A Polysaccharide Isolated from Ecklonia cava Fermented by Lactobacillusbrevis Inhibits the Inflammatory Response by Suppressing the Activation

of Nuclear Factor-jB in Lipopolysaccharide-Induced RAW 264.7 Macrophages

Won-Woo Lee,1 Ginnae Ahn,1 Janaka Priyalal Wijesinghe Arachchillage,1 Young Mog Kim,2

Se-Kwon Kim,3 Bae-Jin Lee,4 and You-Jin Jeon1

1Department of Marine Life Science, Jeju National University, Jeju, Korea.Departments of 2Food Science & Technology and 3Chemistry; 4Marine Bioprocess Co. Ltd.;

Pukyong National University, Busan, Korea.

ABSTRACT We previously reported that the increment of carbohydrate content in the Viscozyme� L (Novozyme Corp.,

Oklahoma City, OK, USA) extract of Lactobacillus brevis–fermented Ecklonia cava affected the inhibition of nitric

oxide (NO) production and that it might be related to the polysaccharide compound. However, there is no report of anti-

inflammatory effects of the polysaccharide or its biological mechanism. Here, we investigated the anti-inflammatory effects of

the polysaccharide and its biological mechanism in lipopolysaccharide (LPS)-activated RAW 264.7 cells. The polysaccharide

isolated from the Viscozyme extract of L. brevis–fermented E. cava (VLFEP) dose-dependently decreased LPS-stimulated NO

production without cytotoxicity. Also, VLFEP significantly decreased the production of prostaglandin E2 (PGE2) at the

100 lg/mL concentration. In addition, VLFEP dose-dependently decreased the protein and mRNA expressions of inducible

NO synthase, whereas it slightly decreased those of cyclooxygenase 2 and only at the 100 lg/mL concentration. Moreover,

VLEFP dose-dependently decreased the productions and/or mRNA expressions of tumor necrosis factor-a and interleukin-6,

compared with those of LPS only–stimulated cells. In further experiments, VLFEP considerably reduced the phosphorylation

and degradation of inhibitory jB as well as the translocation of nuclear transcription factor-jB (NF-jB) p65 into the nucleus,

and its DNA binding was markedly induced by LPS stimulation. This study suggests that VLFEP exerts anti-inflammatory

effects by down-regulating the production and expression of pro-inflammatory cytokines and mediators via inhibiting the NF-

jB pathway in LPS-stimulated RAW 264.7 cells.

KEY WORDS: � anti-inflammatory effect � Ecklonia cava � fermentation � polysaccharide � RAW 264.7 cells

INTRODUCTION

Inflammation, characterized by redness, swelling,pain, and heat, is one of the most important aspects of host

defense mechanisms against invading pathogens.1 Normalinflammatory responses are self-limiting by a process thatinvolves the down-regulations of pro-inflammatory proteinsand the up-regulations of anti-inflammatory proteins.2 Mac-rophages play a central role in inflammation and serve as anessential interface between innate and adaptive immunity.3

Normally, activation of macrophages by stimuli, such aslipopolysaccharide (LPS), increases the production and se-cretion of two pleiotropic inflammatory mediators—induciblenitric oxide (NO) synthase (iNOS) and cyclooxygenase 2(COX2)—and various cytokines, including interleukin (IL)-1b, IL-6, and tumor necrosis factor (TNF)-a.4–6 Also, NO, free

oxygen radical, and prostaglandin E2 (PGE2) are over-produced by iNOS and COX2, and they play a role as cytotoxicagents in pathological processes, particularly in inflammatorydisorders.7 It was previously reported that NO, PGE2, COX2,and iNOS are regulated by various cytokines, includingTNF-a, IL-1b, IL-6, and IL-10, in LPS-activated RAW264.7 cells.8 Moreover, previous studies have demonstratedthat the transcription and secretion of pro-inflammatorymediators and cytokines, for example, iNOS, COX2, TNF-a,and IL-1b, IL-6, and IL-8, were mediated by the activationof inhibitory jB (IjB)/nuclear transcription factor-jB(NF-jB) signal transduction pathway in LPS-activated RAW264.7 cells.9,10 Therefore, it is important to identify sub-stances that can modulate the transcription and secretion ofpro-inflammatory mediators and cytokines via the classicalNF-jB signal transduction pathway. Fungal and bacterialfermentations improve the nutritional and functional prop-erties of foods by increasing the availability of active com-pounds such as polysaccharides and peptides, ingestion rate,and absorption. Our recent studies have reported that the

Manuscript received 27 December 2010. Revision accepted 5 July 2011.

Address correspondence to: Prof. You-Jin Jeon, Ph.D., Department of Marine LifeScience, Jeju National University, Jeju 690-756, Korea, E-mail: youjinj@jejunu.ac.kr

JOURNAL OF MEDICINAL FOODJ Med Food 14 (12) 2011, 1546–1553# Mary Ann Liebert, Inc. and Korean Society of Food Science and NutritionDOI: 10.1089/jmf.2010.1562

1546

major compounds of Ecklonia cava are polysaccharidesand phlorotannins that exert anti-inflammatory propertiesin RAW 264.7 cells and 12-O-tetradecanoylphorbol 13-acetate–induced ear edema mouse model.11–13 It is interest-ing that in our previous studies we showed that fermentingwith three fungi and bacteria (Lactobacillus brevis, Sac-charomyces cerevisiae, and Candida utilis) increased thecarbohydrate contents from E. cava. We also found that thepolysaccharide isolated from the Viscozyme� (NovozymeCorp., Oklahoma City, OK, USA) extract of L. brevis–fermented E. cava (VLFEP) showed the highest NO inhibi-tory effects, compared with the others (its extract, <30-kDafraction, and >30-kDa fraction) and that the fucose andmannose contents affected their NO inhibitory effects.11

However, there is no report about the anti-inflammatoryeffects of the VLFEP and its biological mechanism.

Therefore, the present study investigated whether VLFEPinduces anti-inflammatory effects, including inhibiting theproductions of NO and PGE2, by down-regulating the ex-pressions of pro-inflammatory mediators such as iNOS andCOX2 and pro-inflammatory cytokines such as TNF-a andIL-6 via inhibition of NF-jB activation in LPS-activatedmurine macrophage RAW 264.7 cells.

MATERIALS AND METHODS

Preparation of samples. All samples and their compo-sition were identical with the samples used in our previousstudy.11 VLFEP was isolated according to the identicalmethods indicated by Lee et al.11

Cell culture and sample treatment

The RAW 264.7 murine macrophage cell line was obtainedfrom the Korean Cell Line Bank, Seoul, Korea. These cellswere cultured at 37�C in Dulbecco’s modified Eagle’s mediumsupplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100lg/mL) in a humidified 5% CO2

atmosphere. The cells were treated with VLFEP at variousconcentrations from 12.5 to 100 lg/mL and then stimulatedwith 1lg/mL LPS for the indicated incubation times.

Nitrite assay

The cells were plated at a density of 1 · 105 cells per wellin 96-well plates for 16 hours. The cells were pretreatedwith VLFEP at various concentrations (12.5, 25, 50, and100 lg/mL) and 10 lM l-N6-(1-iminoethyl)-lysine (l-NIL)as a positive control for 2 hours and then stimulated by LPS(1 lg/mL) for 24 hours. After incubation, the culture me-dium (100 lL per well) was mixed with 100 lL of Griessreagent (1% sulfanilamide in 2.5% phosphoric acid and0.1% naphthylenediamine dihydrochloride in distilledwater), an indicator of NO production, for 10 minutes, andthe absorbance of the mixture at 540 nm was measured usingan enzyme-linked immunosorbent assay microplate reader(Amersham Pharmacia Biotech, Buckinghamshire, UnitedKingdom). The nitrite levels were read off a reference to the

standard curve using sodium nitrite. The experimental resultrepresents one of three experiments and is expressed as themean of triplicates.

Lactate dehydrogenase assay

The cells were plated at a density of 1 · 105 cells per wellin 96-well plates for 16 hours. The cells were treated withvarious concentrations of VLFEP (from 12.5 to 100 lg/mL)for 2 hours and then stimulated by LPS treatment or not.After an additional 24-hour incubation period at 37�C, thelactate dehydrogenase level in the culture supernatant wasdetermined by a lactate dehydrogenase cytotoxicity detec-tion kit (Promega, Madison, WI, USA) according to themanufacturer’s instructions.

Determination of PGE2 production

RAW 264.7 cells (1 · 105 cells per well) were pretreatedwith VLFEP (from 12.5 to 100 lg/mL) for 2 hours and thenincubated with LPS (1 lg/mL) for 24 hours. PGE2 levelsin macrophage culture medium were quantified using anenzyme-linked immunosorbent assay kit (Biosource Inter-national, Camarillo, CA, USA), according to the manufac-turer’s instructions.

Determination of TNF-a, IL-6, and IL-1b production

RAW 264.7 cells (1 · 105 cells per well) were pretreatedwith VLFEP (from 12.5 to 100 lg/mL) for 2 hours and thenincubated with LPS (1 lg/mL) for 24 hours. After incuba-tion, the supernatants were collected, and productions ofTNF-a, IL-6, and IL-1b secreted from macrophages weredetermined using mouse enzyme-linked immunosorbentassay kits (R&D Systems Inc., Minneapolis, MN, USA),according to the manufacturer’s instructions.

Preparation of cytoplasmic and nuclear proteins

RAW 264.7 cells (1 · 105 cells per well) were pretreatedwith VLFEP (from 12.5 to 100 lg/mL) for 2 hours and thenincubated with LPS (1 lg/mL). The cells were lysed withbuffer A consisting of 10 mM HEPES (pH 7.9), 10 mM KCl,1.5 mM MgCl2, 1.0% Nonidet P-40, and protease inhibitors(0.5 mM dithiothreitol and 0.1 mM phenylmethylsulfonylfluoride) on ice for 15 minutes. After centrifugation at13,400 g for 10 minutes at 4�C, the supernatant was used asthe cytoplasmic protein, and then the pellet was followed bylysis with buffer B (20 mM HEPES, 20% glycerol, 0.42 mMNaCl, 1 mM EDTA, and protease inhibitors (0.5 mM dithio-threitol and 0.1 mM phenylmethylsulfonyl fluoride) for 30minutes on ice. Finally, nuclear extracts were obtained bycentrifugation at 13,400 g for 15 minutes, and the cytoplasmicand nuclear protein contents were measured using BCA�protein assay kit (Pierce, Rockford, IL, USA). The proteinswere used for western blot analysis.

Western blot analysis

Cytoplasmic proteins (50 lg) from treated and untreatedcell extracts were electrotransferred onto a nitrocellulose

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membrane following separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under denaturing con-ditions. After blocking with 5% nonfat milk for 1 hour, theblots were separately incubated with specific primary rabbitpolyclonal anti-rabbit iNOS (diluted 1:1,000), phospho-IjBa (diluted 1:1,000), or NF-jB p65 (diluted 1:1,000) ormouse monoclonal anti-mouse COX2 (diluted 1:1,000),IjBa (diluted 1:1,000), or b-actin (diluted 1:3,000) anti-bodies for 24 hours and washed twice with Tween 20/Tris-buffered saline. Then, the blots were incubated withhorseradish peroxidase–conjugated anti-mouse or anti-rabbitimmunoglobulin G (diluted 1:2,000) for 60 minutes followedby visualization by using enhanced chemiluminescence re-agents. The protein expressions of iNOS, phospho-IjBa,NF-jB p65, COX2, and IjBa were quantitated relative to b-actin with the Image J program (National Institutes ofHealth, Bethesda, MD, USA).

RNA preparation and reverse transcription–polymerasechain reaction

Total cellular RNA was isolated using TRI� reagent(Molecular Research Center, Inc., Cincinnati, OH, USA),and then the cDNA was synthesized with RNA (1 lg)using a Promega (Madison, WI, USA) A3500 kit, ac-cording to the manufacturer’s instructions, respectively.Polymerase chain reaction of this cDNA and each of theprimers displayed in Table 1 was performed for 40 cycleswith a 45-second denaturing step at 94�C, a 45-secondannealing step at 55–60�C, and a 1-minute extensionphase at 72�C using the TaKaRa polymerase chain reac-tion machine (Takara Bio Inc., Otsu, Japan). Polymerasechain reaction products were electrophoresed on a 1.5%ethidium bromide/agarose gel and visualized by ultravi-olet transillumination. The mRNA expressions of TNF-a,IL-6, and IL-1b were measured relative to b-actin with theImage J program.

Electrophoresis mobility shift assay

To identify whether VLFEP inhibits NF-jB p65 DNAbinding activities in LPS-stimulated RAW 264.7 cells, elec-trophoresis mobility shift assay was performed by followingthe revised method of Ahn et al.,13 NF-jB p65 DNA bindingoligonucleotide was 30-biotinylated and annealed using thebiotin 30-end DNA labeling kit (Pierce). Ten micrograms ofnuclear proteins and 20 fmol of biotin end-labeled target DNAwere used for binding reactions using the LightShift�chemiluminescent electrophoresis mobility shift assay kit(Pierce). The binding mixtures were loaded onto 4% poly-acrylamide gels and electrophoresed at 100 V before beingtransferred onto a positively charged nylon membrane(Hybond�-N + , Amersham Pharmacia Biotech) in 0.5 · Trisborate/EDTA at 380 mA for 1 hour (Mini Trans-Blot� Elec-trophoretic Transfer Cell, Bio-Rad Laboratories, Hercules,CA, USA). Transferred DNA samples were cross-linked anddetected using horseradish peroxidase–conjugated streptavi-din (LightShift chemiluminescent electrophoresis mobilityshift assay kit) according to the manufacturer’s instructions.

Statistical analysis

The results are presented as mean – SE values. Data wereanalyzed using Student’s t test. A P value of <.05 wasconsidered significant.

RESULTS

VLFEP significantly inhibits NO productionin LPS-stimulated RAW 264.7 cells without cytotoxicity

As shown in Figure 1A, LPS stimulation increased NOproduction, whereas the pretreatment with VLFEP dose-dependently decreased the LPS-induced NO production atall concentrations (from 12.5 to 100 lg/mL). l-N6-(1-iminoethyl)-lysine (10 lmol) used as a positive controlinhibited NO production induced by LPS up to 40%, com-pared with LPS only–stimulated cells (data not shown). Inaddition, VLFEP did not show cytotoxicity at all concen-trations. These results indicated that VLFEP has an anti-inflammatory effect via decreasing NO production inLPS-stimulated RAW 264.7 cells.

VLFEP weakly inhibits PGE2 productionin LPS-stimulated RAW264.7 cells

Inhibition of PGE2 production in LPS-stimulated RAW264.7 cells was assessed by measuring PGE2 in culturemedium harvested from the cells treated with or withoutVLFEP (12.5, 25, 50, and 100 lg/mL) and LPS (1 lg/mL).LPS markedly increased PGE2 production, compared withcontrol cells, whereas the pretreatment with VLFEP slightlyinhibited LPS-induced PGE2 production in a dose-dependentmanner (Fig. 1B). In addition, VLFEP at 100 lg/mL showeda weak (20%) inhibition of LPS-induced PGE2 produc-tion. These results indicate that VLFEP might induce anti-inflammatory effects by inhibiting PGE2 production inLPS-stimulated RAW 264.7 cells, although it is weak.

Table 1. Sequences and Size of Primers Used in This Study

Gene Orientation SequenceSize(bp)

iNOS Sense 50-CCCTTCCGAAGTTTCTGGCAGCAGC-30

496

Antisense 50-GGCTGTCAGAGCCTCGTGGCTTTGG-30

COX2 Sense 50-CACTACATCCTGACCCACTT-30 696Antisense 50-ATGCTCCTGCTTGAGTATGT-30

IL-1b Sense 50-CAGGATGAGGACATGAGCACC-30 447Antisense 50-CTCTGCAGACTCAAACTCCAC-30

IL-6 Sense 50-GTACTCCAGAAGACCAGAGG-30 308Antisense 50-TGCTGGTGACAACCACGGCC-30

TNF-a Sense 50-TTGACCTCAGCGCTGAGTTG-30 364Antisense 50-CCTGTAGCCCACGTCGTAGC-30

b-Actin Sense 50-GTGGGCCGCCCTAGGCACCAG-30 603Antisense 50-GGAGGAAGAGGATGCGGCAGT-30

COX2, cyclooxygenase 2; IL, interleukin; iNOS, inducible nitric oxide

synthase; TNF-a, tumor necrosis factor-a.

1548 LEE ET AL.

VLFEP inhibits protein and mRNA expression levelsof LPS-induced iNOS and COX2

To further characterize the mechanisms of VLFEP inhi-bition of LPS-induced NO and PGE2 production, westernblotting and reverse transcription–polymerase chain reac-tion were performed. As illustrated in Figure 2A, the proteinlevels of iNOS and COX2 were markedly increased in LPS-stimulated cells, compared with those of the control cells,but pretreatment with VLFEP significantly inhibited iNOSprotein expression in a concentration-dependent manner,although it only weakly inhibited COX2 at the high con-centration of 100 lg/mL (Fig. 2A). They showed similarresults to the densitometric analysis. It is interesting that theresults of the reverse transcription–polymerase chain reac-tion analysis were similar to the results shown in western

blotting. As shown in Figure 2B, the pretreatment withVLFEP dose-dependently inhibited iNOS mRNA expres-sion at all concentrations, but only slightly inhibited those ofCOX2 only at the high concentration of 100 lg/mL. Theseresults suggest that VLFEP inhibited the production of NOby decreasing iNOS expression at all concentrations andinhibited the production of PGE2 by decreasing COX2 ex-pression, but only at high concentrations, in LPS-stimulatedRAW 264.7 cells.

VLFEP inhibits protein and/or mRNA expression levelsof TNF-a and/or IL-6 in LPS-stimulated RAW 264.7 cells

Because our data showed that VLFEP inhibited the pro-inflammatory mediators, such as NO, PGE2, and iNOS, itseffects on pro-inflammatory cytokines such as TNF-a, IL-1b,and IL-6 were further investigated in LPS-activated macro-phages. RAW 264.7 cells were incubated in the presence ofincreasing concentrations of VLFEP, and quantities of thesecytokines secreted from the cells were monitored by enzyme-linked immunosorbent assay. Pretreatment with VLFEP de-creased the production of TNF-a and IL-6, although it didnot affect production of IL-1b (Fig. 3A). In particular, theprotein levels of TNF-a released from LPS-activated RAW264.7 cells were significantly and dose-dependently decreasedby VLFEP at all concentrations from 12.5 to 100 lg/mL(Fig. 3A). Also, we identified that the pretreatment ofVLFEP weakly decreased IL-6 mRNA production from25 lg/mL to 100 lg/mL, compared with those of LPS only–stimulated RAW 264.7 cells (Fig. 3B). The results on IL-6production in Figure 3B were nearly similar to the resultsshown in Figure 3D. In addition, they were similar to theresults of the densitometric analysis. Taken together, theseresults demonstrate that VLFEP exerts anti-inflammatoryeffects like decreasing NO and/or PGE2 productions bydown-regulating the protein and mRNA expression level ofpro-inflammatory mediators such as iNOS and/or COX2 orpro-inflammatory cytokines such as TNF-a and/or IL-6 inLPS-activated macrophages.

VLFEP inhibits the degradation and phosphorylationof IjBa as well as translocation of NF-jB p65 into nucleusand its DNA binding in LPS-stimulated RAW 264.7 cells

Activated NF-jB, a well-known transcription factor, iscritically required for the activations of iNOS, COX2, TNF-a, IL-6, and IL-1b induced by LPS.9,10 To identify the ef-fects of VLFEP on the degradation and phosphorylation ofIjBa and translocation of NF-jB p65 into nucleus as well asits DNA binding in the classical NFjB pathway, the mac-rophages incubated with VLFEP and/or LPS were examinedby western blot analysis. As Figure 4 illustrates, treatment ofLPS markedly induced the degradation of IjBa at 15 min-utes after LPS stimulation compared with control cells,whereas VLFEP dose-dependently inhibited them. It wassimilar with the result of cytoplasmic NF-jB p65 expres-sion. In addition, in parallel, the LPS-stimulated extensivephosphorylation of IjBa was significantly inhibited bythe treatment with VLFEP as well as its degradation. It is

FIG. 1. Effect of polysaccharide compound of the Viscozyme ex-tract of L. brevis–fermented E. cava (VLFEP) on lipopolysaccharide(LPS)-induced nitric oxide and prostaglandin E2 (PGE2) productionsand lactate dehydrogenase (LDH) release in RAW264.7 cells. (A)Effect of VLFEP on NO production and LDH release was checkedusing the method indicated in Materials and Methods. (B) Inhibitoryeffect of VLFEP on LPS-induced PGE2 production was checked byenzyme-linked immunosorbent assay as indicated in Materials andMethods. Experiments were performed in triplicate, and the data areexpressed as mean – SE values. *P <.05, **P <.01, ***P <.005.

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FIG. 2. Effect of VLFEP on proteinand mRNA expressions of iNOSand COX2 in LPS-stimulated RAW264.7 cells. (A) The expression levelsof iNOS and COX2 proteins wereidentified by western blotting. (B) ThemRNA expression levels of iNOSand COX2 were identified by reversetranscription–polymerase chain reac-tion as indicated in Materials andMethods. The densitometric analysisof iNOS and COX2 expressions on b-actin levels was performed with theImage J program. Experiments wereperformed in triplicate, and the data areexpressed as mean – SE values.

FIG. 3. Effect of VLFEP on pro-tein production and mRNA expressionof TNF-a, IL-6, and IL-1b in LPS-stimulated RAW 264.7 cells. (A–C)Inhibitory effects of VLFEP on pro-duction of (A) TNF-a, (B) IL-6, and(C) IL-1b protein were identified byenzyme-linked immunosorbent assayas indicated in Materials and Methods.(D) Inhibitory effects of VLFEP ontheir mRNA expressions were identifiedby reverse transcription–polymerasechain reaction analysis as indicated inMaterials and Methods. The densito-metric analysis of TNF-a, IL-6, andIL-1b expressions on b-actin wasperformed with the Image J program.Experiments were performed in tripli-cate, and the data are expressed asmean – SE values. *P <.05; **P <.01.

1550 LEE ET AL.

interesting that LPS stimulation increased the translocationof NF-jB p65 into nucleus and its DNA binding, but VLFEPtreatment inhibited the increase by LPS at concentrations of50 lg/mL and 100 lg/mL (Fig. 5). Consequently, VLFEPinhibited the phosphorylation and degradation of IjBa andthe translocation of NF-jB p65 into the nucleus as well as itsDNA binding induced by LPS stimulation. These resultsdenote that VLFEP led to anti-inflammatory effects by in-hibiting NO and PGE2 production by down-regulatingthe expressions of iNOS and/or COX2 and release of pro-inflammatory cytokines such as TNF-a and/or IL-6 via the

inhibition of the classical NF-jB pathway in activatedmacrophages.

DISCUSSION

Although the immunomodulatory and antioxidant activi-ties of E. cava and its compounds have been well establishedfor several decades,14–16 no previous reports have charac-terized fermented E. cava’s participation in the immuneresponse or its underlying mechanism of action.

Normally, fermentation leads to incremental changes inactive compounds such as polysaccharides and peptide, di-gestion rate, and numbers and types of beneficial bacteria inthe human body, all of which can modulate immune re-sponse and oxidative stress in vitro and in vivo.17–22 Wepreviously demonstrated that enzymatic extraction and fer-mentation improved extraction yield and carbohydratecontent of polysaccharides from E. cava.11 Additionally, wefound that the polysaccharides might the active compoundsresponsible for the inhibition of NO production in LPS-stimulated Raw 264.7 cells because the carbohydrate con-centration affected the inhibition of NO production.11

Therefore, we investigated whether VLFEP inhibits NOand/or PGE2 productions and its biological mechanism.Here, we have demonstrated that VLFEP inhibited NO andPGE2 productions, thereby suppressing the pro-inflamma-tory cytokines and mediators via inhibiting the activation ofthe classical NF-jB pathway induced by LPS in murinemacrophage RAW 264.7 cells.

Generally, a pharmacological decrease in LPS-inducedinflammatory mediators (for example, NO, TNF-a, and ILs)is regarded as one of the essential conditions to alleviate avariety of disorders caused by activation of macrophages.Thus, RAW 264.7 macrophages are an excellent model foranti-inflammatory drug screening and for subsequentlyevaluating the inhibitors of the pathways that lead to theproduction and induction of pro-inflammatory cytokines andmediators.23,24 Many researchers have reported that LPSstimulates iNOS transcription and transduction followed byNO production via inducing IjB proteolysis and NF-jBnuclear translocation in RAW 264.7 cells.24 Our present

FIG. 4. Effect of VLFEP on degradation and phosphorylation ofinhibitory jBa (IjBa) and nuclear factor jB (NF-jB) p65 in cytosolof LPS-stimulated RAW 264.7 cells. Effects of VLFEP on activationof the NF-jB pathway induced by LPS were identified by westernblotting as indicated in Materials and Methods. The densitometricanalysis of the protein expressions of IjBa, phospho-IjBa, and NF-jB p65 on b-actin was performed with the Image J program.

FIG. 5. Effect of VLFEP on translo-cation of NF-jB into nucleus and its NF-jB DNA binding in LPS-stimulatedRAW 264.7 cells. (A) Effect of VLFEPon protein expression of NF-jB p65 in-duced by LPS was identified by westernblotting as indicated in Materials andMethods. The densitometric analysis ofnuclear NF-jB p65 expressions on b-actin levels was performed with the Im-age J program. (B) Effect of VLFEP onthe NF-jB DNA binding induced by LPSwas identified by electrophoresis mobil-ity shift assay as indicated in Materialsand Methods. Experiments were per-formed in triplicate.

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study indicated that VLFEP significantly attenuated theproduction of NO and the protein and mRNA expressions ofiNOS in a dose-dependent manner, although it led to weakinhibitory effects on those of PGE2 and COX2 only at highconcentration in LPS-stimulated RAW 264.7 macrophages.These results suggest that the anti-inflammatory effects ofVLFEP depend on reducing NO production by decreasingiNOS expression, rather than blocking of PGE2 by COX2inhibition in LPS-stimulated RAW 264.7 cells.

Normally, TNF-a is a major pro-inflammatory cytokinemainly released by macrophages and plays a considerablerole in the pathophysiology of endometriosis and inflam-matory responses.25 IL-6 is also a pivotal pro-inflammatorycytokine, regarded as an endogenous mediator of LPS-induced fever.26 If VLFEP inhibits the production of TNF-aand/or IL-6 in addition to inhibiting NO, PGE2, iNOS, andCOX2 productions in LPS-stimulated RAW 264.7 cells, itmight be regarded as a potential anti-inflammatory agent.Indeed, the present study found that VLFEP significantlyinhibits the production and expression TNF-a in LPS-stimulated RAW 264.7 cells and weakly inhibited IL-6production and expression. However, we did not identifythe inhibitory effects of VLFEP on the mRNA expressionand protein production of IL-1b in LPS-stimulated RAW264.7 cells. In addition, VLFEP markedly inhibited the IjBdegradation and phosphorylation as well as NF-jB p65translocation into nuclei and its DNA binding, all of whichtypify the classical NF-jB pathway activated by LPS inRAW 264.7 cells. It is interesting that previous studies havereported that a large variety of inflammatory conditions,including bacterial and viral infections, rapidly induce ac-tivation of the NF-jB pathway by activating the IjB kinasecomplex, which phosphorylates IjB, leading to its degra-dation and translocation of NF-jB to the nucleus, where itbinds with DNA and activates the transcription of targetgenes for such pro-inflammatory mediators and cytokinesas iNOS, TNF-a, IL-1b, and IL-6.9,10,27–30 Also, it is wellknown that IjBa phosphorylation at Ser-32 and Ser-36 bythe IjB kinase complex is the critical step in NF-jB acti-vation.31,32 This indicates that the inhibition by VLFEP ofthe NF-jB pathway activation in LPS-stimulated RAW264.7 cells might be related to the inhibition of the IjBkinase activity and other upstream events required for NF-jB activation. Therefore, further studies characterizingthe inhibition of IjB kinase and other upstream events re-quired for NF-jB activation are needed. However, this studyconfirmed that inflammatory responses were significantlyinhibited by VLFEP through the inhibition of NF-jB inLPS-stimulated macrophages.

Taken together, the results of this study suggest thatthe fucose-rich polysaccharide obtained from VLFEPexerted anti-inflammatory effect by inhibiting NO andPGE2 productions and down-regulating the production andexpression of pro-inflammatory cytokines and mediatorsby inactivating the NF-jB pathway in LPS-stimulatedRAW 264.7 cells. We conclude that VLFEP may be a use-ful anti-inflammatory agent for suppressing macrophageactivation.

ACKNOWLEDGMENT

This research was supported by a grant from MarineBioprocess Research Center of the Marine Biotechnologyprogram funded by the Ministry of Land, Transport, andMaritime, Republic of Korea.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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