Food and Chemical Toxicology 66 (2014) 14–22

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Food and Chemical Toxicology

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Toll-like receptor 4-mediated ROS signaling pathway involvedin Ganoderma atrum polysaccharide-induced tumor necrosisfactor-a secretion during macrophage activation

0278-6915/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.fct.2014.01.018

Abbreviations: ATCC, American Type Culture Collection; CM-DCFH, carboxyl-DCFH; CR3, complement receptor 3; FBS, fetal bovine serum; HRP, horseradishperoxidase; IKK, IjB kinase; IRAK, interleukin-1 receptor associated kinase; LPS,lipopolysaccharide; MAPKs, mitogen-activated protein kinases; NAC, N-acetylcys-teine; NF-jB, nuclear factor-jB; PAK-1 PBD, PAK-1 p21-bindng domain agarose;PBS, phosphate buffered saline; PI3K, phosphoinositide 3-kinase; PRRs, pattern-recognition receptors; ROS, reactive oxygen species; TLRs, toll-like receptors; TLR4,toll like receptor 4; TNF-a, tumor necrosis factor a; TRAF6, tumor necrosis factor-associated factor 6.⇑ Corresponding authors at: State Key Laboratory of Food Science and Technol-

ogy, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China. Tel./fax: +86 791 83969009 (M.-Y. Xie). Tel./fax: +86 791 88304452 (S.-P. Nie).

E-mail addresses: spnie@ncu.edu.cn (S.-P. Nie), myxie@ncu.edu.cn (M.-Y. Xie).

Qiang Yu, Shao-Ping Nie ⇑, Jun-Qiao Wang, Peng-Fei Yin, Dan-Fei Huang, Wen-Juan Li, Ming-Yong Xie ⇑State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China

a r t i c l e i n f o

Article history:Received 22 October 2013Accepted 10 January 2014Available online 18 January 2014

Keywords:Ganoderma atrum polysaccharideMacrophage activationROSTLR4TNF-a

a b s t r a c t

Ganoderma atrum has been used as Chinese traditional medicine and healthful mushroom for thousandsof years. The polysaccharide is regarded as the major bioactive substances in G. atrum. To delineate theunderlying mechanism and signaling cascade involved in the immunomodulatory property of G. atrumpolysaccharide (PSG-1). Specifically, this study is designed to examine the possibility of TLR4 as a candi-date receptor interacted with G. atrum polysaccharide (PSG-1) and elucidate the role of reactive oxygenspecies (ROS) in PSG-1-induced tumor necrosis factor-a (TNF-a) production during macrophage activa-tion. Flow cytometric and confocal laser-scanning microscopy analysis showed that fluorescence-labeledPSG-1 bind specifically to the macrophages. Moreover, PSG-1 stimulated TNF-a secretion of peritonealmacrophages from C3H/HeN mice, but not from C3H/HeJ mice. PSG-1-indcued TNF-a production wassuppressed by anti-TLR4 mAb. Furthermore, ROS production was mediated by TLR4, and NADPH oxi-dase-derived ROS act as upstream of phosphoinositide 3-kinase(PI3K)/Akt/mitogen-activated proteinkinases(MAPKs)/nuclear factor(NF)-jB signaling pathway in the regulation of PSG-1 stimulated TNF-aproduction. Taken together, we conclude that PSG-1 induces TNF-a secretion through TLR4/ROS/PI3K/Akt/MAPKs/NF-jB pathways during macrophage activation. Our findings provide a molecular basis forthe potential of PSG-1 as a novel immunomodulatory agent.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Ganoderma atrum has been used as Chinese traditional medi-cine and healthful mushroom for thousands of years. The polysac-charide is regarded as the major bioactive substances in G. atrum(Chen et al., 2007; Paterson, 2006). We recently isolated and puri-fied a polysaccharide from G. atrum, named as PSG-1, with a purityof >99.8%, whose primary structural features and molecular weightwere characterized (Chen et al., 2008; Zhang et al., 2012). Theresults have demonstrated that PSG-1 has potent antioxidation

(Chen et al., 2008), antitumor (Li et al., 2011a,b; Zhang et al.,2013a,b) and cardiovascular protection (Li et al., 2010a, 2009)activities in our previous papers. Especially, it was found thatPSG-1 possesses immunomodulatory effects on macrophages (Yuet al., 2013), and PI3K/Akt/MAPKs/NF-jB signaling pathway playsa key role in macrophage activation by PSG-1 (Yu et al., 2012).However, the membrane receptor and detail signaling pathway in-volved in the activation of macrophages in respond to PSG-1 havenot yet been completely clarified.

Emerging studies have established the essential role of specificcellular receptors on macrophages in response to a wide variety ofmicrobial pathogens with subsequent induction of intracellularsignaling cascade (Ehlers, 2000; Medzhitov and Janeway, 2000;Netea et al., 2006). Toll like receptors are of a family that has beenfound to be crucial for innate immunity (Medzhitov, 2001; Trinchi-eri and Sher, 2007). To date, 10 and 12 functional TLRs have beenidentified in humans and mice, respectively, with TLR1–TLR9 beingconserved in both species. Mouse TLR10 is not functional becauseof a retrovirus insertion, and TLR11, TLR12 and TLR13 have beenlost from the human genome (Akira et al., 2001). Binding of ligandsto TLR leads to the formation of a complex between cytoplasmicregion of TLR and the adaptor protein Myd88 and the IRAK.

Q. Yu et al. / Food and Chemical Toxicology 66 (2014) 14–22 15

This is followed by the activation of TRAF6 and TRAF6 in turn acti-vates IKK complex. The activation of IKK complex leads to the deg-radation of IjB and the activation of NF-jB, which regulates a widespectrum of target genes (Karin and Ben-Neriah, 2000). TLRs areexpressed by various cells of the immune system. Among them,TLR4 has been identified as an important membrane receptor ofmacrophages (Moresco et al., 2011), it mediates macrophage acti-vation by transmitting a variety of extracellular signals. LPS is oneof the best studied components that can interact with TLR4 (Ander-son, 2000), however, the role of TLR4 in response to PSG-1 isunclear.

ROS has been traditionally regarded as toxic by-products ofmetabolism, which could cause damage to lipids, proteins, andDNA (Freeman and Crapo, 1982). However, more and more evi-dences show that ROS is not only injurious by-products of cellularmetabolism but also involved in cell signaling and regulation (Fin-kel, 1998; Rhee, 1999). For example, numerous studies have im-plied that ROS is involved in MAPKs activation after cellstimulation with various agents (Iwaoka et al., 2006). Lots of cellu-lar stimuli that induce ROS production is capable of simultaneouslyactivating MAPKs pathways in various cell types (McCubrey et al.,2006; Rhee, 1999). The prevention of ROS accumulation by antiox-idants inhibits MAPKs activation, indicating the involvement ofROS in activation of MAPKs pathways. Besides MAPKs, other sig-naling molecules, such as protein serine/threonine kinase, andtranscriptional factors can also be activated by ROS (Thannickaland Fanburg, 2000). The PI3K, as well as its downstream serine/threonine kinase Akt, which have been implicated in a number ofcellular responses, including cell migration, phagocytosis andapoptosis (Carpenter and Cantley, s1996), were reported to be acti-vated by ROS (Ushio-Fukai et al., 1999). Moreover, NF-jB is wellknown as a critical transcription factor in the induction of a widevariety of genes, that are involved in regulation of immune andinflammatory responses (Hayden and Ghosh, 2004). A number ofstudies indicate that ROS may serve as common intracellularagents that contribute to the process of NF-jB activation in re-sponse to a diverse range of stimuli (Baeuerle and Henkel, 1994).

Many reports indicated that one of the important sources ofROS in macrophages is NADPH oxidase activation during phagocy-tosis (Bokoch, 1995). NADPH oxidase complex of the macrophagesconsists of four core protein subunits, which are cytochrome b558,along with the cytosolic components p67 phox, p47 phox, and p40phox. Activation of the oxidase, however, requires the additionalparticipation of Rac-1, member of the Ras superfamily of smallGTP-binding proteins. During activation, Rac-1 binds GTP and mi-grates to the plasma membrane along with the cytosolic compo-nents to form the active oxidase complex (Bosco et al., 2009).However, little is known about the potential role of ROS in control-ling PSG-1-mediated intracellular signaling pathways and themechanism by which PSG-1 induces ROS generation inmacrophages.

In this study, the possibility of TLR4 as a candidate receptor forPSG-1-mediated signaling was examined. Moreover, we investi-gated the role of ROS in PSG-1-mediated signal transduction in-volved in the regulation of TNF-a production, as well as themechanism for PSG-1 induction ROS in macrophages.

2. Materials and methods

2.1. Mice

Female C3H/HeN and C3H/HeJ mice (6–8 weeks old) were purchased from theShanghai Laboratory Animal Center, China. Mice were housed at nine per cageand maintained at a constant temperature (25 �C), on a 12/12 h reversed light/darkcycle. All animals used in this study were cared for in accordance with the Guide-lines for the Care and Use of Laboratory Animals published by the National Insti-tutes of Health, Bethesda, MD (NIH Publication 85-23, 1996). All procedures wereapproved by the Animal Care Review Committee, Nanchang University, China.

2.2. Materials and reagents

Cell culture products were obtained from Life Technologies (Paisley, Scotland,UK). ELISA kits were from R&D Systems (Minneapolis, MN, USA). Carboxyl-DCFH(CM-DCFH) was purchased from Molecular Probes, Inc. (Eugene, OR, USA). Dextran(70 kDa), fluoresceinamine, diphenyleneiodonium (DPI) and N-acetylcysteine (NAC)were from Sigma (St. Louis, MO, USA). Antibodies against ERK1/2, phospho-ERK1/2(p-ERK1/2), JNK1/2, phospho-JNK1/2(p-JNK1/2), p38, phospho-p38 (p-p38), Akt,phospho-Akt (p-Akt) and horseradish peroxidase-conjugated goat anti-rabbit IgGwere purchased from Cell Signaling (Beverly, MA, USA). Anti-mouse TLR2, TLR4and complement receptor (CR) 3 mAbs were purchased from eBiosciences (San Die-go, CA, USA) and dialyzed extensively against PBS to remove NaN3 before use in cellculture. pGL4.32 [luc2P/NF-jB-RE/Hygro] Vector, FuGENE� HD transfection reagentand Luciferase Assay System were obtained from Promega (Madison, WI, USA). TheRac-1 activation assay kit was purchased from Millipore Inc. (Bedford, MA, USA).

2.3. Extraction of polysaccharides from G. atrum

The polysaccharide from G. atrum (PSG-1) with a purity of >99.8% was isolatedand purified as described previously (Chen et al., 2008). Briefly, the polysaccharidefractions were prepared from G. atrum, which were collected from Ganzhou, JiangxiProvince, China. All extracts were finally pooled, and the polysaccharide-enrichedfractions were precipitated by the addition of 80% (v/v) ethanol. The polysaccharidefraction was further purified by gel filtration chromatography. Its primary struc-tural features and molecular weight were characterized by infrared spectrometry,gas chromatography, size exclusion chromatography, methylation analysis and1D/2D NMR spectroscopy (Zhang et al., 2012). Our previous study (Yu et al.,2013) showed that the quantity of endotoxin in PSG-1 was less than 0.015 EU/mg(negative), as measured by LAL assay, which demonstrated that PSG-1 is free ofLPS contamination.

2.4. Cell culture

RAW264.7 cells, a macrophage-like cell line, were obtained from the AmericanTissue Culture Collection (ATCC, Rockville, MD, USA). The cells were maintained inRPMI 1640 medium containing 10% heat inactivated FBS, 1% penicillin–streptomy-cin at 37 �C in a humidified atmosphere containing 5% CO2 and 95% air.

2.5. Isolation of mouse peritoneal macrophages

To collect peritoneal macrophages, each mouse received 5 mL ice-cold sterilePBS, the peritoneal cavity was washed carefully and 3 mL was retrieved using a Pas-teur pipette. Peritoneal macrophages were aseptically collected and resuspended inRPMI-1640 containing 10% heat-inactivated FBS, 1% penicillin–streptomycin at37 �C in a humidified atmosphere containing 5% CO2 and 95% air.

2.6. Preparation of fluorescein-labeled PSG-1 and dextran

Fluorescein-labeled PSG-1 and dextran were generated as described previously(Glabe et al., 1983). Briefly, 20 mg/mL PSG-1 or dextran solution was mixed with200 lL 10 mg/mL CNBr in 1 mL water under magnetic stirring, and maintained atpH 11 for 15 min by the addition of 0.2 M NaOH. After dialysis against Na2BO7 buf-fer at pH 8.0 for 20 h, the CNBr-activated PSG-1 or dextran was mixed with 2 mgfluoresceinamine for 10 h in the dark at room temperature. Labeled polysaccharidewas separated by gel filtration chromatography on a Sephadex G-25 column andthe bright yellow fraction was collected. The amount of fluorescence-labeled poly-saccharide was determined by phenol–sulfuric acid assay, and the content of fluo-resceinamine was determined by measuring absorbance at 440 nm.

2.7. Flow cytometry

Cell suspensions were centrifuged, and the cell pellets (1 � 106/tube) wereresuspended with 50 lL of f-PSG-1 or f-dextran (20 lg/mL) and incubated for 1 hat 4 �C. The cells were washed twice in PBS and resuspended in 500 lL of PBS forflow cytometric analysis on FACS Calibur (Becton–Dickinson, San Jose, CA, USA).To perform inhibition assays, the cells (1 � 106/tube) were treated with unlabeledPSG-1 at 160 lg/mL, anti-TLR4, TLR2 or CR3 mAb (20 lg/mL) for 1 h and then f-PSG-1 (20 lg/mL) for another hour at 4 �C. After washes, the cells were resuspendedin 500 lL PBS for flow cytometric analysis.

2.8. Confocal laser microscopy

Living macrophages growing on glass cover slips were incubated with f-PSG-1(20 lg/mL) for 1 h at 4 �C. After a brief wash with PBS, cells were observed throughfluorescence confocal laser microscopy LSM 710 (Zeiss, Jena, Germany) with450 nm excitation and 520 nm emission wavelengths.

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2.9. Cytokine determination by ELISA

Levels of TNF-a in the supernatants were determined by the ELISA kits thatwere specific against murine cytokines. Assays were performed according to themanufacturer’s instructions.

2.10. Measurement of ROS generation

Intracellular ROS was measured by detecting the fluorescent intensity of CM-DCFH oxidized product, CM-DCF. After incubation for indicated times, cells wereharvested and washed with cold PBS. Washed cells were further incubated with10 mM CM-DCFH at 37 �C for 30 min in dark. The relative fluorescent intensity offluorophore CM-DCF was detected at an excitation wavelength of 485 nm and anemission wavelength of 530 nm with Varioskan Flash (Thermo Fisher ScientificInc., Waltham, MA, USA).

2.11. Western blot analysis

Whole cell lysates were denatured by boiling in loading buffer (20 mM Tris–HCl, pH 6.8, 10% glycerol, 4% SDS, 100 mM DTT and 0.04% bromophenol blue). Eachsample (40 lg protein) was loaded onto 10% SDS polyacrylamide gel followed byelectroblotting onto nitrocellulose membrane. After blocking of non-specific bind-ing with 5% nonfat milk (prepared in TBS containing 0.1% Tween 20) for 1 h at37 �C, the membrane was applied with antibodies against ERK1/2, p-ERK1/2,JNK1/2, p-JNK1/2, p38, p-p38, Akt and p-Akt, respectively. And this was followedby incubation with HRP conjugated anti-rabbit antibody. Then the protein was de-tected using ECL chemiluminescence detection kit (Amersham Biosciences, Upp-sala, Sweden). Densitometry was performed using the software Quantity One.

Fig. 1. Polysaccharide labeling with fluoresceinamine. CNBr-activated PSG-1 (A)and dextran (B) were reacted with fluoresceinamine for 10 h at room temperaturein the dark. The mixtures were fractionated on a Sephadex-G 25 column,concentrations (lg/mL) of carbohydrate (j) and fluoresceinamine (N) in each

2.12. Transient transfection and luciferase reporter assay

PGL4.32 [luc2P/NF-jB-RE/Hygro] vector (Promega) contains five copies of anNF-jB response element (NF-jB-RE) that drives transcription of the luciferase re-porter gene luc2P. The RAW264.7 cells were transiently transfected with NF-jB-luc plasmid DNA using FuGENE� HD transfection reagent according to the manufac-turer’s instructions. Transfected cells were subcultured and seeded at 2 � 105 cells/mL in 24-well plates. Cells were pre-incubated with various concentrations of NACfor 30 min, followed by PSG-1 (160 lg/mL) stimulation for an additional 6 h. Afterincubation, cells were washed with PBS, extracted in lysis buffer and luciferaseactivities measured using the Luciferase Assay System according to the manufac-turer’s instructions. The specific transactivation was expressed as the fold inductionover untreated cells.

fraction were determined.

2.13. Measurement of PSG-1-induced Rac-1 activity

The measurement of PSG-1-induced Rac-1 activity followed the manufacturer’sinstructions. Briefly, cells were starved for 18 h and then pretreated with or withoutNAC for 30 min prior to PSG-1 (160 lg/mL) stimulation. The whole cell lysates werecollected at the indicated times and immediately incubated with PAK-1 p21-bindngdomain agarose (PBD) for coprecipitation active PAK-1/Rac-GTP complex. Becausethe Rac-GTP is able to associate with PAK-1, the amount of activated form of Racwas detected via mouse monoclonal IgG2b anti-Rac by Western blot analysis.

2.14. Statistical analysis

Values are expressed as means ± SEM. One-way analysis of variance followed bythe Student–Newman–Keuls test was used to determine the statistical significancebetween various groups. A value of p < 0.05 was considered to be statisticallysignificant.

3. Results

3.1. Fluorescence labeling of PSG-1 and dextran

In order to identify cells expressing specific receptor(s) for PSG-1, PSG-1 was conjugated with fluoresceinamine using the CNBr-activation method as described in Materials and methods. f-dex-tran was constructed as control using the same method. As shownin Fig. 1, a small amount of fluoresceinamine co-eluted with poly-saccharides or dextran, while free fluoresceinamine eluted muchmore slowly. Fluorescence-labeled polysaccharide or dextran frac-tions were mixed and stored at �20 �C for future use.

3.2. TLR4 is the major receptor involved in specific binding of PSG-1 tomacrophages

To demonstrate the specific binding of PSG-1 to macrophages,RAW264.7 cells were stained with f-PSG-1 or f-dextran for 1 h at4 �C, followed by flow cytometric analysis and confocal laser-scan-ning microscopy. As shown in Fig. 2A–C, both methods could de-tect positive staining. However, the staining of f-PSG-1 could benoticeably inhibited by the addition of 8-fold molar excess of unla-beled PSG-1 (Fig. 2D), which demonstrated that f-PSG-1 specifi-cally bound to macrophages. To explore which cell receptorinvolve in the specific binding of PSG-1 to macrophages,RAW264.7 cells were incubated with anti-TLR2, anti-TLR4 oranti-CR3 mAb for 1 h and then with f-PSG-1 (20 lg/mL) for anotherhour at 4 �C. As shown in Fig. 2E and F, anti-TLR4 mAb significantlyinhibited the ability of f-PSG-1 to bind to macrophages, suggestinga direct interaction between PSG-1 and TLR4 on the cell surface. Bycontrast, anti-TLR2 and anti-CR3 mAb did not exhibit any signifi-cant inhibitory effect.

3.3. TLR4-dependent activation of macrophages by PSG-1

To verify if TLR4 was required for PSG-1 activation of macro-phages, we compared the effect of PSG-1 on TNF-a production byperitoneal macrophages from wild-type C3H/HeN mice and func-tional TLR4-deficient C3H/HeJ mice. It was found that C3H/HeJ per-

Fig. 2. TLR4-dependent staining of macrophages with f-PSG-1. (A) RAW264.7 cells were stained with f-dextran or f-PSG-1 for 1 h for flow cytometric analysis. (B and C)RAW264.7 cells stained with f-PSG-1 were observed using a confocal laser-scanning microscope. (D) Competitive inhibition of f-PSG-1 binding to cells by unlabeled PSG-1.RAW264.7 cells were stained with f-PSG-1 (20 lg/mL) in the presence or absence of unlabeled PSG-1 at 160 lg/mL for 1 h at 4 �C for flow cytometric analysis. (E and F)Inhibition of f-PSG-1 binding to macrophages by anti-TLR4 mAb but not by anti-TLR2 or anti-CR3 mAb. RAW264.7 cells were treated with anti-TLR4, anti-TLR2 or anti-CR3mAb (20 lg/mL) for 1 h at 4 �C followed by f-PSG-1 (20 lg/mL) staining for another hour, then measured by flow cytometry.

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itoneal macrophages did not respond to PSG-1 stimulation interms of TNF-a production (Fig. 3A). However, PSG-1 could acti-vate macrophages from C3H/HeN mice. It is likely that TLR4 playsa pivotal role in PSG-1-mediated macrophage activation. To furtherconfirm, we assessed the effect of anti-TLR4 antibody on PSG-1-in-duced TNF-a production by RAW 264.7 cells. The results showedthat pretreatment of anti-TLR4 antibody significantly blockedPSG-1-induced TNF-a production (Fig. 3B). These results suggestthat the PSG-1-mediated activation of macrophages is TLR4-dependent.

3.4. ROS is involved in PSG-1-mediated TNF-a production

To investigate whether ROS is involved in PSG-1-mediated TNF-a production in macrophages, we initially examined whether PSG-1 could induce ROS production in RAW264.7 macrophages. Asshown in Fig. 4A, ROS production was detected in 2 min, and thelevel became 1.5-fold higher when it reached the maximum in15 min, compared to that in untreated control cells. By contrast,pretreatment of RAW264.7 macrophages for 30 min with NAC rap-idly reduced PSG-1-induced ROS production. After 60 min, the dif-ference of PSG-1-induced ROS production in cells treated with orwithout NAC gradually came to a steady level (Fig. 4B). Further-more, we found that NAC was able to elicit a dose-dependent inhi-bition of PSG-1-induced TNF-a secretion, indicating that ROS playsa role in TNF-a secretion in PSG-1-stimulated macrophages(Fig. 4C). Additionally, the role of TLR4 in PSG-1-mediated ROS pro-duction was investigated. Cells pre-incubated with anti-TLR4 mAb

before PSG-1 treatment showed a much lower ROS production,compared to the corresponding PSG-1 treated cells (Fig. 4A andB), which demonstrated that TLR4 is the receptors mediating ROSproduction in response to PSG-1.

3.5. The role of ROS in regulating PSG-1-induced PI3K/Akt, MAPKs, NF-jB pathways

To examine the potential role of ROS in PSG-1-mediated activa-tion of PI3K/Akt and MAPKs pathways, RAW264.7 cells were pre-incubated with and without various concentrations of NAC for30 min and then treated with PSG-1 (160 lg/mL) for an additional30 min. Cell lysates were analyzed by Western blot with anti-p-Akt, anti-p-ERK1/2, anti-p-JNK1/2, or anti-p-p38 antibody. Thephosphorylation state-independent Akt, ERK1/2, JNK1/2 and p38were shown to verify semiquantitative loading for gel analysis perloading lane. As shown in Fig. 5A, in the absence of NAC, PSG-1 in-duced the phosphorylation of Akt by 5.3-fold, compared with thecontrol (Lane 1). In contrast, cells pre-incubated with NAC, at theconcentration as high as 10 mM, followed by treatment with PSG-1, showed an almost completely inhibition. As shown in Fig. 5C,upon PSG-1 stimulation, the level of phosphorylated ERK1/2 inRAW264.7 cells detected in 30 min became about 7-fold higher. Incontrast, ERK1/2 phosphorylation gradually reduced by pretreat-ment with increasing concentration of NAC (Lane 3, 4, and 5 versusLane 2). When pre-incubation of cells with NAC (10 mM), the level ofphosphorylated ERK1/2 decreased to 2.3-fold, as compared withcontrol group. Next, we investigated the effect of ROS on PSG-1 acti-

Fig. 3. Involvement of TLR4 in the activation of TNF-a secretion by PSG-1. (A)Peritoneal macrophages from C3H/HeN and C3H/HeJ mice were stimulated withindicated concentrations of PSG-1 for 6 h. Supernatant TNF-a levels were deter-mined using ELISA. (B) RAW 264.7 cells were pre-incubated with anti-TLR4 mAb(20 lg/mL) or isotype anti-IgG2a mAb for 1 h, and then treated with indicatedconcentrations of PSG-1 for 6 h. After incubation, the released TNF-a in conditionedmedia were measured by ELISA. These data are the means ± SEM of triplicatedeterminations. ⁄p < 0.05 compared with PSG-1 alone.

Fig. 4. Involvement of ROS in the TNF-a secretion induced by PSG-1. (A and B)RAW264.7 macrophages were incubated with PSG-1 (160 lg/mL) in the presence orabsence of NAC (10 mM) or anti-TLR4 mAb (20 lg/mL) for the indicated time, andthen ROS levels were measured by detection of the fluorescence intensity of thefluorophore CM-DCF. The data expressed are one of four representative experi-ments. (C) Cells (1 � 106 mL) were pre-incubated with various concentrations ofNAC for 30 min, followed by PSG-1 stimulation (160 lg/mL) for an additional 6 h.After incubation, the level of released TNF-a in conditioned media was measured byELISA. The data were expressed as the means ± SEM with three separate exper-iments. #p < 0.05 compared with untreated cells, ⁄p < 0.05 compared with PSG-1alone.

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vation of JNK1/2 pathway. Cells incubated with PSG-1 alone showeda 3.5-fold JNK1/2 phosphorylation increase. However, there showeda dose-dependently decrease of JNK1/2 phosphorylation by pre-treatment of NAC (Lane 3, 4, and 5 versus Lane 2), the inducedJNK1/2 phosphorylation returned to the basal level when pretreat-ment with 10 mM NAC. Then, we examined whether ROS couldmediate p38 phosphorylation, another important MAPKs member.PSG-1-induced p38 phosphorylation increased about 5.8-fold com-pared with that of control cells. By contrast, p38 phosphorylationwas dose-dependently inhibited by pretreatment with NAC, it be-came to almost 1-fold by block of NAC (10 mM). Furthermore, to elu-cidate the relationship between the ROS and NF-jB, we investigatedwhether ROS was involved in regulating the transactivation activityof NF-jB. Transient transfection and luciferase reporter assay wasused to examine the ability of NAC to block PSG-1-induced transac-tivation activity of NF-jB. As shown in Fig. 5E, the PSG-1 challengerobustly enhanced luciferase activity, while pretreatment withNAC could significantly reduce luciferase activity. These results indi-cated that ROS acted upstream of PSG-1 activation of PI3K/Akt, MAP-Ks and NF-jB pathways.

Fig. 5. NADPH oxidase-derived ROS acts as upstream of PI3K/Akt/MAPKs/NF-jB signaling pathway. (A and C) Effect of NAC on PSG-1-induced phosphorylation of Akt, ERK1/2,JNK1/2, and p38 in RAW264.7 cells. RAW264.7 cells were pretreated with various concentrations of NAC (1, 3 and 10 mM) for 30 min before stimulation with PSG-1 (160 lg/mL) for an additional 30 min. The cell lysates were collected and analyzed by Western blot with anti-phosohorylated Akt, anti-phosphorylated ERK1/2, anti-phosphorylatedJNK1/2, or anti-phosohorylated p38 antibody respectively. Similar results were obtained in three separate experiments. (E) Effects of NAC on the NF-jB activation in PSG-1-stimulated RAW 264.7 cells. RAW264.7 cells were transiently transfected with luciferase reporter plasmid NF-jB-luc for 48 h, then cells were pretreated with variousconcentrations of NAC for 30 min before incubation with PSG-1 (160 lg/mL) for 6 h. Luciferase activity was measured by luciferase assay. Results are expressed as foldinduction of relative light units (RLU) of treated cells over that of untreated cells. Values are means ± SEM with three separate experiments. #p < 0.05 compared withuntreated cells, ⁄p < 0.05 compared with PSG-1 alone. (F) The role of NADPH oxidase in regulation of PSG-1-mediated Rac1 activity. Cells were pretreated with NADPH oxidaseinhibitor, DPI (5 lM) for 30 min before stimulation of PSG-1 for additional 30 min. The Rac1-GTP complex was coprecipitated from whole cell lysate by PAK-1 PBD as well asactive Rac1. Then the immunoprecipitant was quantitatively measured by western blot using the anti-Rac antibody. (B, D and G) Bar graph represents quantification byQuantity one software. All data of relative activity are expressed as comparison with untreated cells (control cells defined as 1). Similar experiments were repeated threetimes.

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3.6. PSG-1-induced Rac-1 activity and the effect of DPI in Rac-1activation

To further investigate the potential upstream signaling mole-cules involving in PSG-1-mediated ROS production, we focusedattention on a relevant important molecule, Rac-1. The resultsshowed that Rac-1 activity was elevated 4.2-fold (Fig. 5F and G,Lane 2 versus Lane 1). In contrast, DPI, an NADPH oxidase effectiveinhibitor, significantly reduced PSG-1-triggered Rac1 activation by60% as compared with the PSG-1 alone (Fig. 5F and G, Lane 3 versusLane 2).

4. Discussion

A number of studies were well documented that polysaccha-rides cannot penetrate cells due to their large molecular mass, sothe first step in the modulation of cellular activity would be bind-ing to immune cell receptors. Therefore, we hypothesized thatmacrophage activation by PSG-1 starts with specific binding tocells. Our data from flow cytometric analysis and confocal laser-scanning microscopy demonstrated the specific binding of f-PSG-1 to macrophages, which was further confirmed by the competitiveinhibition of f-PSG-1 binding to cells by unlabeled PSG-1.

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The innate immune system identifies infectious agents orcompounds by using of pattern-recognition receptors (PRRs).The best characterized group of PRRs is the evolutionary con-served family of TLRs (Barton and Medzhitov, 2003). It is nowevident that mammalian TLRs play a prominent role in the directactivation of host defense mechanisms. The activation of TLRsstimulates an innate immune response, which involves the pro-duction of direct antimicrobial effector molecules, including ROS,and increases an adaptive immune response by inducing theproduction of TNF-a and IL-1b that augment both cell-mediatedand humoral immune responses. TLR4, a founding member ofthe TLR family, is mainly expressed in macrophages, monocytes,neutrophils, and dendritic cells (Beutler, 2000). Recently, a lot ofstudies have identified that TLR4 is an important membranereceptor of polysaccharides. Polysaccharide from Ganoderma luci-dum and Paecilomyces cicadae interact with TLR4 on macro-phages (Kim et al., 2012; Shao et al., 2004). In addition,polysaccharides from Polyporus umbellatus (Per) Fr, Cordycepsmilitaris, been reported to activate DCs cells through TLR4 (Kimet al., 2010; Li et al., 2010b). These evidences prompted us tostudy TLR4 as the candidate receptor for PSG-1. In the presentstudy, the ability of anti-TLR4 mAb to inhibit f-PSG-1 bindingbut not by anti-TLR2 and anti-CR3 mAb, suggests that TLR4 isthe major receptor involved in PSG-1 binding to macrophages.

Although TLRs are essential for protective immunity againstinfection, inappropriate TLR responses contribute to acute andchronic inflammation, as well as to systemic autoimmune diseases.For example, TLR4 responds to bacterial LPS, which is considered toplay a key role in the septic shock syndrome (Kawai and Akira, 2006).More and more researchers speculate that natural polysaccharidescan directly activate innate immune response by mediating TLR sig-naling, since natural polysaccharides have been safely used as ingre-dients of traditional medicines for a long time in oriental countries,which have not been associated with any detrimental tissue injuriescaused by LPS. Recently, several reports have demonstrated theTLR4-dependent activation of macrophages by mushroom polysac-charides. For instance, Li and Xu (2011) reported that polysaccharidefraction from P. umbellatus (pers.) Fries stimulated TNF-a and IL-1bproduction via TLR4 activation of signaling pathway in macro-phages. Especially, Zhang et al. (2013a) reported that PSG-1 may eli-cit its antitumor effect through TLR4. However, the authors onlypreliminarily investigated the role of TLR4 by measuring the proteinexpression of TLR4, and then suggested that it may be a possiblereceptor for PSG-1. This prompted us to conduct a more systematicaland convincing research to illustrate the role of TLR4 on the activa-tion of macrophage by PSG-1. Specifically, we firstly synthesizedfluorescein-labeled PSG-1(f-PSG-1), and demonstrated the specificbinding of f-PSG-1 to macrophages by flow cytometric analysisand confocal laser-scanning microscopy. Then we used anti-TLR4mAb, anti-TLR2 mAb and anti-CR3 mAb to inhibit f-PSG-1 binding,and found TLR4 is the major receptor involved in PSG-1 binding tomacrophages. Furthermore, our results demonstrated that PSG-1activated peritoneal macrophages from wild-type but not TLR4-deficient mice to produce TNF-a. Moreover, anti-TLR4 mAb couldsignificantly block PSG-1-mediated TNF-a secretion. Based on theseresults, we conclude that TLR4 is the major receptor involved in PSG-1-mediated activation of macrophages.

When the body is stimulated by pathologic stimuli or injury,macrophages protects the host via releasing cytotoxic molecules,such as ROS. Our data showed that PSG-1 is capable of inducingROS production in macrophages by measuring the fluorescent oxi-dative product of CM-DCFH, and this effect was inhibited by pre-treatment of the antioxidant NAC. However, increasing evidencedemonstrated that ROS can not only play key roles in host defense,but also act as a cellular secondary messenger in many biologicalfunctions, such as protein phosphorylation (Ito et al., 2006), tran-

scription factor activation (Kaul et al., 1998), cytokine production(Hong et al., 1997). The present study showed that ROS was in-volved in the regulation of cytokine TNF-a production stimulatedby PSG-1. Furthermore, TLR4 was demonstrated as the criticalreceptor for ROS production stimulated by PSG-1, evidenced bythe inhibition effect of anti-TLR4 mAb on the ROS production.These facts inspired us to explore the role of ROS in TLR4-mediateddownstream signaling pathway in respond to PSG-1.

Our previous work (Yu et al., 2013) demonstrated that PI3K/Akt/MAPKs/NF-jB signaling pathway was involved in the macrophageactivation by PSG-1. To further investigate the upstream pathwayinvolved in the regulation of TNF-a secretion during macrophageactivation, we focus on the effect of ROS on PI3K/Akt/MAPKs/NF-jB pathway. MAPKs are a family of serine/threonine protein ki-nases that include three major groups in mammalian cells, i.e.ERK1/2, JNK1/2, and p38 (Rao, 2001). Extensive literature has doc-umented that ROS can mediate the activation of MAPK pathwaysby a number of cellular stimuli in several cell types, although a re-cent paper (Yang et al., 2011) challenges this concept, the capsularpolysaccharide of pyrogenic liver abscess Klebsiella pneumonia(PLAK. pneumoniae CPS)-mediated activation of ERK1/2, JNK1/2,and p38 is independent of ROS. However, little information is avail-able regarding whether ROS is involved in MAPK activation stimu-lated by PSG-1 in macrophages. Our data showed that inhibition ofROS by NAC diminished PSG-1-induced ERK1/2, JNK1/2 and p38phosphorylation in a dose-dependent manner, which suggestedthat PSG-1-mediated activation of ERK1/2, JNK1/2 and p38 isdependent of ROS. In addition, our previous study demonstratedthat PI3K/Akt signaling pathway acted upstream of MAPKs pathwayin PSG-1-stimulated macrophages. Given our above results demon-strating the activation of MAPKs is mediated by ROS, we nextsought to determine whether ROS also induces the PI3K/Akt signaltransduction pathway. To address this issue, we tested the effect ofNAC on Akt phosphorylation and found that PSG-1 induced Aktphosphorylation was inhibited by pretreatment of NAC in a dose-dependent manner. These results demonstrated that ROS was in-volved in PI3K/Akt pathway in PSG-1 stimulated macrophages.Moreover, NF-jB, a transcription factor that regulates the expres-sion of large numbers of genes involved in innate immunity, hasbeen reported regulated by ROS (Asehnoune et al., 2004; Meyeret al., 1993; Schreck et al., 1992). Based on our previous study,which showed that NF-jB activation by PSG-1 was triggered by up-stream signaling cascades, PI3K/Akt and MAPKs, we hypothesizedthat ROS might be involved in PSG-1-mediated NF-jB activation.The present study found that PSG-1-induced transactivation activ-ity of NF-jB was abrogated by the treatment of NAC, which sug-gested that the induction transactivation activity of NF-jB byPSG-1 was dependent on ROS in RAW 264.7 cells. These results sup-port our hypothesis that NF-jB activation by PSG-1 was triggeredby ROS. Taken together, we come to a conclusion that ROS acts up-stream of PI3K/Akt/MAPKs/NF-jB pathway.

Mounting evidence supports that one of the important sourcesof ROS in macrophages is NADPH oxidase activation during phago-cytosis (Bokoch, 1995), so the most likely origin of ROS followingPSG-1 stimulation in macrophages would be expected to comefrom NADPH oxidase. Given that Rac1 is known as an importantcomponent of functional NADPH oxidase (DeLeo et al., 1998), wetest whether Rac1 activity was elevated following PSG-1 treat-ment. The significant increase of GTP-bound Rac1 after treatmentof RAW264.7 cells with PSG-1 and the DPI reduction of PSG-1-trig-gered Rac1 activation clearly demonstrate that the originality ofPSG-1-induced ROS as a secondary messenger is mainly derivedfrom NADPH oxidase.

In summary, the present study demonstrated that TLR4 is themajor receptor involved in interaction of PSG-1 and macro-phages. Moreover, NADPH oxidase-derived ROS acts as upstream

Fig. 6. The proposed model for PSG-1-mediated ROS and downstream signal transduction pathways in the regulation of TNF-a production in macrophages.

Q. Yu et al. / Food and Chemical Toxicology 66 (2014) 14–22 21

of PI3K/Akt/MAPKs/NF-jB signaling pathway in the regulation ofPSG-1 stimulated TNF-a production. Therefore, we establishedthat a major pathway by which PSG-1 exerted its effects onmacrophage activation via a TLR4/ROS/PI3K/Akt/MAPKs/NF-jBsignal pathway (Fig. 6).

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

Acknowledgements

The financial support for this study by the Key Program of Na-tional Natural Science Foundation of China (No: 31130041), Na-tional Key Technology R & D Program of China (2012BAD33B06),and National Natural Science Foundation of China (31071532,21265011), the Program for New Century Excellent Talents in Uni-versity (NCET-12-0749), Research Program of State Key Laboratoryof Food Science and Technology (SKLF-ZZA-201301), the Project ofScience and Technology of Jiangxi Provincial Education Depart-ment (KJLD13004) and Exploring Project of State Key Laboratoryof Food Science and Technology (SKLF-TS-201107), is gratefullyacknowledged.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.fct.2014.01.018.

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