and 6,6'-bieckol [8,12] by comparing their 1 H-and 13 C-NMR data.

Statistical analysis
Data were expressed as the means ± standard deviations (SDs) from at least three separate experiments unless otherwise indicated. Data were analyzed using one-way analysis of variance. Differences were considered significant at values of p < 0.05. All analyses were performed using SPSS for Windows, version 10.07 (SPSS Inc., Chicago, IL, USA).

Results
Effect of EMM on LPS-induced NO and PGE 2 production NO production, measured as nitrite, was increased by LPS treatment; however, EMM significantly reduced NO levels in LPS-stimulated cells in a dose-dependent manner (p < 0.05, Figure 1A). Increased PGE 2 production by LPS was also significantly suppressed by 100 μg/mL EMM in RAW 264.7 cells ( Figure 1B). To exclude the possibility that the decreased NO and PGE 2 levels were due to cell death, cytotoxicity of EMM was determined by MTS assay. The result demonstrated that EMM showed no cytotoxicity in RAW 264.7 cells up to 100 μg/mL ( Figure 1C). Thus, the inhibitory effects of EMM on NO and PGE 2 production were not due to cytotoxicity.

Effect of EMM on LPS-induced iNOS and COX-2 proteins and mRNA expression
As shown in Figure 2, EMM strongly inhibited iNOS protein production in a dose-dependent manner; however, COX-2 protein production was only inhibited by 100 μg/mL EMM, which is similar pattern observed in the PGE 2 secretion ( Figure 1B). In addition to iNOS protein, EMM also inhibited iNOS mRNA expression in a dose-dependent manner. Like inhibition of COX-2 protein, COX-2 mRNA expression was partially inhibited by 100 μg/mL EMM. These results suggest that the EMM-mediated inhibition of NO and PGE 2 production in LPS-stimulated macrophages is associated with downregulation of iNOS and COX-2 expression at transcriptional level.

Effect of EMM on LPS-induced pro-inflammatory cytokines
Increased levels of IL-1β ( Figure 3A), IL-6 ( Figure 3B), and TNF-α ( Figure 3C) in RAW 264.7 cells by LPS stimulation were dramatically reduced in a dosedependent manner by exposure to EMM (p < 0.05). This result indicates that EMM efficiently suppressed LPSinduced IL-1β, IL-6, and TNF-α release, which supports the hypothesis that EMM inhibits the initial phase of the LPS-stimulated inflammatory response.

Effect of EMM on LPS-induced activation and translocation of NF-κB
Confocal microscopy revealed that NF-κB/p65 was distributed mostly in the cytoplasm in unstimulated cells. After stimulation with LPS, most cytoplasmic NF-κB/p65 were translocated into the nucleus, as shown by strong NF-κB/p65 intensity in the nucleus ( Figure 4A). The level of p65 in the nucleus was markedly reduced by treatment with EMM. Considering the inhibitory effects of EMM on LPS-induced NF-κB translocation into nucleus, we determined the effect of EMM on the promoter activity of NF-κB in LPS-stimulated cells. Data suggested that EMM treatment significantly inhibited LPS-induced NF-κB promoter activity ( Figure 4B, p < 0.05).
LPS treatment resulted in increased IKKβ phosphorylation and IκB-α degradation compared to non-treated control group, and EMM treatment suppressed IKKβ phosphorylation and IκB-α degradation, recovered the control level of cytosolic IκB-α in a dose-dependent manner ( Figure 5). As a result of IκB-α degradation, the increased NF-κB level in nucleus after LPS stimulation was reduced by EMM treatment in a dose-dependent manner ( Figure 5). However, phosphorylation of c-Jun, a component of transcription factor AP-1, was not reduced by EMM treatment.
These results indicate that the EMM-mediated inhibition of iNOS, COX-2, and pro-inflammatory cytokines production was mainly regulated by the transcription factor NF-κB in LPS-stimulated macrophages.

Effect of EMM on LPS-induced phosphorylation of MAPKs and Akt
As shown in Figure 6A, EMM inhibited phosphorylation of JNK, p38 MAPK, ERK, and Akt induced by LPS in RAW 264.7 cells, suggesting the additional characteristics of EMM to regulate NF-κB pathway via blocking the phosphorylation of MAPKs and Akt proteins in response to LPS signal. To further confirm the association of these signaling molecules with the EMM's anti-inflammatory effect, IκB-α phosphorylations in LPS-stimulated RAW 264.7 cells were measured in the presence of PI3K/Akt pathway inhibitor (Wortmannin), ERK inhibitor (PD98059), JNK inhibitor (SP600125), or p38 inhibitor (SB203580). As shown in Figure 6B, IκB-α phosphorylation was remarkably suppressed by p38 inhibitor, which is consistent with the result of Figure 6A. Other inhibitors such as Wortmannin, PD98059, and SP600125, moderately inhibited the phosphorylation of IκB-α. These data confirm additional characteristics of EMM on the phosphorylation of MAPKs and Akt proteins for NF-κB activation in response to LPS.

Effect of EMM on PMA-induced ear edema in mouse
Ear edema was induced by the application of PMA to the mouse ears. As shown in Figure 7, the application of PMA for 6 h significantly increased the weight of ear edema. Indo, a common clinical non-steroidal anti-inflammatory drug, was used as positive control for the inhibitory effect on the ear edema. As shown in Figure 7, pre-treatment of 1 mg/ear of Indo could effectively reduce ear edema after 6 h PMA stimulation (p < 0.05). Similarly, pre-administration of EMM (90 μg/ear) markedly inhibited the PMA-induced ear edema with 66.8% inhibition (p < 0.05).

Isolation of anti-inflammatory compounds from EMM
EMM was further separated by reverse-phase column chromatography ( Figure 8A) for the isolation of anti- inflammatory compound(s). The chemical structures of the isolated compounds were identified as eckol and 6,6'bieckol, which were isolated for the first time from M. myagroides, from the comparison of their NMR spectra with the published spectral data. We obtained 2.5 mg of eckol and 5.6 mg of 6,6'-bieckol from 1 g of EMM. The purified 6,6'-bieckol significantly inhibited LPS-induced iNOS and COX-2 production in a dose-dependent manner (p < 0.05, Figure 8B), however, eckol did not affect on the production of inflammatory proteins. Furthermore, preadministration of 6,6'-bieckol (30 μg/ear) markedly inhibited the PMA-induced ear edema with 64.1% inhibition (Figure 7, p < 0.05). The result indicates that 6,6'-bieckol is one of the anti-inflammatory compounds in EMM.

Discussion
The present study was undertaken to examine the antiinflammatory effect of EMM on LPS-stimulated murine macrophage cells. To further understood the molecular mechanisms of EMM, we investigated the effects of EMM on the secretion of NO, PGE 2 , TNF-α, IL-1β, and IL-6, the expression of iNOS and COX-2, and the activation of NF-κB. Our results indicated that EMM effectively inhibited the secretion of NO, PGE 2 , TNF-α, IL-1β, and IL-6 through a blockade of the NF-κB and MAPK pathways in LPS-stimulated macrophages. The inhibitory effect of EMM on the expression of inflammatory mediators suggested one of the mechanisms responsible for its anti-inflammatory action and its potential for use as a therapeutic agent for treating inflammatory diseases.  Under pathological conditions, excessive inflammatory mediators and pro-inflammatory cytokines produced by activated macrophages cause inflammatory process and act synergistically with other inflammatory mediators [2,4,18]. Compounds able to reduce NO or PGE 2 production may be attractive as anti-inflammatory agents and, for this reason, the inhibitory effects of natural compounds on NO or PGE 2 productions have been rigorously studied to develop therapeutic agent against inflammatory diseases [17,19,20]. Also, excessive production of pro-inflammatory cytokines plays a critical role in acute inflammatory responses as well as chronic inflammatory diseases. Recent studies have shown that in vivo or in vitro treatments of natural compounds are effective in reducing inflammation by the suppression of pro-inflammatory cytokines, which may ameliorate inflammation-related diseases, including atherosclerosis, cancer, and inflammatory arthritis [21,22]. Thus, the regulation of those molecules is important to the inhibition of inflammatory response. Our results indicate that the inhibition of NO and PGE 2 production by EMM in LPS-stimulated macrophage cells is associated with downregulation of iNOS and COX-2 genes (Figure 2), which seems the first addressing transcriptional inhibition of iNOS and COX-2 by EMM.
NF-κB is a transcription factor actively involved in the transcriptional induction of iNOS and COX-2 gene [1,3]. Like NO and PGE 2 produced by iNOS and COX-2, respectively, the release of pro-inflammatory cytokines is regulated by NF-κB pathway and plays an important role in the induction of the innate immune response of the acquired immune response [23]. It has been well studied that the inhibition of NF-κB activation by black tea extract is associated with the phosphorylation, ubiquitination, and subsequent degradation of IκB via ubiquitin-proteosome pathway [24]. Although biochemical actions of EMM on NF-κB regulations remain unknown, the present study showed that EMM potentially inhibits the proteolytic degradation of IκB-α and the NF-κB promoter-driven luciferase expression induced by LPS in RAW 264.7 cells. Therefore, these results demonstrate the ability of EMM to inhibit NF-κB activation in response to LPS signal in RAW 264.7 cells. Hence it is likely that reductions of iNOS, COX-2, and proinflammatory cytokine expression in RAW 264.7 cells are largely associated with activation/deactivation of NF-κB pathway. This report is, to our best knowledge, the novel findings to address the functions of EMM through NF-κB pathway in response to LPS treatment.
NF-κB is also regulated by various signaling kinases including MAPKs (ERK, JNK, and p38) and Akt, which are groups of signaling molecules to play key roles in NF-κB activation [2,3]. MAPKs have been suggested to be involved in pro-inflammatory signaling cascades and in the activation of NF-κB in LPS-stimulated immune cells [2,11]. Therefore, anti-inflammatory mechanisms are closely related with inhibition of MAPKs in activated RAW 264.7 cells. In this study, we found that phosphorylation of MAPKs in response to LPS was inhibited by EMM treatment (Figure 6A). Interesting finding of this study is that the activation of Akt, a downstream regulator of PI3K, was also inhibited by EMM in response to LPS signal in RAW 264.7 cells. Precise mechanisms by which EMM modulate this PI3K/Akt pathway in the LPS stimulation remains unclear. Considering a recent study with a phlorofucofuroeckol A, which indicated a link between ROS and PI3K/Akt pathway in regulations of inflammatory genes [7,11], it is speculated that the antioxidant activity of EMM or unknown components in EMM may be related to inhibition of Akt phosphorylation. Thus, it is likely that inhibition of MAPKs and Akt phosphorylation by EMM may contribute to the EMM-mediated inhibition of NF-κB pathway in LPS-stimulated RAW 264.7 cells.
Topical application of PMA to mouse skin induces inflammatory response and this is a well-established in vivo model for the evaluation of various anti-inflammatory agents. PMA can activate a wide variety of cell types that may contribute to acute inflammation, resulting in an increase in epidermal tissue swelling and infiltration of inflammatory cells [25,26]. The results from our in vivo experiments clearly showed that EMM (90 μg/ear) suppressed 66.8% of PMA-induced skin swelling. Furthermore, 6,6'-bieckol (30 μg/ear) isolated firstly from M. myagroides in this study inhibited 64.1% of PMA-induced skin swelling, indicating that it might be a main antiinflammatory compound in EMM. Although the inhibitory activity of EMM is about one third of 6,6'-bieckol, EMM showed higher anti-edema activity than Indo compared to its concentration. This result demonstrates that EMM can be used as a topical anti-inflammatory agent.
Like other polyphenolic compounds, phlorotannins, a class of compounds polymerized with phloroglucinol units found in brown seaweeds, have strong antioxidant [18,27] and anti-inflammatory [11,28] activities. The multifunctional antioxidant activity of phlorotannins is highly associated with phenol rings which act as electron traps to scavenge peroxy, superoxide-anions, and hydroxyl radicals [29]. Specific mechanisms of phlorotannins on the antiinflammatory actions are not clearly defined. Considering cellular signaling of polyphenols, phlorotannins may not merely exert their effects as free radical scavenger, but may also modulate inflammatory cellular signaling proteins, including NF-κB and AP-1 transcription factors [30]. Recent studies demonstrated that phlorofucofuroeckol A and 6,6'-bieckol isolated from Ecklonia spp. alleviate inflammatory response to LPS by inhibiting NF-κB pathway with its intrinsic antioxidant activity [11,28]. In the present study, the isolated 6,6'-bieckol from EMM remarkably inhibited the production of iNOS and COX-2 proteins, indicating 6,6'-bieckol is one of the principle anti-inflammatory compounds in EMM.

Conclusions
We demonstrated that EMM inhibited the secretion of inflammatory mediators, such as NO and PGE 2 , and pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in LPS-stimulated RAW 264.7 macrophages. Moreover, the inhibitory effect of EMM was associated with inactivation of the NF-κB pathway via blocking the phosphorylation of MAPKs and Akt. In addition, the results of in vivo study showed that EMM can be applicable to a topical anti-inflammatory agent.