Research Article Ganoderma lucidum Polysaccharides Reduce Lipopolysaccharide...

Research ArticleGanoderma lucidum PolysaccharidesReduce Lipopolysaccharide-Induced Interleukin-1120573Expression in Cultured Smooth Muscle Cells and inThoracic Aortas in Mice

Chan-Jung Liang1 Chiang-Wen Lee2 Hsin-Ching Sung13

Yung-Hsiang Chen4 Yao-Chang Chiang5 Hsien-Yeh Hsu6 Ying-Chin Tseng7

Chi-Yuan Li8 Shu-Huei Wang1 and Yuh-Lien Chen1

1 Department of Anatomy and Cell Biology College of Medicine National Taiwan University No 1 Section 1Ren-Ai Road Taipei 100 Taiwan

2Department of Nursing Division of Basic Medical Sciences Chiayi Campus Research Center for Industry of Human EcologyChang Gung University of Science and Technology Taoyuan 303 Taiwan

3Department of Anatomy College of Medicine Chang Gung University Taoyuan 333 Taiwan4Graduate Institute of Integrated Medicine China Medical University Taichung 404 Taiwan5 Center for Drug Abuse and Addiction China Medical University Hospital Taichung 404 Taiwan6 Institute of Biotechnology in Medicine National Yang-Ming University Taipei 112 Taiwan7Department of Obstetrics and Gynecology Hsinchu Cathay General Hospital Hsinchu 300 Taiwan8 Institute of Clinical Medical Science China Medical University No 91 Hsueh-Shih Road Taichung 404 Taiwan

Correspondence should be addressed to Shu-Huei Wang huei112964yahoocomtw and Yuh-Lien Chen ylchenvntuedutw

Received 12 November 2013 Revised 7 January 2014 Accepted 28 January 2014 Published 4 March 2014

Academic Editor Xingjiang Xiong

Copyright copy 2014 Chan-Jung Liang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The expression of inflammatory cytokines on vascular walls is a critical event in vascular diseases and inflammation The aimof the present study was to examine the effects of an extract of Ganoderma lucidum (Reishi) polysaccharides (EORPs) whichis effective against immunological disorders on interleukin- (IL-) 1120573 expression by human aortic smooth muscle cells (HASMCs)and the underlyingmechanismThe lipopolysaccharide- (LPS-) induced IL-1120573 expressionwas significantly reducedwhenHASMCswere pretreated with EORP by Western blot and immunofluorescent staining Pretreatment with 10120583gmL EORP decreased LPS-induced ERK p38 JNK and Akt phosphorylation But the increase in IL-1120573 expression with LPS treatment was only inhibitedby pretreatment with the ERK12 inhibitor while the JNK and p38 inhibitors had no effect In addition EORP reduced thephosphorylation and nuclear translocation of nuclear factor- (NF-) 120581B p65 in LPS-treated HASMCs Furthermore in vivo IL-1120573expression was strongly expressed in thoracic aortas in LPS-treatedmice Oral administration of EORP decreased IL-1120573 expressionThe level of IL-1120573 expression in LPS-treated or in LPSEORP-treated groupwas very low andwas similar to that of the saline-treatedgroup in toll-like receptor 4-deficient (TLR4minusminus) mice These findings suggest that EORP has the anti-inflammatory property andcould prove useful in the prevention of vascular diseases and inflammatory responses

1 Introduction

The inflammatory processes have a key role not only inthe initiation and progression of atherosclerosis but also inthe stability of the established atherosclerotic plaques [1 2]

Cytokines of the interleukin-1 (IL-1) family play a pivotalrole in regulating the immunoinflammatory responses andextensive studies have been performed to determine whetherIL-1 modifies the inflammatory response [3 4] IL-1120573 upreg-ulates a substantial increase in the expression of adhesion

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 305149 13 pageshttpdxdoiorg1011552014305149

2 Evidence-Based Complementary and Alternative Medicine

molecules by vascular smooth muscle cells (VSMCs) in vitroand these factors promotemonocyte recruitment and infiltra-tion into the arterial wall [5 6] and induce VSMC migrationand proliferation [3 7 8] In vivo studies have shownincreased levels of IL-1120573 mRNA in human atheroscleroticlesions [9] and of IL-1120573 mRNA and protein in VSMCsof atherosclerotic arteries from non-human primates [10]Although the relationship between IL-1120573 and cardiovasculardisease has been extensively studied IL-1120573 expression andits related mechanism in LPS-induced human aortic smoothmuscle cells (HASMCs) and in inflammatory vascular wallshave not been studied in detail Moreover reducing IL-1120573expression might be a beneficial approach for inhibiting thedevelopment of vascular inflammation and atherosclerosis[11]

Ganoderma lucidum (G lucidum Reishi) a popular homeremedy has long been known for its beneficial effects onhuman health and longevity and is used to treat chronichepatopathy hypertension hyperglycemia and neoplasia[12 13] Ganoderma lucidum intake caused an increase inplasma antioxidant capacity and that 10 d supplementa-tion was associated with a trend towards a decrease ofplasma lipids in human subjects [14 15] Studies on the roleof G lucidum in regulating various body functions haverevealed that G lucidum polysaccharides are the bioactiveconstituents responsible for many of their health bene-fits such as its antioxidant anticancer anti-inflammatoryand immunomodulatory activities [16ndash18] The effects ofG lucidum on the immune system have been linked tothe regulation of cytokine expression and differentiationof macrophages [19 20] and the maturation of culturedmurine bone marrow-derived dendritic cells [21]G lucidumhave also been shown to suppress TNF-120572 expression fromlipopolysaccharide- (LPS-) stimulated murine RAW 2647cells [22] reduce IL-1120573 secretion from activated microgliaafter LPS treatment [23] and prevent albumin-inducedoxidative damage and chemokines synthesis in human prox-imal tubular epithelial cells [24] Regulation of inflammatorycytokine expression requires a complex array of intracel-lular signaling pathways involving mitogen-activated pro-tein kinases (MAPKs) and transcriptional factors [25 26]Although these multiple signaling molecules have receivedconsiderable attention little is known about the effectsof an extract of Reishi polysaccharides (EORPs) on IL-1120573expression and the mechanisms of these effects and a betterunderstanding of this might provide important insights intothe prevention of cardiovascular diseases and inflammationWe were therefore interested in understanding the mecha-nism of action of EORP on human aortic smooth musclecells (HASMCs) stimulated by inflammatory cytokines andwhether it affects the IL-1120573 expression an important event invascular diseases and inflammation In addition we studiedthe effects of EORP on IL-1120573 expression in thoracic aortas inLPS-treated mice Our results showed that EORP attenuatedIL-1120573 expression both in vitro and in vivo and that thiseffect was mediated at least in part through inhibition ofextracellular signal-regulated kinase (ERK) phosphorylationnuclear factor NF-120581B activation and TLR4 activation

2 Materials and Methods

21 Preparation of EORP A crude powder ofG lucidum pre-pared via alkaline extraction with 01 NNaOH followed byneutralization and ethanol precipitation was obtained fromPharmanex (Provo UT) The biologically active compoundsfrom Reishi are identified as the fucose-containing glyco-protein fraction and named EORPs The molecular structureand the quality control of EORPs were described in detailin the previous reports [13 19] In brief the carbohydratecomposition analyses of crude water soluble Reishi extractindicated that glucose mannose and fucose exist as themajor components The crude extract contains 156 pro-teins The saccharides contain either a polysaccharide back-bone with 120573-13-linkages or a polymannose backbone with120572-14-linkages both with side chains of unknown structureEORPs were prepared as GMP grade and carefullymonitoredto meet the Food and Drug Administration standard TheEORP is currently commercially available as the traditionalChinese medicine

22 HASMC Cultures HASMCs purchased as cryopre-served tertiary cultures from Cascade Biologics (OR USA)were grown in culture flasks in smooth muscle cell growthmedium (M231 Cascade Biologics Inc) The cells were usedbetween passages 4 and 8 All cells were synchronized inserum-free medium for 24 h prior to experiments For alldata shown the experiments were repeated at least 3 times induplicate with different preparations of HASMCs For West-ern blot analysis the graphical analysis represents the resultsfrom three independent experiments and quantification bydensitometry

23 Effect of LPS and EORP on Cell Viability The cellviability was determined by 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich StLouis MO USA) assay HASMCs were plated in 96-wellplates (1 times 104 cellswell) After serum starvation the cellswere treated with different concentrations of LPS or EORPfor 24 h and then MTT solution was added to each well andincubated for another 2 h at 37∘C in 5CO

2

The supernatantwas discarded and dimethyl sulfoxide (DMSO) was addedto dissolve the formazan crystals The cellular viability wasmeasured by ELISA plate reader at 540 nm

24 Western Blot Analysis Western blot analyses were per-formed as described previously [27] In brief samples ofcell lysate (20120583g of protein) were subjected to 12 SDS-PAGE and transferred to PVDF membranes which werethen treated with 5 nonfat milk in 01M phosphate bufferfor 1 h at RT to block nonspecific binding of antibody Tomeasure IL-1120573 levels the membranes were then incubatedwith rabbit anti-human IL-1120573 (1 1000 dilution GeneTexIrvine CA USA) and then with horseradish peroxidase-(HRP-) conjugated goat anti-rabbit IgG antibodies (1 6000dilution KPL Gaithersburg MA USA) and bound antibodywas detected using Chemiluminescence Reagent Plus Inother studies the antibodies used were rabbit anti-humanphospho-Jun N-treminal kinase (JNK) rabbit anti-human

Evidence-Based Complementary and Alternative Medicine 3

phopho-ERK12 rabbit anti-human phospho-p38 (all 1 1000dilution Cell signaling Danvers MA USA) rabbit anti-human phospho-p65 and rabbit anti-human p65 (all 1 1000dilution GeneTex) followed by HRP-conjugated goat anti-rabbit IgG antibodies The intensity of each band was quan-tified using a densitometer Antibodies against 120573-actin 120572-tubulin or GAPDH (all 1 1000 dilution Gene Tex) were usedas loading controls

25 Immunocytochemical Staining HASMCs were plated ata density of 104 cellswell in gelatin-coated coverslips Afterstarvation the cells were treated with various concentrationsand time of LPS and EORP After treatment the cells werefixed in 4paraformaldehyde for 15min at room temperatureand then incubated with IL-1120573 and NF-120581B p65 antibodies at4∘C overnight After incubation the cells reacted with FITC-conjugated anti-rabbit IgG (both from Sigma) for 1 h at roomtemperature DAPI stain was used as the nuclear counterstainand observation was conducted by fluorescence microscope

26 IL-1120573 Enzyme-Linked Immunosorbent Assay (ELISA)The concentrations of IL-1120573 in cultured media of smoothmuscle cell and in plasma from mice were determined usingthe human and mouse IL-1120573 ELISA kits (RampD SystemsMinneapolis MN) respectively The analysis was performedaccording to the instructions from the manufacturer

27 Knockdown of Gene Expression Using Small InterferingRNA Knockdownof ERKgene expressionwas performed bytransfection with small interfering RNA (siRNA InvitrogenCarlsbad CA USA) HASMCs (106) were resuspended in100 120583L of Nucleofector solution (Amaxa Biosystems Ger-many) and gene-specific ERK siRNA oligomers (1 120583M) wereelectroporated according to the manufacturerrsquos instructionmanual The ERK siRNAs (catalog number 10620319 124945F11 and 10620318 124945 F12 Invitrogen) were AUA UUCUGU CAG GAA CCC UGU GUG A and UCA CAC AGGGUU CCU GAC AGA AUA U Cells were transfected for48 h The siRNA results were evaluated by Western blotting

28 RNA Extraction and Reverse Transcription-PolymeraseChain Reaction (RT-PCR) Assay Total RNA was extractedfrom cells using Trizol reagent (Invitrogen) according to themanufacturerrsquos protocol The reverse-transcriptase reactionwas carried out using M-MLV reverse transcriptase (Invit-rogen) Complementary DNA was generated by addition oftotal RNA (1 120583g) to a reaction mixture containing 05120583g120583Loligo-deoxythymidine 20mM dNTP 01M dithiothreitol250mM Tris-HCl pH 83 375mM KCl and 15mMMgCl

2

and reaction at 37∘C for 90min The oligonucleotide primersused were 51015840-CAGACCATGATCACACAGGG-31015840 (forward)and 51015840-TGGAAAGATGGGCCTGTTAG-31015840 (reverse) forERK and 51015840-GTAACCCGTTGAACCCCATT-31015840 (forward)and 51015840-CCATCCAATCGGTAGTAGCG-31015840 (reverse) for 18 Ssubunit ribosomal RNA The PCR conditions were an initialdenaturation at 94∘C for 5min followed by 29 cycles ofdenaturation at 94∘C for 1min primer annealing at 55∘C for1min and extension at 72∘C for 5min The products of PCR

were analyzed by electrophoresis on 2 agarose gels stainedwith ethidium bromide

29 Mouse Model All animal experiments were carried outin accordance with the recommendations in theGuide for theCare andUse of LaboratoryAnimals of theNational Institutesof Health The protocol was approved by the InstitutionalAnimal care and Use Committee of the National TaiwanUniversity (Permit number 20110011) Eight-week-old maleC57BL6J mice and toll-like receptor 4-deficient (TLR4minusminus)mice weighing between 25 and 35 g were used in the presentstudy To examine the effects of EORP administration onthoracic aortas in the LPS-treated mice the C57BL6J micewere divided into two divisions One division is defined asthe acute inflammatory phase (2 days) and the other one isthe chronic inflammatory phase (2 weeks) Each division wasfurther divided into four groups Groups 1 and 2 were givenrespectively either saline (control) or 1mgkgday of LPS onday 1 or on days 1ndash13 by intraperitoneal (ip) injection andgroup 3 was treated with LPS in the same way as group 2but received oral EORP at 60mgkgday on days 0-1 (acuteinflammatory phase) or on days 0ndash13 (chronic inflammatoryphase) and group 4 received only the oral EORP Further-more the TLR4minusminus mice were divided into 3 groups groups1ndash3 were treated the same way as groups 1ndash3 of the chronicinflammatory divisionThe selection of EORPdosewas basedon the previous reports [17 27] At the end of the 2 days or 2weeks of the experiments the animals were anesthetized byip injection of 60mgkg sodium pentobarbital and sacrificedBlood samples were collected and soluble IL-1120573 in theplasma was measured by ELISA One segment of the thoracicaortas was rinsed with ice-cold PBS immersion-fixed with4 buffered paraformaldehyde and paraffin-embedded andthen cross-sectioned for immunohistochemistry while theremaining portion was immediately frozen in liquid nitrogenfor protein extraction The tissue sections (5-6120583m thick)were mounted on poly-l-lysine coated slides deparaffinizedrehydrated and washed with PBS To study cellular expres-sion and localization of IL-1120573 serial sections were incubatedwith 1 hydrogen peroxide in methanol for 10min to blockendogenous peroxidase activity and to permeabilize the cellsthen nonspecific binding was blocked by incubation for 1 h atRT with PBS containing 5mgmL of bovine serum albuminIn the primary antibody step at 37∘C for 1 h the first serialsection was incubated with goat anti-human CD31 antibody(EC marker 1 100 Santa cruz CA USA) the second withgoat anti-human IL-1120573 antibody (1 50) and the third withmouse anti-120572-smooth muscle actin (SMC marker 1 50Dako Cytomation CA USA) The first and second slideswere localized by an indirect immunoperoxidase technique(avidin-biotin-horseradish peroxidase complex) employingdiaminobenzidine (Vector) as chromogen while bound anti-bodies on the third sectionwere then incubated for 15 h at RTwith FITC-conjugated goat anti-mouse secondary antibody(1 400 Sigma Chemical Co St Louis MO USA) andobserved by fluorescent microscopy Each incubation wasfollowed by three times of 5min washes in PBS Negativecontrols were performed by omitting the primary antibody


210 Statistical Analysis Values are expressed as the mean plusmnSEM Studentrsquos 119905-test and one-way ANOVA were performedto compare the two groups and multiple groups respectivelyA 119875 value lt005 was considered statistically significant

3 Result

31 EORP Reduces IL-1120573 Expression in LPS-Treated HASMCsWhen the cytotoxicity of LPS or EORP for HASMCs wasdetected by the MTT assay after 24 h of incubation cellviability was not affected by the presence of 1ndash15 120583gmLof LPSor 1ndash10 120583gmL of EORP (data no shown)

To determine the optimal conditions for LPS-inducedIL-1120573 expression by HASMCs we first performed dose-response studies in which HASMCs were cultured withvarious concentrations of LPS for various time intervalsAs shown in Figure 1(a) IL-1120573 expression in HASMCs with10 120583gmL LPS treatment reached amaximum after 24 h (23plusmn02) IL-1120573 expression was induced in a dose-dependentmanner after treatment with 5 10 or 15120583gmL of LPSfor 24 h (11 plusmn 02 17 plusmn 01 23 plusmn 01 resp of controllevels) (Figure 1(b))The induction caused by the two highestconcentrationswas significant As shown in Figure 1(c) whenthe HASMCs were pretreated with 10 120583gmL of EORP for24 h before incubation with 10 120583gmL of LPS for 24 h LPS-induced IL-1120573 expression was reduced to 09 plusmn 01 of controllevels The effect of EORP on IL-1120573 expression was alsostudied by immunofluorescence staining (Figure 1(d)) Inuntreated cells IL-1120573 expression was weak In contrast cellstreated for 24 h with LPS showed strong IL-1120573 expressionand this effect was inhibited by pretreatment with EORPFurthermore unstimulatedHASMCs produced low amountsof IL-1120573 in the cell culture supernatant (80 plusmn 23 pgmL) and24 h treatment with 10 120583gmL of LPS resulted in a markedincrease in IL-1120573 levels (653 plusmn 90 pgmL) by ELISA Thiseffect was significantly inhibited by 24 h preincubation with10 120583gmL of EORP (398 plusmn 50 pgmL) In all subsequentexperiments unless otherwise specified 10 120583gmL LPS and10 120583gmL EORP were used

32 EORP Attenuated LPS-Induced IL-1120573 Expression is PartlyDependent on Inhibition of ERK12 Phosphorylation Theprevious studies showed that LPS can activate MAPKs andAkt in the signaling pathway leading to inflammation [2628] In the next set of experiments the effects of LPS on theactivation of the MAPK pathway (ERK12 JNK p38) andAkt a signaling cascade contributing to IL-1120573 expression andthe effects of EORP or MAPK inhibitors on LPS-stimulatedIL-1120573 expression were studied As shown in Figures 2(a)ndash2(d) phosphorylation of ERK12 p38 JNK and Akt wassignificantly increased compared with that of control levelsrespectively at 30min after addition of LPS Pretreatmentwith 10 120583gmL EORP decreased LPS-induced ERK p38JNK and Akt phosphorylation As shown in Figure 2(e) theincrease in IL-1120573 expression in response to LPS treatmentwas inhibited by 1 h pretreatment with 30 120583M PD98059 (anERK12 inhibitor) while SP600125 (a JNK inhibitor) orSB203580 (a p38 inhibitor) had no effect The inhibitory

effect of ERK12 was further confirmed by the transfectionof ERK siRNA The effectiveness of the siRNA treatmentwas validated by showing that ERK-specific siRNA caused a50 reduction in ERK protein expression (Figure 2(f)) LPS-induced IL-1120573 expression was inhibited by transfection ofHASMCs with ERK12-specific siRNA (1120583M) (Figure 2(g))Additional experiments on ERK gene expression showed thatit was detected in untreated cells however both LPS andEORP treatments did not affect its expression (Figure 2(h))On the other hand ERK-specific siRNA treatment decreasedERK gene expression Both LPS and EORP treatments alsodid not alter its gene expression These results indicate thatEORP inhibits LPS-induced IL-1120573 expression by preventingLPS-induced phosphorylation of ERK12

33 EORPAttenuates Phosphorylation andNuclear Transloca-tion of NF-120581B p65 in LPS-Treated HASMCs Transcriptionalregulation involving NF-120581B activation has been implicated inthe stimulator-induced expression of inflammatory cytokines[25] As shown in Figure 3(a) low basal levels of NF-120581B p65phosphorylation were detected in control cells and phospho-rylation was significantly increased by 90min treatment withLPS This increase was markedly inhibited by preincubationwith 10 120583gmL EORP for 24 h To determine whether NF-120581Bp65 translocation was involved in the pretranslational effectsof EORP on IL-1120573 expression we also studied NF-120581B p65protein levels in the nuclei of LPS-treated HASMCs byimmunofluorescent staining As shown in Figure 3(b) LPS-stimulated HASMCs showed marked NF-120581B p65 stainingin the nuclei while EORP-pretreated cells showed weakernuclear NF-120581B expression but stronger staining in thecytoplasm Furthermore the stimulatory effect of LPS onIL-1120573 levels was blocked by coincubation with parthenolidea NF-120581B inhibitor (Figure 3(c)) These results suggest thatEORP inhibits LPS-induced IL-1120573 expression by preventingLPS-induced phosphorylation and translocation of NF-120581Bp65

34 EORP Decreases IL-1120573 Protein Expression in Plasmaand in Thoracic Aortas of LPS-Injected Mice To determinethe effect of EORP on IL-1120573 expression in vivo mice wereorally fed with EORP (60mgkgday) prior to treatmentwith ip injection of LPS (1mgkgday) Serial sections ofthoracic aortas were performed to detect IL-1120573 expressionand its association with endothelial cells and smooth musclecells by using CD31 and 120572-actin antibodies as cell mark-ers respectively As shown in Figure 4 in the saline- andEORP-treated groups weak IL-1120573 staining was seen on thevascular wall while in the LPS-treated group strong IL-1120573 staining was seen on the vascular walls at the acuteinflammatory phase IL-1120573 expression was associated withendothelial cells and smooth muscle cells Interestingly IL-1120573 expression was mainly present in smooth muscle cellsof the media rather than in endothelial cells of the intimaIn contrast administration of EORP resulted in weak IL-1120573staining in the LPS-treated mice The corresponding levelsof IL-1120573 expression were similarly found in plasma and inthoracic aortas by ELISA (Figure 4(b)) and by Western blot(Figure 4(c)) respectively Furthermore in the LPS-treated


IL-1120573

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Figure 1 EORP reduces IL-1120573 protein expression in LPS-treated HASMCs (a) and (b) HASMCs were treated with 10 120583gmL of LPS for theindicated time (a) or with the indicated concentration of LPS for 24 h (b) (c) HASMCs were incubated for 24 h with 10120583gmL of EORP andthen for 24 h with 10 120583gmL of LPS in the continued presence of the same concentration of EORP and then IL-1120573 expression was measured incell lysates byWestern blotting 120572-Tubulin was used as the loading control (d)The cells were treated as in panel (c) and then the distributionof IL-1120573 was analyzed by immunofluorescent microscopy IL-1120573 expression is indicated by green fluorescence (FITC) and nuclei by bluefluorescence (DAPI) Bar = 100120583m In panels (a) (b) and (c) the data are expressed as a fold value compared to the control value and are themeans plusmn SEMs (119899 = 3) lowast119875 lt 005 as compared to the untreated (control) cells dagger119875 lt 005 as compared to the LPS-treated cells

group strong IL-1120573 staining was still seen on the vascularwalls at the chronic inflammatory phase as shown in Figure 5The administration of EORP resulted in weak IL-1120573 stainingin the LPS-treated mice ELISA (Figure 5(b)) showed higherlevels of IL-1120573 expression in the LPS-treated group and

EORP treatment notably reduced IL-1120573 expression Theconcentration of IL-1120573 in the chronic inflammatory phasewas much higher than that in the acute inflammatory phaseConsistent with the in situ findings and ELISA Western blot(Figure 5(c)) showed that IL-1120573 was significantly expressed


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Figure 2 EORP-mediated reduction in LPS-induced IL-1120573 expression is partly dependent on inhibition of phosphorylation of ERK (a)ndash(c)Western blot analysis showing the effect of EORP pretreatment on the phosphorylation of (a) ERK12 (b) p38 (c) JNK or (d) Akt in LPS-treated HASMCs HASMCs were incubated for 24 h with or without 10 120583gmL of EORP and then the cells were incubated with 10 120583gmL ofLPS for 30min and aliquots of cell lysate containing equal amounts of protein subjected to immunoblotting with the indicated antibodies(e) Effect of inhibitors of MAPK phosphorylation on IL-1120573 expression in control and LPS-treated HASMCs HASMCs were incubated for 1 hwith 30 120583M PD98059 (an ERK12 inhibitor) SB203580 (a p38 inhibitor) or SP600125 (a JNK inhibitor) and then for 24 h with or without10120583gmL of LPS in the continued presence of the inhibitor and then IL-1120573 expression was measured by Western blotting (f) ERK-specificsiRNA caused a 50 reduction in ERK protein expression byWestern blotting (g)The LPS-induced increase in IL-1120573 expression is inhibitedby transfection of HASMCs with ERK12-specific siRNA (1 120583M) HASMCs were transfected with either control siRNA or ERK12-specificsiRNA (1 120583M) for 48 h then were incubated with 10120583gmL of EORP for 24 h and then with 10120583gmL of LPS for 24 h in the continued presenceof the same concentration of EORP and IL-1120573 expression was measured in cell lysates by Western blotting (h) ERK gene expression underdifferent treatments was analyzed by the RT-PCR assay as described under Section 2 In panels (a)ndash(e) and (g) the data are expressed as afold of the control value and are the means plusmn SEMs (119899 = 3) Total ERK and GAPDH total p38 and GAPDH total JNK and GAPDH totalAkt and GAPDH 120573-actin and 120572-tubulin were used as the loading control for panels (a) (b) (c) (d) (e) (f) and (g) respectively lowast119875 lt 005as compared to the untreated (control) cells dagger119875 lt 005 as compared to the LPS-treated cells

in the LPS-treated group and EORP treatment reduced theexpression

35 EORP Regulating LPS-Induced IL-1120573 Expression WasMediated through the TLR4 Receptor The effects of LPS orEORP on the cytokine expression via TLR4-modulated pro-tein kinase signaling pathways were studied by immunofluo-rescent microscopy In untreated cells TLR4 expression wasweak (Figure 6(a)) whereas cells treated for 24 h with LPSshowed strong TLR4 expression and this effect was reducedby pretreatment with EORP To elucidate whether EORPsmodulate LPS-induced IL-1120573 expression through the TLR4receptor TLR4minusminus mice were used in the present study Asshown in Figures 6(b)ndash6(d) the level of IL-1120573 expressionin LPS-treated or in LPSEORP-treated group was very lowand was similar to that of the saline-treated group examinedby immunofluorescent staining ELISA and Western blotrespectively The expression of TLR4 on TLR4minusminus mice wasconfirmed byWestern blot (Figure 6(e)) Based on these find-ings the LPS-induced IL-1120573 expression was mainly mediatedthrough the activation of TLR4 receptor

4 Discussion

Herein we demonstrated that EORP treatment effectivelyblocked IL-1120573 expression both in vitro in LPS-stimulatedHASMCs and in vivo in thoracic aortas of LPS-treated miceEORP decreased IL-1120573 expression in LPS-treated HASMCsand the effect might be mediated through inhibition ofERK phosphorylation NF-120581B activation and TLR4 receptorpathway

Reishi extract was chosen for testing as it has longbeen known as a healthy food and used as traditionalChinese medicines Its beneficial effects are thought to bedue to its anti-inflammatory antitumor antioxidant andimmunomodulatory actions [16ndash18] A Ganoderma extractprevented albumin-induced oxidative damage of proximaltubular epithelial cells in an experimental setting mimickingthe proteinuric state [24] and reduced LPS-induced super-oxide anion production by macrophages [29] The triterpeneextract from G lucidum (GLT) suppressed the inflammatoryresponse in vitro and in vivo by downregulating the expres-sion of inducible nitric oxide synthase (iNOS) and cyclooxy-genase 2 (COX-2) and the production of TNF-120572 and IL-6


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dagger

(c)Figure 3 EORP-induced reduction in the upregulation of IL-1120573 expression in LPS-treated HASMCs is mediated by inhibition of both NF-120581B p65 phosphorylation and nuclear translocation (a b) Western blotting and immunofluorescent staining for NF-120581B p65 HASMCs wereleft untreated or incubated for 24 h with or without 10120583gmL EORP and then with or without 10120583gmL of LPS for 90min in the continuedpresence of the EORP (a) The phosphorylation of NF-120581B p65 expression is examined by Western blotting (b) NF-120581B p65 expression isindicated by green fluorescence (FITC) and nuclei by blue fluorescence (DAPI) A representative result from three separate experimentsis shown Bar = 100120583m (c) Cells were incubated for 1 h with 30120583M parthenolide (NF-120581B inhibitor) and then coincubated for 24 h with10120583gmL of LPS and then cell lysates were prepared and assayed for IL-1120573 onWestern blotsThe data are the mean plusmn SEM (119899 = 3) lowast119875 lt 005as compared to the untreated (control) cells dagger119875 lt 005 as compared to the LPS-treated cells

in LPS-induced endotoxemic mice [22]Ganoderma lucidumextracts inhibited the production ofmicroglia-derived proin-flammatory and cytotoxic factors including nitric oxideTNF-120572 and IL-1120573 in activated microglia [23] G lucidum

polysaccharide-linked peptide reduced the production ofproinflammatory cytokines (interleukin-6 and monocytechemoattractant protein-1) by activated rheumatoid syn-ovial fibroblasts [30] Our previous report demonstrated


IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)

Figure 4 EORPs reduce IL-1120573 expression in LPS-treated mice at the acute inflammatory phase (2 days) (a) Immunohistochemical stainingfor CD31 (an endothelial cell marker left panels) IL-1120573 (middle panels) and 120572-actin (a smooth muscle cell marker right panels) antibodiesin serial sections of thoracic aortasThe lumen is uppermost in all sectionsThe arrowhead and the arrow indicate IL-1120573-positive overlappingwith endothelial cells and smoothmuscle cells specific staining respectively Bar = 50 120583m (b) IL-1120573 concentration in the plasma was detectedby ELISA The data are the mean plusmn SEM (119899 = 6) lowast119875 lt 005 as compared to the saline-treated mice (control) dagger119875 lt 005 as compared to theLPS-treated mice (c) Western blot analysis of IL-1120573 expression in thoracic aortas 120573-actin was used as the loading control

an EORP-associated protective mechanism against bacteriainfection involving the clearance of LPS by macrophages[31] We also demonstrated that EORP attenuates endotoxin-induced ICAM-1 expression in cultured smooth muscle cellsand in the neointima in mice [27] Moreover EORP pre-vented PDGF-stimulated smooth muscle cell proliferation invitro and neointimal hyperplasia in the endothelial-denudedartery in vivo [32] The present study is the first to reportthat EORP strongly reduces the expression of IL-1120573 proteinin LPS-treated HASMCs

The activation of various intracellular pathways byinflammatory stimuli such as LPS is required for the pro-duction of these adhesion molecules and proinflammatorychemokines [28] LPS-induced inflammatory responses suchas ICAM-1 expression in HASMCs are regulated via TLR4expression [33] Most notably the TLR4-mediated signal-ing pathway for LPS leading to the activation of variousintracellular kinases including MAPKs and transcriptionfactors appears to be critical for the development of vascularinflammation and diseases [26] Our study showed that

LPS caused strong activation of three MAPK subtypes inHASMCs as reported in a previous study [34] However theinvolvement of their activation in the protective mechanismof EORP remains unclear In the present study the increase inIL-1120573 expression induced by LPS was markedly suppressedin the presence of an ERK inhibitor (PD98059) but not ap38 inhibitor (SB203580) or a JNK inhibitor (SP600125)IL-1120573 expression was also inhibited by ERK-specific siRNAEORP decreased LPS-induced ERK phosphorylation Thusone of themechanisms by which EORP reduces LPS-inducedIL-1120573 expression involves a reduction in ERK12 activationConsistent with our results a G lucidum extract inhibitedthe oxidative stress-induced phosphorylation of ERK12 inbreast cancer cells resulting in suppression of IL-8 secretionand finally in inhibition of cell migration [35] Another studyshowed that a G lucidum extract inhibited prostate cancer-dependent angiogenesis by inhibition of phosphorylationof ERK12 and Akt kinases [36] EORP has been shownto inhibit LPS-induced inflammatory cytokine in murineRAW2647 cells by suppression of the phosphorylation of


IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

700

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)

Figure 5 EORPs reduce IL-1120573 expression in LPS-treated mice at the chronic inflammatory phase (2 weeks) (a) Immunohistochemicalstaining for CD31 (left panels) IL-1120573 (middle panels) and 120572-actin (right panels) antibodies in serial sections on thoracic aortasThe lumen isuppermost in all sections The arrowhead and the arrow indicate IL-1120573-positive cells overlapping with endothelial cells and smooth musclecells specific staining respectively Bar = 50 120583m (b) IL-1120573 concentration in the plasma was detected by ELISAThe data are the mean plusmn SEM(119899 = 6) lowast119875 lt 005 as compared to the saline-treated mice (control) dagger119875 lt 005 as compared to the LPS-treated mice (c) Western blot analysisof IL-1120573 protein level in thoracic aortasThe expression ratio (IL-1120573120573-actin) was decreased in the EORP (LPSEORP) group when comparedto the LPS-treated group 120573-actin was used as the loading control

ERK12 and JNK [22] Active lipids of Ganoderma lucidumspores are able to enhance apoptosis in THP-1 cells throughinhibition of ERK12 and Akt and activation of JNK12signaling pathways [32] whereas triterpenes from Gano-derma lucidum induce autophagy in colon cancer through theinhibition of P38MAPK [37] Our previous studies showedthat EORP reduces LPS-induced the ICAM-1 expression bythe decrease of ERK12 activation [27] and prevents thePDGF-stimulated HASMCs proliferation through inhibitionof JNK activation [32] The differences between the aboveresults in terms of the pathways involved may be related tothe different cell types used and the cytokines examined

NF-120581B is one of the most ubiquitous transcription factorsand regulates the genes involved in cellular proliferationinflammatory responses and cell adhesion [25] NF-120581B tran-scriptional activity can be modulated by the phosphorylationof MAPKs LPS-induced IL-1120573 TNF-120572 IL-6 COX-2 andiNOS in Raw2647 macrophages via the NF-120581B activation[38] These findings raised the possibility that EORP reducesIL-1120573 expression through a reduction in NF-120581B activity

Our study demonstrated that the EORP-induced decrease inIL-1120573 expression was mediated through inactivation of NF-120581B binding activity Pretreatment with an NF-120581B inhibitoralso suppressed the LPS-induced increase in IL-1120573 expres-sion This is consistent with a previous report that a Glucidum extract inhibited the proliferation of human breastcancer cells by downregulation of NF-120581B signaling [39]Gan-oderma lucidum polysaccharide peptide (GL-PP) reducedthe production of proinflammatory cytokines in activatedrheumatoid synovial fibroblast by inhibiting the NF-120581Btranscription pathway [30] Another study also showed thata Ganoderma lucidum polysaccharides (Gl-PS) preventedpancreatic islets from alloxan-induced damage by inhibitingactivation of NF-120581B [40] NF-120581B is activated by signalspossibly involving phosphorylation of the I120581B subunit and itsdissociation from the inactive cytoplasmic complex followedby translocation of the active p50p65 dimer to the nucleus[41] We demonstrated that the EORP-induced decrease inIL-1120573 expression was mediated through inhibition of NF-120581Bp65 phosphorylation and translocation


LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)

Figure 6 EORP regulating the LPS-induced IL-1120573 expression was mediated through the TLR4 receptor (a) Immunofluorescent staining forTLR4 HASMCs were left untreated or incubated for 24 h with or without 10120583gmL EORP and then incubated with the 10 120583gmL LPS foranother 24 h TLR4 expression was indicated by green fluorescence (FITC) and nuclei by blue fluorescence (DAPI) The untreated HASMCswere used as the control cells Bar = 100120583m (b) Immunohistochemical staining for IL-1120573 expression in thoracic aortas of TLR4minusminus mice Thelumen is uppermost in all sections Bar = 50120583m (c) IL-1120573 concentration in plasma was detected by ELISA The data are the mean plusmn SEM(119899 = 5) (d)Western blot analysis of IL-1120573 in thoracic aortas of TLR4minusminusmiceThe saline-treated TLR4minusminusmice were used as the control groupin (b) (c) and (d) 120573-actin was used as the loading control (e) Western blot analysis of TLR4 expression in thoracic aortas of C57BL6J (con)and TLR4minusminus mice GAPDH was used as the loading control F CAGACCATGATCACACAGGG R TGGAAAGATGGGCCTGTTAG

LPS-induced systemic inflammatory responses increaseneointimal formation after balloon injury and stent implan-tation and inflammatory cytokines are produced by VSMCsin the neointima [42] In the present study EORP was shownto significantly reduce IL-1120573 expression in thoracic aortas inLPS-treatedmiceOn the basis of the probable involvement ofIL-1120573 expression in migration and proliferation of SMCs ourfindings suggest an additional mechanism by which EORPtreatment may be important in preventing the progression ofcardiovascular disorders and inflammation It has been wellknown that the LPS induces the production of proinflamma-tory molecules through TLR4-activated signaling pathwayand IL-1 was the one of proinflammatory molecules [43]Another study also showed that IL-1120573 expression is mediatedby both TLR4 and Nod1 pathways in the cultured HAPIcells stimulated by LPS [44] In addition LPS-induced TLR4

protein expression and mRNA stabilization in HASMCsare mediated by NADPH oxidase-related ROS productionand MAPK signaling pathways [45] Consistent with theprevious studies LPS significantly induced TLR4 expressionin HASMCs Moreover LPS did not affect IL-1120573 expressionin thoracic aortas in TLR4minusminus mice There was no differenceof IL-1120573 expression in all groups of TLR4minusminus mice by usingimmunohistochemistry cytokine ELISA and Western blotBased on these findings we suggested that EORP suppressedthe LPS-induced IL-1 expression through inhibition of TLR4activation

In conclusion this study provides the first evidence thatEORP reduces IL-1120573 expression in LPS-treated HAMSCsboth in vitro and in vivo The present data suggest that theseeffects might be mediated through inhibition of ERK phos-phorylation and NF-120581B activation The results of the present


study suggest a possible therapeutic role for G lucidumextract in cardiovascular disorders and in inflammatorydiseases

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Chan-Jung Liang Chi-Yuan Li Shu-Huei Wang and Yuh-Lien Chen conceived and designed the experiments Chan-Jung Liang Chiang-Wen Lee Hsin-Ching Sung and Yung-Hsiang Chen performed the experiments Hsin-Ching SungYung-Hsiang Chen Yao-Chang Chiang Hsien-Yeh Hsu andYing-Chin Tseng contributed reagentsmaterialsanalysistools Chi-Yuan Li Shu-Huei Wang and Yuh-Lien Chenwrote the paper

Acknowledgments

This work was supported by research Grants from theNational Science Council (NSC 99-2320-B-002-022-MY3)and the Cooperative Research Program of the NTU andCMUCM (100F008-404) Taiwan

References

[1] R Ross ldquoThe pathogenesis of atherosclerosis a perspective forthe 1990srdquo Nature vol 362 no 6423 pp 801ndash809 1993

[2] F Merhi-Soussi B R Kwak D Magne et al ldquoInterleukin-1plays a major role in vascular inflammation and atherosclero-sis in male apolipoprotein E-knockout micerdquo CardiovascularResearch vol 66 no 3 pp 583ndash593 2005

[3] C A Dinarello ldquoBiologic basis for interleukin-1 in diseaserdquoBlood vol 87 no 6 pp 2095ndash2147 1996

[4] X Wang A M Romanic T-L Yue G Z Feuerstein and E HOhlstein ldquoExpression of interleukin-1120573 interleukin-1 receptorand interleukin-1 receptor antagonist mRNA in rat carotidartery after balloon angioplastyrdquo Biochemical and BiophysicalResearch Communications vol 271 no 1 pp 138ndash143 2000

[5] X Wang G Z Feuerstein J-L Gu P G Lysko and T-L YueldquoInterleukin-1120573 induces expression of adhesion molecules inhuman vascular smooth muscle cells and enhances adhesionof leukocytes to smooth muscle cellsrdquo Atherosclerosis vol 115no 1 pp 89ndash98 1995

[6] D Wu T Koga K R Martin and M Meydani ldquoEffect ofvitamin E on human aortic endothelial cell production ofchemokines and adhesion to monocytesrdquo Atherosclerosis vol147 no 2 pp 297ndash307 1999

[7] J Nilsson ldquoCytokines and smooth muscle cells in atherosclero-sisrdquo Cardiovascular Research vol 27 no 7 pp 1184ndash1190 1993

[8] Z Wang L Kong J Kang et al ldquoInterleukin-l120573 inducesmigration of rat arterial smooth muscle cells through a mech-anism involving increased matrix metalloproteinase-2 activityrdquoJournal of Surgical Research vol 169 no 2 pp 328ndash336 2011

[9] A M Wang M V Doyle and D F Mark ldquoQuantitation ofmRNA by the polymerase chain reactionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 86 no 24 pp 9717ndash9721 1989

[10] C F Moyer D Sajuthi H Tulli and J K Williams ldquoSynthesisof IL-1 alpha and IL-1 beta by arterial cells in atherosclerosisrdquoAmerican Journal of Pathology vol 138 no 4 pp 951ndash960 1991

[11] A Cignarella ldquoTargeting interleukin-1szlig hampers atherosclero-sis progression - Is there great promiserdquoAtherosclerosis vol 217no 1 pp 64ndash66 2011

[12] M-S Shiao ldquoNatural products of the medicinal fungus Gano-derma lucidum occurrence biological activities and pharma-cological functionsrdquo Chemical Record vol 3 no 3 pp 172ndash1802003

[13] Y-Y Wang K-H Khoo S-T Chen C-C Lin C-H Wongand C-H Lin ldquoStudies on the immuno-modulating and antitu-mor activities of Ganoderma lucidum (Reishi) polysaccharidesfunctional and proteomic analyses of a fucose-containing gly-coprotein fraction responsible for the activitiesrdquo Bioorganic andMedicinal Chemistry vol 10 no 4 pp 1057ndash1062 2002

[14] S Wachtel-Galor Y-T Szeto B Tomlinson and I F F Ben-zie ldquoGanoderma lucidum (ldquoLingzhirdquo) acute and short-termbiomarker response to supplementationrdquo International Journalof Food Sciences and Nutrition vol 55 no 1 pp 75ndash83 2004

[15] S Wachtel-Galor B Tomlinson and I F F Benzie ldquoGano-derma lucidum (ldquoLingzhirdquo) a Chinese medicinal mushroombiomarker responses in a controlled human supplementationstudyrdquo British Journal of Nutrition vol 91 no 2 pp 263ndash2692004

[16] Y Gao H Gao E Chan et al ldquoAntitumor activity and under-lying mechanisms of ganopoly the refined polysaccharidesextracted from Ganoderma lucidum in micerdquo ImmunologicalInvestigations vol 34 no 2 pp 171ndash198 2005

[17] S Joseph B Sabulal V George K R Antony and K KJanardhanan ldquoAntitumor and anti-inflammatory activities ofpolysaccharides isolated fromGanoderma lucidumrdquo Acta Phar-maceutica vol 61 no 3 pp 335ndash342 2011

[18] J Sun H He and J X Bi ldquoNovel antioxidant peptidesfrom fermented mushroom Ganoderma lucidumrdquo Journal ofAgricultural and Food Chemistry vol 52 no 21 pp 6646ndash66522004

[19] H-Y Hsu K-F Hua C-C Lin C-H Lin J Hsu and C-H Wong ldquoExtract of Reishi polysaccharides induces cytokineexpression via TLR4-modulated protein kinase signaling path-waysrdquo Journal of Immunology vol 173 no 10 pp 5989ndash59992004

[20] C-W Lieu S-S Lee and S-YWang ldquoThe effect ofGanodermalucidum on induction of differentiation in leukemic U937 cellsrdquoAnticancer Research vol 12 no 4 pp 1211ndash1215 1992

[21] L-Z Cao and Z-B Lin ldquoRegulation on maturation and func-tion of dendritic cells by Ganoderma lucidum polysaccharidesrdquoImmunology Letters vol 83 no 3 pp 163ndash169 2002

[22] S Dudhgaonkar A Thyagarajan and D Sliva ldquoSuppressionof the inflammatory response by triterpenes isolated from themushroom Ganoderma lucidumrdquo International Immunophar-macology vol 9 no 11 pp 1272ndash1280 2009

[23] P Chan R Zhang S Xu Y Cai M Zhou and X Zuo ldquoGan-oderma lucidum protects dopaminergic neuron degenerationthrough inhibition of microglial activationrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID156810 9 pages 2011

[24] K N Lai L Y Y Chan S CW Tang and J C K Leung ldquoGan-oderma extract prevents albumin-induced oxidative damageand chemokines synthesis in cultured human proximal tubularepithelial cellsrdquo Nephrology Dialysis Transplantation vol 21no 5 pp 1188ndash1197 2006


[25] P J Barnes and M Karin ldquoNuclear factor-120581B a pivotal tran-scription factor in chronic inflammatory diseasesrdquo The NewEngland Journal ofMedicine vol 336 no 15 pp 1066ndash1071 1997

[26] A J Muslin ldquoMAPK signalling in cardiovascular health anddisease molecular mechanisms and therapeutic targetsrdquo Clin-ical Science vol 115 no 7-8 pp 203ndash218 2008

[27] C-Y Lin Y-H Chen C-Y Lin et al ldquoGanoderma lucidumpolysaccharides attenuate endotoxin-induced intercellular celladhesionmolecule-1 expression in cultured smoothmuscle cellsand in the neointima in micerdquo Journal of Agricultural and FoodChemistry vol 58 no 17 pp 9563ndash9571 2010

[28] H P A Jersmann C S T Hii J V Ferrante and A FerranteldquoBacterial lipopolysaccharide and tumor necrosis factor alphasynergistically increase expression of human endothelial adhe-sion molecules through activation of NF-120581B and p38 mitogen-activated protein kinase signaling pathwaysrdquo Infection andImmunity vol 69 no 3 pp 1273ndash1279 2001

[29] C W H Woo R Y K Man Y L Siow et al ldquoGanodermalucidum inhibits inducible nitric oxide synthase expression inmacrophagesrdquoMolecular andCellular Biochemistry vol 275 no1-2 pp 165ndash171 2005

[30] YWHo J S L Yeung P K Y Chiu et al ldquoGanoderma lucidumpolysaccharide peptide reduced the production of proinflam-matory cytokines in activated rheumatoid synovial fibroblastrdquoMolecular and Cellular Biochemistry vol 301 no 1-2 pp 173ndash179 2007

[31] K-F Hua H-Y Hsu L K Chao et al ldquoGanoderma lucidumpolysaccharides enhanceCD14 endocytosis of LPS and promoteTLR4 signal transduction of cytokine expressionrdquo Journal ofCellular Physiology vol 212 no 2 pp 537ndash550 2007

[32] S-H Wang C-J Liang Y-W Weng et al ldquoGanodermalucidum polysaccharides prevent platelet-derived growthfactor-stimulated smooth muscle cell proliferation in vitroand neointimal hyperplasia in the endothelial-denuded arteryin vivordquo Journal of Cellular Physiology vol 227 no 8 pp3063ndash3071 2012

[33] S-K Heo H-J Yun E-K Noh W-H Park and S-D ParkldquoLPS induces inflammatory responses in human aortic vascularsmooth muscle cells via Toll-like receptor 4 expression andnitric oxide productionrdquo Immunology Letters vol 120 no 1-2pp 57ndash64 2008

[34] Y-L Chen C-S Hu F-Y Lin et al ldquoSalvianolic acid B attenu-ates cyclooxygenase-2 expression in vitro in LPS-treated humanaortic smooth muscle cells and in vivo in the apolipoprotein-E-deficient mouse aortardquo Journal of Cellular Biochemistry vol 98no 3 pp 618ndash631 2006

[35] AThyagarajan J Jiang A Hopf J Adamec andD Sliva ldquoInhi-bition of oxidative stress-induced invasiveness of cancer cellsby Ganoderma lucidum is mediated through the suppressionof interleukin-8 secretionrdquo International Journal of MolecularMedicine vol 18 no 4 pp 657ndash664 2006

[36] G Stanley K Harvey V Slivova J Jiang and D Sliva ldquoGano-derma lucidum suppresses angiogenesis through the inhibitionof secretion of VEGF and TGF-1205731 from prostate cancer cellsrdquoBiochemical and Biophysical Research Communications vol 330no 1 pp 46ndash52 2005

[37] AThyagarajan A Jedinak H Nguyen et al ldquoTriterpenes fromGanoderma lucidum induce autophagy in colon cancer throughthe inhibition of p38 mitogen-activated kinase (p38 MAPK)rdquoNutrition and Cancer vol 62 no 5 pp 630ndash640 2010

[38] J-HWon H-T Im Y-H Kim et al ldquoAnti-inflammatory effectof buddlejasaponin IV through the inhibition of iNOS and

COX-2 expression in RAW 2647 macrophages via the NF-120581BinactivationrdquoBritish Journal of Pharmacology vol 148 no 2 pp216ndash225 2006

[39] J Jiang V Slivova and D Sliva ldquoGanoderma lucidum inhibitsproliferation of human breast cancer cells by down-regulationof estrogen receptor andNF-120581B signalingrdquo International Journalof Oncology vol 29 no 3 pp 695ndash703 2006

[40] H-N Zhang J-H He L Yuan and Z-B Lin ldquoIn vitro and invivo protective effect ofGanoderma lucidum polysaccharides onalloxan-induced pancreatic islets damagerdquo Life Sciences vol 73no 18 pp 2307ndash2319 2003

[41] S Ghosh and D Baltimore ldquoActivation in vitro of NF-120581B byphosphorylation of its inhibitor I120581BrdquoNature vol 344 no 6267pp 678ndash682 1990

[42] H D Danenberg F G P Welt M Walker III P SeifertG S Toegel and E R Edelman ldquoSystemic inflammationinduced by lipopolysaccharide increases neointimal formationafter balloon and stent injury in rabbitsrdquo Circulation vol 105no 24 pp 2917ndash2922 2002

[43] X Yang D Coriolan V Murthy K Schultz D T Golen-bock and D Beasley ldquoProinflammatory phenotype of vascularsmooth muscle cells role of efficient Toll-like receptor 4signalingrdquo American Journal of Physiology vol 289 no 3 ppH1069ndashH1076 2005

[44] W Zheng X Zheng S Liu et al ldquoTNF120572 and IL-1120573 aremediated by both TLR4 and Nod1 pathways in the culturedHAPI cells stimulated by LPSrdquo Biochemical and BiophysicalResearch Communications vol 420 no 4 pp 762ndash767 2012

[45] F-Y Lin Y-H Chen Y-W Lin et al ldquoThe role of human anti-gen R an RNA-binding protein in mediating the stabilizationof toll-like receptor 4 mRNA induced by endotoxin a novelmechanism involved in vascular inflammationrdquo Arteriosclero-sis Thrombosis and Vascular Biology vol 26 no 12 pp 2622ndash2629 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


molecules by vascular smooth muscle cells (VSMCs) in vitroand these factors promotemonocyte recruitment and infiltra-tion into the arterial wall [5 6] and induce VSMC migrationand proliferation [3 7 8] In vivo studies have shownincreased levels of IL-1120573 mRNA in human atheroscleroticlesions [9] and of IL-1120573 mRNA and protein in VSMCsof atherosclerotic arteries from non-human primates [10]Although the relationship between IL-1120573 and cardiovasculardisease has been extensively studied IL-1120573 expression andits related mechanism in LPS-induced human aortic smoothmuscle cells (HASMCs) and in inflammatory vascular wallshave not been studied in detail Moreover reducing IL-1120573expression might be a beneficial approach for inhibiting thedevelopment of vascular inflammation and atherosclerosis[11]

Ganoderma lucidum (G lucidum Reishi) a popular homeremedy has long been known for its beneficial effects onhuman health and longevity and is used to treat chronichepatopathy hypertension hyperglycemia and neoplasia[12 13] Ganoderma lucidum intake caused an increase inplasma antioxidant capacity and that 10 d supplementa-tion was associated with a trend towards a decrease ofplasma lipids in human subjects [14 15] Studies on the roleof G lucidum in regulating various body functions haverevealed that G lucidum polysaccharides are the bioactiveconstituents responsible for many of their health bene-fits such as its antioxidant anticancer anti-inflammatoryand immunomodulatory activities [16ndash18] The effects ofG lucidum on the immune system have been linked tothe regulation of cytokine expression and differentiationof macrophages [19 20] and the maturation of culturedmurine bone marrow-derived dendritic cells [21]G lucidumhave also been shown to suppress TNF-120572 expression fromlipopolysaccharide- (LPS-) stimulated murine RAW 2647cells [22] reduce IL-1120573 secretion from activated microgliaafter LPS treatment [23] and prevent albumin-inducedoxidative damage and chemokines synthesis in human prox-imal tubular epithelial cells [24] Regulation of inflammatorycytokine expression requires a complex array of intracel-lular signaling pathways involving mitogen-activated pro-tein kinases (MAPKs) and transcriptional factors [25 26]Although these multiple signaling molecules have receivedconsiderable attention little is known about the effectsof an extract of Reishi polysaccharides (EORPs) on IL-1120573expression and the mechanisms of these effects and a betterunderstanding of this might provide important insights intothe prevention of cardiovascular diseases and inflammationWe were therefore interested in understanding the mecha-nism of action of EORP on human aortic smooth musclecells (HASMCs) stimulated by inflammatory cytokines andwhether it affects the IL-1120573 expression an important event invascular diseases and inflammation In addition we studiedthe effects of EORP on IL-1120573 expression in thoracic aortas inLPS-treated mice Our results showed that EORP attenuatedIL-1120573 expression both in vitro and in vivo and that thiseffect was mediated at least in part through inhibition ofextracellular signal-regulated kinase (ERK) phosphorylationnuclear factor NF-120581B activation and TLR4 activation

2 Materials and Methods

21 Preparation of EORP A crude powder ofG lucidum pre-pared via alkaline extraction with 01 NNaOH followed byneutralization and ethanol precipitation was obtained fromPharmanex (Provo UT) The biologically active compoundsfrom Reishi are identified as the fucose-containing glyco-protein fraction and named EORPs The molecular structureand the quality control of EORPs were described in detailin the previous reports [13 19] In brief the carbohydratecomposition analyses of crude water soluble Reishi extractindicated that glucose mannose and fucose exist as themajor components The crude extract contains 156 pro-teins The saccharides contain either a polysaccharide back-bone with 120573-13-linkages or a polymannose backbone with120572-14-linkages both with side chains of unknown structureEORPs were prepared as GMP grade and carefullymonitoredto meet the Food and Drug Administration standard TheEORP is currently commercially available as the traditionalChinese medicine

22 HASMC Cultures HASMCs purchased as cryopre-served tertiary cultures from Cascade Biologics (OR USA)were grown in culture flasks in smooth muscle cell growthmedium (M231 Cascade Biologics Inc) The cells were usedbetween passages 4 and 8 All cells were synchronized inserum-free medium for 24 h prior to experiments For alldata shown the experiments were repeated at least 3 times induplicate with different preparations of HASMCs For West-ern blot analysis the graphical analysis represents the resultsfrom three independent experiments and quantification bydensitometry

23 Effect of LPS and EORP on Cell Viability The cellviability was determined by 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich StLouis MO USA) assay HASMCs were plated in 96-wellplates (1 times 104 cellswell) After serum starvation the cellswere treated with different concentrations of LPS or EORPfor 24 h and then MTT solution was added to each well andincubated for another 2 h at 37∘C in 5CO

2

The supernatantwas discarded and dimethyl sulfoxide (DMSO) was addedto dissolve the formazan crystals The cellular viability wasmeasured by ELISA plate reader at 540 nm

24 Western Blot Analysis Western blot analyses were per-formed as described previously [27] In brief samples ofcell lysate (20120583g of protein) were subjected to 12 SDS-PAGE and transferred to PVDF membranes which werethen treated with 5 nonfat milk in 01M phosphate bufferfor 1 h at RT to block nonspecific binding of antibody Tomeasure IL-1120573 levels the membranes were then incubatedwith rabbit anti-human IL-1120573 (1 1000 dilution GeneTexIrvine CA USA) and then with horseradish peroxidase-(HRP-) conjugated goat anti-rabbit IgG antibodies (1 6000dilution KPL Gaithersburg MA USA) and bound antibodywas detected using Chemiluminescence Reagent Plus Inother studies the antibodies used were rabbit anti-humanphospho-Jun N-treminal kinase (JNK) rabbit anti-human







2






3 Result








IL-1120573

120572-Tubulin

Fold

of c

ontro

l

LPS (h)

25

20

15

10

05

000 6 8 12 24

lowast

(a)

IL-1120573

120572-Tubulin

Fold

of c

ontro

l

25

15

05

000 5 10 15

LPS (120583gmL)

lowast

lowast

20

10

(b)

IL-1120573

120572-Tubulin

Fold

of c

ontro

l

15

05

00

LPSEORP

minus

minus minus

minus+ +

+ +

lowast

dagger

20

10

(c)

Con LPS

LPSEORP EORP

(d)





Fold

of c

ontro

l

25

20

15

10

05

00

lowast

dagger

P-ERK

T-ERK

GAPDH

LPSEORP

minus

minus minus

minus+ +

+ +

(a)

Fold

of c

ontro

l

25

20

15

10

05

00

lowast

dagger

GAPDH

P-P38

T-P38

30

(b)

Fold

of c

ontro

l

20

15

10

05

00

lowast

dagger

GAPDH

P-JNK

T-JNK

(c)

Fold

of c

ontro

l

20

15

10

05

00

lowast

dagger

GAPDH

P-Akt

T-Akt

LPSEORP

minus

minus minus

minus+ +

+ +

(d)

IL-1120573

Fold

of c

ontro

l

LPS

lowast

dagger

Inhibitor

120573-Actin

3

2

1

0

PD SB SPminus

minus minus

+ + + +

(e)

120572-Tubulin

Con

T-ERK

siERK

(f)

Figure 2 Continued


IL-1120573

siERK

120572-Tubulin

lowast

dagger

dagger

daggerDagger

15

10

05

00

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+ +

+

+

+

Fold

of c

ontro

l

(g)

ERK

18S

siERK

LPSEORP

minus

minus

minus

minus

minusminus

minus

minus

minus minus minus

+ +

+ +

+ +

+

+

+

+

(h)




4 Discussion




Fold

of c

ontro

l

15

05

00

20

10

LPS

EORP

minus

minus minus

minus+ +

+ +

p-p65

t-p65

lowast

dagger

(a)

EORP

Con

LPS

LPSEORP

P65 DAPI Merge

100120583m

(b)

Fold

of c

ontro

l

15

05

00

20

10

IL-1120573

120572-Tubulin

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+

+

Parthenolide

lowast

dagger

dagger





IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

700

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2






3 Result








IL-1120573

120572-Tubulin

Fold

of c

ontro

l

LPS (h)

25

20

15

10

05

000 6 8 12 24

lowast

(a)

IL-1120573

120572-Tubulin

Fold

of c

ontro

l

25

15

05

000 5 10 15

LPS (120583gmL)

lowast

lowast

20

10

(b)

IL-1120573

120572-Tubulin

Fold

of c

ontro

l

15

05

00

LPSEORP

minus

minus minus

minus+ +

+ +

lowast

dagger

20

10

(c)

Con LPS

LPSEORP EORP

(d)





Fold

of c

ontro

l

25

20

15

10

05

00

lowast

dagger

P-ERK

T-ERK

GAPDH

LPSEORP

minus

minus minus

minus+ +

+ +

(a)

Fold

of c

ontro

l

25

20

15

10

05

00

lowast

dagger

GAPDH

P-P38

T-P38

30

(b)

Fold

of c

ontro

l

20

15

10

05

00

lowast

dagger

GAPDH

P-JNK

T-JNK

(c)

Fold

of c

ontro

l

20

15

10

05

00

lowast

dagger

GAPDH

P-Akt

T-Akt

LPSEORP

minus

minus minus

minus+ +

+ +

(d)

IL-1120573

Fold

of c

ontro

l

LPS

lowast

dagger

Inhibitor

120573-Actin

3

2

1

0

PD SB SPminus

minus minus

+ + + +

(e)

120572-Tubulin

Con

T-ERK

siERK

(f)

Figure 2 Continued


IL-1120573

siERK

120572-Tubulin

lowast

dagger

dagger

daggerDagger

15

10

05

00

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+ +

+

+

+

Fold

of c

ontro

l

(g)

ERK

18S

siERK

LPSEORP

minus

minus

minus

minus

minusminus

minus

minus

minus minus minus

+ +

+ +

+ +

+

+

+

+

(h)




4 Discussion




Fold

of c

ontro

l

15

05

00

20

10

LPS

EORP

minus

minus minus

minus+ +

+ +

p-p65

t-p65

lowast

dagger

(a)

EORP

Con

LPS

LPSEORP

P65 DAPI Merge

100120583m

(b)

Fold

of c

ontro

l

15

05

00

20

10

IL-1120573

120572-Tubulin

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+

+

Parthenolide

lowast

dagger

dagger





IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

700

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 Result








IL-1120573

120572-Tubulin

Fold

of c

ontro

l

LPS (h)

25

20

15

10

05

000 6 8 12 24

lowast

(a)

IL-1120573

120572-Tubulin

Fold

of c

ontro

l

25

15

05

000 5 10 15

LPS (120583gmL)

lowast

lowast

20

10

(b)

IL-1120573

120572-Tubulin

Fold

of c

ontro

l

15

05

00

LPSEORP

minus

minus minus

minus+ +

+ +

lowast

dagger

20

10

(c)

Con LPS

LPSEORP EORP

(d)





Fold

of c

ontro

l

25

20

15

10

05

00

lowast

dagger

P-ERK

T-ERK

GAPDH

LPSEORP

minus

minus minus

minus+ +

+ +

(a)

Fold

of c

ontro

l

25

20

15

10

05

00

lowast

dagger

GAPDH

P-P38

T-P38

30

(b)

Fold

of c

ontro

l

20

15

10

05

00

lowast

dagger

GAPDH

P-JNK

T-JNK

(c)

Fold

of c

ontro

l

20

15

10

05

00

lowast

dagger

GAPDH

P-Akt

T-Akt

LPSEORP

minus

minus minus

minus+ +

+ +

(d)

IL-1120573

Fold

of c

ontro

l

LPS

lowast

dagger

Inhibitor

120573-Actin

3

2

1

0

PD SB SPminus

minus minus

+ + + +

(e)

120572-Tubulin

Con

T-ERK

siERK

(f)

Figure 2 Continued


IL-1120573

siERK

120572-Tubulin

lowast

dagger

dagger

daggerDagger

15

10

05

00

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+ +

+

+

+

Fold

of c

ontro

l

(g)

ERK

18S

siERK

LPSEORP

minus

minus

minus

minus

minusminus

minus

minus

minus minus minus

+ +

+ +

+ +

+

+

+

+

(h)




4 Discussion




Fold

of c

ontro

l

15

05

00

20

10

LPS

EORP

minus

minus minus

minus+ +

+ +

p-p65

t-p65

lowast

dagger

(a)

EORP

Con

LPS

LPSEORP

P65 DAPI Merge

100120583m

(b)

Fold

of c

ontro

l

15

05

00

20

10

IL-1120573

120572-Tubulin

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+

+

Parthenolide

lowast

dagger

dagger





IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

700

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-1120573

120572-Tubulin

Fold

of c

ontro

l

LPS (h)

25

20

15

10

05

000 6 8 12 24

lowast

(a)

IL-1120573

120572-Tubulin

Fold

of c

ontro

l

25

15

05

000 5 10 15

LPS (120583gmL)

lowast

lowast

20

10

(b)

IL-1120573

120572-Tubulin

Fold

of c

ontro

l

15

05

00

LPSEORP

minus

minus minus

minus+ +

+ +

lowast

dagger

20

10

(c)

Con LPS

LPSEORP EORP

(d)





Fold

of c

ontro

l

25

20

15

10

05

00

lowast

dagger

P-ERK

T-ERK

GAPDH

LPSEORP

minus

minus minus

minus+ +

+ +

(a)

Fold

of c

ontro

l

25

20

15

10

05

00

lowast

dagger

GAPDH

P-P38

T-P38

30

(b)

Fold

of c

ontro

l

20

15

10

05

00

lowast

dagger

GAPDH

P-JNK

T-JNK

(c)

Fold

of c

ontro

l

20

15

10

05

00

lowast

dagger

GAPDH

P-Akt

T-Akt

LPSEORP

minus

minus minus

minus+ +

+ +

(d)

IL-1120573

Fold

of c

ontro

l

LPS

lowast

dagger

Inhibitor

120573-Actin

3

2

1

0

PD SB SPminus

minus minus

+ + + +

(e)

120572-Tubulin

Con

T-ERK

siERK

(f)

Figure 2 Continued


IL-1120573

siERK

120572-Tubulin

lowast

dagger

dagger

daggerDagger

15

10

05

00

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+ +

+

+

+

Fold

of c

ontro

l

(g)

ERK

18S

siERK

LPSEORP

minus

minus

minus

minus

minusminus

minus

minus

minus minus minus

+ +

+ +

+ +

+

+

+

+

(h)




4 Discussion




Fold

of c

ontro

l

15

05

00

20

10

LPS

EORP

minus

minus minus

minus+ +

+ +

p-p65

t-p65

lowast

dagger

(a)

EORP

Con

LPS

LPSEORP

P65 DAPI Merge

100120583m

(b)

Fold

of c

ontro

l

15

05

00

20

10

IL-1120573

120572-Tubulin

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+

+

Parthenolide

lowast

dagger

dagger





IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

700

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Fold

of c

ontro

l

25

20

15

10

05

00

lowast

dagger

P-ERK

T-ERK

GAPDH

LPSEORP

minus

minus minus

minus+ +

+ +

(a)

Fold

of c

ontro

l

25

20

15

10

05

00

lowast

dagger

GAPDH

P-P38

T-P38

30

(b)

Fold

of c

ontro

l

20

15

10

05

00

lowast

dagger

GAPDH

P-JNK

T-JNK

(c)

Fold

of c

ontro

l

20

15

10

05

00

lowast

dagger

GAPDH

P-Akt

T-Akt

LPSEORP

minus

minus minus

minus+ +

+ +

(d)

IL-1120573

Fold

of c

ontro

l

LPS

lowast

dagger

Inhibitor

120573-Actin

3

2

1

0

PD SB SPminus

minus minus

+ + + +

(e)

120572-Tubulin

Con

T-ERK

siERK

(f)

Figure 2 Continued


IL-1120573

siERK

120572-Tubulin

lowast

dagger

dagger

daggerDagger

15

10

05

00

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+ +

+

+

+

Fold

of c

ontro

l

(g)

ERK

18S

siERK

LPSEORP

minus

minus

minus

minus

minusminus

minus

minus

minus minus minus

+ +

+ +

+ +

+

+

+

+

(h)




4 Discussion




Fold

of c

ontro

l

15

05

00

20

10

LPS

EORP

minus

minus minus

minus+ +

+ +

p-p65

t-p65

lowast

dagger

(a)

EORP

Con

LPS

LPSEORP

P65 DAPI Merge

100120583m

(b)

Fold

of c

ontro

l

15

05

00

20

10

IL-1120573

120572-Tubulin

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+

+

Parthenolide

lowast

dagger

dagger





IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

700

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-1120573

siERK

120572-Tubulin

lowast

dagger

dagger

daggerDagger

15

10

05

00

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+ +

+

+

+

Fold

of c

ontro

l

(g)

ERK

18S

siERK

LPSEORP

minus

minus

minus

minus

minusminus

minus

minus

minus minus minus

+ +

+ +

+ +

+

+

+

+

(h)




4 Discussion




Fold

of c

ontro

l

15

05

00

20

10

LPS

EORP

minus

minus minus

minus+ +

+ +

p-p65

t-p65

lowast

dagger

(a)

EORP

Con

LPS

LPSEORP

P65 DAPI Merge

100120583m

(b)

Fold

of c

ontro

l

15

05

00

20

10

IL-1120573

120572-Tubulin

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+

+

Parthenolide

lowast

dagger

dagger





IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

700

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Fold

of c

ontro

l

15

05

00

20

10

LPS

EORP

minus

minus minus

minus+ +

+ +

p-p65

t-p65

lowast

dagger

(a)

EORP

Con

LPS

LPSEORP

P65 DAPI Merge

100120583m

(b)

Fold

of c

ontro

l

15

05

00

20

10

IL-1120573

120572-Tubulin

LPSEORP

minus

minus

minus

minus

minus minus

minus

+ +

+

+

+

Parthenolide

lowast

dagger

dagger





IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

700

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

700

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-1120573CD31

Saline

EORP

LPS

LPSEORP

50120583m

120572-Actin

(a)

Saline

lowast

600

700

500

400

300

200

100

0

IL-1120573

(pg

mL)

EORPLPS LPSEORP

dagger

(b)

120573-Actin

IL-1120573

Saline LPS LPSEORP

(c)






LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LPS LPSEORPCon

TLR4

100120583m

(a)

IL-1120573

Saline LPS LPSEORP

50120583m

(b)

Saline

IL-1120573

(pg

mL)

LPS LPSEORP

120

100

80

60

40

20

0

(c)

Saline LPS LPSEORP

120573-Actin

IL-1120573

(d)

TLR4

GAPDH

Con TLR4minusminus

(e)











Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Ganoderma lucidum Polysaccharides Reduce Lipopolysaccharide...

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