is the most productive edible brown algae in China and has been used as a traditional medicine for thousands of years in China. Nowadays, S. japonica
is a well-known source of bioactive compounds, including mannitol, alginates, fucoidan, and laminarian, in which fucoidan as the major active component has attracted widespread attention in recent years [1
]. Fucoidan is a kind of fucose-containing sulfated polysaccharide, found in the fiber pile cell walls and intercellular spaces of brown seaweeds and echinoderm [3
]. It has a number of attractive biological activities, such as antiviral [4
], anticancer [5
], anti-inflammatory [7
], antioxidant [9
], hypolipidemic [10
], and immunostimulatory effects [12
Inflammation, a host defense response to tissue injuries, infection, stress, and other stimuli, plays a critical role in homeostasis and fine regulation. However, excessive and continuous inflammatory responses turn out to be very harmful to the host, leading to tissue damage from diverse diseases, including obesity, arthritis, cancer, autoimmune diseases, etc. [3
]. The various biological activities of fucoidan, especially in the prevention of inflammation-related diseases and related molecular mechanisms, have attracted great interest. Fucoidan could exert anti-inflammatory effects by inhibition of LPS-induced expression of inflammatory mediators and pro-inflammatory cytokines and down-regulation the protein expression levels of iNOS and COX-2 in macrophage cells. For example, Kang et al., found that the fucoidan extracted from Ecklonia cava
significantly inhibited NO production and prostaglandin-E2 (PGE2) production, and suppressed inducible iNOS and COX-2 expression in LPS-stimulated RAW 264.7 cells. Additionally, the fucoidan from Sargassum horneri
showed high inhibition of NO production in LPS-stimulated RAW 264.7 cells and down-regulated the protein expression levels of iNOS and COX-2 and the production of inflammatory cytokines, including TNF-α and IL-1β [15
The underlying anti-inflammatory mechanism may be correlated with the suppression of the activation of NF-κB and the MAPKs signal pathways [7
]. It was found that fucoidan purified from Fucus vesiculosus
exhibited anti-inflammatory properties by suppression of NF-κB activation and down-regulation of MAPKs and Akt pathways in microglial cells [18
]. In addition, fucoidan isolated from the brown seaweed Padina commersonii
inhibited LPS-induced inflammatory responses via blocking TLR/MyD88/NF-κB signal transduction [7
]. A recent study indicated a fucoidan (LJSF4) purified from S. japonica
was found to show a strong anti-inflammatory effect in LPS-induced RAW 264.7 macrophage cells and zebrafish. The mechanism was revealed to be associated with the down-regulated expression of signal pathways, including MAPK and NF-κB [19
]. The structures of fucoidan vary, including the monosaccharide compositions, the position glycoside bonds, branched chains, sulfate radical content, substitution of sulfate groups position, degree of sulfation, and molecular weight, which are mainly influenced by the species, algae characteristics, geographical location, harvest season, extraction conditions, and other factors [20
]. Considering the above-mentioned influence factors, every new fucoidan obtained could potentially be a new compound with unique structural characteristics and have promising bioactive properties.
In this study, four homogeneous fucoidan were isolated and purified from S. japonica in Fujian Province, southeast of China. Among them, SF6 was observed to have efficient anti-inflammatory effect during the in vitro bioactivity test. On this basis, the preliminary structure of SF6 was characterized by FTIR and 1D-NMR, and the anti-inflammatory effect was investigated in LPS-activated RAW 264.7 macrophage cells. Furthermore, the possible anti-inflammatory mechanism including NF-κB, MAPKs, and JAK2-STAT 1/3 signal pathways were comprehensively investigated.
Studies have found that inflammation, a defensive host reaction responding to pathogenic stimuli, plays a critical role in the pathogenesis of inflammation related diseases [7
]. Our study demonstrated that SF6, a bioactive high sulfate content fucoidan isolated and purified from S. japonica
, could inhibit LPS-activated inflammation via down-regulation of various inflammatory mediators. Recent studies have shown that fucoidan displays a variety of activities, including anticancer, anti-inflammatory, hypolipidemic, and immunostimulatory effects [9
]. For example, Undaria pinnatifida
derived fucoidan attenuated LPS-stimulated inflammation in RAW 264.7 macrophage cells by inhibiting the phosphorylation of MAPK (ERK, p38, and JNK) [17
]. Li et al. found that administration of fucoidan isolated from Laminaria japonica
could regulate the inflammation response via HMGB1 and NF-κB inactivation in the ischemia–reperfusion-induced myocardial damage model [32
]. In addition, fucoidan from sea cucumber Pearsonothuria graeffei
exerts powerful effects in terms of reducing obesity and improving lipid profile by regulating gut microbiota [33
]. A recent study indicated a fucoidan (LJSF4) purified from S. japonica
displayed a strong anti-inflammatory effect in vitro and in vivo by down-regulation of signal pathways, including MAPK and NF-κB [19
]. It is believed that every newly obtained fucoidan could potentially be a new compound with unique structural characteristics and promising bioactive properties; however, the molecular mechanism by which fucoidan from S. japonica
inhibits the inflammatory process remains uncomprehensive. In the present study, SF6 from S. japonica
was found to effectively inhibit inflammatory responses through blocking LPS-induced inflammation pathways, including NF-κB, MAPK, and JAK2-STAT1/3 pathways. These results suggested that SF6 exerted effective pharmacological activities for inflammation inhibition.
In our study, four fractions of fucoidan were separated and purified from S. japonica
cultivated in southeast part of China; among them, SF6 is a fucoidan rich in fucose (24.15%), galactose (41.54%), and sulfate (36.94%). A similar monosaccharide composition was observed in fucoidans of other brown seaweeds. Fucoidans extracted from the Padina commersonii
predominantly contained fucose and galactose, and also contained a sulfate group plus small amounts of mannose, rhamnose, and xylose [7
]. Similarly, fucoidan fractions isolated from Saccharina sculpera
mainly consisted of fucose and galactose, with small amounts of mannose, glucose, rhamnose, xylose, and glucuronic acid [11
]. In addition, Dai et al. found that fucoidan from Hizikia fusiforme
consisted of fucose (37.56%), galactose (38.43%), mannose (22.55%), rhamnose (1.05%), and arabinose (0.40%) [34
]. Sulfate and fucose are two critical factors with which to determine the bioactivity of fucoidan. Recent studies demonstrated that fucoidan with higher contents of sulfates and fucose usually has higher bioactivities than those with lower content [35
]. Alboofetileh et al. found that fucoidan isolated from Nizamuddinia zanardinii
with greater sulfate content had higher anticancer and immunostimulatory activities [37
]. The study of molecular characteristics and anti-inflammatory activity of three fucoidans from Ecklonia cava
suggested that the level of NO released from macrophages was proportionally related to the sulfate and fucose content of the fucoidans [38
]. SF6 has the highest sulfate content in all the four obtained fucoidan fractions. Although the content of fucose of SF6 is not the highest one, SF6 still had better anti-inflammation than the other three fractions. Furthermore, FTIR and NMR were performed to preliminarily characterize the structure of SF6, indicating it presented the specific features of natural fucoidan.
In general, abundance of pro-inflammatory cytokines including TNF-α, IL-6, and IL-1β would be over-expressed during the process of inflammation; therefore, the levels of these pro-inflammatory cytokines are usually regarded as indicators of the degree of inflammation [39
]. Thus, the suppression of excessive production of the inflammatory mediators is considered to be an effective way to prevent the occurrence and development of inflammatory process. Exhilaratingly, our study showed that SF6 obviously inhibited the LPS-induced production of TNF-α, IL-6, and IL-1β in RAW 264.7 cells.
Nitric oxide, an important pro-inflammatory mediator, is mainly synthesized by iNOS enzyme which would excessive express in reaction to various immune and inflammatory stimuli. The excessively produced NO could result in the generation of various pro-inflammatory mediators and cytokines, which would further promote the development of inflammation and finally relate to tissue damage and detrimental inflammatory disorders [40
]. The high level of iNOS is often accompanied with over-expressed COX-2 in the inflammatory process, and COX-2 is the key enzyme required for the conversion of arachidonic acid into PGE2 which is a characteristic marker of inflammatory damage [7
]. In the current study, the excessive level of NO in the LPS-stimulated macrophages was considerably down-regulated by the treatment of SF6 at various concentrations, which could be attributed to the inhibition of protein expression of iNOS and COX-2. Therefore, SF6 could exert its anti-inflammatory effect at least in part through suppression of excessive production of inflammation mediators and pro-inflammation cytokines including TNF-α, IL-6, IL-1β, NO, iNOS, and COX-2.
A number of studies indicated that LPS binding to the surface of macrophages activates a series of complex intracellular inflammatory cascade signaling pathways, including the NF-κB signal pathway [7
]. It plays a critical role in inflammatory diseases because the activation of NF-κB can induce the over-expression of inflammatory genes and further promote the production of inflammatory mediators and pro-inflammation cytokines such as NO, iNOS, COX-2, TNF-α, IL-6, and IL-1β [41
]. Generally, NF-κB, localizing in the cytoplasm, presents as a complex with IκB in an inactive form. Upon inflammatory activation, the upstream kinase IKK mediates the phosphorylation and degradation of IκB, which leads to the release and translocation of NF-κB into the nucleus and results in the inflammatory genes transcription [7
]. In our study, the effect of SF6 on NF-κB signal pathway was examined; the result indicated the phosphorylation level of NF-κB in the nucleus was remarkably increased by LPS stimulation, but nevertheless, pre-treatment with SF6 effectively inhibited the translocation of NF-κB into the nucleus. Therefore, SF6 could effectively block the activation of NF-κB pathway.
The MAPKs, as the upstream inflammatory signal pathway, are also involved in the process of inflammation. Both in vitro and in vivo researches have shown that activation of MAPKs (ERK, JNK, and p38) can mediate the gene transcriptions in the inflammatory responses to LPS [16
]. As reported that the activities of p38 and JNK MAPKs are significantly increased in macrophages stimulated by LPS through enhancing their phosphorylation levels, and then accelerate inflammatory process [42
]. In addition, ERK1/2 is identified to be directly related to NF-κB activation [44
]. The present study displayed that the increased phosphorylation levels of ERK1/2, JNK, and p38 MAPKs were effectively suppressed by SF6 treatment.
In addition to NF-κB and MAPKs signal pathways, previous studies indicated that JAK-STAT pathway is also involved in LPS-induced over-expression of inflammation mediators and pro-inflammation cytokines, especially iNOS, IL-6, and IL-1β, in macrophage cells. Upon inflammatory activation, membrane bound JAK receptor proteins enhance the phosphorylation of the major substrate, the family of STATs, including STAT1 and STAT3. Phosphorylated STAT1/3 can translocate to the nuclear and then result in the expression of pro-inflammatory genes [47
]. In this study, SF6 treatment could significantly down-regulate JAK2 phosphorylation and nuclear translocation of STAT 1/3, indicating that SF6 could prevent the activation of JAK2 and then inhibit JAK2-STAT1/3 pathway.
4. Materials and Methods
Fucoidan is purified from S. japonica, which was collected in September 2016 at Quangang, Fujian province, China. The plant material was authenticated by Prof. Meitian Xiao from Huaqiao University. A voucher specimen (number 20160906) was deposited at the Herbarium of Huaqiao University. LPS (L2880 055: B5), DMSO, dextran analytical standard (410, 270, 150, 50, 25, 5 kDa), d-(+)-glucosamine hydrochloride, d-(+)-galactosamine were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) containing l-glutamine (200 mg/L), fetal bovine serum (FBS), 3-(4, 5-dimethylthiazol-2-yl-)2.5-diphenyltetrazolium bromide (MTT) were purchased from Corning cellgro (Corning, New York, NY, USA). d-(+)-galactose, d-(+)-xylose, l-(+)-rhamnose hydrate, d-(+)-mannose were purchased from Dr. Ehrenstorfer GmbH (Dr. Ehrenstorfer GmbH, Augsburg, Germany), d-(+)-glucose was purchased from Aladdin (Aladdin, Shanghai, China), l-(−)-fucose was purchased from NIFDC (Beijing, China). Other chemicals were of analytical grade. Primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The second antibody was bought from Abcam (Cambridge, UK). Chemiluminescent HRP was purchased from Pierce Scientific (Rockford, IL, USA). Nitric oxide assay kit and the nuclear and cytoplasmic protein extraction kit were purchased from Beyotime Biotech (Guangzhou, China). TNF-α, IL-1β, and IL-6 ELISA kits were from ExCell Bio company (Shanghai, China).
4.2. Isolation and Purification of the Polysaccharide from Saccharina japonica
The S. japonica samples were washed, dried, and ground to powder, 50.0 g of the dried powder was suspended in 95% ethanol for 4 h under boiling point with continuous agitation to remove impurities such as mannitol, ester, and pigment. Then the sample was washed with ethanol for three times. After filtration, the remains were dried in a drying oven. The dried algae were dipped into distilled water and kept at 92 °C for 4 h. After that, the supernatant was separated from algae residues through filtration and gradually treated with 4% CaCl2 facilitating the precipitation of any alginic acid impurities. The supernatant was filtered and concentrated to one-eighth of its original volume by rotary evaporator. Finally, four volumes of 95% ethanol were added to the concentrated solution and kept at 4 °C overnight. Polysaccharides were recovered by centrifugation at 10,000× g and washed with ethyl alcohol for three times. The obtained crude polysaccharide was re-dissolved in distilled water and added with ethanol to a concentration of 30% to remove alginate, and subsequently to 70% to obtain a primary purified fucoidan (SF). SF was fractionated by a Q sepharose Fast flow column with distilled water and 0.1–2.0 M NaCl stepwise gradient solution, giving the fraction of SF3, SF4, SF5, SF6, respectively. The polysaccharide content in each tube was determined by the phenol-sulfuric assay method and pooled based on the total carbohydrate content. The salt in the eluate was removed by using a 3.5 kDa molecular weight cut-off dialysis tubing (Spectra/Por USA) and then lyophilized.
4.3. Homogeneity and Molecular Weight of the Purified Fucoidan
The purity of SF fractions was determined by cellulose acetate membrane electrophoresis (CAME). Briefly, cellulose acetate membranes (2 × 8 cm) were immersed in 0.1 mol/L HCl solution for 30 min. After the excess buffer was removed, 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
The molecular weights (Mw) of the purified fucoidans were determined by high performance liquid gel permeation chromatography (HPGPC) with a Shodex KS-804 column (8 mm × 300 mm, 7 μm) at 80 °C. Additionally, the sample was eluted with highly purified water at a flow rate of 0.5 mL/min. The molecular weight of polysaccharides was calculated by a standard curve obtained using dextran of 410, 270, 150, 50, 25, 5 kDa as reference standards.
4.4. Composition Analysis
Accordingly, phenol-sulfuric method for measuring the polysaccharide content [51
] and barium sulfate precipitation method for sulfate content were used during the analysis [52
]. The amount of proteins was determined according to the folin-phenol assay using the bovine serum albumin (BSA) standard curve [53
]. The composition of monosaccharide was determined by HPLC chromatography after converting them into 1-phenyl-3-methyl-5-pyrazolone (PMP), derivatives [34
]. Briefly, polysaccharide was hydrolyzed in a sealed glass tube with 2M trifluoroacetic acid (TFA) at 110 °C for 2 h; after that, TFA was removed under reduced pressure using a rotary evaporator with methanol and the hydrolysate was evaporated to dryness; 1 mL of distilled water was added to the hydrolysate, 100 μL of hydrolysate was taken into the glass tube and added with 100 μL of 0.5 M PMP in methanol and 0.3 M NaOH; then the solution was incubated at 70 °C for 1 h; after cooling down, 100 μL of 0.3 M HCl was added to adjust the pH to neutrality. Finally, excess chloroform was added; the sample was mixed by shaking; the separation of the two phases was done by allowing the mixture to settle and the organic phase was discarded. Monosaccharide compositions’ identification was done by comparison with reference sugars. Calculation of the molar ratio of the monosaccharides was carried out on the basis of the peak area of the monosaccharides. HPLC chromatograph was performed on an LC-16 (Essentia, Japan) equipped with an Agela Venusil XBP-C18 column (5 μm, 250 × 4.6 mm) (Agela China); 0.1 M phosphate buffer (pH 6.7) and acetonitrile with a ratio of 80:20 at a flow rate of 0.5 mL/min were used as elution solvents and detection wavelength was set at 250 nm; the sample injection volume was 20 μL.
4.5. Fourier Transform Infrared Characterization
The SF6 was analyzed with Fourier transform infrared (FT-IR) spectrometer (Thermo Scientific Nicolet iS 50, MA, USA) by KBr method. Scans were collected with an average of 32 scans and a spectral range and resolution of 400–4000 cm−1 and 4 cm−1, respectively.
4.6. NMR Spectroscopy
NMR spectra were recorded with a Bruker Avance III 500 spectrometer at 293 K. Samples were deuterium-exchanged by lyophilization with D2O, and dissolved in D2O with deuterated acetone as the internal standard. The concentration of the sample was 30 mg of polysaccharide/500 μL D2O.
4.7. Biological Assays
4.7.1. Cell Culture
Mouse mononuclear macrophages leukemia RAW 264.7 cell line was obtained from Chinese Academy of Medical Science and Peking Union Medical College (Beijing, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 °C.
4.7.2. Measurement of Cell Viability of RAW 264.7 Macrophages
The RAW 264.7 cells of logarithmic phase were collected and seeded at a density of 5 × 104 cells/well in 96-well plates and the cell viability was measured using the MTT assay. After pre-incubation of RAW 264.7 cells with different concentrations of sample (50, 100, 150, 200 μg/mL) at 37 °C for 24 h, the cells were incubated with 10 μL of 5 mg/mL MTT in culture medium at 37 °C for another 4 h. Then, 150 μL of DMSO was added and the absorbance was measured at 570 nm. The viability of RAW 264.7 macrophage in each well was presented as percentage of control cells.
4.7.3. Measurement of NO Production
After pre-incubation of RAW 264.7 cells (5 × 104 cells/well in 24-well plates) with LPS (1 μg/mL) and samples at 37 °C for 24 h, then the culture supernatant was mixed with the same volume of Griess reagent and absorbance at 550 nm was measured for analytic purpose of NO release. As an index of NO, the accumulation amount of nitrite was determined from a standard curve with sodium nitrite.
4.7.4. Measurement of Pro-Inflammatory Cytokines Production
The inhibitory effect of fucoidan samples on the pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) production from LPS-induced RAW 264.7 macrophages was determined by using mouse ELISA kits according to the manufacture’s protocol.
4.7.5. Preparation of Cell Lysates
The macrophage cells of logarithmic phase were collected and seeded at a density of 5 × 104 cells/well in 6-well plates. When the density of cells reached 90%, they were pre-treated with filtered culture medium with different concentrations of SF3, SF4, SF5, SF6 (50, 100, 150, 200 μg/mL) or DEM (10 ng/mL) for 2 h, and then treated with LPS (1 μg/mL) for 24 h in a humidified atmosphere of 5% CO2 at 37 °C. Upon completion of the incubation studies the culture supernatant was collected for analysis of cytokine sections, and the cells were washed three times with ice-cold phosphate-buffered saline (PBS, pH 7.2) and then scraped from the plates into ice-cold 1% PMSF lysis buffer. The protein concentration was determined by the bicinchoninic acid (BCA) method. Aliquots were stored at −80 °C for the detection for the MAPK (ERK 1/2, JNK, p38, P-ERK 1/2, P-JNK, P-p38) protein expression level.
4.7.6. Western Blot Analysis
After treated with fucoidan and LPS, RAW 264.7 cells were harvested and washed with ice-cold phosphate-buffered saline (PBS, pH 7.2). The total proteins and nuclear or cytoplasmic proteins were extracted from the cells using Cell Lysis Reagent (Sigma) and ice-cold lysis buffer. The protein content in the supernatant was determined by using the BCA protein assay kit. Equal amounts of proteins were loaded and fractionated by SDS-PAGE and then transferred into polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The membranes were blocked in 5% (w/v) skimmed milk and then incubated with specific primary antibodies overnight at 4 °C followed by incubating with horseradish peroxidase-conjugated secondary antibody. Finally, the blots were probed using enhanced chemiluminescence (ECL) and auto radiographed.
4.8. Statistical Analysis
Data were expressed as means ± SDs and examined for their statistical significance of difference with ANOVA and t-test by using SPSS 16.0. Values of p < 0.05 and p < 0.01 were considered to be statistically significant.