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Immunomodulatory Activity of Polysaccharide-Protein Complex from the Mushroom Sclerotia of Polyporus rhinocerus in Murine Macrophages Chaoran Liu, Jialun Chen, Lei Chen, Xuesong Huang, and Peter C.K. Cheung J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00932 • Publication Date (Web): 07 Apr 2016 Downloaded from http://pubs.acs.org on April 7, 2016

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Journal of Agricultural and Food Chemistry

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Immunomodulatory Activity of Polysaccharide-Protein Complex

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from the Mushroom Sclerotia of Polyporus rhinocerus in Murine

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Macrophages

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Chaoran Liu1, Jialun Chen1, Lei Chen2, Xuesong Huang3, Peter C.K. Cheung*,1

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Hong Kong, Shatin, New Territories, Hong Kong SAR (HKSAR), China

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Resources and Environment, Qingdao Institute of Bioenergy and Bioprocess

Food and Nutritional Sciences, School of Life Sciences, The Chinese University of

Key Laboratory of Biofuels, Qingdao Engineering Research Center of Biomass

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Technology, Chinese Academy of Sciences, Qingdao 266101, China

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3

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China

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Running title: Activation of macrophages by mushroom polysaccharide-protein

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complex

Department of Food Science and Engineering, Jinan University, Guangzhou 510632,

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ACS Paragon Plus Environment


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ABSTRACT: A novel water-soluble polysaccharide-protein complex (PRW1)

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isolated from the sclerotia of an edible mushroom Polyporus rhinocerus which was

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purified by membrane ultrafiltration could significantly activate murine macrophages

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RAW264.7 in vitro. PRW1 had a molecular weight of less than 50 kDa and was found

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to be a highly branched heteropolysaccharide-protein complex composed of 45.7 ±

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0.97% polysaccharide and 44.2 ± 0.41% protein. Based on the results of total acid

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hydrolysis, methylation analysis and Fourier transform infrared spectroscopy, the

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carbohydrate moiety of PRW1 was found to be a β-D-mannoglucan with its backbone

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containing →1)-D-Glcp-(4→, →1)-D-Glcp-(6→ and →1)-D-Manp-(2→ residues

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(molar ratio of 5:4:6) and having terminal D-Glcp as side chain (degree of branching

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of 0.62). In vitro studies showed that PRW1 significantly induced NO production and

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enhanced the release of a variety of cytokines including G-CSF, GM-CSF, IL-6,

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IL12p40/70, MCP-1, MCP-5, MIP-1-α, MIP-2, RANTES, sTNFRI and TNF-α.

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Mechanistically, PRW1 treatment triggered ERK phosphorylation to activate

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macrophages within 15 min and significantly increased the expression level of

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inducible NOS after 6 h. In summary, this study indicates that PRW1 derived from the

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sclerotia of P. rhinocerus is a potential immunomodulatory agent for cancer

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immunotherapy.

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KEYWORDS: β-mannoglucans, macrophage activation, Mushroom sclerotia,

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Polysaccharide-Protein Complex, Polyporus rhinocerus

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INTRODUCTION

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Recently, researches on immunotherapy that are focused on the naturally occurring

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substances which exhibit beneficial therapeutic properties through stimulating the

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immune response mechanism of the host are increasing.1-3 Among the bioactive

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substances targeted in these studies, polysaccharides and polysaccharide-protein

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complex derived from edible and medicinal fungi/mushrooms have attracted the most

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attention as immunomodulators because of their high potency and non-toxicity. The

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most used mushroom polysaccharides or polysaccharide-protein complexes are

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derived from Trametes versicolor, Lentinus edodes and Agaricus brasiliensis which

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are rich in β-glucans.4 Current research results have suggested that the

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immunomodulating function of mushroom polysaccharides or polysaccharide-protein

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complex is associated with their effects on both the innate and adaptive immunity.

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Lentinan, the cell wall (1→3)- β-D-glucan from Lentinus edodes, showed

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immunomodulatory effect by activating nuclear factor-κB p65 and mitogen-activated

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protein kinases in murine RAW264.7 macrophages. The (1→6)- β-D-glucans from A.

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bisporus and A. brasiliensis could stimulate the expression of pro-inflammatory genes

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in human THP-1 derived macrophages.

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Polyporus rhinocerus is a sclerotium-forming mushroom belonging to the

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Polyporacea family.5 Sclerotium is a compact aggregate of mycelia and is one of the

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developmental stages in the mushroom life cycle.6 In China, P. rhinocerus is a

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traditional Chinese medicine in which there has been some preliminary data showing

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their effectiveness in treating liver cancer, chronic hepatitis and gastric ulcer.7 In

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Malaysia, P. rhinocerus is one of the most important medicinal mushroom used to 3



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relieve fever, cough, asthma, cancer, food poisoning and as a general tonic. Generally,

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the sclerotia of P. rhinocerus are considered to be the part with most valuable

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medicinal properties. Recent studies found that the sclerotia of P. rhinocerus were

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rich in non-digestible polysaccharides especially β-glucans but very low in fat.8 The

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potent bioactivity of the water-soluble polysaccharide-protein complex and

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alkali-soluble β-glucan isolated from the sclerotia of P. rhinocerus have been

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demonstrated by our previous studies.5,9,10 These include the remarkable

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host-mediated antitumor activity on the Sarcoma 180 implanted BALB/c mice and the

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anti-proliferative effect on various human leukemic cell lines including HL-60, K562

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and THP-1. Flow cytometric analysis on cell cycle revealed the onset of apoptosis of

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the HL-60 cells by a hot water extract of P. rhinocerus was associated with the cell

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cycle arrest at the G1 phase.9 In vivo study on the immunomodulatory activity of a hot

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water extract from P. rhinocerus demonstrated a significant increase in the spleen

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weight of both healthy BALB/c and healthy athymic nude mice.5 A recent in vitro

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study using RAW 264.7 cells indicated that a cold water extract isolated from the

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sclerotia of P. rhinocerus had significant inhibitory effect on LPS-induced TNF-α

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production and such anti-imflammatory activity was mainly contributed by the protein

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component in a high molecular weight polysaccharide-protein complex.10

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Macrophages, which are distributed throughout the body of the host, are the major

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effector cells of innate immunity by engulfing microbes and secreting

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pro-inflammatory factors, thus constituting a first line of defense against invading

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pathogens.11, 12 Macrophages also play a crucial role to bridge the innate and adaptive 4


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immunity as they function as antigen presenting cells to processes and present

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antigens to T cells to initiate adaptive immune response.12 In addition, macrophages

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are important in regulating tissue development, repair, remodeling and homeostasis by

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producing numerous chemokines and cytokines as well as varieties of growth factors

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and enzymes.11-13 Once activated by various endogenous or exogenous stimuli,

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macrophages will differentiate into classically activated macrophages (M1s) or

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alternatively-activated macrophages (M2s) whose fate is dependent on the cytokines

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environment. Till now, macrophages have been identified as potential target cells for

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antitumor and immunomodulatory drug design.14, 15 Compared with human

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macrophage, murine macrophages are more accessible. RAW264.7 cells are widely

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used as a tool to investigate the mechanisms of macrophages involved in immunity

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regulation.16

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In the present study, we aimed at isolating a novel bioactive component with

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immunomodulating activity on murine macrophages from the sclerotia of P.

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rhinocerus (PRW) and to elucidate the structural characteristics as well as the

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mechanism of macrophage activation exerted by this novel component. As far as we

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know, this is the first study to isolate and chemically characterize a mannoglucan type

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polysaccharide-protein complex from the mushroom sclerotia of P. rhinocerus and to

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demonstrate its immuno-potentiating effect on murine macrophages. Our results

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demonstrated that the mechanism of macrophage activation stimulated by PRW1 is

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via activation of ERK to induce the release of related cytokines.

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MATERIALS AND METHODS

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Chemicals. Analytical grade reagents including ethyl acetate and acetone were

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purchased from Duksan Pure Chemicals Co. and ethanol was purchased from Merck

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Millipore. The bicinchoninic acid (BCA) reagent was purchased from PIERCE.

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Thiazolyl blue tetrazolium bromide (MTT) and Lipopolysaccharide (LPS) were

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purchased from Sigma Aldrich. The Griess reagents were purchased from Promega.

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Isolation and Purification of the Water-Soluble Polysaccharide-Protein

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Complex. Mushroom sclerotia of P. rhinocerus originated from mainland China were

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purchased from Chinese herbal store in Hong Kong. After the removal of the peel, the

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sclerotia were pulverized into powders to pass through a screen with an aperture of

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0.5 mm by using a cyclotech mill (Tecator, Hӧganäs, Sweden). Powder of sclerotia of

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P. rhinoceros were firstly defatted by use of ethyl acetate (1 h, 3 times) followed by

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acetone (1 h, 3 times) at 60oC before being extracted with boiling water for 2h for 3

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times. The hot water-soluble extract was obtained by centrifugation (4000 g for 10

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min). After concentrated in a rotary evaporator under vacuum, the hot water extract

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was subjected to ethanol precipitation by adding of 4 volumes of 96 % ethanol. The

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precipitate was isolated and freeze-dried to give the hot water-soluble polysaccharide

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(PRW). PRW was subjected to membrane ultrafiltration (MUF) with a molecular

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weight cut-off (MWCO) of 1000 Da to remove impurity with low molecular weight.

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Afterward, further purification by MUF with MWCO of 50 kDa was carried out to

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obtain a purified fraction (PRW1) with molecular weight smaller than 50 kDa.

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High Performance Liquid Chromatography (HPLC). The molecular parameters 6


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were determined using a size exclusion chromatograph (SEC) (Waters e2695 HPLC

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equipment system) coupled with a refractive index (RI) detector (Waters 2414, Waters

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Inc. USA) and dual wavelength absorbance detector (Waters 2487, Waters Inc. USA)

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with wavelength at 200 and 280 nm.17 The PRW and PRW1 samples from above were

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dissolved in 0.15 M sodium chloride solution to a concentration of 5 mg/mL and

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filtered through 0.22 mm nylon syringe filter before injection into the SEC system

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with a TSK G-5000 column (30 cm × 7.5 mm i.d., Supelco, USA). A range of dextran

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standards ( 1, 5.9, 12, 25, 50, 80, 112，212, 404, 788, 1400kDa) was used for

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calibration the molecular weight (MW) in the SEC analysis. The pre-degassed 0.15 M

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NaCl aqueous solution was applied as the elution buffer at a flow-rate of 0.7 mL/min.

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One Dimensional Gel Electrophoresis (1-D GE). The SDS-PAGE analysis was

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performed according to the procedures previously reported.18 Briefly, 40 µg PRW1

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sample was loaded into the SDS-PAGE with 5% stacking gel and 12% separating gel.

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After electrophoresis, the gel was stained with Coomassie Brilliant Blue to visualize

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the presence of protein or stained with periodic acid Schiff’s (PAS) buffer to detect

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the presence of polysaccharide.

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Chemical Composition Analysis. The amount of the total carbohydrates and

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proteins in the PRW and the purified PRW1 were determined by the phenol-sulfuric

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acid19 and the bicinchoninic acid protein assay as described previously.20

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The monosaccharide composition of PRW1 was determined by gas

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chromatographic analysis of the alditol acetate derivatives of the sugars formed by

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sequential acid hydrolysis, reduction and acetylation described previously.21 A gas 7



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chromatograph (GC) (6890N, Agilent Technology,USA) fitted with an Alltech

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DB-225 capillary column (15 m × 0.25 mm i.d., 0.25 mm film) and coupled with a

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mass spectrometry (MS) (5973N, Agilent Technology, USA) was used. The GC

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conditions used in these analyses had been reported previously.21

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Linkage Analysis by Methylation. PRW1 was permethylated to partially

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methylated alditol acetates (PMAA) using methyl iodide and solid NaOH in dry

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DMSO as described in a modified protocol reported previously.22 The PMAA samples

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were then analyzed by GC- MS with an Alltech DB-225 capillary column (15 m ×

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0.25 mm i.d., 0.25 µm film) as mentioned above. The oven temperature was increased

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from 130 to 220 °C at a rate of 4 °C/min. The MS detector conditions were the same

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as mentioned above. Each PMAA was identified by matching its mass spectrum with

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the literature database.23

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Cell Culture. The RAW264.7 murine macrophages were purchased from ATCC

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(American Type Culture Collection) and were maintained in DMEM medium

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supplemented with 10% heat-inactivated (56°C, 30 min) FBS (fetal bovine serum),

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penicillin (100 units/mL) and streptomycin (100 units/mL) in the cell culture dish at

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37°C in a humidified incubator with 5% CO2 atmosphere.

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Spectroscopic Methods. The infrared spectrum of PRW1 was recorded with a

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Fourier Transform Infrared Spectrometer (FT-IR, Nicolet 670) in the range of

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4000-400 cm-1 using the KBr-disk method.24 Briefly, the PRW1 sample was ground

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with KBr powder and afterwards pressed into 1 mm pellet for FTIR measurement.

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MTT Assay. Proliferation of RAW264.7 cells was measured based on the 8


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mitochondria-dependent reduction of MTT.25 Briefly, 5×103 cells in 100 µL per well

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were plated in 96-well tissue culture plates for 24 h. Cells were incubated for 76 h in

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the appearance of different concentrations of PRW1 or LPS (100 ng/mL). After that,

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20 µL of MTT solution (5 mg/mL in PBS) were added into each well and followed by

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further incubation for 4-5h. After aspirating the medium, 200 µL of DMSO were

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added into each well to dissolve the formazan crystal. The color intensity of the

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formazan solution, which reflects the cell growth condition, was measured at 570 nm

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using a microplate spectrophotometer (VersaMax).

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Measurement of Concentrations of NO. Nitrite oxide production was measured

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by the Greiss method5 which detects the nitrite presented in the culture medium of

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RAW264.7 cells as a stable and nonvolatile breakdown product of NO. Briefly, cells

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were seeded (5×104 cell/mL) in a 6-well tissue culture plate for 24 h. After stimulated

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with PRW1 for 24 h, the cell culture medium supernatants were collected. Each

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supernatant (50 µL) then was mixed with Sulfanilamide Solution (50 µL) and

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N-1-napthylethylenediamine dihydrochloride (50 µL) Solution and the absorbance at

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520 nm was measured using a microplate spectrophotometer (VersaMax).

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Determination of Cytokine Profile using Cytokine Antibody Array. The

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relative levels of cytokines secreted by RAW264.7 mouse macrophages were detected

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using RayBio ® Mouse Cytokine Antibody Array II (RayBiotech). Briefly, the

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RAW264.7 cells were seeded in a 6-well tissue culture plate at 105 cells/well for 24 h.

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After incubation with 100µg/mL PRW1 for 24h, the cell culture supernatants were

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collected. Firstly, the samples were added to the membrane which was blocked by the 9



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blocking buffer and incubated overnight at 4 °C. After washes with wash buffer for

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three times, the membranes were incubated with biotinylated antibody cocktail

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overnight at 4 °C. After three washes, the membranes were incubated with freshly

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diluted horseradish peroxidase (HRP)-Streptavidin overnight at 4 °C. After washing

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the membrane, 500 µL of detection buffer mixture was added to each membrane and

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incubated for 2 min at RT. Immediately afterwards, the membranes were exposed to

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X-ray film for 0.5 to 5 min and signals were detected using film developer.

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Western Blot Analysis. RAW264.7 murine macrophages (105 cells/well) were

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seeded in a 6-well tissue culture plate and incubated with 100 µg/mL PRW1 for 0.25h,

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0.5h, 1h, 3h, 6h, 12h, 24h. After removing the supernatants, cells were washed twice

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with PBS. The total cellular proteins were extracted by incubating cells in lysis buffer

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(Cell Signaling Technology) on ice and collected by centrifugation (15000 g for 15

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min). After the determination of the protein concentration using bicinchoninic acid

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assay, the lysates were mixed with 2× protein loading buffer and the mixture was

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boiled for 5 min at 95-100 °C. An equal amount of denatured proteins were loaded

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into 10% polyacrylamide gel and separated by SDS-PAGE. Then the separated

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proteins were electrically transferred to a PVDF membrane and blocked with 5% BSA

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in Tris-Buffered-Saline with Tween (TBST) for 1h at RT. After that, the membranes

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were then incubated with primary anti-mouse iNOS, p-NF-κB NF-κB, p-ERK, ERK,

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p-JNK, p-p38, p-AKT, AKT and β-actin antibodies (Cell Signaling Technology,

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Beverly, MA, USA) at 1:1000 dilutions in 5% BSA solution for overnight at 4 °C.

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After being washed by TBST, the membranes were incubated with HRP conjugated 10


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secondary antibody at 1:2000 dilutions for 1h at RT and the reactive bands were

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visualized on an X-ray film using enhanced chemiluminescence (ECL) Western blot

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detection reagent.

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Statistical Analysis. Experiments were carried out in triplicate unless specified

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otherwise, and the results were represented as mean ± SD. Difference between two

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groups was analyzed by ANOVA followed by two-tailed Student’s t test. Difference

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with P

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