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Structural Characterization of a Novel Polysaccharide from Lepidium meyenii (maca) and Analysis of Its Regulatory Function in Macrophage Polarization in vitro Mengmeng Zhang, Wenjia Wu, Yao Ren, Xiao-Feng Li, Yuqian Tang, Tian Min, Furao Lai, and Hui Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05218 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Structural Characterization of a Novel Polysaccharide from

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Lepidium meyenii (maca) and Analysis of Its Regulatory Function in

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Macrophage Polarization in vitro

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Mengmeng Zhang1, Wenjia Wu1, Yao Ren2, Xiaofeng Li1, Yuqian Tang1, Tian Min1, Furao Lai1*, and Hui Wu1*

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Affiliation

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1 College of Food Sciences and Engineering, South China University of Technology,

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Guangzhou, Guangdong 510640, China

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2 College of Light Industry, Textile and Food Engineering, Sichuan University,

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Chengdu, 610065, China

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Short title: Structural Characterization and Regulation in Macrophage Polarization of

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Maca Polysaccharide

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Co-corresponding authors:

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Hui Wu, Department of Food Quality and Safety, South China University of

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Technology, Wushan Road 381, Guangzhou, Guangdong, China.

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Tel: (+86) 20-87112853; E-mail: [email protected]; Fax: (+86)20-87112532

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Furao Lai, Department of Food Quality and Safety, South China University of

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Technology, Wushan Road 381, Guangzhou, Guangdong, China. Tel: (+86) 20-87112373; E-mail: [email protected]; Fax: (+86)20-87112532 1

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ABSTRACT: In our previous study, three novel polysaccharides, named MC-1,

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MC-2, and MC-3, were separated from the roots of maca (Lepidium meyenii), which

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is a food source from the Andes region. The structural information and

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immunomodulatory activity of MC-1 were then investigated. The structure and

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activity of MC-2 is still unknown. In this study, structural characterization revealed

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that MC-2 has an average molecular weight of 9.83 kDa and is composed of arabinose

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(20.9%), mannose (4.5%), glucose (71.9%), and galactose (2.7%). The main linkage

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types of MC-2 were proven to be (1→5)-α-L-Ara, (1→3)-α-L-Man, (1→)-α-D-Glc,

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(1→4)-α-D-Glc, (1→6)-α-D-Glc, and (1→6)-β-D-Gal by methylation and NMR

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analyses. Congo red assay showed that MC-2 possesses a triple helix conformation.

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Immunostimulating assays indicated that MC-2 could induce M1 polarization of

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original macrophages and convert M2 macrophages into M1 phenotype. Although

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MC-2 could not shift M1 macrophages into M2, it could still inhibit inflammatory

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reactions induced by LPS. Furthermore, toll-like receptor 2, toll-like receptor 4,

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complement receptor 3, and mannose receptor were confirmed as the membrane

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receptors for MC-2 on macrophages. These results indicate that MC-2 could

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potentially be used toward hypoimmunity and tumor therapies.

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KEYWORDS: polysaccharides, maca, structure, macrophage , polarization

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INTRODUCTION

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Maca (Lepidium meyenii) is a food source that has been used to enhance fertility for

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centuries in the Andes region.1, 2 Recently, maca has attracted interest as a dietary

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supplement owing to its various pharmacological properties, and as a result, many

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effective compounds in maca were identified, including macaenes, macamides,

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glucosinolates, alkaloid, and flavonolignans.3-7 However, little attention has been

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devoted to the maca polysaccharides. In our previous study, three new

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polysaccharides, named, MC-1, MC-2, and MC-3 were separated from the roots of

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maca. The primary chemical structure and immunomodulatory activity of MC-1 was

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then investigated.8 However, the structure of MC-2 is still unknown.

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Macrophages are an essential component of innate immunity and play a

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central role in inflammation and host defense. Macrophage functions are elicited

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in response to microenvironmental signals, which drive the acquisition of polarized

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programs, whose extremes are simplified in the M1 and M2 dichotomy.9 M1

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macrophages (classically activated) exert pro-inflammatory activity. They have been

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known to be induced by interferon (IFN) or lipopolysaccharide (LPS), and release

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pro-inflammatory cytokines, such as TNF-α and IL-6, and nitric oxide (NO). M1

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macrophages are considered to mediate host defense against microbial infections and

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tumors, but also cause autoimmune tissue damage.10 IL-4 and IL-13 were found to

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induce alternatively activated M2 macrophages, which are involved in inflammation

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resolution and tissue repair.11 IL-10 (anti-inflammation cytokine) and arginase-1

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(Arg-1) represent the signature molecules of M2 macrophages. However, M2 3

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macrophages are also involved in metazoan parasite containment, immune tolerance,

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and tumor progression.12 Macrophage M1 and M2 phenotype responses generate

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opposing activities in terms of killing and repairing processes. An imbalance in

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macrophage M1-M2 polarization is often associated with various diseases or

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inflammatory conditions.13

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Plasticity and flexibility are key features of mononuclear phagocytes and their

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activation states. The phenotype of polarized M1-M2 macrophages can, to some

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extent, be reversed both in vitro and in vivo.14 Polysaccharides obtained from

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natural sources are known to govern the immunomodulatory activity of macrophages.

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Many polysaccharides can induce macrophage-produced pro-inflammatory cytokines

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TNF-α, IL-1b, IL-6, and IL-12.15-17 Conversely, some polysaccharides have been

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shown to have anti-inflammatory activities.18-20 These observations indicate that the

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immunomodulatory activity of polysaccharides is relative to macrophage polarization.

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However, few reports about the immunomodulatory activity of polysaccharides have

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focused on the different macrophage phenotypes. Given the fact that MC-1 has

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stimulating effects on macrophages, we hypothesized that MC-2 displays a similar

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effect.

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In the present study, the primary chemical structure of MC-2 was characterized.

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The regulation of MC-2 on the polarization of RAW 264.7 cells, a murine

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macrophage cell line, was investigated by determining the markers of M1, IL-6 and

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inducible nitric oxide synthase (iNOs), and markers of M2, IL-10 and Arg-1. The

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potential membrane receptors of MC-2 on RAW 264.7 cells were also explored. The 4

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results obtained from this study might provide useful information toward enabling

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further in-depth studies on polysaccharides present in maca.

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MATERIALS AND METHODS

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Chemicals

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The roots of maca were collected from Peru. Myoinositol and standard

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monosaccharides (xylose, rhamnose, arabinose, fucose, mannose, glucose, and

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galactose)

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Diethylaminoethyl (DEAE)-Sepharose Fast Flow was obtained from Shanghai

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Yuanye Bio-Technology Company Limited (Shanghai, China). Sephadex G-50 was

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acquired from GE Healthcare Life Science (Piscataway, NJ). RAW 264.7 cells were

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obtained from the Type Culture Collection of the Chinese Academy of Sciences

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(Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine

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serum (FBS), penicillin, and streptomycin were purchased from Gibco Life

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Technologies (Grand Island, NY). Griess reagent was purchased from Sigma-Aldrich

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(NSW, Australia). Mouse TNF-α, IL-10 and IL-6 detection ELISA kits were

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purchased from R& D Systems. TRIzol was purchased from Invitrogen, USA;

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Transcriptor First Strand cDNA Synthesis Kit and FastStart Universal SYBR Green

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Master (ROX) were purchased from Roche. Anti-scavenger receptor I antibody

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(anti-SR), anti-mannose receptor antibody (anti-MR), anti-beta glucan receptor

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antibody (anti-GR), anti-toll-like 2 antibody (anti-TLR2), anti-complement receptor 3

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antibody (anti-CR3), and anti-toll-like 4 receptor antibody (anti-TLR4) were obtained

were

purchased

from

Sigma-Aldrich

(St.

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Louis,

MO,

USA).

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from Abcam (Cambridge, MA). All of the other chemical reagents used in this study

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were of analytical grade.

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Extraction and Purification of Polysaccharides from the Roots of Maca

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The extraction method was the same as in our previous study.21 Briefly, the powder

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of dried roots of maca was extracted with boiling water at a ratio of 1:30 (w/v) for 2 h,

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and the obtained extract was then centrifuged at 4000 × g for 15 min. After the

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supernatant was concentrated at 60 °C, the protein was removed according to the

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Sevag method. The resulting solution was precipitated with four volumes of 100%

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ethanol at 4 °C for overnight, and then centrifuged at 4000 × g for 15 min to collect

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the precipitates. Finally, the precipitate was redissolved in distilled water prior to

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lyophilization to obtain crude polysaccharides.

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The purification method is similar to that used in the previous study, with some

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modifications.22 A total of 50 mg of crude polysaccharides was dissolved in 10 mL of

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ultrapure water and loaded onto a pre-equilibrated DEAE-Sepharose Fast Flow

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chromatography column (1.6×35 cm) at a flow rate of 1 mL/min, followed by and

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elution step with distilled water. The eluent fractions were collected and concentrated

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at 60 °C by rotary vacuum evaporator, then dialyzed (MW cut off 5.0 kDa) at 4 °C for

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48 h and freeze-dried. In our previous study, a Sephadex G-100 column could not

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totally separate the fractions. Therefore, in this study, a Sephadex G-50

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chromatography column (1.6 × 60 cm) was used to yield a better purification process.

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The fraction (20 mg) was dissolved in distilled water (10 mL) at 25 °C. The column

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was washed with 0.3 L of distilled water at a flow rate of 1 mL/min for 300 min. The 6

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eluent was detected according to the phenol-sulfuric acid method and collected before

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concentrating the polysaccharide solution. As previously, when we studied MC-1,

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three peaks were obtained (Figure 1). In this study, we mainly focused on

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investigating of MC-2.

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MC-2 Molecular Weight Determination

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The weight-average molecular weight of MC-2 was determined through

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high-performance gel permeation chromatography (HPGPC) using a Waters HPLC

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system including two serially linked columns a TSK-GEL G-5000 PWXL column

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(300 mm×7.8 mm inner diameter, 10µm) and a TSK-GEL G-3000 PWXL column

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(300 mm×7.8 mm inner diameter, 6µm), a Waters 2410 differential refractive index

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detector, eluted with 0.02 mol/L KH2PO4 at a flow rate of 0.6 mL/min. MC-2 (2.5 mg)

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was dissolved in 1 mL mobile phase.

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Infrared Spectrum Analysis

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The MC-2 samples (2-3 mg) were analyzed according to the potassium bromate

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pellet method with a Fourier transform infrared (FTIR) spectrophotometer (Bruker,

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Ettlingen, Germany) in the 400-4000 cm-1 vibrations region.23

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Monosaccharide Composition

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A total of 10 mg MC-2 sample was hydrolyzed in 4 mL of 2 mol/L trifluoroacetic

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acid (TFA) for 8 h at 110 °C. Polysaccharides were transformed into alditol acetates

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by hydrolysis. The MC-2 alditol acetates were analyzed by gas chromatography (GC)

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(Agilent, US) fitted with a HP-5 capillary column (30 nm×0.32 mm×0.25 µm,

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160~210 °C at 2 °C /min, and then 210~250 °C at 10 °C /min) equipped with a flame 7

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ionization detector (FID). Glucose, galactose, fucose, rhamnose, mannose, xylose, and

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arabinose were used as the monosaccharide standards. Myoinositol was used as the

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internal reference.

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Methylation Analysis

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The methylation analysis of polysaccharide was performed according to the 21

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method reported by Nie et al

,with some modifications. MC-2 (10 mg) was

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dissolved in 6 mL anhydrous DMSO at 60 °C for 2 h and sonicated for 1 h. NaOH

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(240 mg) was added to the solution, which was subsequently left to react at 60 °C

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overnight. The mixture was added to a 3.6 mL methyliodide solution and stirred for 8

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min. This procedure was repeated three times and stopped by adding 6 mL of distilled

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water. The solution obtained was dialyzed against distilled water for 48 h at 4 °C. The

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methylated polysaccharide was extracted with dichloromethane three times. The

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dichloromethane extract was then dried over sodium sulfate, and evaporated to

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dryness. The dried methylated polysaccharide was hydrolyzed as describe above. The

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hydrolysate was reduced by sodium borodeuteride (70 mg) and acetylated with acetic

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anhydride (0.5 mL). Finally, the resulting product was analyzed by gas

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chromatography (GC) coupled with mass spectrometry (MS) (Agilent, USA) using a

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TR-5MS capillary column (30 m ×0.25 mm ×0.25 µm, 150~180 °C at 10 °C /min, and

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then 180~260 °C at 15 °C /min).

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NMR Spectroscopy

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About 30 mg of MC-2 was dissolved in 0.55 mL of heavy water (D2O) in a NMR 13

C NMR and 1H NMR spectra were recorded on a Bruker 600

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tube and then, the

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MHz NMR apparatus (Bruker Corp, Fallanden, Switzerland) at 60 °C.

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Determination of Triple-helix Structure

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The conformational structure of MC-2 was determined following the Congo red

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method.22

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Measurement of cytokines

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RAW 264.7 cells were incubated at 37°C in a humidified atmosphere with 5%

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CO2. DMEM medium with 10% FBS, 100 µg/mL streptomycin, and 100 units/mL

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penicillin was used as the culture medium. Cells were adjusted to a concentration of 1

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× 106 cells/mL in the exponential phase, loaded onto 96-well or 6-well plates, and

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continuously incubated for 24 h.

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Cells were treated with different concentrations of MC-2 (62.5, 125, 250, 500,

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1000 µg/mL), MC-1 (1000 µg/mL), LPS (1 µg/mL) or IL-4 (20 ng/mL) and incubated

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for 24 h. After that, the cell supernatants were collected and the levels of IL-6 and

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IL-10 were measured using the ELISA kits.

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QPCR Analysis

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RAW 264.7 cells were seeded on 6-well plates at a concentration of 1 × 106

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cells/mL. After 24 h, cells were treated with different concentrations of MC-2 (62.5,

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125, 250, 500, 1000 µg/mL), MC-1 (1000 µg/mL), LPS (1 µg/mL) or IL-4 (20 ng/mL).

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After 12 h, the cells were lysed to isolate total RNA.

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Total RNA was isolated using TRIzol reagent according to the manufacturer’s

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protocol, and the RNA was used for cDNA synthesis using reverse transcriptase. The

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cDNA encoding iNOs and Arg-1 genes was quantified by quantitative real-time PCR

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assay (QPCR). GAPDH was used as the internal reference. The specific primers used

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are described in Table 1. Gene amplification was carried out with the ABI 7500

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sequence detection system (Applied Biosystems, Foster, USA).

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MC-2 Treatment After Polarization

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Cells within the control group were cultured in medium without any treatment.

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Cells in the other groups were polarized to M1 with LPS (1 µg/mL) for 12 h, and then

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treated with combinations of different concentrations of MC-2 (0, 31.25, 125, 500

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µg/mL) and LPS (1 µg/mL) for 24 h. M2 macrophages were polarized by IL-4 (20

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ng/mL) using the same procedure. Following polarization, cells were collected for the

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determination of iNOS and Arg-1 mRNA levels, and the supernatants were used to

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measure the levels of IL-6 and IL-10.

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Investigation of Membrane Receptors

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The cells were pretreated with antibodies (5 µg/mL) against membrane receptors

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(SR, MR, GR, CR3, TLR2 and TLR4) or a mixture of antibodies against MR, CR3,

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TLR4, and TLR2 for 2 h prior to stimulation with MC-2 (125 µg/mL). The group

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treated with only MC-2 (125 µg/mL) was used as the control. The untreated cells were

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used as the negative control group. LPS-treated cells represented the positive control.

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The levels of NO, TNF-α, and IL-6 were measured after 24 h .23 NO, TNF-α, and IL-6

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were measured using a Griess reagent and ELISA kits, respectively. 10

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Statistical Analysis

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Data are expressed as the mean± standard deviation (SD) of three replicates.

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Significant differences between the means of parameters were calculated by Duncan’s

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multiple-range test using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). p