Mannose Receptor Mediates the Immune Response to Ganoderma

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Mannose receptor mediates the immune response to Ganoderma atrum polysaccharides in macrophages Wen-Juan Li, xiao-fang Tang, Xiao-Xue Shuai, Cheng-Jia Jiang, Xiang Liu, Le-Feng Wang, Yu-Fei Yao, Shaoping Nie, and Ming-Yong Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04888 • Publication Date (Web): 08 Dec 2016 Downloaded from http://pubs.acs.org on December 13, 2016

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Journal of Agricultural and Food Chemistry

A schematic representation displaying the mechanism by which MR controls the immune response to PSG-1. TABLE OF CONTENTS GRAPHICS 47x26mm (600 x 600 DPI)

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Mannose receptor mediates the immune response to Ganoderma

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atrum polysaccharides in macrophages

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Wen-Juan Li#, Xiao-Fang Tang, Xiao-Xue Shuai#, Cheng-Jia Jiang#, Xiang Liu, Le-Feng

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Wang, Yu-Fei Yao$, Shao-Ping Nie#, Ming-Yong Xie#*

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#

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East Road, Nanchang 330047, China.

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

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330031, China.

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$

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State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing

School of Basic Medical Sciences, Nanchang University, No. 999 Xuefu Road, Nanchang

Chinese Liberation Army No.94 Hospital, No.1028, Jinggangshan Avenue, Nanchang 330000,

China.

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*Corresponding to:

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Professor Ming-Yong Xie, PhD. State Key Laboratory of Food Science and Technology,

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Nanchang University, 235 Nanjing East Road, Nanchang 330047, China. Tel.: +86 791-3969009;

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Fax: +86 791-3969009; E-mail: [email protected]; [email protected].

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ABSTRACT

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The ability of mannose receptor (MR) to recognize the carbohydrate structures is well

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established. Here we reported that MR was crucial for the immune response to Ganoderma

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atrum polysaccharides (PSG-1), as evidenced by elevation of MR in association with increase of

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phagocytosis and concentrations of IL-1ß and TNF-α in normal macrophages. Elevation of MR

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triggered by PSG-1 also led to control lipopolysaccharide (LPS)-triggered inflammatory

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response via the increase of IL-10 and inhibition of phagocytosis and IL-1ß. Anti-MR antibody

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partly attenuated PSG-1-mediated anti-inflammatory responses, while could not affect TNF-α

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secretion, suggesting that another receptor involved in PSG-1-triggered immunomodulatory

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effects. MR and Toll-like receptor (TLR)4 coordinated the influences on TLR4-mediated

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signaling cascade by NF-κB pathway in LPS-stimulated macrophages subjected to PSG-1.

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Collectively, immune response to PSG-1 required recognition by MR in macrophages. NF-κB

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pathway served as a central role for the coordination of MR and TLR4 to elicit immune response

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to PSG-1.

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KEYWORDS: Mannose receptor; Ganoderma atrum polysaccharides; Macrophage; Toll-like

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receptors; NF-κB signaling pathway



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INTRODUCTION

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Polysaccharides, a class of naturally occurring carbohydrates, are the polymers formed by the

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connection with glycosidic bond from dehydration condensation or dehydration effect of aldose

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or ketose1,2. Currently, the popularity of polysaccharides has received remarkable attention due

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in part to the fact that polysaccharides are the major structural component of cells and exert a

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variety of physiological function. Naturally occurring polysaccharides have been confirmed to

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possess diverse and potentially significant bioactivities like immunomodulatory, anti-

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inflammatory, anti-tumor, cardiovascular support, hypoglycemic and anti-aging activities.

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Among these bioactivities, immunomodulatory effects are considered to be the pivotal role for

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their ability to mediate health benefits 3,4

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Recent studies have reported that polysaccharides stimulate immune system involved in a

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complex mechanism of immune recognition, which rely on germline encoded pattern recognition

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receptors (PRRs). PRRs are a class of receptors which predominantly expressed in the innate

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immune cell surface to identify pathogens. There are several PRRs within the cells, such as

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mannose receptor (MR), Toll-like receptors (TLRs), dendritic cell-associated C-type lectin-1

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(Dectin-1), scavenger receptor (SR), complement receptor 3 (CR3) and so on5,6 Binding of

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polysaccharide to PRRs could promote the synthesis and release of cytokines, chemokines,

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reactive oxygen species and nitrogen intermediate via intracellular signal pathways, which

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mediates the mobilization of immune cells and immune response. C-type lectin family members

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including MR could recognize the carbohydrate structures though carbohydrate recognition

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domains (CRDs) in their extracellular carboxy-terminal domains7-9. MR is present in a wide

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range of immune cells including macrophages, dendritic cells, specific lymphocytes and

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endothelial cells10,11 The receptor could recognize mannose, fucose, and N-acetylglucosamine,


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and plays an essential role in immune homeostasis through enhancing the phagocytic activity of

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macrophages, producing reactive oxygen species (ROS), activating NF-κB and inducing the

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secretion of cytokines.

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Ganoderma atrum is a famous medicinal/nutritional fungus, which is commonly used as a

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natural adjuvant to promote physical fitness. A water-soluble protein-bound polysaccharide

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(namely PSG-1) was isolated from Ganoderma atrum and purified by gel-filtration

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chromatography. Its primary structural feature was determined by methylation analysis and

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1D/2D nuclear magnetic resonance (NMR) spectroscopy. Gas chromatography analysis method

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was used to characterize its monosaccharide composition, and the results showed that PSG-1

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consisted of mannose, galactose and glucose with a molar ratio of 1:1.28:4.91. PSG-1 has been

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demonstrated to trigger the immune modulating effects and subsequent benefits in cancer and

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infection12-14

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Based on the above findings, it is important to explore MR for the purpose of understanding

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the molecular mechanism involved in the immune modulating effects of PSG-1. It is well known

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that MR is primarily expressed on tissue macrophages. Murine MR displays a high degree of

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homology with the human MR in macrophage. Therefore, primary cultures of murine

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macrophages were chosen to investigate MR-mediated immune response to PSG-1 in this work15.

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Additionally, TLRs were found to have an affinity with recognizing polysaccharides among all

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the receptors17,18. Therefore, the aim of the present study was to determine the effect of PSG-1 on

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the regulation of MR in murine macrophage, and examine its effects on the TLRs-mediated

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signal pathway in LPS-induced macrophage.

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MATERIALS AND METHODS



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Reagents. Mannan (from Saccharomyces cerevisiae), FITC-dextran and lipopolysaccharide

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(LPS) were provided by Sigma (St. Louis, MO, USA). RPMI1640 medium was purchased from

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Solarbio Bioscience & Technology Company (Shanghai, China). Fetal bovine serum was

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purchased from HyClone (Logan, UT, USA). Rat anti-mouse MR FITC antibody (CD206) was

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from AbD Serotec (Oxford, United Kingdom). Trizol reagent was from Invitrogen

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(Carlsbad, CA, USA). TNF-α, IL-1β and IL-10 ELISA kits were purchased from Boster Biology

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Engineering Institute (Wuhan, China). Protein extraction and ECL kits were purchased from

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Beyotime Institute of Biotechnology (Shanghai, China). Neutral red was purchased from

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Sinopharm Chemical Reagent Co. (Shanghai, China). Anti-β-actin (mouse monoclonal antibody)

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primary antibody and the HRP-linked secondary antibodies were purchased from Jinqiao

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Biological Technology Co. (Beijing, Shanghai, China). Anti-TLR2 primary antibody (mouse

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monoclonal antibody), anti-TLR4 primary antibody (mouse monoclonal antibody) and anti-

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Histone H2AX primary antibody (rabbit polyclonal antibody), Anti-MR antibody and isotope

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control antibody were purchased from Abcam Biotechnology (Abcam, Cambridge, UK). Anti-

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MyD88, anti-NF-κB p65, anti-total-ERK1/2(t-ERK1/2), anti-phospho-ERK1/2 (p-ERK1/2), anti-

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total-JNK(t-JNK), anti-phospho-JNK (p-JNK), anti-total- p38(t-p38) and anti-phospho-p38 (p-

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p38) primary antibodies (rabbit monoclonal antibodies) were provided by Cell Signaling

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Technology (Beverly, MA, USA).

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Isolation and purification of PSG-1. In this study, the artificial mushroom of Ganoderma

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atrum (commonly known as Hei Lingzhi) was obtained from commercial source, and provided

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by Dongjiangyuan base of strains of the genus Ganoderma in Dongjiangyuan’s precious edible

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fungus factory (Xunwu county Gannan, Jiangxi Province, China). The fruit bodies of

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Ganoderma atrum are dried in the sun and cut into small pieces, then smashed to irregular


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particles by using a crushing machine over and over again prior to experiment for extracting

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polysaccharides. The crude Ganoderma atrum polysaccharide was obtained by water extraction

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and alcohol precipitation. Then the Sevag method was used to remove protein from the crude

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polysaccharide, and the samples without proteins were subjected to distilled water dialysis and

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concentration. Then 80% ethanol was used for precipitation, washing, and lyophilization to get

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refined polysaccharide. Finally, the purified polysaccharide of Ganoderma atrum (PSG-1) was

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obtained by gel filtration chromatography on SephadexG-200 column. The polysaccharide was

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found to be mainly composed of mannose, galactose and glucose in a molar ratio of 1:1.28:4.91,

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with an average molecular weight of approximately 1013 kD, by infrared spectroscopy, gas

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chromatography, molecular size exclusion chromatography, amino acids analysis and high

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performance liquid chromatography.12

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Experimental animals. BALB/c clean mice (20-24 g) were purchased from School of

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Medical Sciences, Nanchang University. Animals were housed in air-conditioned green house,

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with a temperature of 22 ±2 ºC, relative humidity of 50-60% and 12 h/12 h light/dark cycle, with

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free feeding and water. These conditions were kept for 1 week before the conduct of the

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experiments. Animals were carried out in this work referring to the Guide for the Care and Use

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of Laboratory Animals published by the United States National Institute of Health (NIH

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Publication No. 85-23, revised 1996), and all procedures were approved by Nanchang University

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Medical College Animal Care Review Committee.

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Primary culture of mouse peritoneal macrophages. BALB/c mice were sacrificed by

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cervical dislocation, soaked in 75% alcohol for approximately 5 min and then placed in a clean

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sterile petri dish. Mouse abdomen was intraperitoneally injected with 6-8 mL pre-cooled RPMI

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1640 medium, and abdominal fluid was aspirated with a pipette and placed into centrifuge tubes.



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Abdominal fluid was collected after the treatment with RPMI 1640 medium, then filtered and

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centrifuged for 5 min (1,400 × g), and finally resuspended in a complete medium consisting of

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RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (100 U/ml) and

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streptomycin (100 μg/ml). The culture solution was gently removed after cultivated in carbon

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dioxide for 4 h, and the complete RPMI 1640 medium was slowly added along the side wall of

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the flask. The flasks were gently shaken to wash away the non-adherent cells, and the purified

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peritoneal macrophages were obtained. Its activity was assessed by trypan blue exclusion

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(>95%). Finally, macrophages were seeded onto 6-well cell culture plate with 1×106 cells per

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well.

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Analysis of MR in mouse peritoneal macrophages. The expression of MR was measured by

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staining with FITC-labeled rat anti-mouse CD206 antibody. Macrophages were treated with

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various concentrations of PSG-1 (20-160 μg/mL) for 24 h. After treatment, cells were collected

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in EP tubes and centrifuged at 1,400 × g for 5 min. The supernatant was removed, cells were

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harvested and washed twice with PBS and then 100 μL PBS was added to resuspend the cells.

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Ten μL of FITC-labeled rat anti-mouse CD206 antibody was added into the cells. Meanwhile, 10

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μL FITC-IgG2a were incubated with the cells as an isotype control. The cells were then

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incubated for 30 min in the dark and washed twice with PBS. Finally the cells were resuspended

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into single cell suspension, and the percentage of positive cells for each specimen was measured

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by flow cytometry.

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Analysis of MR in LPS-stimulated mouse peritoneal macrophages. Purified peritoneal

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macrophages were seeded onto 6-well cell culture plates, and LPS (1 μg/mL) and different

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concentrations of PSG-1 were added to the macrophages, and then incubated for 24 h.

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Simultaneously, control group and LPS-stimulated group were set up in the same way.


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Experimental groups were as follows: (1) Control group, served as a blank with the same amount

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of culture solution; (2) LPS (1μg/mL); (3) LPS (1μg/mL) + PSG-1 (20 μg/mL); (4) LPS (1μg/mL)

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+ PSG-1 (40 μg/mL); (5) LPS (1μg/mL) + PSG-1 (80 μg/mL); (6) LPS (1μg/mL) + PSG-1 (160

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μg/mL). The cells in each group were collected and the expression of MR was determined using

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flow cytometry.

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Analysis of phagocytosis in mouse peritoneal macrophage. After the macrophage were

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pretreated with mannan (2.5, 5, 10 mg/mL) for 30 min and then exposed to PSG-1 (160 μg/mL).

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After 24 h incubation, culture solution was removed. The cells were harvested and washed twice

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with PBS. FITC-dextran fluorescence intensity was determined by flow cytometry.

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Analysis of phagocytosis in LPS-stimulated mouse peritoneal macrophage. Peritoneal

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macrophages were seeded onto 24-well cell culture plates, and cells are grouped as follows: (1)

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Control group, with the same amount of culture solution; (2) LPS (1 μg/mL); (3) Isotype control

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antibody + LPS: hatched in isotype control antibody (10 μg/mL) for 30 min, further treatment

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with LPS (1μg/mL); (4) Anti-MR antibody + LPS: hatched in anti-MR antibody (10 μg/mL) for

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30 min, further treatment with LPS (1 μg/mL); (5) LPS + PSG-1: treatments with LPS (1 μg/mL)

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and PSG-1 (160 μg/mL); (6) Isotype control antibody + LPS + PSG-1: hatched in isotype control

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antibody (10 μg/mL) for 30 min, further treatments with LPS (1μg/mL) and PSG-1 (160 μg/mL);

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(7) Anti–MR antibody + LPS + PSG-1: hatched in anti-MR antibody (10 μg/mL) for 30 min,

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further treatments with LPS (1 μg/mL) and PSG-1(160 μg/mL). After 24 h incubation, the

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culture solution was removed and washed twice with PBS. The cells were collected and

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cultivated in neutral red saline solution (1 mg/mL) for 4 h. The supernatant was removed and

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washed three times with PBS. 1 ml cell lysates was added to each well and placed at room



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temperature for 2-3 h to lysis the cells completely. Absorbance at 540 nm (A540) was measured

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on a multifunction microplate reader.

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Measurement of TNF-α and IL-1β levels in mouse peritoneal macrophage. Cells were

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handled the same way as Section 2.7, and the macrophage supernatant was collected. The levels

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of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were measured by ELISA kits

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following the instructions of manufacturers. The absorbance at 570nm (A570) was measured on a

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multifunction microplate reader, and the cytokine concentrations were determined from the

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standard curve.

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Determination of TNF-α, IL-1β and IL-10 levels in LPS-stimulated mouse peritoneal

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macrophage. Purified peritoneal macrophages were seeded onto 6-well cell culture plates and

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divided into 7 grouped as Section 2.8. The macrophage supernatant was collected after

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stimulated for 24 h. The levels of TNF-α, IL-1β and IL-10 were determined by ELISA kits

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following the instructions of manufacturers. Absorbance at 570 nm (A570) was measured on a

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multifunction microplate reader and the cytokine concentrations were determined according to

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the standard curve.

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Analysis of TLR2 and TLR4 protein expression in the LPS-stimulated mouse peritoneal

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macrophages. Purified peritoneal macrophages were incubated with or without LPS (1μg/mL)

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and different concentrations of PSG-1 for 24 h in 6-well cell culture plates. Cells were grouped

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as follows: (1) Control group, with the same amount of culture solution; (2) LPS (1 μg/mL); (3)

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LPS (1μg/mL) + PSG-1 (20 μg/mL); (4) LPS (1 μg/mL) + PSG-1 (40 μg/mL); (5) LPS (1 μg/mL)

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+ PSG-1 (80 μg/mL); (6) LPS (1 μg/mL) + PSG-1 (160 μg/mL). The culture solution was

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removed when the stimulation was finished. Cells were washed once with PBS, transferred into

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EP tubes, and centrifuged at 1,400 × g for 5 min. The supernatant was removed and washed


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twice with pre-cooled PBS. After centrifugation, the cell precipitate was used for the isolation of

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membrane proteins and cytoplasmic proteins using extraction kits. Equivalent amounts of

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proteins (30 μg) were separated by 12% SDS-PAGE and transferred to PVDF membranes. After

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blocking with 5% non-fat milk, the PVDF membranes were blotted with primary antibodies and

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horseradish peroxidase linked secondary antibodies. The specific bands were determined through

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ECL kit.

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Analysis of TLR4-associated proteins in LPS-stimulated mouse peritoneal macrophages

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in the presence of anti-MR antibody. Peritoneal macrophages were seeded onto 6-well cell

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culture plates, and cells are grouped as follows: (1) Control group, with the same amount of

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culture solution; (2) LPS (1μg/mL); (3) Isotype control antibody + LPS: hatched in isotype

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control antibody (10 μg/mL) for 30 min, further treatment with LPS (1 μg/mL); (4) Anti-MR

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antibody + LPS: hatched in anti-MR antibody (10 μg/mL) for 30 min, further treatment with LPS

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(1 μg/mL); (5) LPS + PSG-1: treatments with LPS (1 μg/mL) and PSG-1(160 μg/mL); (6)

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Isotype control antibody + LPS + PSG-1: hatched in isotype control antibody (10 μg/mL) for 30

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min, further treatments with LPS (1μg/mL) and PSG-1 (160 μg/mL); (7) Anti–MR antibody +

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LPS + PSG-1: hatched in anti-MR antibody (10 μg/mL) for 30 min, further treatments with LPS

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(1μg/mL) and PSG-1 (160 μg/mL). After administration, the expression levels of the proteins

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were determined by Western blot analysis.

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Statistical analysis. WinMDI 2.9 (specialized data analysis software) was used to convert the

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fluorescence data into the histograms for statistical analysis. One-way analysis of variance,

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followed by LSD test using SPSS 11.5 software, was performed to evaluate the effects on

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macrophage MR-mediated the immune response to PSG-1. The Bonferroni was used for Post-

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hoc test..The value of P

Mannose Receptor Mediates the Immune Response to Ganoderma

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