Separation, purification, structural analysis and immune-enhancing activity of sulfated polysaccharide isolated from sea cucumber viscera

Journal Pre-proof Separation, purification, structural analysis and immuneenhancing activity of sulfated polysaccharide isolated from sea cucumber viscera

Dongda Yang, Fudi Lin, Yayan Huang, Jing Ye, Meitian Xiao PII:

S0141-8130(19)35632-6

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.064

Reference:

BIOMAC 13845

To appear in:

International Journal of Biological Macromolecules

Received date:

19 July 2019

Revised date:

26 October 2019

Accepted date:

7 November 2019

Please cite this article as: D. Yang, F. Lin, Y. Huang, et al., Separation, purification, structural analysis and immune-enhancing activity of sulfated polysaccharide isolated from sea cucumber viscera, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.11.064

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© 2019 Published by Elsevier.

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Separation, purification, structural analysis and Immune-enhancing activity of sulfated polysaccharide isolated from sea cucumber viscera

Dongda Yang1, Fudi Lin1, Yayan Huang1, Jing Ye1, 2 *, Meitian Xiao1, 2 *

Xiamen Engineering and Technological Research Center for Comprehensive Utilization of

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College of Chemical Engineering, Huaqiao university of China, Xiamen, 361021, China

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Marine Biological Resources, Xiamen 361021, Fujian, China

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* Corresponding author: Jing Ye E mail: [email protected] Meitian Xiao E mail: [email protected]

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Abstract A novel sulfated polysaccharide (SCVP-1) was isolated from sea cucumber viscera and purified to elucidate its structure and immune-enhancing ability. SCVP-1 was found to be a homogeneous polysaccharide with a relative molecular weight of 180.8 kDa and composed of total sugars (60.2±2.6%), uronic acid (15.3±1.8%), proteins (6.8±0.8%), and sulfate groups (18.1±0.9%). SCVP-1 consisted of mannose, glucosamine, glucuronic acid, N-acetyl-galactosamine, glucose, galactose and fucose at an approximate molar ratio of 1.00: 1.41: 0.88: 2.14: 1.90: 1.12: 1.24. The fourier transform infrared spectra (FT-IR) and nuclear magnetic resonance (NMR) analyses showed that SCVP-1 was a kind of glycosaminoglycan. And the sulfation patterns of the fucose branches were Fuc2,4S, Fuc3,4S and Fuc0S. The surface morphology of SCVP-1 presented loose and irregular sheet structure formed by aggregation of polysaccharide molecules with spherical structure. Moreover, SCVP-1 promoted the production of nitric oxide (NO) and cytokines (IL-1β, IL-6 and TNF-α) by RAW264.7 cells as well as the expression of related genes (iNOS, IL-1β, IL-6 and TNF-α) and also enhanced their phagocytic activity through TLR4-mediated activation of the MAPKs and NF-κB signaling pathways. This study suggests that sea cucumber viscera are good sources of polysaccharides and SCVP-1 might be a novel immunomodulator.

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Keywords: Sea cucumber viscera, Sulfated polysaccharide, Structure, Immune enhancement, RAW264.7 macrophages

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1.Introduction Sea cucumber, belonging to the class Holothuroidea, is a valuable marine animal for both medicine and food industry. In China, sea cucumber has been considered for a long time a traditional and restorative food with high nutritional and pharmaceutical value [1,2]. Some bioactive ingredients extracted from sea cucumber such as polysaccharides and polypeptides exhibit strong biological activity [3]. In recent years, most of the studies on sea cucumber have used the body wall as raw material. In fact, there are only a few functional studies on sea cucumber viscera, especially in the field of health care and medicine. The viscera of sea cucumber contain proteins, amino acids, polysaccharides, fatty acids, saponins, and other components similar to the body wall [4]. In addition, it contains many active components such as pigments, trace elements, enzymes and beneficial bacteria [3,5]. The viscera of sea cucumber not only have immune-enhancing [6], anti-oxidation [7], anti-fatigue [8] and anti-cancer [9], but also exert hypoglycemic, hypolipidemic, blood nourishing, and kidney-tonifying effects [10–13]. In industrial processing, the viscera of sea cucumber are often considered sea cucumber waste, resulting in the waste of resources and environmental pollution. If the viscera of sea cucumber would be comprehensively utilized, the value of this animal would increase. Polysaccharides are one of the most important bioactive components of sea cucumber. As a by-product of sea cucumber industry, the viscera of sea cucumber contain a certain amount of polysaccharides but has not attracted enough attention yet. In recent years, some researchers have isolated and purified polysaccharides from the intestinal tract of Apostichopus japonicas and presumed that they are chondroitin sulfate polysaccharides [14]. In addition, some studies have demonstrated that Apostichopus japonicas spawn polysaccharides have the ability to inhibit tumor proliferation [15]. However, there is a lack of in-depth studies on the systematic structure

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analysis and related active mechanisms of polysaccharides from sea cucumber viscera. Some studies showed that acidic polysaccharides are one of the most important components of sea cucumber body wall [16] and have abundant biological activities including anti-cancer [17], anti-coagulant [18–20], anti-inflammatory [21], anti-thrombotic [22] and hypolipidemic activities [23]. There are two types of acidic polysaccharides isolated from sea cucumbers: fucosylated chondroitin sulfate (fCS) and sulfated fucans [24]. The fCS has a chondroitin core [→4)-β-D-GlcA-(1→3)-β-D-GalNAc-(1→]n and a unique sulfated fucose side chain attached to the O-3 position of GlcUA residues, and the sulfation patterns of the fucose branches of fCS are the main differences in their structures [25]. In contrast to fCS, sulfated fucans from sea cucumber are linear polysaccharides consisting of α1→3 linked fucose repeating units with various sulfation patterns [26,27]. Although these two polysaccharides have different glycosyl groups，some hydroxyl groups in the polysaccharide chain are sulfated. The structure of the two polysaccharides is unique in the sea cucumber. The level of biological effects of sea cucumber polysaccharides significantly depends on their structural features [28]. Therefore, the characterization of these biological macromolecules, such as monosaccharide composition, molecular weight, pattern of sulfation and types of glycosidic linkages, is particularly important. Till now, many types of polysaccharides from different sources have shown immunoregulatory effects in vitro and vivo and were able to activate the proliferation and cytokine production of mouse B cells and macrophages [29–31]. Immunostimulation is an important strategy to enhance the body’s defense systems. Macrophages play a crucial role in immune defense, self-stabilization and immune surveillance [32]. Activated macrophages can directly kill pathogenic microorganisms, eliminate apoptotic cells and mutant cells, and secrete nitric oxide (NO), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and interleukin 1-β (IL-1β), which play an important role in the innate immune defense and acquired immune response [33–35]. It has been reported that Toll-like receptors (TLRs) are essential for many natural polysaccharide-induced activations of macrophages [36] and, when they bind to polysaccharides, activate multiple signals including the nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathway, further promoting the secretion of cytokines such as IL-1β, IL-6, TNF-α and NO [37,38]. These immunologically active molecules act as endogenous signals of intercellular interactions, secondarily induce the production of other cytokines (IL-2, IL-8, IL-12, Ig-G), and play an important regulatory role in the immune response [39]. In this study, therefore, we first extracted and purified a novel polysaccharide (SCVP-1) from the viscera of sea cucumber. Its structure was characterized by a combination of chemical and instrumental analysis of monosaccharide composition, UV-vis spectra, fourier transform infrared spectra (FT-IR), high performance liquid gel permeation chromatography (HPGPC), and nuclear magnetic resonance (NMR) spectroscopies. Its morphology was further analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). In addition, we examined whether SCVP-1 has the capacity to enhance the immune function of macrophages. Moreover, we investigated the role of TLR4 as a candidate receptor in SCVP-1-induced immunomodulation of macrophages and its related signaling pathways. 2. Materials and methods 2.1 Materials and chemicals

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Sea cucumber viscera was provided from Xiapu sea cucumber breeding base in Fujian city of the Fujian province of China. DEAE (diethylaminoethyl)-52 and Sephadex G-200 were purchased from Pharmacia Chemical Co (Uppsala, Sweden). The dextran standards with molecular masses of 410,000, 270,000, 150,000, 50,000, 25000 and 5,000 respectively) , monosaccharide standards of D-mannose (Man), D-galactose (Gal), D-glucose (Glc), L-fucose (Fuc), D-xylose (Xyl), D-Glucuronic acid (GlcUA), N-Acetyl-D-Galactosamine hydrochloride (GalNAc) and D-Glucosamine (GlcN), Lipopolysaccharide (LPS), polymyxin B (PMB) and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St.Louis, USA). 1-Phenyl-3-methyl-5-pyrazolone (PMP, 99%) was purchased from Sigma Chemical Co. (China). RAW264.7 cells were purchased from the cell bank of Shanghai Institute of Cell Biology (Shanghai, China). Fetal bovine serum (FBS) and DMEM (Dulbecco’s modified eagle medium) high glucose medium were obtained from Yeasen (Shanghai, China) and GE Healthcare Life Sciences (Beijing, China). Assay kits for 1L-1β, TNF-ɑ, IL-6 were purchased from ExCell technology Co. (Shanghai, China). Antibodies against ERK1/2, p-ERK1/2, P38, p-P38, JNK, p-JNK, P65 and p-P65 were purchased from Affinit Bioscience Co. (Cincinnati, OH, USA). GAPDH and goat anti-rabbit IgG-HRP were obtained from Cell Signaling Technology (Danvers, MA, USA). TLR4 inhibitor (TAK-242), p38 MAPK inhibitor (SB239063), ERK1/2 inhibitor (CI-1040), JNK1/2 inhibitor (SP600125) and NF-κB (BAY11-7082) were obtained from MedChem Express (NJ, USA). All other chemical reagents were of analytical grade and obtained locally.

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2.2 Polysaccharide extraction and purification Polysaccharides of sea cucumber viscera were prepared according to the method described by Chang et al. with little modifications [40]. Briefly, the viscera of sea cucumber were washed, lyophilized, and ground to powder. Then, the dried sample was dissolved in 30 × (v/w) of 0.1 M sodium acetate buffer, pH 6.0, containing 5% papain, 5 mM EDTA, and 5 mM cysteine, and hydrolyzed at 60℃ for 24 h. The mixture was centrifuged (2,000 × g for 15 min), and the clear supernatant was added to 5% cetylpyridinium chloride and kept at room temperature for 24 h. The precipitate was collected by centrifugation (2,000 × g for 15 min) and dissolved in a solution of 3 M NaCl: ethanol (100:15, v/v). After mixing it with 4 × 95% ethanol, the mixture was kept at 4℃ for 24 h. The precipitate was collected by centrifugation (2,000 × g for 15 min), washed with 80% and 90% ethanol for some times, and then dialyzed, lyophilized to yield crude polysaccharides. The crude polysaccharides were then applied to a DEAE-52 cellulose chromatography column (2.4 × 40 cm) at a flow rate of 1 mL/min; elution was performed with 0–1.0 M NaCl sequentially. All fractions were confirmed by the phenol-sulfuric acid assay. The fraction with the highest total sugar content was collected, further purified by the Sephadex G-200 column (1.5 × 100cm), and eluted with 0.5 M NaCl at a rate of 0.5 mL/min. Finally, the purified polysaccharide fraction was collected, dialyzed, lyophilized and designated as SCVP-1. Total carbohydrates were determined using the phenol-sulfuric acid colorimetric method [41]. The uronic acid content was assessed using the carbazole-sulfuric acid method [42]. The protein content was determined using the Bradford method [43]. The amount of sulfate was estimated using the BaCl2 gelatin method after hydrolysis of the polysaccharides with 0.5 M HCl [44].

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2.3 Homogeneity and molecular weight The purity of polysaccharide fraction SCVP-1 was determined by cellulose acetate membrane electrophoresis (CAME). Briefly, a cellulose acetate membrane (2 × 8 cm) was immersed in 0.1 mol/L HCl solution for 30 min. The excess buffer was removed with two layers of filter paper. The electrophoresis was carried out under 2.5 V/cm, 0.4–0.6 mA/cm for 200 min. The membrane was stained with 0.2% Alcian blue solution containing 10% ethanol, 0.1% glacial acetic acid and 0.03 M MgCl2 [45]. The molecular weight of SCVP-1 was evaluated by high performance liquid gel permeation chromatography (HPGPC) on an Agilent 1250 Ⅱ system (Palo Alto, CA, USA) with a refractive index detector and equipped with a Shodex Sugar KS-804 column (8 × 300 mm, 7 μm) using ultrapure water as mobile phase. The SCVP-1 concentration was 5 mg/mL, and the injection volume was 20 μL. The column temperature was set at 60℃, and the flow rate was 0.5 mL/min. Dextran standards with molecular masses of 410,000, 270,000, 150,000, 50,000, 25000 and 5,000 were used to calibrate the column and establish a standard curve.

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2.4 Monosaccharide composition analysis SCVP-1 (0.5 mg) was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110℃ for 2 h in a sealed test tube. The acid was removed by repeated evaporation with methanol for three to four times. After evaporation, the dried samples were reconstituted in deionized water. One hundred microliters of the sample solution, 100 μL 0.5 M 1-Phenyl-3-methyl-5-pyrazolone (PMP), and 100 μL 0.3 M NaOH solution were mixed and incubated at 70℃ for 1 h. After cooling, the pH was adjusted to 7.0 with 0.3 M HCl, and 1 mL of chloroform was added to extract PMP three times. The top aqueous phase was collected for High Performance Liquid Chromatography (HPLC) analysis. A Waters SunFire C18 column (4.6 × 250 mm, 5 µm) with UV detection at 252 nm was used for HPLC analyses. The mobile phase A and B (v/v, 76:24) contained 0.1 M phosphate buffer (pH 6.7) and acetonitrile, respectively. The flow rate was 0.5 mL/min, and the injection volume was 20 μL [46]. 2.5 UV and FT-IR spectral analysis The UV scanning spectra were determined by spectrophotometer (UV-1800, Metash, China) at the wavelength of 190–350 nm. The SCVP-1 solution (0.1 mg/mL) was prepared for ultraviolet scanning. The FT-IR spectra of SCVP-1 (in KBr pellets, 3 mg of sample and 250 mg KBr) was measured with a Thermo Scientific Nicolet iS50 instrument in a range of 4,000-400 cm-1. 2.6 NMR spectroscopy SCVP-1 was treated with deuterium (D2O, 99.9%) and lyophilized with D2O for three times to exchange protons. After the treatment, SCVP-1 was placed in a 5 mm NMR tube and dissolved in 0.5 mL of D2O. NMR-spectra were recorded with a Brucker AVANCE III 850 MHz NMR (Karlsrhue, Germany). The analysis included a 1D spectrogram (1H, 13C) and 2D spectrogram (1H-1H COSY, 1H-13C HSQC). 2.7 Surface morphology observation The powder of SCVP-1 was placed on the sample stage, and coated with a thin layer of gold.

Journal Pre-proof It was examined by a Sigma 300 scanning electron microscope (ZEISS Company, Germany). The sample was shot in enlargement multiple 500, 2,000 and 5,000. SCVP-1 was diluted to the final concentration of 5 μg/mL in distilled water. The atomic force microscopy (AFM) was manufactured by Nano-Wizard Ⅱ(JPK, Germany). 5 μL of solution were dropped onto freshly cleaved mica and air dried for more than 1.5 h. The newly prepared samples were mounted on the AFM platform and imaged under tapping mode in air.

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2.8 Cell culture The murine macrophage cell line RAW264.7 was cultured in Dulbecco's Modified Eagle Medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). The RAW 264.7 cells were maintained at 5% CO2 in a humidified incubator at 37℃.

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2.9 Cell viability assay The effect of different concentrations of SCVP-1 on the viability of RAW264.7 cells was measured by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-bromo diphenyltetrazolium (MTT) method. As lipopolysaccharide (LPS) is one of the most effective stimulators of the immune system, this molecule was used as a positive control. RAW264.7 cells were cultured in 96-well plates at 4.5×105 cells/well and incubated for 24 h at 37℃. Then, cells were treated with various concentrations of SCVP-1 (50, 100, 200 and 400 μg/mL), LPS (1 μg/mL) as a positive control, or medium alone for the control group for 24 h. After this period, cells were incubated with 20 μL MTT solution (0.5 mg/mL) in each well for 4 h in the dark; the medium was then discarded, and 200 μL Dimethyl sulfoxide (DMSO) was added into each well to dissolve the formazan crystals. Absorbance was recorded at a wavelength of 570 nm.

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2.10 Assay for nitric oxide and cytokine secretion The RAW264.7 cells (4.5×105 cells/well) were seeded in 24-well plates for 24 h and then treated with different concentrations of SCVP-1 (50,100, 200 and 400 μg/mL) and LPS (1 μg/mL) for 24 h. After incubated, nitrite levels were measured by the Griess assay. The supernatants were mixed with an equal volume of Griess reagent, followed by reaction for 10 min at room temperature. The absorbance was evaluated at 540 nm by a microplate reader. The level of NO secretion was measured by a sodium nitrite standard curve. In addition, the level of cytokines (IL-1β, IL-6 and TNF-α) in the supernatants were determined by ELISA Kits according to manufactures’ instructions. Polymyxin B (PMB), a specific LPS antagonist, can bind to lipid portion of LPS to inhibit its activity. Therefore, in order to verify if the effects of SCVP-1 were due to endotoxin contamination, PMB was used. Briefly, cells were treated with SCVP-1 (200 μg/mL) or LPS (1 μg/mL) alone or with PMB (30 μg/mL) for 24 h. After incubation, the level of NO production was measured by Griess assay. 2.11 Phagocytosis assay RAW264.7 cells (4.5×105 cells/well) were seeded in 96-well plate. After incubation for 24 h, the medium was discarded, and cells were treated with virous concentrations of SCVP-1 (50, 100, 200 and 400 μg/mL), LPS (1 μg/mL) as a positive control, or medium alone for the control group.

Journal Pre-proof After 24 h, the supernatant was discarded. 100 μL of neutral red solution (0.1%, w/w) were added to each well and incubated at 37℃ for 1 h. Then the supernatant was discarded, followed by washing with phosphate-buffered saline (0.01 M, pH 7.4) three times. 100 μL of cell lysis solution (ethanol/acetic acid, 1:1, v/v) were added into each well and kept at room temperature for 2 h. The absorbance was measured at a wavelength of 540 nm using a microplate reader.

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2.12 Quantitative analysis of cytokine mRNA expression The mRNA expression of IL-1β, IL-6, iNOS, and TNF-α was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). RAW264.7 cells were seeded at 5×105 cells/well in 6-well plate. After 24 h, cells were incubated with SCVP-1 (50, 100, 200 and 400 μg/mL) or LPS (1 μg/mL) for another 24 h. Then, total RNA was extracted using Beyozol reagent (Beyotime, Shanghai, China). The purity and concentration of RNA were measured by a spectrophotometer. The construction of cDNA was performed using a Revert Aid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. The following sequences for PCR primers from 5’ to 3’ end were used: IL-1β: forward, 5’-ATGGCAACTATTCCTGAACTCAACT-3’, IL-1β: reverse, 5’-CAGGACAGGTATAGATTCTTTCCTTT-3’; IL-6: forward, 5’-TTCCTCTCTGCAAGAGACT-3’, IL-6: reverse, 5’-TGTATCTCTCTGAAGGACT-3’; iNOS: forward, 5’-CCCTTCCGAAGTTTCTGGCAGCAGC-3’, iNOS: reverse, 5’-GGCTGTCAGAGCCTCGTGGCTTTGG-3’; TNF-α: forward, 5’-ATGAGCACAGAAAGCATGATC-3’, TNF-α: reverse, 5’-TACAGG CTTGTCACTCGAATT-3’; β-actin: forward, 5’-TGGAATCCTGTGGCATCCATGAAAC-3’, β-actin: reverse, 5’-TAAAACGCAGCTCAGTAACAGTCCG-3’. The real-time quantitative PCR reaction was performed with the Fast Start DNA Master SYBR Green I kit according to the manufacturer’s instructions. The β-actin gene was used as an internal reference.

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2.13 Western blot analysis After being treated with SCVP-1 (50, 100, 200 and 400 μg/mL) or LPS (1 μg/mL ) for 24 h, RAW264.7 cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% Sodium dodecyl sulfate (SDS), pH 7.8) containing 0.1mM phenyl methyl sulfonyl fluoride (PMSF). The extract was centrifuged at 4°C and 12,000 × g for 15 min, and the supernatant was used for Western blot analysis. The amount of protein was determined by BCA Protein Assay Kit (Pierce, Brooks, USA). The protein was separated through 10% SDS-polyacrylamide gel electrophoresis (PAGE). Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, p65, phosphorylated ERK, phosphorylated JNK, phosphorylated p38, and phosphorylated p65 were evaluated by Western blot analysis with anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38, anti-phospho-p65, anti-ERK, anti-JNK, anti-p38, and anti-p65 antibodies. The target bands were detected by an enhanced chemiluminescence (ECL) kit according to the manufacturer’s instructions and imaged with Image Lab software under ChemiDoc TM imaging system. 2.14 Inhibition of TLR4, MAPKs and NF-κB using specific inhibitors In order to verify the effect of TLR4 in SCVP-1-induced RAW264.7 cells, the cells were incubated with or without TLR4 inhibitor TAK-242 (10 μg/mL) for 1 h. Besides, to identify the

Journal Pre-proof signal transduction pathways underlying SCVP-1-induced macrophage activation, the cells were treated with or without ERK1/2 inhibitor CI-1040 (40 μM), JNK1/2 inhibitor SP600125 (40 μM), p38 inhibitor SB239063 (40 μM), and NF-κB inhibitor BAY11-7082 (20 μM) for 2 h. After this pretreatment, SCVP-1 (200 μg/mL) or LPS (1 μg/mL) were added to RAW264.7 cells. The secretion of NO and cytokines were determined by Griess reagent and ELISA kit, as previously described in subsection 2.10.

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2.15 Statistical analysis All experiments were repeated at least three times. The data are expressed as means ± standard deviation (SD) and were analyzed by one-way analysis of variance (ANOVA) using SPSS software. Analyses of multiple comparisons between two groups were performed using the Student’s t-test. p＜0.05 was considered statistically significant and p＜0.01 was regarded as highly significant.

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3. Results and discussion 3.1 Polysaccharide isolation and chemical analysis At present, the chemical and enzymatic hydrolysis methods are commonly used to extract polysaccharides from sea cucumber [47,48]. However, chemical hydrolysis may further degrade polysaccharides by alkali and easily desulfurize them. On the contrary, enzymatic hydrolysis has the advantages of being performed under mild production conditions, high safe, and low cost, neither polluting the environment nor producing organic residues [49]. Therefore, in this study, we used papain hydrolysis combined with ethanol precipitation and quaternary ammonium salt precipitation to extract polysaccharides from the viscera of sea cucumber. The yield of crude polysaccharides obtained from sea cucumber viscera was approximately 4.9%. Then, crude polysaccharides were separated by a DEAE-52 cellulose column and eluted with distilled water and 0.1 M, 0.3 M, 0.5 M, 0.7 M and 1.0M NaCl. As shown in Fig 1, the elution curve of 0.7 M NaCl has a single and symmetrical peak, presenting a high yield and better immune activation activity in vitro than the other fractions in our pre-experiment of biological activity (Data were shown in Fig. S1a, b, c). Hence, we chose the fraction eluted by 0.7 M NaCl for further purification by gel filtration chromatography using Sephadex G-200 (Fig. 2). The main fraction of the viscera of sea cucumbers was collected and named SCVP-1. SCVP-1 contained different amounts of total sugar (60.2±2.6%), uronic acid (15.3±1.8%), proteins (6.8±0.8%), and sulfate groups (18.1±0.9%). The purity and molecular weight of SCVP-1 were confirmed by CAME and HPGPC. Fig. 3A shows that SCVP-1 exhibited a single dot and migrated faster than chondroitin sulfate A. Thus, we speculated that the sulfate content of SCVP-1 was higher than chondroitin sulfate A. The HPGPC chromatograms of SCVP-1 (Fig. 3B) show that this fraction has a single and symmetrical peak, with a peak area of 92.51%. Both Fig. 3A and Fig. 3B indicate that SCVP-1 was homogeneous. The average molecular weight was calculated as approximately 180.8 kDa with reference to the dextran series standard samples of known molecular weight. After hydrolysis of SCVP-1 by 2 M TFA and derivatization by 0.1 M PMP, we analyzed the monosaccharide composition of SCVP-1 through HPLC. As shown in Fig.4B, SCVP-1 was mainly composed of Man, GlcN, GlcUA, GalNAc, Glc, Gal, and Fuc with a molar ratio of 1.00: 1.41: 0.88: 2.14: 1.90: 1.12: 1.24, respectively, based on the retention time of monosaccharide standards (Fig.4A). The results of monosaccharide composition of SCVP-1 were similar to those

Journal Pre-proof of a previous study in which polysaccharides from the sea cucumber A. japonicus were isolated from the body wall [48]; however, the monosaccharide content was slightly different, probably because Liu et al. isolated polysaccharides from the body wall of the sea cucumber, whereas we extracted them from the viscera.

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3.2 Preliminary structural characterization As shown in Fig. 5, there was no obvious absorption detected at 280 and 260 nm, indicating the proteins and nucleic acids had been basically eliminated in SCVP-1. The FT-IR spectrum of SCVP-1 is shown in Fig. 6. The signal at 3,400 cm-1 has a broad intense peak that is attributed to the stretching of O-H. The band at 2,930 cm-1 reflects the stretching vibration of C-H representing the fucose methyl group. Bands at 1,650 cm-1 and 1,070 cm-1 were assigned to the asymmetric stretching vibration of C=O (-COO-) and C-O-C, respectively, that indicated the presence of uronic acid. The signal at 1,410 cm-1 was attributed to the stretching of C-N indicating the acetamido group of the N-acetyl-D-galactosamine. The absorptions at 1,260 cm-1, 854 cm-1 and 584 cm-1 revealed the presence of sulfate in SCVP-1, and the band at 1,260 cm-1 was assigned to the asymmetric stretching vibration of S=O, which at 584 cm-1 was caused by the stretching vibration of S-O. Besides, the peak at 854 cm-1 was attributed to the stretching of the symmetric C-O-S of sulfate at C-4 axial might indicate the pattern of the sulfates, which represented the presence of 4-O-sulfated Fuc and/or GalNAc. These results confirmed that the polysaccharides of the viscera of sea cucumber were substituted by sulfate esters. In the 1D 1H NMR spectra of SCVP-1 (Fig. 7A), the strong signals at 1.98 ppm and 1.27 ppm were easily identified as the characteristic signals of methyl protons of GalNAc and Fuc, respectively. The area ratio of GalNAc and Fuc signals was 1.00:0.75, which was obtained from the integration of the signals. The chemical shifts at 5.0–5.8 ppm were due to the anomeric proton signals, which indicated the presence of the α-configuration of sulfated fucose residues with different sulfation patterns. The three major signals at 5.61 ppm, 5.32 ppm and 5.09 ppm were assigned to 2,4-O-disulfated fucose (Fuc2,4S), 3,4-O-disulfated fucose (Fuc3S) and non-sulfated fucose (Fuc0S), respectively. Moreover, the ratio of Fuc2,4S, Fuc3,4S and Fuc0S in SCVP-1 was 0.05:0.12:0.07. Other proton signals at the region of 3.5–4.5 ppm were attributed to protons of the C2-C6 of the glycosidic ring. However, these signals were ambiguous and overlapping, which rendered difficult the obtention of accurate information. Therefore, 1H-1H COSY spectra of SCVP-1 (Fig. 7B) was shown to confirm various proton correlations of different fucose residues. SCVP-1 showed two clearly distinguishable signals from the sulfated fucose in the anomeric region. The cross-peaks between anomeric protons H1 and H2 of different sulfated fucose, e.g. a1/a2 from Fuc2,4S, b1/b2 from Fuc3,4S and c1/c2 from Fuc0S. The signals at 5.61/4.39 ppm and 5.32/3.72 ppm in the 1H spectra of SCVP-1 were assigned to H1/H2 of Fuc2,4S and Fuc3,4S, which were related to 1H-1H COSY spectra. The data of the remaining proton signals in the glycosidic ring were summarized in Table 1. In the 13C NMR spectra of SCVP-1 (Fig. 7C), the anomeric carbon signals at 103.82 and 99.74 ppm were assigned to GlcUA and GalNAc, respectively. However, the Fuc signal was very fuzzy and overlapped in the 95-105 ppm region, due to its complex sulfation patterns. A signal at 51.37 ppm was attributed to C-2 of pyranosyl ring connected with N-acetyl group. The chemical shift at 174.37 ppm was assigned to carbonyl carbon of β-GlcUA and/or β-GalNAc. Signals at 22.59 ppm and 15.98 ppm were attributed to methyl carbon of N-acetyl group and methyl carbon

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of fucose residues, respectively. The signal at 66.50 ppm was attributed to the sulfated C-6 of GalNAc. In addition, a signal at 61.02 ppm indicated a non-sulfated C-6 in GalNAc. Moreover, the backbone carbon signals observed in the spectra of SCVP-1 was very similar to those obtained previously for many other fucosylated chondroitin sulfates isolated from the sea cucumber body wall [50, 51]. Heteronuclear singular quantum correlation (HSQC) can be used to determine the results of 1H-1H COSY spectra. In order to obtain more accurate structure information of SCVP-1, the chemical shifts of carbon in different monosaccharide residues were demonstrated by HSQC spectra based on the correlation signals produced by adjacent proton and carbon. As shown in Fig. 7D, signals d1, d2, d3, d4 and d5 were from (H-1, C-1), (H-2, C-2), (H-3, C-3), (H-4, C-4) and (H-5, C-5) correlations of GlcUA. Signals e1, e2, e3, e4, e5 and e6 were from (H-1, C-1), (H-2, C-2), (H-3, C-3), (H-4, C-4), (H-5, C-5) and (H-6, C-6) correlations of GalNAc. In addition, the signal of e’6 was attributed to non-sulfated GalNAc. The chemical shifts of H-4 and H-6 in GalNAc were in the down-field, indicated that they were sulfated at the 4-O- and 6-O- positions. The data of C/H chemical shifts of SCVP-1 were presented in Table 1. Overall, according to the above structural analysis, SCVP-1 contained three main monosaccharides, including GlcUA, GalNAc and Fuc, and the backbone structure of SCVP-1 was very similar to some fucosylated chondroitin sulfates isolated from the body wall of sea cucumber. Therefore, we speculated that SCVP-1 is a kind of glycosaminoglycan which has a chondroitin sulfate-like structure and the sulfation occurred at the 4-O- and 6-O- positions of GalNAc. Moreover, SCVP-1 has fucose branched chains with different sulfation patterns (Fuc2,4S, Fuc3,4S and Fuc0S).

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3.3 Surface morphology observation of SCVP-1 We further observed the surface morphology of SCVP-1 by SEM and AFM. SEM mainly uses secondary electron signal imaging to observe the surface morphology of samples [52]. The SEM micrograph of SCVP-1 is presented in Fig. 8, SCVP-1 presented loose and irregular sheet structure under the 500-fold magnification condition (Fig. 8a). At the 2000-fold magnification, the surface morphology of SCVP-1 was rough and presented a state of curl and aggregation by many point-like polysaccharides molecules (Fig. 8b). After further magnification to 5000-fold, several irregular spherical lumps gathered on the surface of SCVP-1 (Fig. 8c). These loose sheet-like microstructures of SCVP-1 explain the spongy morphology under macroscopic conditions. As a novel material structure analysis method, AFM has gradually become an important means to characterize the advanced structure of biological macromolecules such as DNA and polysaccharides. AFM enables the observation of the surface morphology of substances and quantitative analysis of nano-scale protuberances on the surface of substances [53]. Moreover, it can be used to study the macromolecular structure of organisms. The AFM diagram and the amplitude error diagram of SCVP-1 are presented in Fig. 9a and Fig. 9b, respectively. It is clearly observed that SCVP-1 was formed by aggregation of polysaccharide molecules with spherical structure，suggesting that SCVP-1 may be a spherical chain conformation. In the 3D AFM diagram of SCVP-1(Fig. 9c), it can be observed that the size of polysaccharides is not completely uniform. A number of polysaccharides forms an aggregate and becomes a surface protuberance. The diameter of the aggregates is approximately 258.6–911.8 nm and their height is approximately 50.1–100.1 nm. However, in general, the height of a single polysaccharide

Journal Pre-proof chain is 0.1-1 nm. The SCVP-1 molecular aggregation may occur due to the fact that the SCVP-1 molecules contain a large amount of uronic acid and have a negative charge. Mica sheet itself also has negative charge. At a higher concentration, both of them have a greater repulsion force, which leads to the aggregation of SCVP-1. In addition, SCVP-1 itself contains a large number of hydroxyl groups, and the intramolecular forces further promote the formation of aggregates of SCVP-1.

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3.4 Effects of SCVP-1 on cell viability of RAW264.7 cells Macrophages are the main components of the mononuclear phagocyte system. They can not only initiate innate immune response, but also participate in cellular immune responses. The activated macrophages can phagocytose pathogenic microorganisms, process and present antigens, and simultaneously synthesize and secrete chemokines and cytokines to enhance the body's immune defense capabilities [33,34,36]. Therefore, the viability of RAW264.7 macrophages treated with various concentrations of SCVP-1 (50, 100, 200, and 400 μg/mL) for 24 h was evaluated by the MTT assay. As shown in Fig. 10A, both SCVP-1 and LPS exhibited no cytotoxic effect on RAW264.7 cells within the tested concentration range, which indicated that these concentrations of SCVP-1 can be used for further studies. Besides, SCVP-1 increased the proliferation of RAW264.7 cells in a dose-dependent manner. At the SCVP-1 concentration of 400 μg/mL, cell viability reached a maximum of 119.2±3.6%, which enhanced cell growth by nearly 20%.

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3.5 Effects of SCVP-1 on production of NO and cytokines in RAW264.7 cells Activated macrophages can secrete a series of chemokines and cytokines, which play an important role in activating adaptive immune responses and regulating other immune responses [32]. Thus, the Griess and ELISA assays were used to quantify NO and IL-1β, IL-6 and TNF-α secretion, respectively, in the culture medium of RAW264.7 cells. Fig. 10B shows the effects of SCVP-1 on the level of NO of RAW264.7 cells. SCVP-1 significantly induced NO secretions in a concentration-dependent manner as compared to control group (p ＜0.05, p＜ 0.01). In particular, the NO production in high-dose (400 μg/mL) SCVP-1-treated groups was 37.32±0.97 μM, which was slightly lower than that of the positive control LPS (1 μg/mL)-treated group (42.11±1.29 μM). In addition, SCVP-1 dramatically promoted IL-1β (Fig. 10C), IL-6 (Fig. 10D) and TNF-α (Fig. 10E) secretion in a dose-dependent manner as compared with control group (p ＜0.05, p＜0.01). Importantly, IL-1β, IL-6, and TNF-α secretion after treatment with a high-dose (400 μg/mL) of SCVP-1 was notably increased to 28.79±1.80, 289.36±8.77, and 673.56±15.72 pg/mL, respectively, which were the closest concentrations to those of the positive control LPS (1 μg/mL)-treated group. Taken together, these results indicated that SCVP-1 might display immune-enhancing activity by inducing the production of NO and cytokines in RAW264.7 cells. Furthermore, PMB was used to exclude the possibility of contamination of SCVP-1 with LPS by determining the production of NO in RAW264.7 cells after treatment with SCVP-1 (200 μg/mL) in the presence or absence of PMB. As shown in Fig. 10F, the combination of PMB and LPS significantly inhibited LPS-induced NO production in RAW264.7 cells (p＜0.01), whereas PMB did not affect the activation of macrophages by SCVP-1. These results demonstrated that SCVP-1 itself induced NO production in macrophages, rather than LPS endotoxin contamination.

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3.6 Effects of SCVP-1 on the phagocytic activity of RAW264.7 cells Macrophages are highly conserved phagocytes in the process of continuous evolution. Phagocytosis is one of the most basic defense mechanisms [54]. As a research hotspot in the field of immunology, the phagocytosis ability can reflect the activity of macrophages and play an extremely important role in the defense response against pathogenic bacteria and the maintenance of homeostasis. Neutral red is an acid-base indicator of living cells; when it enters the cells, it reacts with lysosomes, producing a red color. The neutral red test is based on the neutral red dye intake, thus reflecting the phagocytic activity of the cell [55]. Therefore, effects of SCVP-1 on the phagocytic activity of RAW264.7 cells were investigated by the neutral red uptake assay. As shown in Fig. 11, SCVP-1 distinctly augmented the rate of phagocytic in a dose-dependent manner as compared with control group (p＞0.05), which indicated that SCVP-1 has a macrophage phagocytosis-enhancing function. The phagocytic rate at the high-dose (400 μg/mL) of SCVP-1 was approximately 13% higher than that of the control groups and slightly higher than that of the positive control LPS (1 μg/mL)-treated group.

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3.7 Effects of SCVP-1 on iNOS, IL-1β, IL-6 and TNF-α mRNA expression in RAW264.7 cells The activation of macrophages is closely related to gene expression of immune-related cytokines. To further verify the effects of SCVP-1 on NO, IL-1β, IL-6 and TNF-α at the molecular level, the expression of iNOS, IL-1β, IL-6 and TNF-α was determined by RT-PCR. As shown in Fig. 12A, the mRNA expression level of iNOS was significantly increased in a dose-dependent manner as compared with control group (p＜0.05, p＜0.01) when RAW264.7 cells were treated with SCVP-1. Besides, a previous study reported that polysaccharides could induce NO production by RAW264.7 cells by up-regulating the iNOS mRNA expression [56], which is in accordance with the results described in Section 3.5. In addition, the mRNA expression of cytokines in RAW264.7 cells had a similar trend to that of iNOS mRNA expression, with SCVP-1 also promoting IL-1β, IL-6, and TNF-α mRNA expression in a dose-dependent manner (Fig. 12B–D). Treatment with a high-dose (400 μg/mL) of SCVP-1 increased 8–10-fold the mRNA expression levels of IL-1β, IL-6, and TNF-α compared with that of the control group (p＜0.01). These results showed that SCVP-1 induced the secretion of cytokines (NO, IL-1β, IL-6, and TNF-α) by up-regulating the expression of iNOS, IL-1β, IL-6, and TNF-α. 3.8 TLR4 may participate in SCVP-1-induced macrophages activation TLRs are a class of important pattern recognition receptors. They are composed of type I transmembrane proteins, transmembrane domains, and cytoplasmic Toll interleukin-1 receptor domains, which mediate the recognition of pathogen-associated molecular patterns [57]. TLRs are widely distributed and can be expressed on the surface of mammalian cells such as macrophages, dendritic cells, NK cells, and lymphocytes [58]. Among TLRs, many studies have shown that TLR4 could mediate the activation of macrophages upon polysaccharide induction [59,60]. To verify whether TLR4 was involved in SCVP-1-induced macrophage activation, we observed the effects of TLR4 inhibitor TAK-242 on NO and cytokines secretion induced by SCVP-1 and LPS. As shown in Fig. 13A, after treatment with TAK-242, NO production of RAW264.7 cells induced by LPS (1 μg/mL) and SCVP-1 (200 μg/mL) was significantly

Journal Pre-proof decreased 7.26±0.31 and 6.52±0.45 μM, respectively (p＜0.01) compared with that of the positive control LPS-treated group (16.63±0.59 μM) and SCVP-1-treated group (9.18±0.61 μM). A significant decrease was also observed in the secretion of IL-1β and IL-6 by RAW264.7 cells treated with LPS or SCVP-1 and TAK-242 in comparison to that of LPS- or SCVP-1-treated groups (Fig. 13B and Fig. 13C). When RAW264.7 cells were treated with SCVP-1 and TAK-242, relatively less IL-1β (11.87±0.55 pg/mL) and IL-6 (98.32±3.16 pg/mL) were secreted (p＜0.01) compared with the levels secreted by the SCVP-1-treated group (21.27±1.02 and 256.44±6.89 pg/mL, respectively). A similar trend was also observed in TNF-α secretion by RAW264.7 cells treated with LPS or SCVP-1 and TAK-242 (Fig. 13D). Altogether, these results demonstrated that TLR4 participated in SCVP-1-induced macrophage activation.

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3.9 MAPKs pathway participates in SCVP-1-induced macrophage activation MAPKs is a serine/threonine protein kinase widely expressed in mammals. Classical MAPKs include three main subunits: ERK1/2, p38, and JNK1/2. They participate in the basic processes of cell proliferation, differentiation, stress response, and apoptosis, and can phosphorylate related cytoplasmic proteins and activate the expression of related genes, thus participating in a series of cell responses [61]. Moreover, MAPKs play an important role in the transcriptional activation of NF-κB. To elucidate the molecular mechanism underlying SCVP-1-induced macrophage activation, the protein expression and phosphorylation of ERK1/2, p38, and JNK1/2 were detected by western blot. As shown in Fig. 14A, compared with the GAPDH, LPS (1 μg/mL) and SCVP-1 (50, 100, 200, and 400 μg/mL) treatment had no significant effect on the expression of total ERK1/2, p38, and JNK1/2 but the phosphorylated ERK1/2, p38, and JNK1/2 protein content was significantly different. The phosphorylation levels of ERK1/2, p38, and JNK1/2 increased with different concentrations of SCVP-1, showing a dose-dependent manner increase at SCVP-1 concentrations of 100, 200 and 400 μg/mL. When SCVP-1 concentration was 400 μg/mL (Fig. 14B), the expression level ratio of p-ERK1/2/ERK1/2, p-p38/p38, and pJNK1/2/JNK1/2 were approximately 1.63, 1.43 and 1.51-fold higher (p＜0.01), respectively, than that of the control group. Additionally, the phosphorylation level of these three proteins increased significantly under LPS (1 μg/mL) treatment. Furthermore, we used inhibitors of specific MAPKs signaling pathways to further validate their involvement in SCVP-1-induced macrophage activation. As shown in Fig. 14C, ERK1/2 (CI-1040), p38 (SB239063), and JNK1/2 (SP600125) inhibitors significantly blocked NO production induced by SCVP-1 (200 μg/mL) (p＜0.01). Similarly, the addition of these inhibitors significantly reduced NO release induced by LPS (1 μg/mL). In addition, these three inhibitors dramatically reduced the amount of IL-1β (Fig. 14D), IL-6 (Fig. 14E), and TNF-α (Fig. 14F) secreted by RAW264.7 cells treated with SCVP-1 (p＜0.01). Therefore, it can be inferred that SCVP-1 played an immune-stimulating role by increasing ERK1/2, p38, and JNK1/2 phosphorylation and signaling the MAPKs pathways. 3.10 NF-κB pathway participates in SCVP-1-induced macrophage activation As a major regulator of innate immunity, adaptive immunity, and inflammation, NF-κB is a protein family composed of complex polypeptide subunits and is expressed in many kinds of cells. As a hub of signal transduction pathways, NF-κB is closely involved in immune responses, tumorigenesis and development, apoptosis regulation, and embryonic development, and is an

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important nuclear transcription factor [62]. MAPKs and NF-κB signal transduction pathways are two important intracellular signaling pathways with complex interactions that can be widely involved in a series of physiological and pathological reactions. To investigate whether the NF-κB pathway participates in SCVP-1-induced macrophage activation, the expression of phosphorylated p65 in nucleus was detected by Western blot. According to the Fig. 15A, the expression level of p-p65 in the nucleus was significantly increased after treatment with SCVP-1 at the concentrations of 200 and 400 μg/mL compared with that of the control groups (p＜0.01). However, when cells were treated with SCVP-1 concentrations of 50 and 100 μg/mL, there was no significant difference in the expression of p-p65 protein in the nucleus. The expression level ratio of p-p65/p65 reached 0.92±0.09 and 0.99±0.03 when SCVP-1 was added at 200 and 400 μg/mL, respectively, that of the positive control LPS-treated group was 1.21±0.05, and that of the control group was 0.52±0.03 (Fig. 15B). We further investigated the involvement of NF-κB on SCVP-1-induced macrophage activation by assessing NO and cytokines release by RAW264.7 cells upon treatment with the NF-KB inhibitor BAY11-7082. As shown in Fig. 15C–F, when both BAY11-7082 and SCVP-1 or LPS were added to the cells, the production of NO, IL-1β, IL-6, and TNF-α was remarkably decreased as compared to that induced by SCVP-1 or LPS alone (p ＜0.01). These results are consistent with those of the western blot experiments, suggesting that the NF-κB pathway is involved in SCVP-1-induced activation of macrophages.

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4. Conclusions The viscera of sea cucumber are usually considered waste material during sea cucumber processing but the viscera are actually good sources of polysaccharides. In this study, a sulfated polysaccharide (SCVP-1) was isolated from sea cucumber viscera and further purified by DEAE-52 cellulose and Sephadex G-200 column. The SCVP-1 was a homogeneous polysaccharide with relative molecular weight of 180.8 kDa and composed of various ratios of total sugars (60.2±2.6%), uronic acid (15.3±1.8%), proteins (6.8±0.8%), and sulfate groups (18.1±0.9%). The results of monosaccharide composition indicated that SCVP-1 consisted of mannose, glucosamine, glucuronic acid, N-acetyl-galactosamine, glucose, galactose and fucose at a molar ratio of 1.00: 1.41: 0.88: 2.14: 1.90: 1.12: 1.24, respectively. The FT-IR and NMR spectra demonstrated that SCVP-1 was a kind of glycosaminoglycan which was similar to some fucosylated chondroitin sulfates isolated from the body wall of sea cucumber. And the sulfation patterns of the fucose branches were Fuc2,4S, Fuc3,4S and Fuc0S. The surface morphology of SCVP-1 showed irregular sheet structure and was composed of many spherical molecules, suggesting that SCVP-1 may have a spherical chain conformation. Moreover, SCVP-1 exhibited strong immune stimulation activity. Our study demonstrated that SCVP-1 was able to induce macrophages to produce considerable amounts of NO and cytokines (IL-1β, IL-6, and TNF-α) and enhance the phagocytic activity of macrophages. Moreover, SCVP-1 significantly stimulated the expression of related genes (iNOS, IL-1β, IL-6, and TNF-α). TLR4 acted as a cell membrane target receptor for SCVP-1, activating the intracellular MAPKs and NF-κB signaling pathways and, consequently, activating macrophages. In addition, the MAPKs and NF-κB blocking experiments showed that SCVP-1 significantly activated macrophages due to the involvement of MAPKs and NF-κB signaling pathways through up-regulating the phosphorylation levels of ERK1/2, p38, JNK1/2, and p65. As illustrated in Fig. 16, our results suggest that TLR4 may be involved in the activation of macrophages induced by SCVP-1 through the MAPKs and NF-κB

Journal Pre-proof signaling pathways. Therefore, our findings shine light on the added value of sea cucumber, providing a new direction for the development and utilization of the viscera of this organism.

Declaration of competing interest The authors declare that there are no conflicts of interest.

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Acknowledgements This work was funded by the National Nature Science Foundation of China (grant number 81703390), Major Project on the Integration of Industry and Education of Fujian Province (grant number 2018N5008), Subsidized Project for Postgraduates’ Innovative Fund in Scientific Research of Huaqiao University, Fujian Marine High-tech Industry Development Project (Fujian Marine High-tech [2013] 19), Huaqiao University Support Program for Science and Technology Innovation Young teachers (grant number ZQN-PY515), and China scholarship Council (No. 201807540001).

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[51] Panagos C G, Thomson D S, Moss C, et al. Fucosylated Chondroitin Sulfates from the Body Wall of the Sea Cucumber Holothuria forskali: Conformation, Selectin Binding, and Biological activity[J]. Journal of Biological Chemistry, 289(41) (2014)28284-28298. [52] Rosenberg M, Isaiah J.K, Yeshayahu T. A Scanning Electron Microscopy Study of Microencapsulation[J]. Journal of Food Science, 50(1) (2010)139-144. [53] Franz J. Giessibl. Advances in atomic force microscopy[J]. Review of Modern Physics, 75(3) (2003)949-983. [54] Bai Y, Zhang P, Chen G, et al. Macrophage immunomodulatory activity of extracellular polysaccharide (PEP) of Antarctic bacterium Pseudoaltermonas sp. S-5[J]. International Immunopharmacology, 12(4) (2012)611-617. [55] Weeks B A, Anisa S. Keisler, Quentin N. Myrvik, et al. Differential uptake of neutral red by macrophages from three species of estuarine fish[J]. Developmental & Comparative Immunology, 11(1) (1987)117-124. [56] Bogdan C. Nitric oxide and the immune response[J]. Nature Immunology, 2(10) (2001)907-916. [57] Yu Q, Nie S P, Wang J Q, et al. Toll-like receptor 4-mediated ROS signaling pathway involved in Ganoderma atrum polysaccharide-induced tumor necrosis factor-α secretion during macrophage activation[J]. Food and Chemical Toxicology, 66(2014)14-22. [58] Hirschfeld M, Ma Y, Weis J H, et al. Cutting Edge: Repurification of Lipopolysaccharide Eliminates Signaling Through Both Human and Murine Toll-Like Receptor 2[J]. The Journal of Immunology, 165(2) (2000)618-622. [59] Zhang X, Qi C, Guo Y, et al. Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible interaction models[J]. Carbohydrate Polymers, 149(2016)186-206. [60] Takeuchi O, Akira S. Toll-like receptors; their physiological role and signal transduction system[J]. International Immunopharmacology, 1(4) (2001)625-635. [61] Wang M, Yang X B, Zhao J W, et al. Structural characterization and macrophage immunomodulatory activity of a novel polysaccharide from Smilax glabra Roxb[J]. Carbohydrate Polymers, 156(2017)390-402. [62] Siebenlist U, Franzoso G, Brown K. Structure, Regulation and Function of NF-κB[J]. Annual Review of Cell Biology, 10(1) (1994)405-455.

Journal Pre-proof Table 1 Chemical shifts of proton and carbon in SCVP-1 Residue H1/C1 H2/C2 H3/C3 H4/C4 H5/C5 H6/C6 CH3 C=O

GlcUA

Fuc2,4S

Fuc3,4S

4.46/99.74 3.86/51.37 3.65/76.60 4.32/75.60a 3.77/71.74 4.36/66.71 1.98/22.37 -/174.37

4.40/103.82 4.05/76.19 3.88/72.01 4.42/77.58 3.81/76.17 -/174.37

5.61/96.17 4.39/75.08 4.07/65.71 4.61/76.31 4.33/75.62 1.27/15.85 -

5.32/98.36 3.72/60.97 3.95/72.11 4.15/77.27 3.71/68.64 1.27/16.18 -

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Chemical shifts of the sulfation sites were highlighted in bold.

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GalNAc

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Figure captions

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Fig.1 Elution curve of crude polysaccharide from sea cucumber viscera on DEAE-52 cellulose column

Fig.2 Purification of SCVP-1 on gel filtration chromatography (Sephadex G-200)

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(A)

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11.559

(B)

< 19.980

RID1 A, Refractive Index Signal (F:\DATA\XMT\CHUNDU 2018-07-19 11-27-14\005-P1-A5-1.D) nRIU

9000

8000

7000

6000

5000

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0 2

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0

19.194

1000

12.308

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2000

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Fig.3 (A)The cellulose-acetate membrane electrophoresis of SCVP-1. Lane 1: chondroitin sulfate A; lane 2: SCVP-1. (B) GPC chromatograms of SCVP-1 (A)

(B)

Fig.4 HPLC analyse for the monosaccharide composition. (A) HPLC chromatogram of derivatives of mixed standard monosaccharides. (B) HPLC chromatogram of derivatives of SCVP-1 1.D-(+)-Mannose；2.D-(+)-Glucosamine；3.D-(+)-Glucuronic acid；4.D-(+)-Galactosamine； 5.D-(+)-Glucose；6.D-(+)-Galactose；7.D-(+)-Xylose；8.L-(+)-Fucose

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Fig.5 UV-vis spectrum of SCVP-1

Fig.6 FT-IR spectrum of SCVP-1

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B

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Fig.7 (A) 1H NMR spectra of SCVP-1. (B) 1H-1H COSY spectra of SCVP-1. (C) 13C NMR spectra of SCVP-1. (D) 1H-13C HSQC spectra of SCVP-1. Signals designated with a, b and c refer to those produced by Fuc2,4S, Fuc3,4S and Fuc0S, respectively; signals designated with d, e and f refer those produced by GlcUA, GalNAc and GalNAc (CH3), respectively.

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Fig.8 SEM images of SCVP-1 at 500×(a), 2000×(b) and 5000×(c)

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Fig.9 (a) AFM image of SCVP-1. (b)The amplitude error image of SCVP-1. (c)Three-dimensional AFM image of SCVP-1.

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Fig.10 The activating effect of SCVP-1 on RAW264.7 cells. RAW264.7 cells were treated with different concentrations of SCVP-1 and LPS (1 μg/mL) for 24 h. (A) Effect of SCVP-1 on cell viability of RAW264.7 cells analyzed by MTT. (B) Effect of SCVP-1 on the production of NO in RAW264.7 cells detected by Griess reagent. (C-E) The level of IL-1β, IL-6 and TNF-α were measured by ELISA kits. (F) Endotoxin contamination tests using PMB in SCVP-1/LPS-treated RAW264.7 cells. The RAW264.7 cells were incubated with SCVP-1 (200 μg/mL) or LPS (1 μg/mL) which was pretreated with or without PMB for 24 h. The results were expressed as means ± SD (n = 3). (B-E) *p<0.05, **p<0.01 vs. control group. (F) **p<0.01 vs. PMB treated group.

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Fig.11 Effects of SCVP-1 on phagocytosis of RAW264.7 cells B

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Fig.12 The effects of SCVP-1 on the mRNA expression of cytokines in RAW264.7 cells. mRNA expression of (A) iNOS, (B) IL-1β, (C)IL-6, and (D) TNF-α. β-actin was used as an internal reference. Values were presented as means ± standard deviation (n=3). *p<0.05, **p<0.01 vs. control group.

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Fig.13 Effect of TLR4 on SCVP-1-induced activation of RAW264.7 cells (A-D) Effect of TLR4 inhibitor on SCVP-1/LPS-induced NO, IL-1β, IL-6 and TNF-α production in RAW264.7 cells. The results were expressed as means ± SD (n = 3). **p<0.01 vs. without TLR4 inhibitor treated group.

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Fig.14 Effect of MAPKs signaling pathways on SCVP-1-induced macrophage activation. (A) The effect of SCVP-1 on the level of phosphorylation of ERK1/2, p38 and JNK1/2 in RAW264.7 cells by Western blotting. (B) Relative quantitative analysis of p-p38/p38, p-ERK/ERK and p-JNK/JNK protein phosphorylation levels. (C-F) Effect of specific MAPKs inhibitors (ERK1/2 inhibitor: CI-1040, p38 inhibitor: SB239063 and JNK1/2 inhibitor: SP600125) on SCVP-1/LPS-induced NO, IL-1β, IL-6 and TNF-α production of RAW264.7 cells. The results were expressed as means ± SD (n = 3). (B) *p<0.05, **p<0.01 vs. control group. (C-F) *p<0.05, **p<0.01 vs. without anti-MAPKs antibodies treated group A

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Fig.15 Effect of NF-κB signaling pathway on SCVP-1-induced macrophage activation. (A) The effect of SCVP-1 on the level of phosphorylation of p65 in RAW264.7 cells by Western blotting. (B) Relative quantitative analysis of p-p65/p65 protein phosphorylation levels. (C-F) Effect of specific NF-κB inhibitors (BAY11-7082) on SCVP-1/LPS-induced NO, IL-1β, IL-6 and TNF-α production of RAW264.7 cells. The results were expressed as means ± SD (n = 3). (B) *p<0.05, **p<0.01 vs. control group. (C-F) *p<0.05, **p<0.01 vs. without anti- NF-κB antibodies treated group

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Fig.16 Possible signal transduction pathways involved in macrophage activation by SCVP-1