Inhibition of dextran sodium sulfate-induced colitis in mice by baker’s yeast polysaccharides

Carbohydrate Polymers 207 (2019) 371–381

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Inhibition of dextran sodium sulfate-induced colitis in mice by baker’s yeast polysaccharides ⁎

Ying Suna, Xiaodan Shib, Xing Zhenga, Shaoping Nieb, , Xiaojuan Xua, a b

T

⁎

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, China State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Yeast glucan Structure Anti-inflammation Inflammatory bowel disease

Most of the reported yeast polysaccharides are a mixture of chitin, β-glucan and mannoprotein, leading to different biological activities. Herein, we report the structures and the anti-inflammation of the purified baker’s yeast polysaccharides (BBG1-BBG4). Experimental data indicated that BBG1 was a highly branched β-(1,6)glucan linked to mannoprotein; BBG2 was a linear β-(1,3)-glucan; BBG3 and BBG4 were mixtures of a β-(1,6)branched β-(1,3)-glucan and a linear β-(1,3)-glucan. Of these, BBG1 exhibited stronger inhibition of pro-inflammatory mediators of NO/iNOS, IL-6, IL-1β, etc. at protein and/or mRNA levels in LPS-stimulated RAW264.7 cells through inhibiting MAPK signalling pathways. Orally administered BBG1 and BBG2 significantly decreased the pro-inflammatory mediators of IL-6, iNOS and IL-1β at protein and/or mRNA levels, as well as colonic mucosal damage and macrophages infiltration in DSS-induced colitis mice. All these findings suggest that yeast polysaccharides have potentials as anti-inflammatory drugs or adjuvants in the intestinal inflammation therapy.

1. Introduction Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease, is a chronic idiopathic inflammatory disease of the gastrointestinal tract affecting significant population numbers in the world (Burisch & Munkholm, 2013). IBD is characterized by chronic inflammatory disorders of the gastrointestinal tract (Xavier & Podolsky, 2007). During the continued inflammatory process, activated and infiltrated leukocytes produce pro-inflammatory cytokines like tumor necrosis factor alpha (TNF-α), a molecule known to play an important role in inflammation of the intestinal mucosa (Monteleone, Caruso, & Pallone, 2014). Meanwhile, increased levels of interleukins (IL) such as IL-1β, IL-6 and IL-8 have been also reported in UC patients, and their tissue levels are correlated with the degree of inflammation similarly to TNF-α (Biesiada et al., 2012). In patients with active IBD, there is a strong expression of inducible nitric oxide synthase (iNOS) at the apical side of epithelial cells, by which nitric oxide (NO) is produced in

inflammatory and autoimmune diseases, involving in nonspecific immunity and in the complex mechanism of tissue injury as a major mediator of inflammatory processes and apoptosis (Zamora, Vodovotz, & Billiar, 2000). A large number of evidences indicate that many inflammatory diseases, such as colitis and gastritis, as well as many types of cancer, occur through sustained and elevated activation of iNOS. And iNOS expression correlates well with the severity of the colitis (Kubes & McCafferty, 2000). For IBD therapy, suppressing the inflammatory mediators is the first priority to prevent the progression of the disease. Based on the central pro-inflammatory role that TNF-α plays, an antiTNF-α antibody strategy has been developed for treatment of IBD (Billiet, Rutgeerts, Ferrante, Van Assche, & Vermeire, 2014). Nonetheless, several problems have been reported as a consequence of antiTNF-α antibody therapy, including the high cost and side effects (Shepela, 2008). Therefore, it is of great interest to identify alternative and/or complementary medicinal molecules regulating specific targets associated with IBD.

Abbreviations: BBG, baker’s yeast-derived β-glucan; CCL5, chemokine (C-C motif) ligand 5; DSS, dextran sodium sulfate; DAI, disease activity index; ELISA, enzymelinked immunosorbent assay; ERK1/2, extracellular signal-regulated kinase 1/2; FBS, fetal bovine serum; GC, gas chromatography; GC–MS, gas chromatography–mass spectrometry; HPAEC, high performance anion exchange chromatography; H&E, hematoxylin and eosin; IBD, inflammatory bowel disease; IL-1β, interleukin-1β; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; IHC, immunohistochemical; JNK1/2, c-Jun N-terminal kinase 1/2; LPS, lipopolysaccharide; MDA, malondialdehyde; MAPK, mitogen-activated protein kinase; MEK1/2, MAP kinase kinase 1/2; NO, nitric oxide; PBS, phosphate-buffered saline; RT-PCR, quantitative real time reverse transcription polymerase chain reaction; SAS, Psalicylazosulfapyridine; SEC-MALS, size-exclusion chromatography-multi-angle light scattering; TNF-α, tumor necrosis factor alpha; UC, ulcerative colitis ⁎ Corresponding authors. E-mail addresses: [email protected] (S. Nie), [email protected] (X. Xu). https://doi.org/10.1016/j.carbpol.2018.11.087 Received 6 August 2018; Received in revised form 7 November 2018; Accepted 27 November 2018 Available online 27 November 2018 0144-8617/ © 2018 Published by Elsevier Ltd.

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was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies of rabbit polyclonal phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), β-actin, anti-rabbit IgG, and anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and iNOS, phosphorylated MAP kinase kinase 1/2 (pMEK1/2), p-p38 and phosphorylated c-Jun N-terminal kinase 1/2 (pJNK1/2) were purchased from Cell Signaling Technology (Beverly, MA, USA). Inhibitors of U0126 (MEK/ERK inhibitor), SP600125 (JNK inhibitor) and SB239063 (p38 inhibitor) were purchased from Med Chem Express (MCE, USA). Griess reagent was purchased from Promega (Promega Biotech Co., Ltd, USA), and DSS with molecular weight of 36–50 kDa was purchased from MP Biomedical (Santa Ana, CA, USA). Monosaccharide standards including fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), mannose (Man), fructose (Fru), xylose (xyl), galacturonic acid (GalA) and glucuronic acid (GlcA), methyl iodide (CH3I), sodium borodeuteride (NaBD4), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich chemical Co. (St. Louis, MO, USA) for sugar component analysis of the purified BBG samples.

Natural polysaccharides with anti-inflammatory activity provide a new way in reducing side effect of IBD therapy. β-Glucan is known to have anti-inflammatory properties (Ukai, Hara, Kuruma, & Tanaka, 1983) and dietary β-Glucans have been demonstrated to have beneficial effects on IBD (Ye et al., 2011). Different glucans from varied sources like mushrooms (Nosal’ova, Bobek, Cerna, Galbavý, & Stvrtina, 2001; Shi et al., 2016), oat (Thies, Masson, Boffetta, & Kris-Etherton, 2014), seaweed (O’Shea et al., 2016), bacteria (Lee et al., 2014) etc. have been reported to play positive roles in IBD therapy that were attributed to the crosstalk between the immune cells, epithelial cells, gut microorganisms, and the main mechanisms involve promoting immune system and reducing inflammation as well as modulating gut microbiota (Nie, Lin, & Luo, 2017). The bioactivities of yeast-derived glucan have been studied for a long history due to its rich source and multiple activities, among which the anti-inflammation and immunomodulatory activity are the most representative functions of yeast glucan. Dietary with yeast glucans can enhance the host immune system and increase resistance against invading pathogens that is associated with the crosstalk with intestinal immune cells (Tsukada et al., 2003). However, yeastderived glucans are usually extracted with heterogeneous composition. Many reported yeast-derived glucans are mixture of β-(1,3)-glucan with or without β-(1,6)-linked branches, β-(1,6)-glucan, mannoprotein and chitin (Bacon, Farmer, Jones, & Taylor, 1969; Lipke & Ovalle, 1998). In our previous work, baker’s yeast-derived polysaccharide particles with ∼75% of β-glucans were demonstrated to reduce lipopolysaccharide (LPS)-induced NO production in RAW264.7 macrophages (Xu, Yasuda, Mizuno, & Ashida, 2012), showing that yeast glucan is a good candidate to inhibit inflammatory modulators. Usually, the bioactivity of yeast polysaccharide is owned to the β-(1,3)-linked part, but the function and the exact composition of other components such as β-1,6-linked parts, possessing an important role in the yeast cell wall, are not fully known (Lesage & Bussey, 2006). In this work, the crude baker’s yeast polysaccharide with major component of β-glucan (denoted as BBG) was carefully purified successively by hot water and alkali of NaOH to get four fractions (coded as BBG1, BBG2, BBG3, and BBG4) with different compositions. The chemical structures of the components and anti-inflammation in LPS-stimulated RAW264.7 cells and dextran sodium sulfate (DSS)-induced colitis mice were studied to evaluate the active components in BBG. Consequently, the water extract fraction BBG1 composed of glucose, mannose and protein showed much higher anti-inflammatory activity than BBG2-BBG4 composed of glucose owing much to its chemical structure and lower molecular weight as well as its better water-solubility; oral administration of the purified fractions of BBG1 and BBG2 significantly ameliorated the intestinal inflammation in mice characterized by down-regulation of iNOS and the pro-inflammatory cytokines. This work provides a scientific basis for better understanding the correlation between chemical structure and biological activity of yeast polysaccharides.

2.2. Characterization High performance anion exchange chromatography (HPAEC). HPAEC was used to determine the monosaccharide composition of BBG1 according to the reported method (Shi et al., 2017). A detailed description is provided in the Supporting Information. Gas chromatography (GC). The components of BBG samples were analyzed by gas chromatography according to the reported method (Zheng et al., 2016). The detailed description is provided in the Supporting Information. Gas chromatography-mass spectrometry (GC–MS). The glycosidic linkages of BBG1 was analyzed by the methylation analysis combined with a gas chromatography-mass spectrometer. The detailed description is provided in the Supporting Information. Fourier transform infrared spectroscopy (FTIR). FTIR spectra of BBG fractions were recorded on a Nicolet 170SX FT-IR spectrometer (Spectrum One, PerkinElmer Co., Madison, WI) in the wavenumber range of 4000–400 cm−1. Samples were dried prior to tableting with KBr powder. Nuclear magnetic resonance (NMR) Spectroscopy. 13C NMR spectra of BBG fractions, HSQC and 1H-1H COSY spectra of BBG3 were acquired at 25 °C on a DRX-Bruker spectrometer operating at a proton frequency of 500 MHz. 2.3. Cell culture and animal experiments Cell culture. Murine RAW264.7 macrophages from the China Center for Typical Culture Collection (Wuhan, China) were maintained in DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS). The cells were incubated at 37 °C under a humidified atmosphere of 95% air and 5% CO2. For the inflammatory factors assay, RAW264.7 cells (5 × 105 cells/ mL) were seeded in 24-well plates and incubated for 24 h before stimulation. Cells were rinsed with phosphate-buffered saline (PBS), and the medium was exchanged to DMEM without FBS, followed by exposure to BBG1 (200 μg/mL), BBG2 (200 μg/mL) or LPS (500 ng/mL) and incubation for desired time. The conditioned media were collected and centrifuged for NO, IL-1β and IL-6 determination. Animal experiments. Seven-week-old male C57BL/6 mice weighing 24–26 g were purchased from the Animal Experiment Center of Wuhan University (Wuhan, China), and all animal protocols were approved by the Wuhan University Center for Animal Experiment/Animal Biosafety Level-III laboratory (Hubei Province, China). Mice were housed under the controlled room temperature (22 ± 2 °C) and humidity (55 ± 5%) on a 12 h light-dark cycle with free access to water and diets, and were randomly divided into 5 groups (the control group; 2% DSS group; 2%

2. Materials and methods 2.1. Materials and reagents The crude BBG with purity of ∼75% from S. cerevisiae was kindly provided by Prof. Hitoshi Ashida in Kobe University, which was a gift from Oriental Yeast Co., Ltd (Tokyo, Japan). It was further purified according to the procedures in Fig. 1 to obtain four fractions coded as BBG1, BBG2, BBG3 and BBG4, respectively. The details on preparation are provided in Supporting Information. The chemicals used in this work are listed as follows. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 and salicylazosulfapyridine (SASP) were purchased from Sigma (St. Louis, MO, USA). Mouse interleukin (IL)-1β and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Dakewe Biotech Corporation (Beijing, China), and malondialdehyde (MDA) assay kit 372

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Fig. 1. Purification of the crude BBG.

DSS + BBG1; 2% DSS + BBG2 group; 2% DSS + SASP group, 7 mice/ group). The mice in the control group were supplied with distilled water, whereas all other experimental groups were given 2.0% (w/v) DSS from day 4 to day 9. Besides, the mice in the SASP (50 mg/kg/ day)-, BBG1 (50 mg/kg/day)- and BBG2 (50 mg/kg/day)-treated groups were administered by gavage with saline from day 1 to day 15, respectively. The schematic presentation of experimental schedule is shown in Fig. S1. The animal experiment mentioned above was carried out in accordance National Research Council’s Guide for the care and use of laboratory animals. Evaluation of disease activity index (DAI). The DAI was determined by scoring the changes of body weight loss, diarrheal condition and fecal bleeding. The DAI is the sum of the score of these three parameters. Each score was determined as shown in Table S1 according to the reported method (Mu et al., 2016). At the end of the experiment, all mice were sacrificed; colons were dissected from each mouse, and the lengths between the ileo-cecal junction and anal verge were measured. Histological and immunohistochemical (IHC) analysis. The harvested colons of mice were gently washed with ice-cold PBS, fixed in 4% paraformaldehyde overnight and embedded in paraffin. After slicing, hematoxylin and eosin (H&E) staining and IHC staining were performed. The histological scoring system is shown in Table S2 determined according to the reported method (Williams et al., 2001). The detailed procedures of IHC analysis are provided in the Supporting Information. The ratio of the macrophages area/colon area in IHC image was digitally quantified using Image Pro Plus 6.0 software.

instructions (Nanjing Jiancheng Bioengineering institute).

2.4. Cytokine assay

3.1. Chemical structure of BBG fractions

NO concentration in the medium supernatant of RAW264.7 cells was determined by the Griess reaction. IL-1β and IL-6 cytokines were assessed using ELISA kits according to the manufacturer’s instructions (Dakewe Biotech Corporation). MDA in colon tissue slurry supernatant was measured by MDA assay kit according to the manufacturer’s

Yeast glucan has been shown to be heterogeneous, and is a mixture of two structurally related β-glucans. Of these, the major component (∼85%) is a branched β-(1,3)-glucan containing 3% of β-(1,6)-glucosidic interchain linkages, and the minor component is a highly branched β-(1,6)-glucan containing 5–10% of β-(1,3)-glucosidic interchain

2.5. Quantitative real time reverse transcription polymerase chain reaction (RT-PCR) assay Total RNA from RAW264.7 cells and colon tissues from mice were extracted and analyzed by RT-PCR assay according to the reported method (Cao, Sun, Zou, Li, & Xu, 2017; Xu, Yasuda, Nakamura-Tsuruta, Mizuno, & Ashida, 2012). A detailed description is provided in the Supporting Information, and the sequences of primers used are listed in Table S3. 2.6. Western blotting analysis Total proteins from RAW264.7 cells and colon tissues from mice were extracted and analyzed by Western blotting. A detailed description is provided in the Supporting Information. 2.7. Statistics All data are presented as the mean ± standard error (S.E.) from at least three independent experiments unless specified otherwise. Student’s t-test was performed, and the differences were considered statistically significant at p < 0.05. 3. Results and discussion

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linkages and 15–25% of the glucose residues on the backbone triply linked at C1, C3 and C6 (Manners, Masson, & Patterson, 1973). According to the cell wall architecture of yeast, mannan or mannoprotein is also one of major components of the cell wall (Lipke & Ovalle, 1998). Moreover, the components change with different yeast strains and cultivation conditions (Bacon et al., 1969). In this work, the crude BBG was purified according to the procedures in Fig. 1, and the chemical structures of these fractions were studied. The sugar and protein analysis data indicate that BBG fractions had much higher content of sugars than the crude BBG, and BBG1 contained 6.1% protein (Table S4). In particular, BBG2-BBG4 were almost pure carbohydrates, and BBG2 with the yield of 63.5% was the major component of the crude BBG. The SEC patterns of BBG fractions in DMSO (Fig. S2) suggest that BBG1 and BBG2 were homogeneous with an almost symmetric single peak, while both BBG3 and BBG4 showed a shoulder besides the major peak, suggesting a heterogeneous fraction with two components. BBG2, the same fraction reported in our previous work (Zheng et al., 2016), has been determined to be a linear β-(1,3)-glucan, while other three fractions were not yet known. Therefore, BBG1, BBG3 and BBG4 were used for structure analysis in the following. HPAEC data (Fig. 2A) shows that BBG1 consisted of two monosaccharide units of glucose and mannose, and the mass contents were estimated to be 74.5% for glucose and 25.5% for mannose. The GC traces of monosaccharides indicate that both BBG3 and BBG4 were composed of only glucose (Fig. 2C). Clearly, BBG1 had more complex structure than BBG2-BBG4. As reported, mannoproteins are located in the outer layer of yeast cell walls, which are linked to β-1,6-glucan by a glycosyl phosphatidylinositol (GPI) anchor or to β-1,3-glucan by alkalisoluble bonds (Lesage & Bussey, 2006). In Table S4, the protein content measured by bicinchoninic acid (BCA) assay shows that there was 6.1% protein in BBG1, and no proteins were detected in other BBG fractions. In combination with the SEC pattern (Fig. S4), it can thus be speculated that BBG1, the hot-water extracts, might be a β-glucan as the major component (74.5%) linked to mannoproteins as the minor one (25.5%), but not a mixture of the two components. IR spectra (Fig. 2B) confirm

Table 1 Methylation analysis and linkages of sugars in BBG1. Sugar

Linkage type

Amount (mol/100 mol)

2,3,4-tri-O-methyl Glc 2,3,4,6-tetra-O-methyl Glc 3-O-methyl Glc 2-O-methyl Glc 2,3-di-O-methyl Glc 4-O-methyl Man 2,4,6-tri-O-methyl Man 2, 6-di-O-methyl Man

1,6-Glc T-Glc 1,2,4,6-Glc 1,3,4,6-Glc 1,3,6-Glc 1,2,3,6-Man 1,3-Man 1,3,4-Man

36.6 18.4 10.2 4.8 4.3 17.5 4.6 3.6

the existence of mannose in BBG1 characterized by a weak characteristic peak of 810 cm−1 (Zhao et al., 2014) and β-glucans of BBG3 and BBG4 with the characteristic β-configuration peak at 890 cm−1 (Wang, Yuan, & Yue, 2014). To clarify the linkages in BBG1, methylation and GC–MS were performed. As shown in Table 1, mainly alditol acetates for 2,3,4-tri-Omethyl Glc (1,6-linked, 36.6%), followed by 2,3,4,6-tetra-O-methyl glucose (permethylated terminal glucose, 18.4%), 3-O-methyl Glc (1,2,4,6-linked, 10.2%), 2-O-methyl Glc (1,3,4,6-linked, 4.8%), 2,4-diO-methyl Glc (1,3,6-linked, 4.3%), 4-O-methyl Man (1,2,3,6-linked, 17.5%), 2,4,6-tri-O-methyl Man (1,3-linked, 4.6%), and 2,6-di-O-methyl Man (1,3,4-linked, 3.6%) species were clarified in BBG1. The approximate molar ratio of glucose to mannose was 2.9:1.0, consistent with the HPEAC result in Fig. 2A. Aimanianda et al. have demonstrated that the cell wall β-(1,6)-glucan of S. cerevisiae is on average branched at every fifth residue with one or two β-(1,3)-linked glucose units in the side chain (Aimanianda et al., 2009). Herein, the glucan component in BBG1 contained 1,6- and 1,3,6-linkages as well as 1,2,4,6- and 1,3,4,6linakges, showing more branches than the reported β-(1,6)-glucan (Aimanianda et al., 2009). Additionally, the terminal glucose residues accounted for 18.4% as the second highest content in BBG1. It can thus be deduced that the glucan component in BBG1 was a highly branched

Fig. 2. Component analysis of BBG fractions. (A) HPAEC spectra of BBG1 and monosaccharide standards. (B) FTIR spectra of BBG fractions. (C) GC spectra of BBG3 and BBG4. 374

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Fig. 3. NMR spectra of BBG fractions. 13C NMR spectra of BBG1 (A), BBG3 (B) and BBG4 (C), HSQC (D) and COSY (E) NMR spectra of BBG3. NMR experiments were performed in DMSO-d6 at 25 °C. Chemical shifts are expressed in δ ppm.

usually have a similar overall architecture, consisting of α-1,6-linked mannose backbone decorated with side chains of different composition and structure such as short α-1,2- and α-1,3-linked side chains (Lipke & Ovalle, 1998; Vinogradov, Petersen, & Bock, 1998); however, the exact structure of the mannoprotein is largely unknown. As shown in Table 1, there were not 1,3- and 1,4- linked glucose residues, suggesting no β(1,3)-glucan or chitin linked to BBG1; however, there were 1,2,3,6-, 1,3-, and 1,3,4-linked mannose residues in BBG1, suggesting that the mannan component was linked to the major component of (1,6)-glucan. According to the amount of the linkage type, it was deduced that 1,6-

(1, 6)-glucan with branches at C2, C3, and C4 of (1,6)-linked glucose units. If these branching points were all linked to glucose residues, there should be 34.3% of terminal glucoses according to the amount of 1,3,6-, 1,2,4,6- and 1,3,4,6-linakges, much higher than 18.4%. Therefore, some other substituted sugar units should be linked to the (1,6)-glucan besides glucose. As reported, β-(1,6)-glucan stabilizes the cell wall, and plays a central role as a linker for specific cell wall components, including β-(1,3)-glucan, chitin, and mannoproteins (Aimanianda et al., 2009); on the outer surface of the wall are mannoproteins, which are extensively O and N glycosylated; the N-glycosylated oligosaccharides 375

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3.2. BBG fractions decrease inflammatory cytokines expression in LPSstimulated RAW264.7 cells

Table 2 Chemical shifts (δ/ppm) of β-(1,6)-glucan, β-(1,3)-glucan, mannoprotein and BBG fractions. C1

C2

C3

C4

C5

C6

Source

β-(1→6)-glucan

103.1

73.3

76.5

69.8

75.4

68.3

β-(1→3)-glucan

103.2

73.2

86.5

68.9

76.7

61.4

Mannoprotein

102.8 101.3 99.1

81.2 70.9 70.4

67.8

74.0

67.3 67.0 66.6 61.9

104.6 103.8 103.5 103.2 102.3 101.1 98.8 103.5

74.0 73.3 72.8 77.3 78.9 71.3

79.3 79.0 78.7 72.0 71.3 77.1 86.8 88.1

Smiderle et al. (2013) Watanabe et al. (2014) Gonçalves et al. (2002)

70.5 68.6 76.7

77.1 76.1

69.0 67.7 61.6

75.5 70.5

74.4

73.3

79.1 71.3 71.9 86.7

68.9

76.8

67.1 66.2 61.6 61.3

103.5 103.7 103.5

73.3 73.9 73.3

86.7 76.9 86.7

68.9 70.2 68.9

76.8 76.9 76.8

68.9 61.6 61.6

BBG1

β-1,6-glucan part

Mannoprotein part BBG2 BBG3 BBG4

As reported, inflammatory cytokines of IL-1β, TNF-α, IL-6, etc. contribute to the pathogenesis of many diseases, such as rheumatoid arthritis, Crohn’s disease and encephalomyelitis (Ishihara & Hirano, 2002). LPS is an active substance of gram-negative bacteria, a wellknown activator of macrophage to initiate Toll-like receptor 4 (TLR4), that can induce macrophage RAW264.7 cells to produce large amounts of NO and other inflammatory factors such as IL-1β, IL-6, etc. (Laskin & Pendino, 1995; Wang, Jiang, Zhang, Qian, & Du, 2012) It is worth noting that treatment with BBG and its fractions alone did not induce NO, IL-6 and IL-1β production in resting macrophages (data are not shown), indicating that the endotoxin level in BBG samples is negligible. The LPS-stimulated RAW264.7 cells were thus used to investigate the anti-inflammatory activity of BBG fractions. As shown in Fig. 4A and B, LPS stimulation for different time significantly enhanced NO production in RAW264.7 cells, and the crude BBG and its fractions significantly inhibited NO production, similar to our previously reported result (Xu, Yasuda et al., 2012). BBG2-BBG4 fractions with the same composition of glucose showed almost the same inhibitory effect, and BBG1 composed of glucose, mannose and a few proteins exhibited higher inhibition of NO secretion. As discussed above, BBG1 was a highly branched β-(1,6)-glucan linked to mannoprotein extracted from the dilute phase of water-suspensions, and BBG2-BBG4 fractions were β-(1,3)-glucans with or without β-(1,6)-linked glucose residues as branches from alkaline suspensions. And the noticeable NO inhibition difference between BBG1 and BBG2-BBG4 fractions was possibly attributable to the different water-solubility and chemical composition. It seems that the side glucose linkages showed little effect on NO inhibition. As shown in Fig. S2, BBG3 and BBG4 are heterogeneous indicated by the shoulder peak in SEC-MALS, which may create controversy in discussion structure-function relationship. Based on these results, BBG1 and BBG2 composed of homogeneous components with different sugar compositions were thus chosen for further anti-inflammatory study in the following experiments. As shown in Fig. 4C and D, both BBG1 and BBG2 could significantly inhibit IL-6 and IL-1β production in LPS-stimulated RAW264.7. Similar to the result of NO, BBG1 showed higher inhibition of IL-6 and IL-1β than BBG2. To confirm these results, the mRNA expression changes of inflammation-associated factors of iNOS, IL-1β, and IL-6 were measured by RT-PCR as well as other cytokines/chemokines (Fig. 4E). Consequently, BBG1 and BBG2 significantly down-regulated the high mRNA expression of iNOS, IL-6, chemokine (C-C motif) ligand 5 (CCL5), IL-10, IL-27 and IL-1ra induced by LPS except IL-1β and IL-1α. As well known, iNOS, IL-6, CCL5 and IL-27 are closely associated with inflammation progression (Aktan, 2004; Murooka, Rahbar, Platanias, & Fish, 2008; Neurath & Finotto, 2011; Pflanz et al., 2002), suggesting BBG1 and BBG2 really inhibited LPS-induced inflammation. IL-10, an anti-inflammatory cytokine, the level of which increased with increasing inflammatory reaction, could inhibit LPS-induced production of IL-1, IL6, and TNF-α significantly in macrophage cell lines (Fiorentino, Zlotnik, Mosmann, Howard, & O’Garra, 1991); IL-1rα is IL-1 receptor antagonist, regulating IL-1α and IL-1β pro-inflammatory activity by competing with them for binding sites of the receptor (Dinarello, 2002). Suppression of IL-10 and IL-1rα also reflected the reduced inflammatory level (Fiorentino et al., 1991). In combination with the results of cytokine assay, it can be obtained that inhibition of NO and IL-6 is through suppression of their respective mRNA expression by BBG fractions.

This work

Zheng et al. (2016) This work This work

linked mannose might be the backbone with branches at C2, C3 and C4. As shown in Table S4, BBG1 contained proteins, suggesting that it was mannoprotein linked to the (1,6)-glucan. The structural features of BBG1 were further elucidated by NMR spectral analysis. The 13C NMR spectrum of BBG1 (Fig. 3A) contained more carbon peaks than the linear β-1,3-glucan and β-1,6-glucan. The chemical shifts of carbons were assigned according to the reported βglucan (Synytsya & Novák, 2013) and mannan obtained directly from S. cerevisiae yeast (Vinogradov et al., 1998) and summarized in Table 2 as well as those of the linear β-1,3-glucan (Watanabe, Tamura, Saito, Habu, & Isogai, 2014), β-1,6-glucan (Smiderle et al., 2013) and mannoprotein (Gonçalves, Heyraud, de Pinho, & Rinaudo, 2002). The chemical shifts of anomeric carbons indicate that the glucose was βconfiguration, and mannose was α-configuration. All these data conformed the linkages in Table 1, and BBG1 was a composite of highly branched β-(1,6)-glucan linked to mannoprotein. As shown in Table 2 and Fig. 2C, BBG3 and BBG4 were pure polymers consisting of glucose, having relatively simple chemical structure. BBG4 had only six signals corresponding to the six carbons of (1,3)linked glucose (Fig. 3C), suggesting the linear β-(1,3)-glucan as the major component according to the chemical shifts (Table 2), similar to BBG2 (Zheng et al., 2016). In contrast, BBG3 showed several weak signals besides six major signals (Fig. 3B). The chemical shifts of BBG3 were assigned and summarized in Table 2 by using 2D HSQC (Fig. 3D) and COSY (Fig. 3E) as well as 1D 13C NMR (Fig. 3B). As a result, the carbon chemical shifts of BBG3 were similar to those of (1,6)-branched β-(1,3)-glucans such as scleroglucan (Falch, Espevik, Ryan, & Stokke, 2000), schizophyllan (Zhang, Kong, Fang, Nishinari, & Phillips, 2013), and Lentinan (Xu, Chen, Zhang, & Ashida, 2012). In combination of the weak signals of the substituted carbons, BBG3 was mainly a β-(1,3)glucan with a small amount of (1,6)-linked glucose residues as side chains. From size-exclusion chromatography-multi-angle light scattering (SEC-MALS), the Mw of BBG fractions were estimated and summarized in Table S4. Mw increased with the sample number, that is, BBG1 had the lowest Mw and BBG4 had the highest Mw. BBG2 was the precipitate of BBG/NaOH suspension, indicative of the worst water-solubility, consistent with the linear chemical structure analyzed above. Due to the presence of branching, BBG3 and BBG4 had relatively higher watersolubility than BBG2, although both of them had higher Mw than BBG2. Therefore, branching is benefit for enhancing the water-solubility of polysaccharides with the same composition.

3.3. BBG fractions suppress the activation of MAPK in LPS-stimulated RAW264.7 cells Mitogen-activated protein kinase (MAPK) signaling pathway is considered as the important pathway mediating LPS-induced 376

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Fig. 4. BBG fractions reduced inflammation in LPS-stimulated RAW264.7 macrophages. RAW264.7 macrophages were stimulated by LPS (500 ng/mL) and BBG fractions (200 μg/mL) for a desired time, and the supernatant was collected for determining NO (A, B), IL-1β (C) and IL-6 (D) levels. Total RNA was isolated from cells and subjected to RT-PCR analysis (E). The LPS-stimulation time was 36 h in (A) and 12 h in (E). **p＜0.01, ***p＜0.001 vs. LPS. ap＜0.01 and bp＜0.001 vs. LPS.

inflammation-associated diseases.

inflammation factors secretion through activation (phosphorylation) of ERK, JNK and p38 (Guha & Mackman, 2001) and subsequently promoting transcription of cytokines. To verify the relationship between NO production and MAPK signaling pathway, we evaluated the effect of specific inhibitors of p38, JNK1/2 and ERK1/2 on LPS-induced NO production. As a result, treatment with SB239063 (10 μM, p38 inhibitor), SP600125 (10 μM, JNK inhibitor) and U0126 (30 μM, MEK/ ERK inhibitor) all significantly decreased the production of NO induced by LPS (Fig. 5A). In particular, p38 and JNK1/2 inhibitors showed stronger inhibition than the ERK1/2 inhibitor, suggesting that p38 and JNK-dependent pathways played the major role in NO production. The western blotting results showed that LPS enhanced the expression of pERK1/2, p-MEK1/2, p-p38, and p-JNK1/2, while BBG1 and BBG2 significantly inhibited their high expression (Fig. 5C). Moreover, BBG1 and BBG2 significantly inhibited the production of iNOS (Fig. 5B), and the inhibition rate of BBG1 was higher than BBG2, consistent with the results in NO production and iNOS mRNA expression. These data indicate that BBG1 and BBG2 inhibited LPS-induced NO production via an inhibition of the ERK1/2, p38 and JNK signaling pathways. Therefore, BBG has a great potential for prevention and treatment of

3.4. BBG fractions reduce DSS-induced colitis in mice Since BBG1 and BBG2 had a good effect on LPS-induced cell inflammation by suppressing the phosphorylation of MAPK signaling pathways, we then investigated the anti-inflammatory activity of BBG1 and BBG2 in DSS-induced colitis mice. SASP was utilized as a reference drug. As shown in Fig. 6A, the loss of body weight induced by DSS was significantly reduced in mice treated with BBG1 and BBG2 at the dosage of 50 mg/kg comparing with the untreated colitis mice. The severity of clinical symptoms in mice with DSS-induced colitis is usually reflected by the scores of DAI (Mu et al., 2016). As shown in Fig. 6B, the DAI scores of mice treated with BBG1 and BBG2 as well as SASP were significantly lower than those of the DSS group. Additionally, shortening of colon length was significantly attenuated by BBG1 and BBG2 (Fig. 6C and D). Spleen is a very important organ for immunity, the size of which is related to the severity of inflammation. Usually, splenomegaly occurs during the progress of colitis. In Fig. 6C, spleen sizes of mice treated with BBG1 and BBG2 were smaller than the DSS group, 377

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Fig. 5. BBG1 and BBG2 inhibited the activation of MAPK pathway in RAW264.7 macrophages induced by LPS. RAW264.7 macrophages were incubated with LPS (500 ng/mL) and SB239063 (p38 inhibitor, 10 μM), SP600125 (JNK inhibitor, 10 μM), U0126 (MEK/ERK inhibitor, 30 μM), respectively for 48 h, and the supernatants were collected and used to determine NO (A). RAW264.7 macrophages were incubated with LPS (500 μg/ml) and glucan (200 μg/ml) for 24 h (B) and 30, 60, 90 min (C), then total proteins of the cell were extracted and used to determine the level of iNOS expression and MAPK pathway protein. B1 + L, i.e., BBG1 + LPS. B2+L, i.e., BBG2 + LPS. **p＜0.01, ***p＜0.001 vs. LPS.

Fig. 6. BBG1 and BBG2 reduced the inflammation in colitis mice. Colitis was induced in all groups except the control group. The change in body weight (A), stool consistency and stool bleeding were recorded daily. The DAI score (B) was determined by combining scores of (i) body weight loss, (ii) stool consistency and (iii) stool bleeding. On day 15, all the mice were sacrificed and the colons and spleens were pictured (C). The lengths of colons were measured (D). Data are expressed as mean ± SD, n = 7 (*p < 0.05, **p < 0.01 vs. DSS group).

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Fig. 7. BBG1 and BBG2 ameliorated the inflammation in colon of 2% DSS induced colitis. On day 15, the mice were sacrificed and the colons were harvested. H&E staining (A) and IHC staining (B) for macrophages marked by CD68 of the colon sections were conducted. Histological score (C) and quantitative analysis of macrophages infiltration (D) of the colon tissue were carried out. Labels (in A): arrow 1 indicates the loss of crypts; arrow 2 indicates necrosis; arrows 3, 6 and 7 indicate and accumulation of inflammatory cells; arrows 4 and 5 indicate intact crypts. The scales at bottom left are 200 μm. Am/Ac, i.e., the ratio of macrophages area to colon section area. Data are expressed as mean ± SD, n = 3 (*p < 0.05, **p < 0.01 vs. DSS group).

& Fiocchi, 2016), and the recruitment of macrophages will aggravate the colon inflammation. Fig. 7B shows the IHC staining of CD68-positive macrophages indicated by brown color in colon sections. Obviously, BBG2 (c) and BBG1 (d) group with less brown color exhibited less macrophages infiltration than DSS (b). Meanwhile, macrophages infiltration was quantified by calculating the ratio of the macrophages area/colon area (Fig. 7D). These findings turned out that BBG1 and BBG2 remarkably reduced inflammation-induced macrophages infiltration. Inflammation in the intestinal mucosa contains a complex array of inflammatory mediators, including iNOS and cytokines that are correlated with the degree of inflammation. The inflamed intestines of patients with IBD are massively infiltrated by inflammatory cells that release a large number of pro-inflammatory mediators, such as cytokines like IL-6, IL-1β and NO (Soufli, Toumi, Rafa, & Touil-Boukoffa, 2016). Moreover, it was reported that the overexpression of iNOS and the increased concentration of NO are related to the severity of IBD (Avdagić et al., 2013). IL-6 has been identified as a crucial regulator of IBD pathogenesis and patients with IBD have also shown augmented IL6 serum levels (Neurath & Finotto, 2011). Several studies have proved

indicating that BBG1 and BBG2 suppressed the inflammation. All these data demonstrate that BBG fractions reduced the clinical symptoms, and promoted recovery of colitis mice, which can thus be used as a potential anti-inflammation drug candidate. To further confirm the above results, H&E and IHC staining analysis of colons were performed. As shown in Fig. 7A, comparing with the normal mice (a), DSS caused severe mucosal damage in colonic tissue (b) such as a loss of crypts (arrow 1), necrosis (arrow 2), and focal influx of inflammatory cells (arrow 3). In contrast, mice in BBG2 (c) and BBG1 (d) treatment groups exhibited more intact crypts (arrows 4 and 5) and less influx of inflammatory cells (arrows 6 and 7), indicating that BBG1 and BBG2 significantly ameliorated the signs and symptoms of colonic inflammation. Inflammation and crypt damage were assessed using a validated scoring scheme (Table S2) as reported previously (Williams et al., 2001). The histological evaluation of colons (Fig. 7C) from BBG1 and BBG2 treated mice revealed a pronounced reduction in the inflammatory response resulting in a decreased microscopic damage score, compared with colons from DSS mice. Macrophages are considered as an important source of pro-inflammatory mediators, such as NO and IL-6, playing a key role in the pathophysiology of IBD (De Souza

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Fig. 8. BBG1 and BBG2 reduced the inflammatory mediators in DSS induced colitis mice. On day 15, the mice were sacrificed, the colons and serum were harvested. The serum IL-6 (A), colon iNOS (B), colon mRNA of iNOS, IL-6, IL-1β expression (C) and colon MDA activity (D) were determined. Data are expressed as mean ± SD, n = 3 (*p < 0.05, ap＜0.01 vs. DSS group).

showed higher inhibition than other three BBGs composed of glucose. Additionally, BBG1 and BBG2 inhibited other pro-inflammatory mediators such as iNOS, IL-6, IL-1β, IL-27, CCL-5 etc. at protein and/or mRNA levels in vitro. Furthermore, BBG1 and BBG2 reduced the clinical symptoms and promoted recovery of mice with colitis indicated by suppressing inflammatory cytokines like IL-6, IL-1β and inflammatory mediator iNOS expression at protein and/or gene levels. Taken together, BBG showed good anti-inflammatory effect in vitro and in colitis mice. Our findings reveal a good prospect of yeast polysaccharides as an anti-inflammatory drug or adjuvant as well as a functional food in the intestinal inflammation treatment.

that blockade of IL-6 signaling in chronic intestinal inflammation caused significant suppression of colitis activity (Atreya et al., 2000; Noguchi et al., 2007; Yamamoto, Yoshizaki, Kishimoto, & Ito, 2000). These inflammatory mediators impair the function of the intestinal epithelia, leading to a robust recruitment of immune cells to the site of injury; the immune cells then produce additional pro-inflammatory cytokines that increase inflammation further. As shown above, BBG fractions could reduce the expression of NO, IL-1β and IL-6 in LPS-stimulated RAW264.7 cells significantly in vitro. In DSS-induced colitis, BBG fractions not only suppressed the serum IL-6 titer, reflecting the suppression of systemic inflammation (Fig. 8A), but also inhibited iNOS protein expression (Fig. 8B) as well as mRNA levels of iNOS, IL-6 and IL-1β in colon tissues (Fig. 8C). These findings indicate that BBG1 and BBG2 could reduce DSS-induced colitis effectively. MDA is one of the well-known end-products of lipid peroxidation induced by ROS, reflecting free radical induced damage and are therefore useful in assessing the membrane-damaging role of free radicals in patients with IBD (Baskol, Baskol, Yurci, Ozbakir, & Yucesoy, 2006). Herein, BBG1 and BBG2 obviously down-regulated the level of MDA (Fig. 8D), suggesting the reduced mucosal damage, consistent with the result in Fig. 7A. Collectively, all these results provide evidence for the potential use of BBG fractions as a preventive measure for patients with colitis. Yeast is generally considered as healthy food ingredient and used in food industry for centuries. Therefore, yeast glucan is a promising medicinal food addition for IBD patients.

Acknowledgments We gratefully acknowledge the financial supports from the National Key Research and Development Plan of China (2016YFD0400202), and National Natural Science Foundation of China (21574102 and 21274114). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2018.11.087. References Aimanianda, V., Clavaud, C., Simenel, C., Fontaine, T., Delepierre, M., & Latgé, J. P. (2009). Cell wall β-(1, 6)-glucan of Saccharomyces cerevisiae-structural characterization and in situ synthesis. The Journal of Biological Chemistry, 284(20), 13401–13412. Aktan, F. (2004). iNOS-mediated nitric oxide production and its regulation. Life Sciences, 75(6), 639–653. Atreya, R., Mudter, J., Finotto, S., Müllberg, J., Jostock, T., Wirtz, S., ... Strand, D. (2000). Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: Evidence in Crohn disease and experimental colitis in vivo. Nature Medicine, 6(5), 583. Avdagić, N., Zaćiragić, A., Babić, N., Hukić, M., Šeremet, M., Lepara, O., & Nakaš-Ićindić, E. (2013). Nitric oxide as a potential biomarker in inflammatory bowel disease. Bosnian Journal of Basic Medical Sciences, 13(1), 5. Bacon, J. S. D., Farmer, V. C., Jones, D., & Taylor, I. F. (1969). The glucan components of the cell wall of baker’s yeast (Saccharomyces cerevisiae) considered in relation to its ultrastructure. The Biochemical Journal, 114(3), 557–567. Baskol, G., Baskol, M., Yurci, A., Ozbakir, O., & Yucesoy, M. (2006). Serum paraoxonase 1 activity and malondialdehyde levels in patients with ulcerative colitis. Cell

4. Conclusion In summary, the different components (BBG1-BBG4) of yeast cell wall glucan were isolated, and the chemical structures were determined by FTIR, GC, GC–MS and NMR. The results indicate that BBG1 and BBG2 were composed of homogeneous components, while BBG3 and BBG4 were heterogeneous. In brief, BBG1 was a highly branched β(1,6)-glucan linked to mannoprotein; BBG2 was a linear β-(1,3)-glucan; BBG3 was a β-(1,3)-glucan branched at β-(1,6) contaminated with a small amount of linear β-(1,3)-glucan, and BBG4 was composed of a linear β-(1,3)-glucan as a major and a β-(1,6)-branched β-(1,3)-glucan as a minor. Four BBG fractions inhibited NO production in LPS-stimulated RAW264.7 cells through suppressing the activated MAPK pathway by LPS, and BBG1 composed of glucose, mannose and a few proteins 380

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