Immunomodulatory Effects of Alginate Oligosaccharides on Murine

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Immunomodulatory Effects of Alginate Oligosaccharides on Murine Macrophage RAW264.7 Cells and Their Structure−Activity Relationships Xu Xu,† Xiaoting Wu,† Qingqing Wang,‡ Nan Cai,† Hanxue Zhang,† Zedong Jiang,#,Δ Min Wan,*,§ and Tatsuya Oda*,# †

College of Life Science, Shenzhen Key Laboratory of Marine Bioresources and Ecology, Shenzhen University, Shenzhen 518060, China ‡ College of Life Science, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen 518060, China § Division of Physiological Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm 17177, Sweden Δ College of Biological Engineering, Jimei University, Jimei, Xiamen 361021, China # Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852 8521, Japan ABSTRACT: This study evaluated the immunomodulatory activities, including regulation of nitric oxide (NO), reactive oxygen species (ROS), and tumor necrosis factor (TNF)-α production in RAW264.7 murine macrophages, of alginate oligosaccharides (AOS) and investigated their structure−activity relationships. Our results revealed that unsaturated guluronate oligosaccharide prepared by enzymatic degradation (GOS-ED) induced NO production and inducible nitric oxide synthase (iNOS) expression, dose and time dependently, and stimulated ROS and TNF-α production; however, other AOS prepared by different ways or polymers showed very low and even no such effects. Moreover, GOS-ED induced macrophage activation to release the abovementioned mediators partly involved in nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase signaling pathways. We also show that the structural characteristics of AOS, especially the unsaturated terminal structure, molecular size, and M/G ratio, play important roles in determining the macrophage-activating effects. GOS-ED could be applicable for agriculture, drug, and food industry as a potent immune-modulatory agent. KEYWORDS: guluronate oligosaccharide, mannuronate oligosaccharide, macrophage activation, nitric oxide, ROS, TNF-α, NF-κB, MAP kinase

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Fucus vesiculosus9 and ascophyllan isolated from Ascophyllum nodosum10 were reported to induce NO production in RAW264.7 cells via the NF-κB- and mitogen-activated protein (MAP) kinase-dependent signaling pathways. Alginate is a natural acidic unbranched polysaccharide, extracted from marine brown algae, composed of α-(1−4)-Lguluronate (G) and β-(1−4)-D-mannuronate (M) residues. These residues are arranged in homopolymeric stretches (G blocks and M blocks) or random heteropolymeric stretches (GM blocks).11 Alginate has been used for a wide range of commercial applications including the food, medical, cosmetic, and textile-processing industries.12 The alginate oligosaccharides (AOS) from alginate polymers depolymerized by bacterial alginate lyase showed various physiological activities, such as enhancement of growth of human endothelial cells,13 induction of cytokine production,14 enhancement of protection against infection with pathogens,15 antioxidant,16 and neuroprotective activity.17 Furthermore, structure−activity relationships of the alginate have been investigated from a wide variety of viewpoints. The differences

INTRODUCTION The mammalian immune system is composed of innate and acquired immune systems. The innate immune system is the first line of host defense against pathogens and is mediated by phagocytes including macrophages and neutrophils.1 Macrophages are involved in tissue remodeling during embryogenesis, wound repair, clearance of apoptotic cells, and hematopoiesis.2 The activation process of macrophages includes the production of various inflammatory mediators and cytokines such as interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), nitric oxide (NO),3 and reactive oxygen species (ROS).4 Recently, it has been reported that some immune-enhanced polysaccharides and oligosaccharides from food origin showed potential as immunotherapeutic agents.5 For instance, chitosan and chitooligosaccharides were reported to be capable of stimulating macrophage activation to produce various inflammatory mediators, including IL-1, IL-6, TNF-α, NO, and granulocyte macrophage colony stimulating factor (GM-CSF).6,7 Further study reported that chitooligosaccharide composed of sugars with a degree of polymerization (DP) of 1−6 induced NO production through the activation of the nuclear factor kappa B (NF-κB) signaling pathway mediated by the receptors CD14, TLR4, and CR3 in RAW264.7 macrophages.8 In addition, the sulfated polysaccharides fucoidan isolated from © 2014 American Chemical Society

Received: Revised: Accepted: Published: 3168

December March 10, March 15, March 16,

15, 2013 2014 2014 2014

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in the molecular weight, M/G ratio, MG sequence, and the entire molecular conformation of alginates seem to be responsible for their diversity of physicochemical properties and bioactivities.16 It has been reported that unsaturated AOS promote the growth of bifidobacteria and induce the cytotoxic cytokines in human mononuclear cells and murine macrophage RAW264.7 cells, whereas the original alginate polymers or saturated AOS have no such effects.18,19 Further study showed that unsaturated AOS improved the growth rate of Chlamydomonas reinhardtii and increased the levels of the fatty acids C16:0, C18:2 cis, and C18:3 n-3 but not saturated AOS.12 Similar structure−activity relationship profiles were also found in the inducing effects of AOS on IL-1α, IL-1β, and IL-6 secretion from RAW264.7 cells, and the most potent activities in G8 and M7 oligomers were observed.18 Moreover, the M/G ratio also seems to influence these activities, besides the molecular sizes.20 In the present study, we investigated the mechanisms of the immunomodulatory effects of alginate oligosaccharides on murine macrophage RAW264.7 cells and their structure− activity relationships from the following aspects: (1) the effect of several AOS, such as guluronate oligosaccharides and mannuronate oligosaccharides prepared by enzymatic degradation, acid hydrolysis and oxidative degradation, respectively, and also the effect of G2−G9 from GOS-ED on NO production in RAW264.7 cells; in addition, we investigated (2) the potent stimulatory effect of GOS-ED on RAW264.7 macrophages activation to induce the production of NO, ROS, and TNF-α and the activation of NF-κB and MAP kinase signaling pathways.

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MATERIALS AND METHODS Reagent. Sodium alginate (1000-cps grade) was purchased from Nuotai (Shanghai, China) and used without further purification. Bacterial alginate lyase from Pseudoalteromonas sp. strain no. 272 was prepared as described previously.21 Trizol reagent was purchased from Invitrogen Co. (Carlsbad, CA). 2′, 7′-Dichlorofluorescein diacetate (DCF-DA) was from Beyotime Inst Biotech (Jiangsu, China). Pyrrolidine dithiocarbamate (PDTC) and NG-nitro-L-arginine methyl ester (L-NAME) were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were commercially available products with the highest grade. Preparation of Alginate Oligosaccharides (AOS). Polyguluronate (PG) and polymannuronate PM (DP = 20−24) were prepared from sodium alginate as described previously.22 The homogeneity of the prepared polyuronates was confirmed by the analysis of circular dichroic (CD) spectrum using a Jasco spectropolarimeter J-815 based on the method of Morris et al.23 The chemical structures of PG and PM were analyzed by the infrared (IR) spectra with Bruker Vertex 70 according to the method of Linker et al.24 PG and PM were digested with bacterial alginate lyase21 to produce GOS-ED and MOS-ED, which have an unsaturated terminal structure with a double bond. GOS-AH and MOS-AH, which lack the unsaturated terminal structure with a double bond, were prepared from PG and PM by the acid hydrolysis method.12 To prepare GOS-OD and MOS-OD, which have a carboxyl group at the 1-position of the reducing end, PG and PM were incubated with 5% H2O2 solutions at a final concentration of 8% at 90 °C for 2 h. Before use, all alginate samples were filtered through an endotoxin-removing filter (0.22 μm) (Millipore Co., Billerica, MA). The schematic representations of

Figure 1. Schematic representation of chemical structures of guluronate and mannuronate oligosaccharides (enzymatic degradationacid hydrolysis and oxidative degradation) used in this study.

chemical structures of AOS using three different preparation ways were shown in Figure 1. TLC Analysis. Thin-layer chromatography (TLC) analysis was used to analyze the size and the DP of each AOS. The reaction product mixtures were loaded onto a precoated Silica Gel 60 TLC aluminum plate (2.5 cm × 7.0 cm, Merck, Germany) and developed with a solvent system consisting of n-butanol/formic acid/water (2:2:1 or 6:6:1, v/v/v). The developed plate was stained by dipping in a diphenylamine−aniline− phosphoric acid reagent (1 mL of 37.5% HCl, 2 mL of aniline, 10 mL of 85% H3PO4, 100 mL of ethyl acetate, and 2 g of diphenylamine) at RT for 3 s and then heated at 100 °C for 10 s. Isolation and Purification of GOS-ED. The isolation and purification of each oligosaccharide derived from GOS-ED were conducted by a P-6 Bio Gel chromatography (Bio-Rad Labs, Richmond, CA) (2.6 × 100 cm) as described previously.21 The phosphate in each fraction was removed by gel-filtration chromatography equipped with a P-2 Bio Gel (Bio-Rad Labs, Richmond, CA) (1.6 × 60 cm). Cell Culture. Murine macrophage RAW264.7 cells, purchased from Shanghai Inst of Biochem and Cell Biol (Shanghai, China), were cultured at 37 °C in RPMI-1640 medium supplemented with 10% FBS, 100 IU/ml penicillin, and 100 μg/mL streptomycin in humidified atmosphere with 5% CO2. 3169

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Cytotoxicity. The cytotoxic effects of saccharide samples were assessed by measuring cell viability using a tetrazolium salt (WST-8)-based colorimetric assay in the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan).25 NO Assay. To estimate NO level in RAW264.7 cells, nitrite, a stable reaction product of NO with molecular oxygen, was measured by Griess assay as described previously.10 Briefly, adherent RAW264.7 cells in 96-well plates (2 × 105 cells/well) were treated with saccharide samples at the concentration of 1 mg/mL in the growth medium at 37 °C. After 24 h of treatment, 50 μL of each cultured supernatant was collected and mixed with 100 μL of Griess reagent.10 After 10 min of incubation at RT, the optical density was measured at 540 nm using a Spectra Max microplate reader (Molecular Devices, LLC, Sunnyvale, CA). A calibration curve was made with a known concentration of NaNO2 standard solution. For the time-course analysis of NO production, RAW264.7 cells were treated with tested samples at the concentration of 1 mg/mL in the growth medium at 37 °C, and nitrite levels in each cultured supernatant were measured at 3, 6, 12 and 24 h, respectively. For the concentration-dependent analysis of NO production, RAW264.7 cells were treated with varying concentrations of tested samples (0−1 mg/mL) for 24 h in the growth medium at 37 °C, and then the nitrite levels in each well were measured. LPS (1 μg/mL), which can significantly induce NO production in RAW264.7 cells, was used as positive control. RNA Isolation, cDNA Synthesis, and RT-PCR for iNOS mRNA. Total cellular RNA was extracted from treated cells with Trizol reagent (RNAfast200, Fastagen, China). Total RNA (1 μg) was reverse transcribed with an oligo dT primer in a 10 μL using PrimeScript first strand cDNA Synthesis Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s instruction. PCR was performed with 1 cycle of 3 min at 95 °C, 25 cycles of 55 s at 93 °C, 45 s at 60 °C, 40 s at 72 °C and 1 cycle of 100 s at 72 °C, in a 50 μL reaction mixture containing 25 μL of Premix Taq (TaKaRa Biotechnology Co., Ltd., Dalian, China), 1 μL of forward and reverse inducible nitric oxide synthase (iNOS) primers (20 μM each) or β-actin primers (20 μM each), 5 μL of first strand cDNA, and 18 μL of nuclease-free water. The primer sequences used were 5′-CAACCAGTATTATGGCTCCT-3′ (forward) and 5′-GTGACAGCCCGGTCTTTCCA-3′ (reverse) for mouse iNOS and 5′-GGAGAAGATCTGGCACCACACC-3′ (forward) and 5′-CCTGCTTGCTGATCCACATCTGCTGG3′ (reverse) for mouse β-actin.10 The β-actin primer was used as an internal control. Each PCR reaction (6 μL) product was run on 1% agarose gels containing 0.1 μg/mL ethidium bromide, and the amplified products (845 bp for iNOS, 850 bp for β-actin) were observed by an G: BOX Syngene imaging system (Gene Company Ltd., Hong Kong). Western Blot Analysis. After different treatments, cells (3 × 106 cells) were lysed in radio immunoprecipitation assay lysis buffer (Biocolors, Shanghai, China) at 4 °C for 30 min. Cell lysates (30 μg of protein for each sample) were separated by 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech., England, U.K.), blocked with 5% skim milk, and hybridized with antimouse iNOS antibody (dilution, 1:1,000) (Cell Signaling Technology, Inc., Beverly, MA). After incubation with a horseradish-peroxidase-conjugated secondary antibody at RT, immune-reactive proteins were detected by using a chemiluminescent ECL assay kit (Amersham Pharmacia Biosciences, England, U.K.) according to the

manufacturer’s instructions. Western blot bands were visualized using a LAS3000 Luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan). The analyses of IκB-α and phospho-IκB-α in cytosolic extracts and NF-κB p65 in nuclear extracts were performed. Adherent RAW264.7 cells (4 × 106 cells) were incubated with 1 mg/mL of saccharides at 37 °C for 1 h. Thereafter, the cells were washed three times with ice-cold PBS and incubated with 100 μL ice-cold cytosol extraction buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2% Igepal CA-630, 1 mM dithiothreitol, 20 mM β-glycerophosphate, 1 mM sodium orthoranadate, 0.5 mM phenylmethysulfonyl fluoride, 1 μg/mL leupeptin, and 1 μg/mL aprotinin) for 25 min on ice. The cytosolic extracts were collected after centrifugation at 7000g for 5 min at 4 °C. The nuclear pellets were resuspended in 30 μL of ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.45 M NaCl, 25% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethysulfonyl fluoride, 1 μg/mL leupeptin, and 1 μg/mL aprotinin) and incubated on ice for 25 min. The nuclear extracts were then obtained after centrifugation at 15 000g for 10 min at 4 °C. After protein quantitation, the nuclear and cytosolic extracts were separated by 12.5% SDS-PAGE. The Western blot analysis was conducted similar to the method described above with anti-NF-κB p65, anti-IκB-α, and antiphospho-IκBα antibodies (Cell Signaling Technology, Inc., Beverly, MA). For the analysis of MAP kinases, adherent RAW264.7 cells (3 × 106 cells) were treated with 1 mg/mL of each saccharide in the serum-free medium at 37 °C for 10 min. The whole-cell lysates were prepared from the treated cells. Samples containing 30 μg of proteins were subjected to SDS-PAGE in 12.5% polyacrylamide gel, and Western blot analysis using appropriate antibodies against phosphorylated and nonphosphorylated p38, JNK, and ERK MAP kinases (Cell Signaling Technology, Beverley, MA) were carried out as described above. Intracellular ROS Measurement. The fluorescent probe DCF-DA was used to measure intracellular accumulation of ROS as described previously.26 For this purpose, DCFH-DA solution (10 μM) was added to the suspension of each saccharide-treated cells (5 × 105 cells/ml), and the mixture was incubated at 37 °C for 20 min. Cells were then washed three times with PBS by centrifugation (1500g), and finally the fluorescence intensity of cell suspension was measured by the fluorescence activated cell sorting (FACS) system (Becton Deckinson, San Jose, CA) with excitation and emission wavelengths of 488 and 525 nm, respectively. Cytokine Measurement. Adherent RAW264.7 cells in 96-well plates (2 × 105 cells/well) were treated with saccharides at the concentration of 1 mg/mL for 24 h in the growth medium at 37 °C. The levels of TNF-α in the culture medium were measured using a mouse TNF-α quantikine ELISA kit (R&D Systems, Inc., Minneapolis, MN), following the manufacturer’s instructions. Statistical Analysis. All of the experiments were repeated at least three times. The data are expressed as the means ± standard deviation (SD) and analyzed using a t-test or one-way ANOVA to determine any significant differences. A value of p < 0.05 was considered statistically significant.

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RESULTS Preparation and Structure Analysis of AOS. The CD analysis showed a high homogeneity of polyguluronate (PG) and polymannuronate (PM) (data not shown). The IR spectra 3170

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The average DP of all kinds of AOS was measured by TLC analysis. As shown in Figure 2A−C, in all lanes, the spots corresponding to the oligosaccharides having 2−7 saccharide units were detected, whereas the spots at the origin corresponding to polysaccharides and oligosaccharides with >16 saccharide units were the most intense, indicating that AOS with average DP of 5 were obtained by three preparation ways used in this study. GOS-ED Induces NO Production in RAW264.7 Cells. None of alginate polysaccharides (PG and PM) or AOS (-ED, -AH, and -OD) showed cytotoxic effects on RAW264.7 cells even up to a concentration of 1 mg/mL in WST-8 assay (data not shown). Hence, it is considered that any observed changes on NO production in RAW264.7 cells or related events in the following studies are not due to the cytotoxicity of alginatederived saccharides. Next, we investigated NO production induced by PG, PM, and AOS (GOS-ED, -AH, -OD and MOS-ED, -AH, -OD) in RAW264.7 cells. As shown in Figure 3A, only GOS-ED significantly enhanced NO production within all tested alginate polysaccharides and oligosaccharides, whereas all other saccharides showed no significant activity. Furthermore, GOS-ED-induced NO production in a time- and concentration-dependent manner, and the nitrite level reached the maximum in RAW264.7 cells treated with 1 mg/mL of GOS-ED for 24 h (Figure 3B,C). To further analyze the structure−activity relationship of GOS-ED, the enzymatically digested PG was applied to a column Bio Gel P-6. The elution profile shown in Figure 4A

Figure 2. TLC of AOS prepared by different ways. The oligosaccharide solutions (25 mg/mL, 0.5 μL of each) were loaded onto a precoated Silica Gel 60 TLC aluminum plate and developed with a solvent system consisting of n-butanol/formic acid/water (2:2:1 or 6:6:1, v/v/v). The developed plate was stained by dipping in diphenylamine−aniline−phosphoric acid reagent (1 mL of 37.5% HCl, 2 mL of aniline, 10 mL of 85% H3PO4, 100 mL of ethyl acetate, and 2 g of diphenylamine) for 3 s and heated at 100 °C for 10 s. The average DP of GOS-ED and MOS-ED (A), GOS-AH and MOS-AH (B), and GOS-OD and MOS-OD (C) was measured using TLC analysis.

of PG and PM are very similar, but they show differences in the absorption peaks at 789 cm−1 and 821 cm−1, indicating the unique distinction of PG and PM (data not shown).

Figure 3. Effects of GOS (-ED, -AH, -OD), MOS (-ED, -AH, -OD), PG, and PM on NO production in RAW264.7 cells. Adherent RAW264.7 cells (2 × 105 cells/well) were cultured in 96-well plates. (A) RAW264.7 cells were incubated with 1 mg/mL of GOS (-ED, -AH, -OD), MOS (-ED, -AH, -OD), PG, and PM at 37 °C in the growth medium, respectively. After 24 h, the nitrite levels in the culture medium were measured by Griess assay, as described in Methods. (B) RAW264.7 cells were incubated with 1 mg/mL of GOS-ED for 3, 6, 12, and 24 h. After that, the nitrite levels in the culture medium were measured. (C) RAW264.7 cells were incubated with GOS-ED in different concentrations (0.1, 0.5, and 1 mg/mL). After 24 h, the nitrite levels in the culture medium were measured. LPS (1 μg/mL) was used as a positive control. Each value represents mean ± SD of triplicate measurements. * indicates significant differences between various time groups and 0 time group, * p < 0.05, ** p < 0.01, and *** p < 0.001, respectively. # indicates significant differences between time groups and previous time group, ##p < 0.01 and ###p < 0.001, respectively. 3171

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indicated that GOS-ED was a mixture of oligomers from dimer to nonamer (G2−G9), and the fractions from G2 to G9 were obtained as relatively purified fractions. As shown in Figure 4B, purified G2, G6, and G7 fractions showed even higher activity than GOS-ED, and the highest activity was observed in G6 fraction. Meanwhile, G3 and G5 fractions showed nearly equal activity to GOS-ED, although the G4 fraction had almost no activity. The other notable founding was that G8 and G9 fractions were detected as precipitation with Griess reagent, so they were not suitable for this analysis. GOS-ED Induces iNOS Expression in RAW264.7 Cells. As we have known, NO is produced by the activation of iNOS from L-arginine in macrophages.27 As shown in Figure 5A−C, GOS-ED significantly induced iNOS mRNA expression in RAW264.7 cells in a time- and concentration-dependent manner. Consistent with mRNA expression, iNOS protein expression was also detected in GOS-ED-treated RAW264.7 cells (Figure 5D). In addition to GOS-ED, MOS-ED also induced iNOS mRNA and protein expression, although the expression was very low and even trace levels (Figure 5A,D). Furthermore, NO production by GOS-ED-treated RAW264.7 cells was significantly suppressed by L-NAME, a NOS inhibitor (Figure 5E). GOS-ED Induces ROS Production in RAW264.7 Cells. As shown in Figure 6A, the intensity of the DCF fluorescence was significantly increased in GOS-ED-stimulated RAW264.7 cells compared to control cells, indicating that GOS-ED enhanced intracellular ROS production. In contrast, other saccharides including GOS (-AH, -OD), MOS (-ED, -AH, -OD), and original polymers (PG/PM) showed no statistical effect on ROS production. As shown in Figure 6B, a similar tendency was observed with the effects of G2−G9 on NO production. The purified G2, G3, and G6−G9 fractions showed higher ROSinducing activity than GOS-ED, and the highest activity was observed in the G9 fraction. Meanwhile, the G5 fraction showed

Figure 4. Effects of guluronate dimer to heptamer (G2−G7) on NO production in RAW264.7 cells. (A) GOS-ED solution (3 mL, 50 mg/mL in distilled water) was put on a column of Bio Gel P-6 equilibrated with 50 mM phosphate buffer, pH 6.5. Eluates were examined for absorbance at 235 nm. Gal UA: monomer; G2−G9: guluronate dimer-nonamer. (B) RAW264.7 cells were incubated with 0.5 mg/mL of GOS-ED and G2−G7, respectively. After 24 h, the nitrite levels in culture medium were measured. LPS (1 μg/mL) was used as a positive control. Each value represents mean ± SD of triplicate measurements.

Figure 5. Effects of GOS (-ED, -AH, -OD), MOS (-ED, -AH, -OD), PG, and PM on iNOS expression in RAW264.7 cells. (A) Adherent RAW264.7 cells (2 × 106 cells/well in 6-well plates) were incubated with 1 mg/mL of GOS (-ED, -AH, -OD), MOS (-ED, -AH, -OD), PG, and PM at 37 °C in the culture medium, respectively. After 12 h of incubation, iNOS gene levels in RAW264.7 cells were analyzed by RT-PCR. (B) iNOS gene expression after 4, 8, and 12 h of stimulation with 1 mg/mL of GOS-ED was measured. (C) iNOS gene expression after 12 h stimulation with various doses (0.1, 0.5, and 1 mg/mL) of GOS-ED was measured. (D) Adherent RAW264.7 cells (2 × 106 cells/well in 6-well plates) were incubated with 1 mg/mL of GOS-ED, MOS-ED, PG, and PM at 37 °C, respectively. After 24 h of incubation, the whole-cell lysates were prepared from the treated cells and analyzed by Western blot analysis. (E) Adherent RAW264.7 cells were incubated with 1 mg/mL of GOS-ED for 24 h by the addition of the NOS inhibitor L-NAME (100 μM). The nitrite levels in the culture medium from the treated cells were measured by Griess assay. Each value represents mean ± SD of triplicate measurements. The * is representative of p < 0.05, when compared to control group. 3172

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Figure 7. Effects of GOS-ED, MOS-ED, PG, and PM on TNF-α secretion in RAW264.7 cells. Adherent RAW264.7 cells (2 × 105 cells/ well in 96-well plates) were incubated with various concentrations (0.1, 0.5, and 1 mg/mL) of GOS-ED, MOS-ED, PG, and PM at 37 °C, respectively. After 24 h of incubation, the TNF-α levels in the culture medium were measured by ELISA. Each value represents mean ± SD of triplicate measurements.

Figure 6. Effects of alginate oligosaccharides and polysaccharides on ROS production in RAW264.7 cells. (A) Adherent RAW264.7 cells (5 × 105 cells/well in 24-well plates) were incubated with 1 mg/mL of GOS (-ED, -AH, -OD), MOS (-ED, -AH, -OD), PG, and PM at 37 °C, respectively. After 24 h, intracellular ROS were measured by flow cytometry utilizing an oxidation-sensitive fluorescent probe, DCFH-DA. LPS (1 μg/mL) was used as a positive control. (B) Adherent RAW264.7 cells were incubated with 0.5 mg/mL of GOS-ED and G2−G9, respectively. After 24 h, intracellular ROS were measured. LPS (1 μg/mL) was used as a positive control. Each value represents mean ± SD of triplicate measurements. The * is representative of p < 0.05 and *** is representative of p < 0.001, when compared to the control group.

in GOS-ED-induced production of NO, ROS, and TNF-α, we used a NF-κB inhibitor PDTC to block the NF-κB signaling pathway. Our results demonstrated that PDTC significantly inhibited the productions of NO (Figure 8B), ROS (Figure 8C), and TNF-α (Figure 8D) in GOS-ED-treated cells, suggesting that GOS-ED-induced NO, ROS, and TNF-α production in RAW264.7 cells were partly mediated through the NF-κB signaling pathway. GOS-ED Activates the MAP Kinase Signaling Pathway in RAW264.7 Cells. To further investigate the involvement of MAP kinase signaling pathway in immunomodulatory effects of enzymatically degraded alginate oligosaccharides and polysaccharides, RAW264.7 cells were treated with each saccharide and the levels of phosphorylated and nonphosphorylated ERK, p38, and JNK MAP kinases were analyzed by Western blot. As shown in Figure 9, a significant increase in the phosphorylation levels of ERK, p38, and JNK MAP kinases were observed in GOS-ED-treated RAW264.7 cells, whereas the phosphorylation levels of these three MAP kinases induced by MOS-ED were evidently lower than that induced by GOS-ED.

this activity nearly equal to that of the GOS-ED, whereas the G4 fraction had almost no activity. GOS-ED Induces TNF-α Secretion in RAW264.7 Cells. As displayed in Figure 7, the levels of TNF-α secretion in the culture medium from GOS-ED- and MOS-ED-treated RAW264.7 cells were elevated in a concentration-dependent manner. A significant level of TNF-α secretion was capable to be detected at 0.1 mg/mL of GOS-ED and MOS-ED, and further increased levels were detected at 0.5 and 1 mg/mL of GOS-ED and MOS-ED. Meanwhile, the slightly stronger TNFα-inducing activity of GOS-ED than MOS-ED was observed. On the contrary, PG or PM before depolymerization had much lower effect than oligomers on TNF-α secretion. GOS-ED Activates the NF-κB signaling Pathway in RAW264.7 Cells. As shown in Figure 8A, the nuclear level of NF-κB p65 protein increased in GOS-ED- and MOS-EDtreated RAW264.7 cells with a greater extent than those induced by PG and PM. In addition, GOS-ED and MOS-ED, but not PG or PM, also induced both the phosphorylation and degradation of IκB-α. As the results indicated, GOS-ED and MOS-ED could induce the NF-κB activation in RAW264.7 cells. To test the potential involvement of the NF-κB activation

DISCUSSION Many polysaccharides isolated from seaweeds have been recently reported to induce immune activation as molecules target to immune cells in a host immune system, such as macrophages, lymphocytes, and natural killer cells, and stimulate cytokines release and other immune responses.28 Alginate, currently used widely in commercial enterprises, has been known to induce cytokine production via NF-κB signaling pathway from murine macrophage RAW264.7 cells29 and exert antitumor activities against Sarcoma-180 solid tumor transplanted in mice without toxicity via the activation of the host immune system.30 Although the properties of alginate have been widely investigated, the actual application of alginate polymers into in vivo system is limited due to the higher molecular weight. It is likely that the depolymerization of polymers is one possible way to overcome this drawback of alginate.18 Our previous studies have reported that AOS-ED could stimulate RAW264.7 macrophages to produce cytokines14 and induce multiple immunocompetent cytokines in mice via intraperitoneal injection,31 suggesting that AOS-ED were potentially capable of stimulating macrophages and the host immune system activation, but the detailed mechanisms and the structure−activity relationships are unclear so

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Figure 8. Effects of GOS-ED, MOS-ED, PG, and PM on the activation of NF-κB signaling pathway in RAW264.7 cells and the involvement of NFκB in GOS-ED-induced NO, ROS, and TNF-α production. (A) RAW264.7 cells were treated with 1 mg/mL of GOS-ED, MOS-ED, PG, and PM, respectively. Afterward, Western blot analysis was conducted to detect the expression of NF-κB p65 in the nuclear extracts and the expression of IκB-α and phosphorylated IκB-α in the cytosolic extracts. (B) Adherent RAW264.7 cells were incubated with 1 mg/mL of GOS-ED for 24 h by the addition of the NF-κB signaling pathway inhibitor PDTC (50 μM). The nitrite levels in the culture medium from the treated cells were measured by Griess assay. (C) Adherent RAW264.7 cells were incubated with 1 mg/mL of GOS-ED for 24 h by the addition of the NF-κB signaling pathway inhibitor PDTC (50 μM). Intracellular ROS were measured by a FACS analysis. (D) Adherent RAW264.7 cells were incubated with 1 mg/mL of GOS-ED for 24 h by the addition of the NF-κB signaling pathway inhibitor PDTC (50 μM). The TNF-α levels in the culture medium of the treated cells were measured by ELISA. Each value represents mean ± SD of triplicate measurements. The *, **, and *** are representative of p < 0.05, p < 0.01, and p < 0.001, respectively, when compared to control group.

revealed that GOS-ED showed significant NO-inducing activity in RAW264.7 cells in a time- and concentration-dependent manner, and this activity of GOS-ED was much higher than other oligosaccharides (GOS-AH, -OD and MOS-ED, -AH, -OD) and original polysaccharides (PG and PM). Consistent with this finding, significantly increased iNOS gene and protein expression levels were observed in GOS-ED-treated RAW264.7 cells in a time- and concentration-dependent manner, whereas MOS-ED only slightly induced iNOS gene expression. Other oligosaccharides (GOS-AH, -OD and MOS-AH, -OD) and polysaccharides (PG and PM) showed no such effects on the expression of iNOS gene and protein (Figure 5A−D). Because L-NAME, a NOS inhibitor, significantly inhibited GOS-EDinduced NO production in RAW264.7 cells (Figure 5E), GOSED is considered to be a potent macrophage activator capable of inducing NO production through the induction of iNOS expression. Next, the ROS and TNF-α production induced by these saccharides on RAW264.7 cells was evaluated. Our results indicated that GOS-ED significantly stimulated ROS production in RAW264.7 cells, but no significant effect was observed in other tested saccharides (Figure 6). Interestingly, contrary to the NO- and ROS-inducing activities, both kinds of AOS-ED (GOS-ED and MOS-ED), but not polysaccharides (PG and PM), significantly induced TNF-α secretion in RAW264.7 cells in a concentration-dependent manner, even though MOS-ED evidently showed lower activity than GOS-ED (Figure 7). These findings further support the notion that GOS-ED is a potent macrophage activator.

Figure 9. Effects of GOS-ED, MOS-ED, PG, and PM on the activation of MAP kinases in RAW264.7 cells. Adherent RAW264.7 cells (3 × 106 cells/3.5 cm dishes) were incubated in serum-free medium with 1 mg/mL of GOS-ED, MOS-ED, PG, and PM, respectively. The whole-cell lysates were prepared from the treated cells, and the levels of both phosphorylated and nonphosphorylated MAP kinases were analyzed by Western blot.

far. Here, we prepared GOS-ED, -AH, -OD and MOS-ED, -AH, -OD using three different depolymerized ways to compare their macrophage-activating activities and provided key insights into potential mechanisms. Macrophages play important roles in host defense systems, and activated macrophages can kill tumor cells and pathogens either by direct contact or by releasing diffusible cytotoxic molecules, such as NO, ROS, and TNF-α.32 As we have known, NO plays a critical role in host defense during pathogen infection and tissue damage during autoimmune responses.33 The expression of iNOS is responsible for NO production in activated macrophages.34 In the present study, our results 3174

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MOS-ED showed much higher activities on the induction of NO, ROS, and TNF-α production in RAW264.7 cells than other AOS (GOS-AH, -OD, and MOS-AH, -OD) and polysaccharides (PG and PM), and these effects induced by GOS-ED were evidently greater than those of MOS-ED (Figures 3A, 6, and 7). These findings provided valuable evidence to the idea that unsaturated end structure and the composition (M/G ratio) were important structural elements for their biological activities. Previous structural studies have proposed that mannuronate and guluronate adopt different chair conformations; mannuronate block predominately forms an extended ribbon structure, whereas guluronate block forms a buckled chain.18 Moreover, the highest NO-inducing activity among unsaturated G2−G7 fractions was found in G6 and G7 fractions (Figure 4B), which suggested that G6 and G7 oligomers were the most effective structure or molecular size in the specific activities. Interestingly, the smaller oligomer G2 also showed relatively high cytokineinducing activities. The reason for this is uncertain now, but the finding may provide a clue to understand the recognition site or mechanism on RAW264.7 cells by alginate oligomers. In conclusion, these results first revealed that AOS-ED but not AOS-AH, AOS-OD, or polysaccharides activated the NF-κB and MAPK signaling pathways, resulting in significant induction of NO, ROS, and TNF-α production. Our findings suggested that the structural characteristics of AOS, especially the unsaturated terminal structure, molecular size, and M/G ratio, are important in RAW264.7 macrophage activation. On the basis of these findings, it is worthwhile to develop GOS-ED as a promising agent used to regulate immune responses and vascular relaxation in functional foods and pharmacological fields.

NF-κB regulates many important biological and pathological processes in cells, and it is also a key to modulate various gene expressions involved in immune and inflammatory responses, including the production of NO, ROS, and TNF-α in macrophages.35 In our present studies, it was found that GOS-ED showed much higher RAW264.7 macrophage-stimulating activity than other saccharides derived from alginate. Meanwhile, MOS-ED exhibited less activity on macrophage activation than GOS-ED, but the activity was higher than other saccharides, especially the induction of TNF-α production. Although alginate has been reported to stimulate RAW264.7 cells activation to release immune cytokines via the NF-κB signaling pathway, there has been no available information on the precise action mechanisms of AOS-ED on macrophagestimulating activity so far. Our results showed that the NF-κB signaling pathway was potently activated in GOS-ED- and MOS-ED-treated RAW264.7 cells, whereas the activation levels in PG- or PM-treated cells were hardly distinguishable from the unstimulated control level (Figure 8A). Interestingly, the activation level of NF-κB in MOS-ED-treated RAW264.7 cells seems to be higher than that of GOS-ED-treated cells (Figure 8A). As we have known, the MAP kinase signaling pathway plays an important role in NO, ROS, and TNF-α production in activated macrophages. Our previous report showed that AOSED were capable of activating JNK and p38 MAP kinases involved signaling pathway leading to TNF-α secretion from RAW264.7 cells.20 Considering the differences between GOSED and MOS-ED in macrophage-stimulating activities and the NF-κB-activated effects, it is speculated that the MAP kinase signaling pathway involved in GOS-ED- and MOS-ED-induced macrophage activation might be different. Our results showed that ERK, p38, and JNK MAP kinase signaling pathways were potently activated in GOS-ED-treated RAW264.7 cells, whereas the activation levels of these MAP kinases in MOS-ED-treated cells were quite low compared to the levels of unstimulated control (Figure 9), considering NO, ROS, and TNF-α production and NF-κB signaling pathway activation, suggesting that the underlying mechanisms of macrophage activation activity were different between GOS-ED and MOS-ED. To determine the possible involvement of NF-κB signaling pathway in the induction of NO, ROS, and TNF-α production by GOS-ED, NF-κB inhibitor PDTC was used to block the NFκB activation. Our results showed that significantly inhibitory effects of PDTC on GOS-ED-inducing NO, ROS, and TNF-α production were observed (Figure 8B−D), further demonstrating that the NF-κB signaling pathway is responsible for the production of NO, ROS, and TNF-α in GOS-ED-treated RAW264.7 cells. The results obtained in this study may provide valuable basic information to the mechanisms of AOS-induced macrophage activation, especially in terms of NO, ROS, and TNF-α production in RAW264.7 cells. It has been reported that enzymatically depolymerized alginate oligomers were capable to enhance the growth of bifidobacteria,36 plant roots,21 and green alga.12,37 Our previous reports revealed that unsaturated AOS-ED were highly active inducers for cytokine production in RAW264.7 cells, whereas saturated AOS-AH showed smaller effects.18 It seems that unsaturated terminal structure with double bonds of alginate oligomers play important roles in their biological activities, including inducing cytokines production. In fact, we presented an idea that the biological activities of AOS are strongly influenced by their molecular size, M/G composition, and the entire molecular conformation.16 This study revealed that GOS-ED and

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: (M.W.) [email protected]. Fax: +46-8-7360439. Tel.: +46-8-52487623. *E-mail: (T.O.) [email protected]. Fax: +81-95-8192799. Tel.: +81-95-8192831. Author Contributions *

Corresponding authors contributed equally.

Funding

This work was supported financially by the project supported by the National Natural Science Foundation of China (grant no. 31000770) and Knowledge Innovation Program in Shenzhen Scientific Research and Development Funding Program (grant no. JCYJ20130329111455027). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Mrs. Lijun Yao and Dr. Liyan Wang from Shenzhen University and Mr. Yi Liu from Guangdong Medical College for their technical assistance.

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ABBREVIATIONS USED AH, acid hydrolysis; AOS, alginate oligosaccharide; DP, degree of polymerization; ED, enzymatic degradation; ELISA, enzymelinked immunosorbent assay; GOS, guluronate oligosaccharide; iNOS, inducible nitric oxide synthase; MAP kinase, mitogenactivated protein kinase; MOS, mannuronate oligosaccharide; NF-κB, nuclear factor-κB; NO, nitric oxide; OD, oxidative degradation; PG, polyguluronate; PM, polymannuronate; ROS, reactive oxygen species; TNF, tumor necrosis factor 3175

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