Structural characterization and immunomodulatory activity of a new polysaccharide from jellyfish

Accepted Manuscript Title: Structural characterization and immunomodulatory activity of a new polysaccharide from jellyfish Author: Qiang-Ming Li Jing-Fei Wang Xue-Qiang Zha Li-Hua Pan Hai-Lin Zhang Jian-Ping Luo PII: DOI: Reference:

S0144-8617(16)31407-2 http://dx.doi.org/doi:10.1016/j.carbpol.2016.12.031 CARP 11838

To appear in: Received date: Revised date: Accepted date:

8-11-2016 6-12-2016 15-12-2016

Please cite this article as: Li, Qiang-Ming., Wang, Jing-Fei., Zha, Xue-Qiang., Pan, Li-Hua., Zhang, Hai-Lin., & Luo, Jian-Ping., Structural characterization and immunomodulatory activity of a new polysaccharide from jellyfish.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.12.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Original research

Structural characterization and immunomodulatory activity of a new polysaccharide from jellyfish

Qiang-Ming Li#1, Jing-Fei Wang#1,2, Xue-Qiang Zha1,3*, Li-Hua Pan1, Hai-Lin Zhang1, Jian-Ping Luo1*

1School

of Food Science and Engineering, Hefei University of Technology, Hefei 230009, Anhui

Province, People’s Republic of China 2Students’

Affairs Division, Qilu University of Technology, Jinan 250300, Shandong Province,

People’s Republic of China 3School

of Biological and Medical Engineering, Hefei University of Technology, Hefei 230009,

Anhui Province, People’s Republic of China

*Correspondence: (1) Prof. Dr. Xue-Qiang Zha, School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China. Telephone: +86-551-62901537; Fax: +86-551-62901516. E-mail: [email protected]; (2) Prof. Dr. Jian-Ping Luo, School of Food Science and Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China. Telephone: +86-551-62901539; Fax: +86-551-62901516. E-mail: [email protected]

#

These authors contributed equally to this work.

1

Highlights (1) A homogenous polysaccharide (JSP-11) was isolated from jellyfish. (2) The structural features of JSP-11 were characterized. (3) The activation of macrophages by JSP-11 and the related mechanism were studied.

ABSTRACT A new polysaccharide (JSP-11) with a molecular weight of 1.25 × 106 Da was extracted and purified from jellyfish. Monosaccharide analysis showed that JSP-11 was composed of mannose, galactose and glucuronic acid with a molar ratio of 2.18:1.00:1.94. According to the analysis of fourier transform-infrared spectroscopy, methylation analysis, and NMR spectroscopy, JSP-11 was determined to contain a linear backbone which consisted of (1→3,6)-linked β-D-Manp and (1→6)-linked β-D-Galp. The branch of (1→)-linked α-D-GlcpA was attached to the C-3 position of (1→3,6)-linked β-D-Manp in the backbone. The immunomodulatory assay exhibited that JSP-11 could significantly enhance the viability of RAW 264.7 macrophage cells, and promote the release of NO, TNF-α, and IL-1β via activating NF-κB, MAPKs and PI3K/Akt signal pathways.

Keywords: Jellyfish; Polysaccharide; Structural characterization; Immunomodulatory activity.

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1. Introduction Rhopilema esculetum Kishinouye, a marine plankton belonging to the Rhopilema family, is the most abundant jellyfish specie distributed in the areas of South China Sea, Bohai Sea and Yellow Sea. In China, jellyfish has been regarded as a nutritional seafood since it is rich in proteins, amino acids, vitamins and inorganic elements (Ding et al., 2011; Liu, Guo, Yu, & Li, 2012). Besides, jellyfishes were also recorded as an effective folk medicine in the ancient literatures of traditional Chinese medicine to treat hypertension, arthritis, tracheitis, asthma and ulcers for more than one thousand years (Ding et al., 2011). The active collagen (Fan, Zhuang, & Li, 2013; Morishige et al., 2011) and venom (Kang et al., 2009; Xiao et al., 2010) of jellyfish have attracted strong interests from pharmacologists and chemists in recent decades. Natural polysaccharides are widely distributed in animals, plants and microorganisms. Some of them have been proved to exhibit various bioactivities, such as anti-oxidative (Jin et al., 2016; Tian, Zha, Pan, & Luo, 2013), anti-fatigued (Chi et al., 2015; Zhao et al., 2015), immunomodulatory (Jing et al., 2014; Li et al., 2015; Liao et al., 2015; Lin, Liao, & Ren, 2016; Xie et al., 2016; Yuan et al., 2015; Zha et al., 2014; Zhang, Wang, Lai, & Wu, 2016), anti-atherosclerotic (Zha et al., 2012; Zha et al., 2015), and hepatoprotective (Tian, Zha, & Luo, 2015; Wang, Luo, Chen, Zha, & Pan, 2015; Wang, Luo, Chen, Zha, & Wang, 2014) activities. Moreover, the structures of these polysaccharides have been well characterized. To the best of our knowledge, although polysaccharides have been observed in jellyfish, their structural information and bioactivities are still not well known to us.

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For this purpose, a homogeneous polysaccharide was extracted and purified from jellyfish in the present work. The structure features were elucidated using gas chromatograph-mass spectrometer (GC-MS) and nuclear magnetic resonance spectrometer

(NMR).

Moreover,

the

immunomodulatory

activity

of

this

polysaccharide was investigated in vitro using RAW 264.7 macrophage cells. The underlying immunomodulatory mechanism of this polysaccharide was further clarified from the aspects of signaling pathways. These results would provide valuable information about the structure and bioactivity of the jellyfish polysaccharide, which will be useful for potentially commercial use of the polysaccharide as functional food supplement with immunomodulatory activity.

2. Materials and methods 2.1. Materials and reagents Fresh jellyfish (containing about 95% water) was obtained from Dalian Shuiyuan Seafood Co. Ltd., China. Glucose (Glu), rhamnose (Rha), arabinose (Ara), xylose (Xyl),

mannose

(Man),

galactose

(Gal),

lipopolysaccharide

(LPS),

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Sephacryl S-500 and DEAE-Cellulose-52 were purchased from Sigma-Aldrich (MO, USA). Fetal bovine serum, DMEM medium, streptomycin, and penicillin were obtained from Hyclone Co. (UT, USA). ELISA kits of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were obtained from R&D System (Minneapolis, MN, USA). Nitric oxide (NO) kit was supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The

4

primary antibodies against extracellular regulated protein kinase 1/2 (ERK1/2), phosphor-ERK1/2, c-Jun N-terminal kinase (JNK), phosphor-JNK, p38, phosphor-p38, nuclear factor kappa B (NF-κB) p65, phosphor-NF-κB p65, Akt, phosphor-Akt, inhibitor of κB (IκB), phosphor-IκB and β-actin were obtained from Cell Signal Technology Inc. (Beverly, MA). The secondary antibody anti-rabbit IgG was purchased from Boster Co. (Wuhan, China). 2.2. Extraction and purification The crude jellyfish polysaccharide (JSP) powder was extracted via the optimized method described in our previous report (Zhang et al., 2014). The JSP powder (175 mg) was dissolved in 5 ml double-distilled H2O (ddH2O) and loaded on a DEAE-cellulose-52 column (5.0 × 70.0 cm), followed by stepwise elution with ddH2O, 0.1 M, 0.2 M, 0.3 M and 0.4 M aqueous sodium chloride solution at a flow rate of 5 mL/min, giving four polysaccharide fractions of JSP-1, JSP-2, JSP-3 and JSP-4 (Fig.S1 in supporting information), respectively. The main polysaccharide fraction, water-eluted polysaccharide JSP-1, was further purified by a Sephacryl S-500 column (1.6 × 80.0 cm) eluting with water to afford a purified polysaccharide (JSP-11). 2.3. Characterization of JSP-11 2.3.1. Determination of purity and molecular weight The purity and molecular weight were determined by high performance liquid chromatography system (HPLC, 1260 Infinity, Agilent Technologies) equipped with a refractive index detector. The columns of TSK G4000 PWxl (7.8×300 mm) and

5

G5000 PWxl (7.8×30.0 mm) were selected as the separating medium, which were connected in series. The sample was eluted with ddH2O at a flow rate of 0.5 mL/min. To obtain the molecular weight information of JSP-11, standard T-series dextrans (T-700, T-580, T-110, T-80, T-70, T-40 and T-11) were also passed through the columns, and a calibration curve was thus plotted between the retention time and the logarithms of standard molecular weights. The molecular weight of JSP-11 was calculated according to the calibration curve. 2.3.2. General analysis The carbohydrate content was quantified according to the phenol-sulfuric acid method using glucose as a standard (Tong et al., 2009). Total uronic acid content was determined by m-hydroxydiphenyl method using glucuronic acid as the standard (Tong et al., 2015). Fourier transform infrared (FT-IR) spectroscopy was recorded using a Nicolet 67 Thermo instrument. The sample was ground with KBr powder and then pressed into pellets for FT-IR measurement in the frequency range of 4000−400 cm−1. 2.3.3. Monosaccharide analysis According to published literature (Shao, Shao, Han, Lv, & Sun, 2015), the carboxyl of uronic acid in JSP-11 was reduced to primary alcohol to obtain the reduction product JSP-11R. Ten milligrams of JSP-11 and JSP-11R were transferred to a test tube and hydrolyzed in 4 mL of 2 M trifluoroacetic acid (TFA) at 120 °C for 4 h, respectively. The hydrolysates were subsequently converted into alditol acetates (Li et al., 2015), and then analyzed by gas chromatography (7890A GC system, Agilent)

6

fitted with a HP-5 capillary column (30 mm × 0.32 mm × 0.25 µm, 150−200°C at 10°C/min, and then 200−270°C at 4°C/min). The standard monosaccharides were treated and measured using the same procedure as described above. 2.3.4. Methylation analysis Methylation analysis of JSP-11 and JSP-11R were performed according to the modified Ciucanu method (Liu, Sun, Yu, & Liu, 2012). The methylated products were monitored by FT-IR until the stretching band of OH ranging from 3200 to 3700 cm−1 disappeared. After the methylated products were hydrolyzed at 120°C in an oil bath by 2 mL TFA (2 M) for 3 h, the hydrolysates were further reduced with NaBH4 and acetylated with acetic anhydride–pyridine. The resulting aditol acetates of partially methylated monosaccharides were identified by GC and GC-MS. 2.3.5. NMR spectroscopy The dried JSP-11 (100 mg) was dissolved with 0.55 mL of D2O in a NMR tube and then the 1H-NMR,

13

C-NMR, HSQC, HMBC and 1H-1H COSY were recorded at

27°C on a Bruker Avance DPX-400 instrument (Fällanden Switzerland). 2.4. Measurement of the immunomodulatory activity 2.4.1. Cell line and cell culture The RAW 264.7 macrophage cell line was supplied by Prof. Jian Liu, Hefei University of Technology. The cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 µg/mL streptomycin, and 100 U/mL penicillin at 37 °C in a humidified incubator with 5% CO2. 2.4.2. Analysis of cell viability

7

The effect of JSP-11 on the viability of RAW 264.7 cells was analyzed in vitro using MTT assay (Zha et al., 2015). The cells were loaded into the 96-well plate with a density of 5 × 104 cells/mL and incubated for 4 h at 37°C in a humidified incubator with 5% CO2. The cells were then treated with JSP-11 at different concentrations ranged from 10 to 400 µg/mL. After 24 h incubation, 20 µL of MTT-PBS solution (5 mg/mL) was added into each well. The plates were further incubated for another 4 h followed by addition of 100 µL of DMSO to each well. The absorbance was measured at 570 nm on the Bio-Rad model 680 Microplate Reader (Pennsylvania, USA). 2.4.3. Determination of NO and cytokines Cells were treated with LPS at 5 μg/mL and JSP-11 at 10, 50 and 200 μg/mL, respectively. After 24 h incubation, the supernatants were collected to determine the levels of NO, TNF-α and IL-1β using commercial kits. 2.4.4. RT-PCR analysis RAW 264.7 cells were seeded in a 24-well plate at a density of 1×106 cells/mL. After 24 h incubation, cells were treated with JSP-11 at the concentration of 10, 50 and 200 μg/mL or LPS at the concentration of 5 μg/mL, respectively. After 12 h incubation, the conditioned cells were harvested for the preparation of total RNA. Total RNA was extracted using TRIZOL reagent (Gibco, BRL), and the RNA was used for cDNA synthesis using iScriptTM cDNA Synthesis kit according to the manufacturer’s instructions. The quantitative real-time PCR was performed on a BIO-RAD MyIQ2 Real Time PCR system (California, USA). The reaction mixture contained 10 μL 2×iTaqTM Universal SYBR® Green supermix, 0.4 μL forward primer,

8

0.4 μL reverse primer, 1 μL cDNA and 8.2 μL PCR grade sterile water. The thermal cycling procedures were set to the initial denaturation at 94°C for 1 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 15 s, and extension at 72°C for 6 s. The cycle threshold (Ct) values were normalized to the expression of GAPDH.

All

primer

sequences

were

5′-GTTGGATTTGGAGCAGAAGTG-3′

listed

as

follows:

(upper

5′-TCTTGTATTGTTGGGCTGAGAA-3′

(lower

5′-TCTTTTGGGGTCCGTCAACT-3′

primer) primer)

(upper

5′-GCAACTGTTCCTGAACTCAACT-3′

(low

5′-AGATAGCAAATCGGCTGACG-3′

(lower

5′-GGTGAAGGTCGGTGTGAACG-3′

(upper

;

primer) primer);

(upper

5′-ACGGCATGGATCTCAAAGAC-3′

(1)

primer) primer)

; primer)

iNOs, and IL-1β, and TNF-α, and

GAPDH, and

5′-CTCGCTCCTGGAAGATGGTG-3′ (lower primer). 2.4.5. Western blot analysis The RAW 264.7 cells were seeded in a 6-well plate at a density of 2 × 106 cells/well and incubated for 4 h. The cells were then treated with JSP-11 at the concentration of 10, 50 and 200 µg/mL or LPS at the concentration of 5 µg/mL for 2 h, respectively. At the end of incubation, the cells were harvested for protein extraction. The proteins in nucleus and cytoplasm were extracted with nuclear and cytoplasmic extraction reagents (Sangon biotech Co. Ltd., Shanghai, China), respectively. The total proteins were extracted with radio immunoprecipitation assay lysis buffer. The protein concentration was determined using the Bradford protein assay reagent (Sangon

9

biotech Co. Ltd., Shanghai, China). Western blot was performed to analyze the levels of ERK1/2, phospho-ERK1/2, JNK, phospho-JNK, p38, phospho-p38, nuclear NF-κB p65, cytoplasmic NF-κB p65, Akt, phospho-Akt, IκB and phospho-IκB according to the previous reports (Li et al., 2015; Xie et al., 2016). 2.4.6. Statistical analysis All experiments were carried out independently in triplicates and the data were expressed as mean ± SEM values. All data were analyzed statistically using one-way analysis of variance. Significant differences were set at p < 0.05

3. Results and discussion 3.1. Structural characterization of JSP-11 3.1.1. Physicochemical properties of JSP11 As shown in Fig.1, JSP-11 exhibited a symmetrical and narrow peak on the size exclusion chromatographic column, indicating this polysaccharide obtained in the present work was a homogeneous fraction. The content of polysaccharide and uronic acid of JSP-11 were determined as 96.7% and 37.8% by phenol-H2SO4 method and m-hydroxydiphenyl

method,

respectively.

The

molecular

weight

of

this

polysaccharide was calculated to be 1.25 × 106 Da. In the FT-IR spectrum of JSP-11 (Fig.S2 in supporting information), the characteristic peaks at 3326, 2938 and 1654 cm−1 corresponded to O-H bending, C-H bending, and C=O bending, respectively. The weak peak at 1401 cm-1 suggested the presence of uronic acid.

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3.1.2. Monosaccharide composition Fig.2 showed the monosaccharide composition of JSP-11. As a result, mannose and galactose were identified with a molar ratio of 2.18:1.00 in the hydrolysate of JSP-11. To determine the type of uronic acid, the carboxyl of uronic acid in polysaccharides was reduced to primary alcohol, giving the reduction product JSP-11R. Results showed that mannose, galactose and glucose were observed with a molar ratio of 2.01:1.00:1.94 in the hydrolysate of JSP-11R. It was interesting that the content of glucose in JSP-11R (39.2%) was in accordance with that of uronic acid in JSP-11 (37.8%), indicating the uronic acid in JSP-11 was glucuronic acid. According to the data above, we can conclude that JSP-11 was mainly composed of mannose, galactose and glucuronic acid. 3.1.3. Methylation analysis To ascertain the composition of glycosidic linkages, JSP-11 and JSP-11R were completely methylated, hydrolyzed and converted to alditol acetates before GC-MS analysis. As the data summarized in Table 1, two types of alditol acetates were observed in the hydrolysate of methylated JSP-11. The two alditol acetates were identified to be 2,4-Me2-Manp and 2,3,4-Me3-Galp in a molar ratio of 1.98:1.00.

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Compared to JSP-11, three alditol acetates of 2,4-Me2-Manp, 2,3,4,6-Me4-Glcp and 2,3,4-Me3-Galp were included in the hydrolysate of methylated JSP-11R with a molar ratio of 2.14:2.03:1.00. According to these data, mannose, galactose and glucuronic acid were deduced to be linked by (1→3,6), (1→6) and (1→) glycosidic bond in JSP-11, respectively. 3.1.4. NMR analysis The details of JSP-11’s structure were further characterized by NMR. The spectra of 1H-NMR,

13

C-NMR, 1H-1H COSY, HSQC and HMBC were shown in Fig.S3-S7

(supporting information). According to the published data (Cong et al., 2014; Li et al., 2015; Li et al., 2014), the 1H-NMR signals at δ 5.35, 4.60 and 4.45 ppm were assigned to the anomeric proton of (1→)-linked α-D-GlcpA, (1→3,6)-linked β-D-Manp and (1→6)-linked β-D-Galp. According to the order of decrease in the chemical shifts of anomeric proton, the (1→)-linked α-D-GlcpA, (1→3,6)-linked β-D-Manp and (1→6)-linked β-D-Galp were assigned as A, B and C, respectively. In the HSQC spectrum, the cross peaks of δ 5.35/97.66, 4.60/101.55 and 4.45/102.12 ppm suggested that the chemical shifts at δ 97.66, 101.55 and 102.12 ppm in the 13

C-NMR spectrum were assigned to the anomeric carbon of residues A, B and C,

respectively. Similarly, according to the analysis of 2D NMR spectra, the other C and H signals were further assigned and shown in Table 2, which were very close to those published in the literatures (Cong et al., 2014; Li et al., 2015; Li et al., 2014; Nie et al., 2011; Smiderle, Sassaki, Van Griensven, & Iacomini, 2013; Wang, Zha, Luo, & Yang, 2010; Zhang, Nie, Yin, Wang, & Xie, 2014). Moreover, three cross-peaks were 12

obviously observed in HMBC spectrum, including A H-1 (δ 5.35 ppm) and B C-3 (δ 72.55 ppm), B H-1 (δ 4.60 ppm) and C C-6 (δ 72.90 ppm), and C H-1 (δ 4.45 ppm) and B C-6 (δ 75.10 ppm). Therefore, the sugar residue A, B and C could be linked by a s equ en c e s how n in Fi g.3 t o f orm the mol ec ul e o f J SP -11.

3.2. Immunomodulatory activity of JSP-11 3.2.1. Cell toxicity of JSP-11 to macrophages As we know, macrophages, an important immunomodulatory effector cells, play a key role in the immune system by keeping homeostasis and providing defense against pathogen invasion (Gamal-Eldeen & Amer, 2007). MTT assay showed that JSP-11 stimulated the proliferation of RAW 264.7 cell over the entire tested concentration range from 10 to 400 µg/mL (Fig.S8 in supporting information). 3.2.2. JSP-11 activates NO, TNF-α and IL-1β secretion from macrophages It is generally acknowledged that pro-inflammatory mediator NO and cytokines TNF-α and IL-1β secreted by activated macrophages were directly involved in defending against pathogen invasion (Commins, Borish, & Steinke, 2010; Kim et al., 2012; Lee et al., 2013). Therefore, the effects of JSP-11 on the production of NO, TNF-α and IL-1β in RAW 264.7 cells were investigated. As shown in Fig.4, the release of NO, TNF-α and IL-1β were significantly promoted by JSP-11 in a dose-dependent manner. When the polysaccharide dosage reached 200 µg/mL, the production of NO, TNF-α and IL-1β increased 140.1 % (Fig 4A), 77.4 % (Fig 4B) and 53.8% (Fig 4C) than those of control group, respectively. These results suggested that 13

macrophages can be activated by JSP-11 in the tested concentration range.

3.2.3. JSP-11 activates mRNA expression of iNOS, TNF-α and IL-1β in macrophages To confirm the activation of macrophages by JSP-11, quantitative RT-PCR analysis was further employed to analyze the mRNA levels of iNOS, TNF-α and IL-1β in macrophages. It has been reported that the induced NO synthase (iNOS) is a critical controller responsible for a major amount of NO synthesis in macrophages (Pacher, Beckman, & Liaudet, 2007). Results showed that the mRNA expression of iNOS was enhanced by JSP-11 in a dose-dependent manner (Fig.4D). Similarly, the mRNA expression of TNF-α and IL-1β were also promoted by JSP-11 in a dose-dependent manner (Fig.4E and 4F). These results further supported the conclusion that macrophages can be activated by JSP-11 in the tested concentration range. 3.2.4. JSP-11 activates NF-κB, MAPKs and PI3K/Akt signaling pathways in macrophages. Nuclear factor κB (NF-κB) is a ubiquitous transcription factor that plays a critical role in the host defenses via regulating inflammation, immune response, cell division and cell apoptosis (Vermeulen, De Wilde, Notebaert, Vanden Berghe, & Haegeman, 2002; Xie et al., 2016). Under normal conditions, most NF-κB keeps in the cytoplasm in its inactive form via binding with IκB. Upon stimulus-induced IκB phosphorylation and degradation, the NF-κB would translocate to the nucleus and thus activate a series of dependent genes encoding iNOs and pro-inflammatory cytokines. As shown in Fig.5A, compared to the cells of normal control, the levels of nuclear NF-κB p65 were

14

remarkably increased and cytoplasmic NF-κB p65 were decreased after the cells were treated with JSP-11, suggesting the fact that JSP-11 stimulated the translocation of NF-κB p65 from cytoplasm to nucleus in a dose-dependent manner. It has been reported that mitogen-activated protein kinases (MAPKs) are evolutionarily-conserved protein kinases that control a series of cellular events such as proliferation, differentiation, growth, migration and apoptosis (Guha & Mackman, 2001; Zha et al., 2015). In mammalian cells, the extracellular signal regulated kinase(ERK1/2), C-Jun-N-terminal kinase (JNK) and p38 are the most representative MAPKs (Guha & Mackman, 2001; Zha et al., 2015). As shown in Fig.5B, the phosphorylation of ERK1/2, JNK1/2 and p38 were elevated by JSP-11 in a dose-dependent manner. The phosphoinositide 3-kinase (PI3K) and the downstream serine/threonine kinase Akt is another signaling pathway involve in the regulation of inflammatory response, cellular activation and apoptosis (Zhang et al., 2008). The present data revealed that the levels of phospho-Akt were dose-dependently enhanced by JSP-11 (Fig.5C). Taken together, these results indicated that JSP-11 activated RAW 264.7 cells to produce NO, TNF-α and IL-1β via regulating NF-κB, MAPKs and P I 3 K / A k t

s i g n a l i n g

p a t h w a y s

( F i g . 6 ) .

4. Conclusion In summary, a new jellyfish polysaccharide was extracted, purified and characterized in the present study. The pharmacological test showed that this polysaccharide has the ability to stimulate immune response via regulating NF-κB,

15

MAPKs and PI3K/Akt signaling pathways. These results suggested that the jellyfish polysaccharide might be a good candidate for the development of new immunomodulatory functional food supplement.

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 31271814), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20130111110001), and the Fundamental

Research

Funds

for

the

Central

Universities

(Grant

No.

2014HFCH0011).

Supplementary data Supplementary data associated with this article can be found in the online version.

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Fig. 1. High performance liquid chromatogram of JSP-11 extracted from jellyfish.

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Fig. 2. Gas Chromatogram of standard monosaccharides (A), JSP -11 (B) and JSP-11R (C). In each figure, the numbers of 1, 2, 3, 4, 5 and 6 represent Rha, Ara, Xyl, Man, Glc and Gal, respectively.

Fig. 3. Predicted structure of the repeating unit of JSP-11.

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Fig. 4. Effects of JSP-11 on production levels of NO (A), TNF-α (B), IL-1β (C) and mRNA levels of iNOS (D), TNF-α (E), IL-1β (F) in RAW 264.7 cells. The group without JSP-11 was used as the normal control, and LPS (5 μg/mL) was used as the positive control group. The data shown are means ± SEM (n = 3). All data were analyzed statistically using one-way analysis of variance. (*) p < 0.05 and (**) p < 0.01 compared with the normal control, respectively.

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A

B

C

Fig. 5. Effect of JSP-11 on NF-κB (A), MAPKs (B) and PI3K/Akt (C) signaling pathway. The group without JSP-11 was used as the negative control, and LPS (5 μg/mL) was used as the positive control group. The data shown are means ± SEM (n = 3). All data were analyzed statistically using one-way analysis of variance. (*) p < 0.05 and (**) p < 0.01 compared with control, respectively.

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Fig. 6. Possible mechanism of JSP-11 activating RAW 264.7 cells.

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Table 1. Methylation analysis data of JSP -11 and JSP-11R. Molar ratio Methylated sugar

Linkage types

Mass fragments(m/z)

2,4-Me2-Manp

1,3,6-Manp

43,59,71,87,101,113,117,129,161,173

2,3,4,6-Me4-Glcp

1-Glcp

43,71,87,101,117,129,145,161,205

2,3,4-Me3-Galp

1,6-Galp

43,58,71,87,101,117,129,161,189,233

Table 2. 1 H NMR and

13 C

Sugar residues (A) α-D-GlcpA-(1→

(B)→3,6)-β-D-Manp-(1→

(C)→6)-β-D-Galp-(1→

JSP-11

JSP-11R

1.98

2.14

-

2.03

1.00

1.00

NMR chemical shifts for the polysaccharide JSP-11. 1

2

3

4

5

6

1H

5.35

3.51

3.72

3.49

4.46

nd

13C

97.66

71.36

72.90

71.66

72.09

177.87

1H

4.60

3.67

3.62

3.51

3.50

3.49/3.67

13C

101.55

75.10

72.55

71.36

71.66

75.10

1H

4.45

3.53

3.72

3.92

3.70

3.61/3.65

13C

102.12

71.55

72.90

68.64

75.10

72.90

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