Physicochemical characterization of Sargassum fusiforme fucoidan fractions and their antagonistic effect against P-selectin-mediated cell adhesion

International Journal of Biological Macromolecules 133 (2019) 656–662

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Physicochemical characterization of Sargassum fusiforme fucoidan fractions and their antagonistic effect against P-selectin-mediated cell adhesion Siya Wu a,1, Xu Zhang a,1, Jian Liu a,1, Jianxi Song b, Ping Yu a, Peichao Chen a, Zhiyong Liao a, Mingjiang Wu a,⁎, Haibin Tong a,⁎ a b

College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, China Analytical and Testing Center, Beihua University, Jilin 132013, China

a r t i c l e

i n f o

Article history: Received 2 January 2019 Received in revised form 28 March 2019 Accepted 28 March 2019 Available online 29 March 2019 Keywords: Sargassum fusiforme Fucoidan P-selectin

a b s t r a c t P-selectin, mediated adhesion between endothelium and neutrophils, is a promising target for the therapeutics of acute inflammatory-related diseases. It is reported that brown algal fucoidans can antagonize P-selectin function. However, the fractionation and physicochemical characterization of Sargassum fusiforme fucoidan, and the screening of fucoidan fractions with P-selectin antagonistic capability have not been investigated. In this study, we isolated and fractionated systematically the S. fusiforme fucoidan by ion-exchange chromatography and size exclusion chromatography to obtain eight fucoidan fractions. Their physicochemical characterization was determined by chemical methods, HPLC and FT-IR. The inhibitory capacity of the fucoidan fractions in Pselectin-mediated leukocyte adhesion was evaluated by static adhesion assay and parallel-plate flow chamber. Results showed that fucoidan fractions possessed distinct physicochemical properties, including total carbohydrate, uronic acid and sulfate contents, molecular weight, and monosaccharide compositions. Among all the fucoidan fractions, SFF-32 and SFF-42 showed better blocking ability against P-selectin-mediated cell adhesion. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Inflammation is a complex event of immune system responsible to deliver the appropriate defense, including leukocytes and cytokines, to the injured sites and eliminate harmful stimuli [1]. Leukocyte recruitment from bloodstream into injured or infected tissues is an essential step of the development of inflammation. The excessive inflammatory response is harmful, therefore, inflammation is terminated once the harmful stimuli have been removed [2,3]. However, the inappropriate recruitment and immoderate activation of leukocytes will cause serious damage [4]. Thus, blocking the excessive and inappropriate recruitment of leukocytes has been considered as an effective strategy for inflammation-related diseases [5,6]. Leukocyte recruitment is mediated by the selectin family of adhesion molecules on the endothelium. P-selectin is stored in granules of endothelial cells and is rapidly translocated to the cell surface by inflammatory stimulation. P-selectin regulates the initial leukocyte rolling on the activated endothelium, and these events are controlled by a series of glycoconjugate carbohydrates, especially those coating the leukocyte ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Wu), [email protected] (H. Tong). 1 Contributed equally to this work.

https://doi.org/10.1016/j.ijbiomac.2019.03.218 0141-8130/© 2019 Elsevier B.V. All rights reserved.

surfaces [7]. Therefore, P-selectin becomes a promising therapeutic target for interfering with inflammation-related pathological injuries, causing strong interest searching for high-affinity glycoconjugate ligands from natural resources as antagonists against the initial recruitment of leukocyte to the endothelium mediated by P-selectin [8–10]. The carbohydrate moiety, recognized by P-selectin and mediated the binding between P-selectin and its physiological ligand, is essentially the O-linked tetrasaccharide Sialyl-LewisX motif of P-selectin glycoprotein ligand-1 (PSGL1) on the leukocyte surface [11]. Fucoidan extensively distributed in the brown algae and several marine invertebrates is a family of sulfated homo- and heteropolysaccharides, with a high percentage of fucose and galactose residues [12]. Growing evidence indicates that exogenous sulfated glycan, such as fucoidan, can lead to an anti-inflammatory effect through the competitive inhibition of the molecular interactions between P-selectin and PSGL1 [13–15]. Sargassum fusiforme, an edible brown alga that belongs to the Sargassaceae family, is extensively distributed in eastern Asian countries, particularly China, Japan, and South Korea [16,17]. S. fusiforme is functional seaweed as healthy food, also utilized as a traditional Chinese medicine for thousands of years [18,19]. Fucoidan is one of the most important bioactive macromolecules in S. fusiforme, which possesses multiple medicinally effects, such as anti-inflammatory, anti-tumor, antihyperlipidemia, and immunomodulatory, and antioxidant activity

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[20–22]. Although, the relative selectin blocking effects of nine different fucoidans from brown seaweeds was investigated by Cumashi et al. [23], whether the anti-inflammatory mechanism of S. fusiforme fucoidan is related to the antagonism of P-selectin function is still unclear. In addition, the procedure of extraction and purification might also affect the physicochemical properties and biological activities of S. fusiforme fucoidan. Therefore, the systematic fractionation and physicochemical properties of S. fusiforme fucoidan are indispensable for clarifying their structure-activity relationship. Thus, in the present study, the entire fucoidan fractions were purified by ion exchange and size exclusion chromatography according to their distinct characteristics in charge and molecular weight. We further evaluated the effects of fucoidan fractions on the antagonistic capacity against P-selectin-mediated cell adhesion. The present findings will provide theoretical support for S. fusiforme fucoidan as anti-inflammatory agent. 2. Materials and methods 2.1. Materials and chemicals S. fusiforme was collected in October 2017 from the East China Sea on the coast of Dongtou District, Wenzhou City (Zhejiang Province, China). The specimen was deposited at the College of Life and Environmental Science, Wenzhou University, with voucher specimen number 2017–0195. P-selectin blocking mAb (9E1) and non-blocking mAb (AC1.2) were purchased from BD PharMingen (Franklin Lakes, New Jersey, USA). Sepharose CL-6B was purchased from Amersham Pharmacia Co. (Sweden). T-series dextrans were purchased from Fluka. DEAEcellulose, dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Trifluoroacetic acid (TFA) and standard monosaccharides [including arabinose (Ara), rhamnose (Rha), xylose (Xyl), fucose (fuc), mannose (Man), galactose (Gal), glucose (Glc), glucuronic acid (GlcA), and galacturonic acid (GalA)] were purchased from Aladdin Reagent Int (Shanghai, China). All other chemical reagents used were analytical grade. 2.2. Extraction of S. fusiforme fucoidan The dried alga of S. fusiforme was ground into powder, and then defatted with 80% ethanol for 12 h. The fucoidan from defatted algal powder was extracted by soaking 0.01 M HCl (1:20, w/v), stirred for 6 h and filter through a mesh nylon sieve. Then, the filtrate was added with 4 M CaCl2, incubated for 30 min, and then centrifuged at a speed of 12,000 rpm for 15 min. Supernatant was dialyzed (MWCO 1000 Da) against deionized water for 48 h. The sample was then concentrated and precipitated with four volumes of 95% ethanol, kept at 4 °C for 12 h. The precipitate was collected by centrifugation and further deproteinated by a freeze–thaw process and Sevag method [24]. Then, the sample was collected, dialyzed, and lyophilized to yield crude S. fusiforme fucoidan, coded as cSFF.

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2.4. Physicochemical characterization of S. fusiforme fucoidan fractions Total carbohydrate content was quantified according to Dubois methods [25] using phenol sulfuric acid method with L-fucose as the standard. The sulfate content was quantified based on the BaCl2gelatin method using K2SO4 as the standard [26]. Uronic acid content was measured by m-hydroxydiphenyl method using D-glucuronic acid as standard [27]. Protein content was determined by the Bradford's method [28]. Fourier transform-infrared spectroscopy (FT-IR) was recorded on a BRUKER Tensor 27 FT-IR spectrometer in the range of 400–4000 cm−1, using KBr-disk method.

2.5. Molecular weight determination Molecular weight of fucoidan fractions was determined by HPLC. The samples were dissolved in distilled water, applied to a Waters 1525 HPLC system equipped with a TSK-GEL G5000 PWXL column (7.8 × 300 mm, TOSOH, Japan), eluted with 0.1 M Na2SO4 solution and detected by a Waters 2424 Refractive Index Detector. Dextran standards with different molecular weights (2000 kDa, 150 kDa, 41.1 kDa, 21.4 kDa, 7.1 kDa, 4.6 kDa and 180 Da) were to calibrated the column and establish a standard curve using linear regression.

2.6. Monosaccharide composition analysis The monosaccharide composition was determined by HPLC using a 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method [29] with some modification. Briefly, fucoidan fractions (4 mg) were hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120 °C for 3 h. After removed excess acid, 100 μL of 0.3 M NaOH and 100 μL of 0.5 M PMP solution were added into the reaction mixture and incubated at 70 °C for another 30 min. After neutralized by HCl solution, equal volume of chloroform was added into the reaction mixture. The aqueous phase was filtered for HPLC analysis using a Waters 1525 HPLC system with a Hypersil ODS-2 column (5 μm, 4.6 mm × 250 mm) detected at the wavelength of 254 nm. The mobile phases were 0.05 M phosphate buffer solution (pH 6.8) and acetonitrile (83: 17, v/v), at a flow rate of 0.8 mL min−1.

2.7. Cell culture Chinese hamster ovary (CHO) cells and Human promyelocytic leukemia (HL-60) cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China). CHO-P cells stably expressing human P-selectin were obtained by transfecting CHO cells with the full-length human P-selectin cDNA. All cells were cultured in IMDM (Gibco) supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin. The cell cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2.

2.3. Fractionation of S. fusiforme fucoidan by chromatography cSFF (1 g) was dissolved in 10 mL distilled water and applied on a DEAE-cellulose column (2.6 × 50 cm) pre-equilibrated with distilled water. This column was initially eluted with distilled water, and a fraction (SFF-1, 231 mg) was recovered. The bound fucoidan was then eluted by 0.1 M (SFF-2, 35 mg), 0.3 M (SFF-3, 87 mg), 0.5 M (SFF-4, 132 mg) and 1 M (SFF-5, 155 mg) NaCl solution, respectively. Each eluent was collected and further fractionated using Sepharose CL-6B column (2.5 cm × 90 cm) and eluted with 0.15 M NaCl. The eluate was analyzed using phenol sulfuric acid method [25], and the appropriate fractions were collected to produce: SFF-11, SFF-12, SFF-2, SFF-31, SFF32, SFF-41, SFF-42 and SFF-5. The procedure for the extraction and fraction of S. fusiforme fucoidan is illustrated in Fig. 1.

2.8. Static adhesion assay Static adhesion assay was carried out as previously described [9]. Briefly, CHO or CHO-P cells were seeded in 24-well plates overnight to form monolayers. Then, cells were pre-incubated with blocking mAb 9E1, non-blocking mAb AC 1.2, or 100 μg/mL of S. fusiforme fucoidan fractions at 37 °C for 1 h. After incubation with 5 μM Calcein-AM at 4 °C for 30 min, HL-60 cells were added to the pre-treated CHO-P or CHO monolayers and incubated for another 1 h. After gently washed with PBS, the fluorescence intensity was measured with a Tecan Infinite M200 microplate reader with excitation and emission wavelengths set at 485 nm and 530 nm, respectively.

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Fig. 1. Extraction and fractionation scheme of S. fusiforme fucoidan.

2.9. Parallel-plate flow chamber assay

2.10. Statistical analysis

Parallel-plate flow chamber assay was carried out as previously described [9]. Briefly, CHO or CHO-P cells were seeded in 35-mm culture dishes and incubated at 37 °C overnight to form monolayers. Cells were pre-treated with 9E1, AC 1.2, or 100 μg/mL of S. fusiforme fucoidan fractions at 37 °C for 1 h. The culture dishes were then assembled in a parallel-plate flow chamber (GlycoTech, Rochville, MD, USA) and mounted onto an inverted microscope. After washed with PBS, 2 × 106 cells/mL HL-60 cells were perfused through the flow chamber at a shear stress of 1.2 dyn/cm 2 driven by a syringe pump. The observation fields were randomly selected and recorded for 3 min via a CCD-camera. The number of HL-60 cells rolling on the CHO or CHO-P monolayers was calculated from three independent experiments.

Data were expressed as means ± SD (standard deviations). Statistical comparison with multiple groups was made by one-way ANOVA, followed by a Tukey post hoc test using SPSS version 13.0. P values b 0.05 were considered statistically significant. The significant differences were indicated by different letters (p b 0.05); the same letters meant no statistical significances. 3. Results and discussion 3.1. Isolation and purification of fucoidan fractions from S. fusiforme In the last decades, fucoidans from the various species of brown alga have been discovered as an antagonist of P-selectin, making it a

S. Wu et al. / International Journal of Biological Macromolecules 133 (2019) 656–662

candidate of anti-inflammatory agent [23]. When making comparisons of their inhibitory capability, one challenge lies in many variations to physicochemical characterization of those fucoidan fractions. Fractions vary widely in terms of purity, composition, molecular weight and sulfation even from the same species of brown alga, and less pure fractions can deliver confounding data [30–32]. Many researches have proven that fucoidan from S. fusiforme possesses various biological activities. Unfortunately, in most cases, the comprehensive fractionation of S. fusiforme fucoidan and their physicochemical characterization are not fully elucidated. In this study, a Lisure Polysaccharide Purifier purification system, equipped with a APPS 10D pump, UV–Vis/ORP PC3110-RS monitor, EC-4110-RS conductivity detector and APPS PS 10DT fraction collector, was employed to purify all the fucoidan fractions from S. fusiforme with DEAE-cellulose ion exchange chromatography and Sepharose CL-6B gel filtration chromatography. The yield of crude S. fusiforme fucoidan (cSFF) was 7.3% of dried material (w/w). After freeze–thaw process and deproteinization by a combination of proteinase and Sevag method, cSFF was loaded onto the DEAE-cellulose column (Fig. 2A) and eluted with a stepwise gradient elution of NaCl solution (0, 0.1, 0.3, 0.5, 1 M). Five main fractions were collected based on their elution profiles, designated as SFF-1, SFF-2, SFF-3, SFF-4 and SFF-5, respectively. These fucoidan fractions were further purified using Sepharose CL-6B column eluted with 0.15 M NaCl

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solution, giving eight fractions, SFF-11, SFF-12, SFF-2, SFF-31, SFF-32, SFF-41, SFF-42 and SFF-5 (Fig. 2B–F).

3.2. Physicochemical characterization of S. fusiforme fucoidan fractions 3.2.1. Contents of carbohydrate, protein, uronic acid and sulfate The molecular weight, contents of total carbohydrate, uronic acid and sulfate of the S. fusiforme fucoidan fractions are summarized in Table 1. The average molecular weights were determined to be 348.4 kDa for SFF-11, 187.1 kDa for SFF-12, 1.7 kDa for SFF-2, 601.5 kDa for SFF-31, 47.3 kDa for SFF-32, 424.9 kDa for SFF-41, 98.6 kDa for SFF-42 and 697.6 kDa for SFF-5. The contents of total carbohydrate evaluated in SFF-11, SFF-12, SFF-31, SFF-32, SFF-41, SFF-42 and SFF-5 were 69.88%, 76.84%, 70.50%, 64.06%, 79.66%, 71.69% and 63.37%, respectively. SFF-2 has low total carbohydrate content (22.91%), indicating the presence of non-sugar impurities. All fucoidan fractions had negative response to Bradford's assay, indicating that protein was completely removed by a freeze–thaw process and Sevag method. Furthermore, SFF-32 and SFF-41 had the highest uronic acid contents (28.21% for SFF-32 and 24.51% for SFF-41) among all the fractions. SFF-41, SFF-42 and SFF-5 contained higher sulfate group (16.93%, 16.63% and 23.99%, respectively) than other fractions.

Fig. 2. Fractionation of S. fusiforme fucoidan by ion exchange chromatography and gel filtration chromatography. (A) The elution profile of cSFF on DEAE-cellulose column, eluted with a stepwise gradient elution of NaCl solution (0, 0.1, 0.3, 0.5, 1 M). (B\ \F) The elution profiles of samples on sepharose CL-6B column, eluted with 0.15 M NaCl.

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Table 1 Physicochemical characterization of S. fusiforme fucoidan fractions.

Molecular weight (kDa) Total carbohydrate (%) Uronic acid (%) Sulfate (%)

SFF-11

SFF-12

SFF-2

SFF-31

SFF-32

SFF-41

SFF-42

SFF-5

348.4

187.1

1.7

601.5

47.3

424.9

98.6

697.6

69.88 7.33 9.35

76.84 12.39 11.46

22.91 12.62 3.81

70.50 13.83 10.03

64.06 28.21 5.43

79.66 24.51 16.93

71.69 11.83 16.63

63.37 9.66 23.99

3.2.2. Monosaccharide compositions The monosaccharide composition analysis of S. fusiforme fucoidan fractions is an important procedure to control quality standard and exhibit basic physicochemical information. According to HPLC analysis with a PMP pre-column derivatization method, the monosaccharide compositions of all the S. fusiforme fucoidan fractions are shown in Table 2. Overall, the data indicated all the fucoidan fractions appeared to be heteropolysaccharides, and composed of mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, and fucose, as well as trace galacturonic acid. Fucose, mannose and galactose were the major monosaccharides, similar to the fucoidans isolated from other brown algae previously reported [15,33], and other monosaccharides exhibited distinct ratios in different fractions. For example, the monosaccharide composition of SFF-11 was the simplest among all the fractions, only contains fucose, mannose and galactose. In addition, galacturonic acid was only detected in SFF-12 with a small amount. Considering the physicochemical characterization of SFF-2, including low carbohydrate content (22.91%) and sulfate content (3.81%), low molecular weight (1.7 kD) and complex monosaccharide composition, it is speculated that SFF-2 is more likely to be a compound of oligomeric fragment fractured from fucoidan fractions. 3.2.3. Spectroscopic analysis The FT-IR spectra (Fig. 3) of all fractions revealed a typical major broad stretching peak at around 3400 cm−1 for the hydroxyl group, and a weak band at 2930 cm−1 showing the C\\H stretching vibration. The strong absorption at 1250 cm−1 could be attributed to asymmetric O=S=O stretching vibration of sulfate esters. In the finger print region (750–960 cm−1), it was supported by the appearance of a peak at 836 cm−1 that corresponds to sulfate at equatorial position, assuming that sulfate group binds to both of C-2 and C-4 of fucopyranose residues to form sulfate fucose. The band at 1641 cm−1 corresponded to asymmetric stretching of carboxylate anions groups, indicating the presence of carboxyl groups. A strong extensive absorption in the region around 1050 cm−1 for stretching vibrations of C-O-H side groups and the CO-C glycosidic band vibrations were observed in the spectra.

development of acute inflammatory diseases [34]. Thus, the agents that can block these two activities of P-selectin would be considered as ideal anti-inflammatory compounds. The antagonistic effects of S. fusiforme fucoidan fractions against P-selectin were carried out by determining their abilities to block P-selectin-mediated adhesive function. The data from static adhesion assay are shown in Fig. 4. Compared with control group, both of the total fucoidan SFF and fractions, at the concentration of 100 μg/mL, exhibited significant blocking effects against P-selectin-mediated adhesion between HL-60 cells and CHO-P cells under static condition, but the blocking effect of fucoidan fractions was distinct. The percentage of adherent HL-60 cells on CHO-P monolayers treated with SFF reduced to 48.5% compared with control group, and after fractionation, SFF-2, SFF-32 and SFF-42 showed better blocking ability than other fractions. The percentage of adherent cells was decreased in the order of SFF-11 (86.9%), SFF-12 (77.3%), SFF-31 (64.1%), SFF-5 (54.4%), SFF-41 (42.8%), SFF-32 (35.9%), SFF-2 (27.9%) and SFF-42 (27.5%). Parallel plate flow chamber was utilized to investigate the blocking capacity of P-selectin-mediated dynamic adhesion. The initial adhesion of HL-60 cells on CHO-P monolayers and their subsequent rolling along cell monolayers were observed and recorded with an inverted microscope equipped with a CCD-camera, once the HL-60 cells rolled through the surface of CHO-P monolayer. At the concentration of 100 μg/mL, SFF significantly (p b 0.05) reduced the percentage of HL-60 cells rolling on the CHO-P cell monolayer, causing as much as 56% reduction compared to the control (Fig. 5). SFF-2, SFF-32, SFF-41 and SFF-42 exhibited approximative (p N 0.05) inhibitory effects against the rolling of HL-60 cells, the percentage of rolling HL-60 cells reduced to 33.3%, 30.0%, 38.7% and 28.9%, respectively. The data from parallel-plate flow chamber assay (Fig. 5) exhibited a similar pattern as the data from static SFF-11 SFF-12

SFF-2 SFF-31

3.3. S. fusiforme fucoidan fractions inhibits P-selectin-mediated adhesion SFF-32

As an important cell adhesion molecule, P-selectin is involved in the initial attachment of neutrophil to and its subsequent rolling on activated vascular endothelial cells, both of which are critical to the

SFF-41 SFF-42 SFF-5

Table 2 Monosaccharide compositions of S. fusiforme fucoidan fractions.

Man Rha GlcA GalA Glc Gal Xyl Fuc a

SFF-11

SFF-12

SFF-2

SFF-31

SFF-32

SFF-41

SFF-42

SFF-5

0.19 nda nd nd nd 0.42 nd 1.00

0.51 0.32 0.36 0.47 1.42 0.93 nd 1.00

0.79 0.66 0.32 nd 1.39 0.64 0.09 1.00

0.48 0.04 0.14 nd 0.04 0.17 0.23 1.00

1.25 0.86 0.46 nd 0.27 0.36 0.45 1.00

0.84 0.12 0.20 nd nd 0.48 0.14 1.00

0.92 0.38 0.37 nd 0.09 0.59 0.20 1.00

0.21 0.05 0.08 nd 0.06 0.62 0.06 1.00

nd: not detected.

836 1641

3446

4000

3500

3000

2500

2000

1404

1500

1250 1050

1000

500

Wave number cm-1 Fig. 3. FT-IR spectra of S. fusiforme fucoidan fractions. FT-IR was recorded on a BRUKER Tensor 27 FT-IR spectrometer in the range of 400–4000 cm−1, using KBr-disk method.

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Fig. 4. Inhibition of HL-60 cell adhesion to CHO-P cells by S. fusiforme fucoidan fractions under static condition. CHO or CHO-P monolayers were pretreated with mAb or fucoidan fractions. CHO and untreated CHO-P monolayers were used as negative and positive control, respectively. Fluorescent-labeled HL-60 cells were added to the monolayers. After gentle washing, the fluorescence intensity was measured and expressed as the percentage relative to the positive control. Data are the means ± SD from three independent experiments. Alphabet indicates a significant difference at p b 0.05.

adhesion assay (Fig. 4), suggesting S. fusiforme fucoidan fractions have a relatively stable blocking capacity against P-selectin-mediated adhesion under both dynamic and static conditions. Fucoidans from brown algal are the mostly studied algal sulfated glycans possessing the anti-inflammatory activity, which has been proven by numerous in vitro and in vivo studies of inflammation. For example, Park et al. [35] reported fucoidan from Fucus vesiculosus can inhibit LPS-induced inflammation in microglial cells via inhibition of NF-κB, MAPK and Akt activation. Lee et al. [36] reported the fucoidan isolated from the brown alga Ecklonia cava was able to significantly inhibit NO production in LPS induced Raw 264.7 macrophage cells by down-regulating the expression of iNOS, cyclooxigenase-2, and proinflammatory cytokines such as TNF-a, IL-6, and IL-1β. Therefore, fucoidans affect multiple targets in the inflammatory progression, such as inhibiting the inflammatory signaling pathway and modulating pro−/anti-inflammatory cytokine secretion profiles. Currently, one of

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the interests in fucoidans deemed as anti-inflammatory agents is demonstrated by their ability to interfere with the migration of leukocytes to inflammatory sites [37]. Carvalho et al. [38] reported that intravenous delivery of Fucus vesiculosus fucoidan significantly alleviated the acute pancreatitis-related pathological process through blocking neutrophil infiltration. Consistent with our present findings, the essential molecular mechanism of Fucus vesiculosus fucoidan in ameliorating the pancreatitis is due to this fucoidan was able to act as inhibitor of P-selectin in this inflamed model. This action of natural carbohydrates blocking the interaction between P-selectin and its ligands on leukocyte, and further inhibiting selectin-mediated leukocyte migration and infiltration, eventually leads to the decrease of the systemic inflammation [8,39]. Our present study indicated that the potential anti-inflammatory activity of S. fusiforme fucoidans is conferred by their abilities to mimic the ligands of P-selectin, effectively blocking the interaction between Pselectin and its native ligands. It is speculated that the interaction pattern in the antagonistic ability of those fucoidan fractions could due to the synergism of their molecular weight and sulfate group. SFF-32 and SFF-42 possessing better antagonistic effects are characterized by appropriate molecular weight and high sulfate content. Although SFF-5 also has a higher sulfate group, its huge molecular weight may affect its binding ability to P-selectin. Fucoidans are usually the most complex sulfated glycans in terms of structure [40], which is directly dependent on the brown algal species from which they are synthesized, even though they are mostly composed of L-Fucose units. The presence of additional monosaccharide types associated with occasional sparse branches enhances structural complexity. The occurrence of repetitive units in S. fusiforme fucoidans is somewhat yet uncertain. Further evidence of structure is still needed to elucidate the structure-activity relationship in the blockage of P-selectin and ligand binding by S. fusiforme fucoidans. 4. Conclusion In the present study, the crude fucoidan (cSFF) was extracted from S. fusiforme, and then fractionated systematically to obtain eight fractions (SFF-11, SFF-12, SFF-2, SFF-31, SFF-32, SFF-41, SFF-42 and SFF-5). Chemical and instrumental analysis shows that these fucoidan fractions possess distinct physicochemical properties in total carbohydrate, uronic acid and sulfate contents, molecular weight, and monosaccharide compositions. The fucoidan fractions, SFF-32 and SFF-42, could effectively block the interaction between P-selectin and its native ligand, and inhibit P-selectin-mediated cell adhesion, showing promising anti-inflammatory potential. These findings are expected to be the basis for future research on structure-activity relationship of S. fusiforme fucoidan, as well as promote its exploitation and utilization as promising candidates for amelioration of inflammation-related diseases. Conflict of interest The authors declare no conflict of interest. Acknowledgement

Fig. 5. Inhibition of the interaction between HL-60 cells and CHO-P cells by S. fusiforme fucoidan fractions evaluated by parallel plate flow chamber. The rolling of HL-60 cells on CHO cell or CHO-P cell monolayer preincubated with mAb or fucoidan fractions. at 1.2 dyn/cm2 using parallel plate flow chamber was recorded by videomicroscopy. Data, expressed as the percentage relative to the positive control, are the means ± SD from three independent experiments. Alphabet indicates a significant difference at p b 0.05.

This work was financially supported by the National Natural Science Foundation of China (41876197, 31470430 and 81872952), the Natural Science Foundation of Zhejiang Province (LY18C020006 and LGN18C020004), Science and Technology Program of Wenzhou (Y20180210), the Scientific Foundation of Education Department of Zhejiang Province (Y201737374). References [1] R. Medzhitov, Origin and physiological roles of inflammation, Nature 454 (2008) 428–435.

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