research article ikk -targeted anti-inflammatory activities of a

Research ArticleIKK120573-Targeted Anti-InflammatoryActivities of a Butanol Fraction of Artificially CultivatedCordyceps pruinosa Fruit Bodies

Han Gyung Kim1 Woo Seok Yang1 Gi-Ho Sung2 Ji Hye Kim1 Gwang-Soo Baek1

Eunji Kim1 Sungjae Yang1 Yung Chul Park3 Jae Mo Sung4

Deok Hyo Yoon5 Tae Woong Kim5 Sungyoul Hong1 Jong-Hoon Kim6 and Jae Youl Cho1

1 Department of Genetic Engineering Sungkyunkwan University Suwon 440-746 Republic of Korea2Mushroom Research Division National Institute of Horticultural and Herbal Science Rural Development AdministrationEumseong 369-873 Republic of Korea

3 Department of Forest Environment Protection Kangwon National University Chuncheon 200-701 Republic of Korea4 Institute of Mushroom Mushtech Chuncheon 200-180 Republic of Korea5 Department of Biochemistry Kangwon National University Chuncheon 200-701 Republic of Korea6Department of Veterinary Physiology College of Veterinary Medicine Biosafety Research InstituteChonbuk National University Jeonju 561-756 Republic of Korea

Correspondence should be addressed to Jong-Hoon Kim jhkim1jbnuackr and Jae Youl Cho jaechoskkuedu

Received 25 April 2014 Accepted 30 June 2014 Published 15 July 2014

Academic Editor Youn Chul Kim

Copyright copy 2014 Han Gyung Kim 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 inhibitory activities of the Cordyceps pruinosa butanol fraction (Cp-BF) were investigated by determining inflammatoryresponses of lipopolysaccharide (LPS)-treated RAW2647 macrophage cells and by evaluating HClethanol (EtOH)-triggeredgastric ulcers inmiceThemolecular mechanisms of the inhibitory effects of Cp-BF were investigated by identifying target enzymesusing biochemical and molecular biological approaches Cp-BF strongly inhibited the production of NO and TNF-120572 release ofreactive oxygen species (ROS) phagocytic uptake of FITC-dextran andmRNAexpression levels of interleukin (IL)-6 inducibleNOsynthase (iNOS) and tumour necrosis factor-alpha (TNF)-120572 in activated RAW2647 cells Cp-BF also strongly downregulated theNF-120581Bpathway by suppressing IKK120573 according to luciferase reporter assays and immunoblot analysis Furthermore Cp-BF blockedboth increased levels of NF-120581B-mediated luciferase activities and phosphorylation of p65p50 observed by IKK120573 overexpressionFinally orally administered Cp-BF was found to attenuate gastric ulcer and block the phosphorylation of I120581B120572 induced byHClEtOH Therefore these results suggest that the anti-inflammatory activity of Cp-BF may be mediated by suppression ofIKK120572 and its downstream NF-120581B activation Since our group has established the mass cultivation conditions by developing cultureconditions for Cordyceps pruinosa the information presented in this study may be useful for developing new anti-inflammatoryagents

1 Introduction

Inflammation is a complicated defensive response againstvarious infecting pathogens and toxins and serves as one ofthe bodyrsquos innate immunity barriers As a major inflamma-tory cell population macrophages respond to inflammatoryevents by phagocytosing infected materials and producing

cytokines such as interleukin (IL)-1 and tumour necrosisfactor (TNF)-120572 as well as inflammatory mediators such asnitric oxide (NO) and prostaglandin E

2(PGE2) For these

responses macrophages need to activate cell surface recep-tors (eg toll-like receptors) by interacting with their ligandssuch as lipopolysaccharide (LPS) from Gram (minus) bacteriaExtracellular signals received by plasmamembrane receptors

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 562467 12 pageshttpdxdoiorg1011552014562467

2 Evidence-Based Complementary and Alternative Medicine

will then generate intracellular signalling events throughnonreceptor type protein tyrosine kinases including Syk Srcphosphoinositide-3-kinase (PI3K) and mitogen activatedprotein kinase (MAPK) upstream of extracellular signal-related kinase (ERK) p38 and c-Jun N-terminal kinase(JNK) Ultimately all of these intracellular responses reachactivation of transcription factors including CREB nuclearfactor-120581B (NF-120581B) and activator protein-1 (AP-1) [1 2] Theoutcomes of such activation are linked to the expression ofvarious proinflammatory genes and inflammatory mediators[3ndash5] In addition recent findings have raised the possibilitythat sustained inflammatory responses can cause seriousdiseases such as septic shock cancer diabetes gastritis andatherosclerosis[5ndash7]Therefore development of stronger andsafer anti-inflammatory remedies may contribute to morepromising strategies to effectively treat inflammation-deriveddiseases

The genus Cordyceps which includes Cordyceps sinen-sis Cordyceps militaris Cordyceps pruinosa and Cordycepsbassiana comprises ethnopharmacologically valuable mush-rooms in Korea China and Japan [8] The genus Cordycepshas been traditionally used for the treatment of variousinflammatory and infectious diseases such as eczema skindiseases chronic bronchitis asthma and tuberculosis orused as a tonic for longevity endurance and vitality [8 9]ThemushroomCordycepswas also described in ancient tradi-tional Chinese medicine literatures as a medicinal ingredientprescribed for relieving chronic bronchitis chronic obstruc-tive pulmonary disease and tuberculosis [10 11] Additionalstudies have found that these mushrooms have numerousbiological activities based on antioxidative anticancer antidi-abetes antibacterial antifungal and antifatigue properties[12 13] Fromphytochemical approaches it has been reportedthat cordycepin ergosterol peroxide and polysaccharides arethe major components responsible for the various pharma-cological activities of Cordyceps [14] However in spite oftheir excellent pharmaceutical and nutraceutical potential itis difficult to mass produce these mushrooms To overcomethe limitations of mass production of Cordyceps species ourgroup has developed an artificial culturing condition witha grain rice-enriched medium that is able to achieve massproduction of fruit bodies of Cordyceps militaris Cordycepspruinosa and Cordyceps bassiana under defined cultureconditions

Among the mushrooms that we demonstrated to havegrown in a mass production setting we focused especiallyon Cordyceps pruinosa (Figure 1(a)) as only a few papershave reported its pharmacological activities towards cancerand inflammation [15 16] and the cultivation rate of its fruitbody is much higher than other species Although the anti-inflammatory activity of the methanol extract of Cordycepspruinosa has been suggested previously by determination ofIL-1120573 TNF-120572 NO and PGE

2levels [15] the exact molec-

ular mechanism for these effects has not been fully elu-cidated Therefore in this studywe aimed to explore anti-inflammatory mechanism of Cordyceps pruinosa butanolfraction by using kinase assays luciferase reporter assaysmolecular binding assays and overexpression strategies aswell as a mouse model of gastritis

Table 1 PCR primers used in this study

Name Sequence (51015840 to 31015840)

iNOS F CCCTTCCGAAGTTTCTGGCAGCAGR GGCTGTCAGAGCCTCGTGGCTTTGG

TNF-120572 F TTGACCTCAGCGCTGAGTTGR CCTGTAGCCCACGTCGTAGC

IL-6 F GTACTCCAGAAGACCAGAGGR TGCTGGTGACAACCACGGCC

GAPDH F CACTCACGGCAAATTCAACGGCAR GACTCCACGACATACTCAGCAC

2 Materials and Methods

21 Materials Artificially cultivated fresh fruiting bodiesof Cordyceps pruinosa were obtained from Mushtech Co(Chuncheon Korea) andwere authenticated byDr JM Sung(Mushtech Chuncheon Korea) A voucher specimen(EFCC 11968) was deposited in the EntomopathogenicFungal Culture Collection Kangwon National UniversityKorea Forskolin cordycepin (3-45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) H

2DCFDA

fluorescein isothiocyanate (FITC)-dextran phorbol 12-myristate 13-acetate (PMA) and LPS from Escherichia coli0111B4 were purchased from Sigma-Aldrich (St Louis MO)BAY 11-7082 was obtained from Calbiochem (La Jolla CA)Luciferase constructs containing binding promoters for NF-120581B CREB and AP-1 were gifts from Professor Chung HaeYoung (Pusan National University Pusan Korea) and ManHee Rhee (Kyungpook National University Daegu Korea)Enzyme immunosorbent assay (ELISA) kits for determininglevels of TNF-120572 were purchased from Amersham (LittleChalfont Buckinghamshire UK) Foetal bovine serum(FBS) and RPMI1640 were obtained from GIBCO (GrandIsland NY) RAW2647 and HEK293 cells were purchasedfrom ATCC (Rockville MD) All other chemicals were ofSigma-Aldrich grade Phosphospecific and total antibodiesfor transcription factors (p65 p50 and c-Jun) MAPK (ERKp38 and JNK) I120581B120572 IKK120573 AKT lamin AC and 120573-actinwere obtained from Cell Signalling Technology (BeverlyMA) Primers (Table 1) were designed in our laboratory andwere synthesized by Bioneer (Daejeon Korea)

22 Preparation and Characteristics of Cp-BF After dryingthe fruiting bodies of Cordyceps pruinosa at 50∘C an ethanolextract was prepared and several solvent fractions weresubsequently prepared with n-hexane n-butanol and ethylacetate After evaporation of the fractions under reducedpressure each fraction was dried using a freeze-dryer to givesolid subfractions Among the various fractions the butanolfraction (Cp-BF) gave a 7 yieldThe phytochemical charac-teristics of Cp-BF were identified by high performance liquidchromatography (HPLC) analysis as reported previously [17]the system was equipped with KNAUER (Wellchrom HPLC-pump K-1001 Wellchrom fast scanning spectrophotometerK-2600 and 4 channel degasser K-500) The elution solventsconsisted of distilled water and acetonitrileThe gradient step

Evidence-Based Complementary and Alternative Medicine 3

(a)

Cordyceps pruinosa (Cp-EE) (X2 EtOH 80) RT

Residue

n-Hexane fr Residue

EtOAc fr

Cp-EE (500 g)n-Hexane (800mL) X3

(476 g)

(167 g) EtOAc (800mL) X3

n-BuOH (800mL) X3

n-BuOH fr (Cp-BF)(35 g)

(75 g)

H2O

H2O

H2OCH2Cl2 fr

CH2Cl2 (800mL) X3

(b)

Cordycepin

Rt (min) [area]

Cordycepin

Cp-BF

148min [127]

153min [112]

Abso

rban

ce at

260

nm

100

50

0

0 10 20 30 40

Rt (min) [area]

Abso

rban

ce at

260

nm

100

50

0

0 10 20 30 40

Rt (min) [area]

Abso

rban

ce at

260

nm

100

50

0

0 10 20 30 40

(c)

Figure 1 Description andHPLC-analysis of the butanol fraction of cultivatedCordyceps pruinosa (a) Photos of the artificially cultivated formof Cordyceps pruinosa (b) Experimental procedure for preparation of the butanol fraction from Cordyceps pruinosa (Cp-BF) (c) Cp-BF andcordycepin were analysed by high performance liquid chromatography (HPLC) equipped with KNAUERThe elution solvents were distilledwater and acetonitrile The gradient step of the solvent was ldquowater to acetonitrile 1minrdquo performed using a Vydac C18 Column

for solvent elution was ldquowater to acetonitrile 1minrdquo using aVydac C18 column

23 Mice Six-week-old male ICR mice (6ndash8 weeks old 17ndash21 g) were obtained fromDaehan Biolink (Osong Korea) andmaintained in plastic cages under conventional conditionsWater and pelleted diets (Samyang Daejeon Korea) weresupplied ad libitum Studies were performed in accordancewith guidelines established by the Sungkyunkwan UniversityInstitutional Animal Care and Use Committee

24 Cell Culture RAW2647 and HEK293 cells were cul-tured with RPMI1640 medium supplemented with 10

heat-inactivated FBS glutamine and antibiotics (penicillinand streptomycin) at 37∘C in a 5 CO

2atmosphere For each

experiment cells were detached with a scraper Examinationof cell densities at 2 times 106 cellsmL revealed that the propor-tion of dead cells was consistently lt1 according to Trypanblue dye exclusion as the criterion for viability

25 NO and TNF-120572 Production After preincubation ofRAW2647 cells (1 times 106 cellsmL) for 18 h cells were pre-treated with Cp-BF (0ndash200 120583gmL) for 30min and thenincubated with LPS (1120583gmL) for an additional 24 h Theinhibitory effect of Cp-BF on NO and TNF-120572 produc-tion was determined by analysing NO and TNF-120572 levels


with Griess reagent and ELISA kits as described previously[18 19]

26 Determination of Phagocytic Uptake Phagocytic activityof RAW2647 cells was determined as described previouslywith some modifications [20] Briefly RAW2647 (5 times 104)cells treated with Cp-BF (0ndash200120583gmL) were resuspended in100 120583L PBS containing 1 human AB serum and incubatedwith fluorescein isothiocyanate (FITC)-dextran (1mgmL) at37∘C for 2 h Incubations were stopped by adding 2mL ice-cold phosphate-buffered saline (PBS) containing 1 humanserum and 002 sodium azide The cells were then washedthree times with cold PBS-azide and analysed on a FACScanflow cytometer as reported previously [21]

27 Determination of Reactive Oxygen Species GenerationLevels of intracellular ROS were determined by analysingthe change in fluorescence resulting from the oxidation ofthe fluorescent probe H

2DCFDA Briefly 5 times 105 RAW2647

cells were exposed to Cp-BF (0ndash100120583gmL) for 30min Afterincubation cells were incubated with LPS (1120583gmL) as aninducer of ROS production at 37∘C for 6 h Cells were thenincubated with 50120583MH

2DCFDA for 1 h at 37∘C The degree

of fluorescence corresponding to the level of intracellularROS was determined using a FACScan flow cytometer(Beckton-Dikinson San Jose CA USA) as reported previ-ously [16]

28 Flow Cytometric Analysis RAW2647 cells (2 times106 cellsmL) treated with Cp-BF (0ndash100 120583gmL) and FITC-dextran or H

2DCFDA were washed with a staining buffer

(containing 2 rabbit serum and 1 sodium azide in PBS)and incubated with directly labelled antibodies for a further45min on ice After washing three times with stainingbuffer cells were analysed on a FACScan flow cytometer(Becton-Dickinson)

29 Cell Viability Test After preincubation of RAW2647cells (1 times 106 cellsmL) for 18 h the Cp-BF (0ndash200 120583gmL)was added to the cells and incubated for an additional 24 hThe cytotoxic effect of the Cp-BF was then evaluated by aconventionalMTT assay as described previously [22] Briefly3 h prior to culture termination 10 120583L of a MTT solution(10mgmL in phosphate buffered saline pH 74 PBS) wasadded and cells were continuously cultured until terminationof the experiment Incubation was halted by the addition of15 sodium dodecyl sulphate into each well to solubilise theproduced formazan [23] The absorbance at 570 nm (OD

570)

was measured using a Spectramax 250 microplate reader

210 mRNA analysis by Reverse Transcription (RT) Poly-meraseChainReaction (PCR) Todetermine cytokinemRNAexpression levels total RNA was isolated from LPS-treatedRAW2647 cells with TRIzol Reagent (Gibco BRL) accordingto the manufacturerrsquos instructions Total RNA was storedat minus70∘C until use Determination of mRNA levels wasperformed using RT-PCR as reported previously [24] Results

are expressed as the ratio of optimal density to glyceraldehyde3-phosphate dehydrogenase (GAPDH)The sequences of theprimers used in this analysis are shown in Table 1

211 Transfection of DNA and Luciferase Reporter GeneActivity Assay HEK293 or RAW2647 cells (5times 106 cellsmL)were transfected with 1 120583gmL of empty vectors or FLAG-IKK120573 Transfections were performed with lipofectamine2000 (Invitrogen Grand Island NY) in 100mm cell culturedishes in the presence or absence of MHNC For luciferaseassays HEK293 cells (1 times 106 cellsmL) were transfected with1 120583g of plasmids containing NF-120581B-Luc CREB-Luc or AP-1-Luc as well as 120573-galactosidase using the PEI method [25 26]in 12-well plates according to the manufacturerrsquos protocolCells were used for experiments 48 h after transfectionLuciferase assays were performed using the Luciferase AssaySystem (Promega Madison WI) as reported previously [27]

212 Preparation of Total Lysates and Nuclear FractionsImmunoblotting and Immunoprecipitation Stomach tissuesor cultured cells (HEK293 and RAW2647) (5 times 106 cellsmL)were washed three times in cold PBS with 1mM sodiumorthovanadate and lysed in lysis buffer (20mM Tris-HClpH 74 2mM EDTA 2mM ethylenediaminetetraacetic acid50mM 120573-glycerophosphate 1mM sodium orthovanadate1mM dithiothreitol 1 Triton X-100 10 glycerol 10 120583gmLaprotinin 10 120583gmL pepstatin 1mM benzamide and 2mMphenylmethanesulfonyl fluoride) for 30min with rotation at4∘CNext lysates were clarified by centrifugation at 16000timesgfor 10min at 4∘C and stored at minus20∘C until needed

Nuclear lysates were prepared in a three-step procedure[28] After treatment cells were collected with a rubberpoliceman washed with 1 times PBS and lysed in 500120583L of lysisbuffer on ice for 4min Cell lysates were then centrifugedat 19326timesg for 1min in a microcentrifuge In the secondstep the pellet (the nuclear fraction) was washed once inwashing buffer which was the same as the lysis buffer withthe omission of Nonidet P-40 In the final step nuclei weretreated with an extraction buffer containing 500mM KCl10 glycerol and the inhibitors noted in the lysis buffer Thenucleiextraction buffer mixture was frozen at minus80∘C andthen thawed on ice and centrifuged at 19326timesg for 5minFinally the supernatant was collected as the nuclear extract

For immunoprecipitation cell lysates containing equalamounts of protein (500120583g) from RAW2647 cells (1 times107 cellsmL) treated with or without LPS (1 120583gmL) for25min were pre-cleared with 10120583L of protein A-coupledSepharose beads (50 vv) (Amersham BuckinghamshireUK) for 1 h at 4∘C Precleared samples were then incubatedwith 5120583L of anti-IKK120573 antibody overnight at 4∘C Immunecomplexes were mixed with 10 120583L of protein A-coupledSepharose beads (50 vv) and rotated for 3 h at 4∘C

Soluble cell lysates or boiled beads used for immunopre-cipitation were subjected to immunoblotting for detection ofphosphorylated or total levels of transcription factors (p65p50 and c-Jun) MAPK (ERK p38 and JNK) I120581B120572 IKK120572120573IKK120573 Akt 120574-tubulin lamin AC and 120573-actin Immunoblotswere visualized as previously reported [29]


213 IKK120572 and IKK120573 Kinase Assays To evaluate the abilityof extracts to inhibit IKK120572 and IKK120573 kinase activities usingpurified enzymes a kinase profiler service from Millipore(Billerica MA) was used In a final reaction volume of 25 120583LIKK120572 or IKK120573 (human 1ndash5mU) was incubated with thereaction buffer and the reaction was initiated by the additionof MgATP After incubation for 40min at room temperaturethe reaction was stopped by the addition of 5mL of 3phosphoric acid solution Next 10 120583L of the reaction productwas spotted onto a P30 filtermat and washed three timesfor 5min each in 75mM phosphoric acid The filtermatswere washed once in methanol dried and then subjected toscintillation counting

214 HClEthanol (EtOH)-Induced Gastritis Inflammationof the stomach was induced with HClEtOH according to apublished method [30] Fasted ICR mice were orally treatedwith Cp-BF (200mgkg) or ranitidine (40mgkg) twice perday for 3 days Thirty min after the final injection 400 120583Lof 60 EtOH in 150mM HCl was administered orally Eachanimal was anesthetized with an overdose of urethane 1 hafter the administration of necrotizing agents The stomachwas then excised and gently rinsed under running tap waterAfter opening the stomach along the greater curvature andspreading it out on a board the area (mm2) of mucosalerosive lesions was measured using a pixel-counter underblind condition as reported previously [31]

215 Statistical Analyses Data are expressed as the meanplusmn standard deviation (SD) calculated from at least threeindependent experiments each performed in triplicate orrepresentative of three different experiments with similarresults For statistical comparisons results were analysedusing analysis of varianceScheffersquos post hoc test and Kruskal-WallisMann-Whitney test Values of 119875 lt 005 were taken toindicate statistically significant differences All statistical testswere carried out using the SPSS computer program (SPSSChicago IL)

3 Results

31 Cp-BF Suppresses the Inflammatory Responses ofMacrophages We first tested the effect of Cp-BFin macro-phages As shown in Figures 2(a) and 2(b) Cp-BF dose-dependently suppressed the production of NO and TNF-120572stimulated from LPS-treated RAW2647 cells In particular200120583gmL of Cp-ME greatly inhibited the production ofNO up to 95 and the release of TNF-120572 by 43 In additionthis fraction significantly decreased the uptake of FITC-dextran by up to 41 at 100 120583gmL (Figure 2(c)) MoreoverCp-BF almost completely suppressed the generation ofROS stimulated by LPS at 50 and 100 120583gmL (Figure 2(d))Importantly Cp-BF at 200120583gmL did not affect the viabilityof either RAW2647 or HEK293 cells (Figure 2(e))

32 Cp-BF Suppresses the Inflammatory Responses at the Tran-scription Level To determine the mechanism by which Cp-BF exerts its anti-inflammatory effects we next analysed the

mRNA levels of genes involved in inflammation As shownin Figure 3(a) Cp-BF strongly suppressed the expressionof iNOS and IL-6 between 100 and 200 120583gmL while thisfraction weakly inhibited the mRNA expression of TNF-120572 at these concentrations The regulatory role of Cp-BFon the activation of inflammatory transcription factors wasdetermined by luciferase assay Thus under the describedconditions the activity of luciferase was clearly enhancedby PMA treatment for NF-120581B and AP-1 activation and byforskolin exposure for CREB (Figure 3(b)) Interestingly Cp-BF remarkably diminished the NF-120581B-mediated luciferaseactivity in a dose-dependent manner whereas others weremarginally suppressed (Figure 3(b)) In agreement withthis result treatment with 200120583gmL Cp-BF decreased thenuclear translocation of p50 but not p65 triggered by LPStreatment in RAW2647 cells (Figure 3(c))

33 Cp-BF Inhibits NF-120581B Activation Pathway by SuppressingIKK120573Activity Immunoblot analysis revealed thatCp-BFwasable to suppress the upstream signalling pathway for NF-120581Bactivation Thus Cp-BF clearly blocked the phosphorylationof I120581B120572 at 5 30 and 60min (Figure 4(a) left panel) Incontrast this fraction did not block the phosphorylation ofp38 JNK and ERK from 5 to 60min (Figure 4(a) left panel)In addition there was no alteration of phosphorylation levelsof Akt and IKK120572120573 between 2 to 5min (Figure 4(a) rightpanel) Since there was no inhibition of IKK120572120573 phospho-rylation but suppression of I120581B120572 phosphorylation Cp-BFwas assumed to have dampened the enzymatic activities ofIKK120572 and IKK120573 In order to test our theory we employedkinase assays to evaluate IKK120572120573 kinase activity As shownin Figure 4(b) Cp-BF significantly suppressed the kinaseactivity of IKK120573mdashbut not IKK120572mdashup to 44 at 200120583gmLSimilarly overexpression of IKK120573-induced luciferase activityand phosphorylation of I120581B120572 was completely diminished byCp-BF (50 to 200 120583gmL) (Figure 4(c)) In agreement withthis result the phosphorylation of NF-120581B subunits (p65 andp50) and I120581B120572 induced by IKK120573 overexpression in HEK293cells was strongly suppressed by Cp-BF (Figure 4(d)) Finallywe found that Cp-BF was able to block the molecularinteraction between IKK120573 and I120581B120572 (Figure 4(e))

The importance of IKK in inflammatory responses wasalso demonstrated using the strong IKK inhibitor BAY11-7082 Specifically BAY11-7082 inhibited the release of NOand suppressed the phagocytic uptake of FITC-dextran(Figure 4(f))

34 Cp-BF Ameliorates HClEtOH-Induced Gastritis viaSuppression of NF-120581B Pathway To demonstrate the anti-inflammatory activity of Cp-BF in vivo we analysed theeffects of orally administered Cp-BF in a model of gastritisAs shown in Figures 5(a) and 5(b) orally administered Cp-BF ameliorated the inflammatory symptoms of HClEtOH-induced gastritis in vivo Cp-BF (200mgkg) clearly sup-pressed the formation of inflammatory lesions in stomachduring HClEtOH treatment and was more effective thanranitidine (40mgkg) Interestingly Cp-BF also suppressedthe phosphorylation of I120581B120572 induced by gastric damage(Figure 5(c))


NO

pro

duct

ion

( o

f con

trol)

Cp-BF (120583gmL)+ +

50+

100+

200+25

0

20

40

60

80

100

120

LPS (1120583gmL) minus

minus minus

lowastlowast

lowastlowastlowastlowast

(a)

TNF-120572

pro

duct

ion

( o

f con

trol)

+ +100

+200

0

20

40

60

80

100

120

Cp-BF (120583gmL)LPS (1120583gmL) minus

minus minus

lowastlowast

(b)

0

20

40

60

80

100

120

140

Phag

ocyt

ic u

ptak

e (

of c

ontro

l)

50 10025125Cp-BF (120583gmL)

minus minus

lowast

FITC-dextran (1mgmL)

(c)

0

200

400

600

800

ROS

gene

ratio

n (M

FI)

50 100Normal minus minus

Cp-BF (120583gmL)LPS (1120583gmL)


H2DCFDA (50120583M)

(d)

0

20

40

60

80

100

120

140

RAW2647 cellsHEK293 cells

Cel

l via

bilit

y (

of c

ontro

l)

0 50 100 20025Cp-BF (120583gmL)

(e)

Figure 2 Effect of Cp-BF on production of inflammatory mediators release of reactive oxygen species and phagocytic uptake ((a) and (b))Levels of NO and TNF-120572 were determined by Griess assay and ELISA from culture supernatants of RAW2647 cells treated with Cp-BF andLPS (1120583gmL) for 24 h (c) The effect of Cp-BF on phagocytic uptake of RAW2647 cells was determined by treatment with FITC-dextran(1mgmL) for 2 h and the uptake of which was determined by flow cytometric analysis (d) The effect of Cp-BF on reactive oxygen species(ROS) generation in LPS-treated RAW2647 cells was determined by incubation with H

2DCFDA (50120583M) and flow cytometric analysis (e)

Cell viability of RAW2647 and HEK293 cells as determined by MTT assay lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control


IL-6

+50

+100

+200

+

GAPDH

iNOS

minusminus minusCp-BF (120583gmL)

LPS (1120583gmL)6h

TNF-120572

(a)

0

2

4

6

8

10

12

14

16

AP-1CREB

Stimulus

Luci

fera

se ac

tivity

(fol

d in

crea

se)

+ +50

+100

+200

+25


NF-120581B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

p65

+ + + + + ++ + +

p50

Lamin ACc-Jun

LPS (1120583gmL) minusminus minus minus minusCp-PBF (200120583gmL)

15min 30min 60min

(c)

Figure 3 Effect of Cp-BF on the transcriptional regulation of inflammatory genes (a) The mRNA levels of iNOS IL-6 and TNF-120572 weredetermined by semiquantitative RT-PCR (b) HEK293 cells cotransfected with plasmid constructs NF-120581B-Luc CREB-Luc or AP-1-Luc (each1 120583gmL) and 120573-gal (as a transfection control) were treated with Cp-BF in the presence or absence of PMA (100 nM for NF-120581B and AP-1-Luc) or forskolin (100 nM for CREB-Luc) Luciferase activity was measured by a luminometer (c) Levels of nuclear NF-120581B (p65 and p50)or AP-1c-Jun were determined by immunoblot analysis of the nuclear fractions of LPS-treated RAW2647 cells lowast119875 lt 005 and lowastlowast119875 lt 001compared to control

4 Discussion

As part of the strategy to develop fruit bodies of artificiallycultured Cordyceps species Cordyceps pruinosa was selectedand cultivation conditions were established with grain rice-containing medium Recently our group succeeded in man-ufacturing fruit bodies of Cordyceps pruinosa (Figure 1(a)) atthe mass production level and as the next step we decidedto explore its pharmacological activity Thus in the presentstudy we evaluated the anti-inflammatory mechanism of abutanol extract (Cp-BF) of the Cordyceps pruinosa fruit body

Similar to a previous study of a methanol extract ofthe Cordyceps pruinosa fruit body we found that Cp-BFcould suppress the functional activation of macrophagesin inflammatory responses Thus Cp-BF suppressedthe essential outcomes of inflammatory process frommacrophage activation such as production of NO and TNF-120572(Figures 2(a) and 2(b)) ROS generation (Figure 2(c))and phagocytic uptake (Figure 2(d)) without altering cellviability (Figure 2(e)) [32 33] Considering the importanceof macrophages in inflammation these data strongly impliedthat Cp-BF could negativelymodulatemacrophage-mediated

inflammatory responses These results are consistent withthe previous reports that extracts of Cordyceps militariscan diminish inflammatory gene expression in LPS-treatedRAW2647 cells [34] Furthermore a chemical derivativeof militarin from Cordyceps militaris exhibits strong anti-inflammatory properties in LPS-treated macrophages [35]The ethanol extract of Cordyceps bassiana also blocksthe production of IL-12 in LPS-treated RAW2647 cells[36] Since the majority of inflammatory diseases such ashepatitis gastritis colitis and nephritis are known to becaused by overactivated macrophages our data stronglysuggest that the inhibitory activity of macrophage functionmay contribute to the immunopharmacological action ofCordyceps In fact the curative efficacy of these mushroomsagainst macrophage-mediated diseases has been previouslydemonstrated in a number of different disease animal models[37 38]

Understanding the molecular mechanism of Cp-BF mediated suppression of macrophage inflammatoryresponses is a significant goal for immunopharmacologicalresearch of the genus Cordyceps However only a few reportsto date have attempted to explore the molecular target of


+ + + ++ + + + + + + +

p-ERKERK

p-JNK

p-p38p38

JNK

LPS (1120583gmL) minusminus minus minus minusminusCp-BF (200120583gmL)

15min 30min 60min5min

p-I120581B120572I120581B120572

120573-Actin

+ + + + + ++ + +

p-Akt

Akt

LPS (1120583gmL)minusminusminusminus

minusCp-BF (200120583gmL)

120573-Actin

IKK120572120573

p-IKK120572120573

2min 3min 5min

(a)

0

20

40

60

80

100

120

140

Cp-BF

Kina

se ac

tivity

( o

f con

trol)

+ +(200120583gmL)

minusIKK120573IKK120572

lowast

(b)

0

50

100

150

200

250

300

(fold

incr

ease

)

0+

200+

100+

50+

minus

minus

Cp-BF (120583gmL)

p-I120581B120572

120573-Actin

NF-120581

B lu

cife

rase

activ

ity

IKK120573 (05 120583g)

lowastlowastlowastlowastlowastlowast

(c)

p-p65

+ +100

p-p50

HEK293 cells

p-I120581B120572120573-Actin

Cp-BF (120583gmL)IKK120573 minus

minus minus

(d)

WB

+ ++

+IgG Input

+

LC

minusminus minus minus minus

I120581B120572Cp-BF (100120583gkg)

FLAG-IKK120573 (1120583gmL)IP FLAG

(e)

0

20

40

60

80

100

120

NO

pro

duct

ion

and

phag

ocyt

ic

upta

ke (

of c

ontro

l)

0 10 15 0 10 15

NO production Phagocytosis

BAY11-7082 (120583M)


lowastlowast

lowastlowast

(f)

Figure 4 Effect of Cp-BF on the upstream signalling for NF-120581B activation (a) Phosphoprotein or total protein levels of I120581B120572 Akt p38 ERKJNK IKK120572120573 and 120573-actin from cell lysates were determined by phosphospecific or total protein antibodies (b) Kinase activities of IKK120572 andIKK120573 were determined by a direct kinase assay using purified enzymes The control was set as 100 for each enzyme activity obtainedwith vehicle treatment (c) NF-120581B-mediated luciferase activity in IKK120573-transfected HEK293 cells was measured using a luminometerPhosphoprotein levels of I120581B120572 were determined by immunoblot analysis (d) Phosphoprotein levels of I120581B120572 p50 and p65 from IKK120573-transfected RAW2647 cells were determined by immunoblot analysis (e) Binding of IKK to I120581B120572 was determined by immunoprecipitationand immunoblot analysis ofwhole cell lysates of LPS-treatedRAW2647 cells (5times 106 cellsmL) (f) Level ofNOwas determined byGriess assaywith culture supernatants of RAW2647 cells treated with BAY11-7082 and LPS (1 120583gmL) for 24 h The effect of BAY11-7082 on phagocyticuptake of RAW2647 cells treated with FITC-dextran (1mgmL) was determined by flow cytometric analysis lowast119875 lt 005 and lowastlowast119875 lt 001compared to control


Normal Vehicle Cp-BF Ranitidine

EtOHHCl

(200mgkg) (40mgkg)

(a)

0

20

40

60

80

100

120

Normal Vehicle CPBF Ranitidine

Size

of b

lood

spot

s (

of c

ontro

l)

(200mgkg) (40mgkg)

lowastlowast

lowastlowast

(b)

EtOHHCl + ++

Stomach

minusminusminus

p-I120581B120572

120573-Actin

Cp-BF (200mgkg)

(c)

Figure 5 Effect of Cp-BF on inflammatory stomach lesions in HClEtOH-treated mice ((a) and (b)) Mice administered orally with Cp-BF(200mgkg) or ranitidine (40mgkg) for 3 days were treated with HClEtOH delivered orally After 1 h gastric lesions in the stomach werephotographed and measured with a ruler Gastric lesions after treatment with inducer alone were set as 100 (c) Phosphoprotein levels ofI120581B120572 from tissue lysates were determined using phosphospecific and total protein antibodies 120573-actin was used as a control lowastlowast119875 lt 001compared to control

Cordyceps species with respect to their anti-inflammatoryactions Specifically we previously suggested that thephosphorylation of p38 might target the inhibition ofIL-12 expression in Cordyceps bassiana [36] Cordycepinwas found to suppress the phosphorylation of Akt forNF-120581B inhibition in LPS-treated conditions [39] It was alsoreported that NF-120581B could be targeted by a methanol extractof Cordyceps pruinosa although no further evaluationof its upstream signalling enzymes was performed [15]Importantly none of these reports clearly identified a targetprotein responsible for the activity of Cordyceps as they onlytested the phosphorylation levels of relevant pathways Toimprove such methodological limitations we employed adirect kinase assay followed by an overexpression approachfor target proteins to confirm their involvement Throughthis approach we identified IKK120573 as a new potential targetenzyme of Cp-BF

We next analysed the mRNA levels of inflammatorygenes (Figure 3(a)) luciferase promoter activity (Figure 3(b))and nuclear levels of transcription factors (Figure 3(c))Our results suggested that Cp-BF was able to suppressmacrophage-mediated inflammatory responses at the tran-scriptional level by suppression of the NF-120581B pathwayThrough determination of upstream phosphorylation pat-terns regulating the activation and translocation of NF-120581Bwe confirmed the presence of a target event between IKKand I120581B120572 based on clear suppression of the phosphorylationof I120581B120572 by Cp-BF (Figure 4(a) left panel) and lack of

inhibition of upstream phosphorylation events for the acti-vation of IKK and Akt (Figure 4(a) right panel) Althoughthe inhibitory activity was not strong direct kinase assay withIKK120573 indicated that approximately 40 of IKK120573 enzymeactivity was suppressed by Cp-BF (200120583gmL) implying thatIKK120573 may be a potential target enzyme Indeed additionalexperiment data strongly supported the idea Cp-BF treat-ment remarkably blockedNF-120581B-mediated luciferase activityinduced by overexpressed IKK120573 (Figure 4(c)) upregulationof p65p50 phosphorylation (Figure 4(d)) and molecularbinding between IKK and I120581B120572 (Figure 4(e)) However thediscrepancy between kinase assay (Figure 4(b)) and IKKoverexpression experiments (Figures 4(c) and 4(d)) shouldbe further explored Presently we speculate that there mightbe different pharmacological sensitivities between purifiedhuman IKK120573 used for kinase assays and transfected IKK120573expressed in HEK293 cells

The ethnopharmacological value of Cordyceps is nowgreatly accepted in Asian countries since these mushroomshave been traditionally prescribed for the treatment of var-ious inflammatory and metabolic diseases Unlike ginsengone of most famous herbal plants worldwide for whichcultivation methods have been traditionally established themajority of Cordyceps products are still harvested in natureAlthough natural fruit bodies of Cordyceps may be best interms of pharmacological activities large-scale productionremains a key issue Therefore recent studies with Cordycepsspecies have focused on the development of cultural methods


LPS

TLR4 TLR3TRIF MyD88 TRAF6

SrcSyk

p85PI3K

PDK1AKT

AP-1 CREB

MKK36 MKK47

ERK p38

c-fos c-Jun

MEK12

iNOS

NO

HClEtOH

Cp-BF

IL-6Pro-TNF-120572

NF-120581B

TNF-120572

I120581B120572

IKK120572120573

JNK

Figure 6 Putative inhibitory pathway of the Cp-BF-mediated anti-inflammatory response

for mass production of Cordyceps In fact our group hasestablished a suitable cultivation method and plant manu-facturing system that achieves high productivity ofCordycepsfruit bodies [40] Using the approach we analysed the phar-macological efficacies of artificially cultivated fruit bodiesof Cordyceps militaris Cordyceps bassiana and Cordycepspruinosamaintained under identical conditions

To specifically test the activity of Cordyceps pruinosa weemployed a mouse in vivo disease model with HClEtOH-induced gastritis under oral administration of Cp-BF Inter-estingly this fraction strongly ameliorated gastritis symptoms(Figures 5(a) and 5(b)) and suppressed the phosphorylationof I120581B120572 increased by HClEtOH treatment (Figure 5(c))These results strongly implied that the artificially cultivatedfruit bodies exhibit similar pharmacological activity as thenatural forms of this mushroom Indeed we previouslyobserved that the artificially cultivated fruit body of Cordy-ceps bassiana can effectively treat symptoms of atopic der-matitis [41] Moreover the artificially cultivated fruit body ofCordyceps militaris possesses antihepatitis activity (data notshown) Taken together these results strongly suggest thatthe artificially cultivated fruit bodies of Cordyceps pruinosacan be developed for the purpose of pharmaceutical andnutraceutical remedies

To date little information on the phytochemical charac-teristics of Cordyceps pruinosa has been reported A totalof 5 alcohols 21 amino acids 15 organic acids 4 purines 3pyrimidines 7 sugars 11 fatty acids and 5 other metabolitesin Cordyceps pruinosamycelia cultivated with various media

have been characterized by metabolic profile analysis [42]This same study also showed thatCordyceps pruinosamyceliacontains a high phenolic content and exhibits good antioxida-tive properties although the specific compounds responsiblefor this effect have not yet been identified [42] According tothe analysis of cordycepin by HPLC this compound mightnot be included in this mushroom (Figure 1(c)) Thereforefuture studies will need to include the understanding ofactive components with anti-inflammatory activity in thismushroom

In summary we have shown that Cp-BF can blockmacrophage-mediated inflammatory responses such as NOand TNF-120572 production ROS generation and phagocyticuptake at the transcriptional level Orally administered Cp-BF clearly ameliorates gastric ulcer formation induced byHClEtOH treatment By analysing transcription factors andintracellular signalling enzymes our results suggest that Cp-BF can directly inhibit IKK linked to the suppression ofNF-120581B pathway as summarized in Figure 6 The data fromthe present study strongly support the pharmaceutical andnutraceutical values of artificially cultivated fruit bodies ofCordyceps pruinosa

Abbreviations

Cp-BF Butanol fraction of Cordyceps pruinosaNO Nitric oxideiNOS Inducible NO synthaseTNF-120572 necrosis factor-alphaERK Extracellular signal-related kinase


TLR Toll-like receptorMAPK Mitogen activated protein kinaseNF-120581B Nuclear factor-120581BAP-1 Activator protein-1JNK c-Jun N-terminal kinaseAkt Protein kinase BIKK I120581B120572 kinaseELISA Enzyme-linked immunosorbent assayMTT 3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromidePI3K Phosphoinositide 3-kinasesLPS LipopolysaccharideRT-PCR Reverse transcriptase-polymerase chain

reaction

Conflict of Interests

The authors report no conflict of interests The authors aloneare responsible for the content and writing of the paper

Authorsrsquo Contribution

Han Gyung Kim Woo Seok Yang and Gi-Ho Sung equallycontributed to this work

Acknowledgment

Thisworkwas performedwith the support of theCooperativeResearch Program for Agriculture Science amp TechnologyDevelopment (Project no PJ009241) Rural DevelopmentAdministration Korea

References

[1] Y Sekine T Yumioka T Yamamoto et al ldquoModulation of TLR4signaling by a novel adaptor protein signal-transducing adaptorprotein-2 in macrophagesrdquo Journal of Immunology vol 176 no1 pp 380ndash389 2006

[2] K Takeda and S Akira ldquoRoles of Toll-like receptors in innateimmune responsesrdquo Genes to Cells vol 6 no 9 pp 733ndash7422001

[3] B Bresnihan ldquoPathogenesis of joint damage in rheumatoidarthritisrdquo Journal of Rheumatology vol 26 no 3 pp 717ndash7191999

[4] G R Burmester B Stuhlmuller G Keyszer and R W KinneldquoMononuclear phagocytes and rheumatoid synovitis master-mind or workhorse in arthritisrdquoArthritis and Rheumatism vol40 no 1 pp 5ndash18 1997

[5] J A Gracie R J Forsey W L Chan et al ldquoA proinflammatoryrole for IL-18 in rheumatoid arthritisrdquo Journal of ClinicalInvestigation vol 104 no 10 pp 1393ndash1401 1999

[6] B Stuhlmuller U Ungethum S Scholze et al ldquoIdentification ofknown and novel genes in activated monocytes from patientswith rheumatoid arthritisrdquo Arthritis and Rheumatism vol 43no 4 pp 775ndash790 2000

[7] E Michaelsson M Holmdahl A Engstrom H Burkhardt AScheynius and R Holmdahl ldquoMacrophages but not dendriticcells present collagen to T cellsrdquo European Journal of Immunol-ogy vol 25 no 8 pp 2234ndash2241 1995

[8] T B Ng and H X Wang ldquoPharmacological actions ofCordyceps a prized folk medicinerdquo Journal of Pharmacy andPharmacology vol 57 no 12 pp 1509ndash1519 2005

[9] X Zhou Z Gong Y Su J Lin and K Tang ldquoCordyceps funginatural products pharmacological functions and developmen-tal productsrdquo Journal of Pharmacy and Pharmacology vol 61no 3 pp 279ndash291 2009

[10] J Zhu G M Halpern and K Jones ldquoThe scientific rediscoveryof a precious ancient Chinese herbal regimen cordyceps sinen-sis Part IIrdquo Journal of Alternative and Complementary Medicinevol 4 no 4 pp 429ndash457 1998

[11] J S Zhu G M Halpern and K Jones ldquoThe scientific rediscov-ery of an ancient Chinese herbal medicine cordyceps sinensispart IrdquoThe Journal of Alternative and ComplementaryMedicinevol 4 no 3 pp 289ndash303 1998

[12] KWojcikowski DW Johnson and G Gobe ldquoHerbs or naturalsubstances as complementary therapies for chronic kidneydisease ideas for future studiesrdquo Journal of Laboratory andClinical Medicine vol 147 no 4 pp 160ndash166 2006

[13] S Patel and A Goyal ldquoRecent developments in mushrooms asanti-cancer therapeutics a reviewrdquo 3 Biotech vol 2 no 1 pp1ndash15 2012

[14] K Yue M Ye Z Zhou W Sun and X Lin ldquoThe genus Cordy-ceps a chemical and pharmacological reviewrdquo Journal of Phar-macy and Pharmacology vol 65 no 4 pp 474ndash493 2013

[15] K Kim Y Kwon H Chung et al ldquoMethanol extract ofCordyceps pruinosa inhibits in vitro and in vivo inflammatorymediators by suppressing NF-120581B activationrdquo Toxicology andApplied Pharmacology vol 190 no 1 pp 1ndash8 2003

[16] H G Kim H Song D H Yoon et al ldquoCordyceps pruinosaextracts induce apoptosis of HeLa cells by a caspase dependentpathwayrdquo Journal of Ethnopharmacology vol 128 no 2 pp 342ndash351 2010

[17] D Jeong W S Yang Y Yang et al ldquoIn vitro and in vivoanti-inflammatory effect of Rhodomyrtus tomentosa methanolextractrdquo Journal of Ethnopharmacology vol 146 no 1 pp 205ndash213 2013

[18] M Kim and J Y Cho ldquo20S-dihydroprotopanaxatriol modulatesfunctional activation of monocytes and macrophagesrdquo Journalof Ginseng Research vol 37 no 3 pp 300ndash307 2013

[19] S H Lee E Lee and Y T Ko ldquoAnti-inflammatory effects of amethanol extract fromPulsatilla koreana in lipopolysaccharide-exposed ratsrdquo BMB Reports vol 45 no 6 pp 371ndash376 2012

[20] J W Jeon B C Park J G Jung Y S Jang E C Shin and Y WPark ldquoThe soluble form of the cellular prion protein enhancesphagocytic activity and cytokine Production by human mono-cytes via activation of ERK and NF-120581Brdquo Immune Network vol13 no 4 pp 148ndash156 2013

[21] B H Kim Y G Lee J Lee J Y Lee and J Y Cho ldquoRegulatoryeffect of cinnamaldehyde on monocytemacrophage-mediatedinflammatory responsesrdquoMediators of Inflammation vol 2010Article ID 529359 9 pages 2010

[22] Y Kwon J Jung N Park et al ldquoAnnexin A5 as a new potentialbiomarker for cisplatin-induced toxicity in human kidneyepithelial cellsrdquo Biomolecules andTherapeutics vol 21 no 3 pp190ndash195 2013

[23] M Kim and J Y Cho ldquo20S-dihydroprotopanaxadiol a ginseno-side derivative boosts innate immune responses of monocytesandmacrophagesrdquo Journal of Ginseng Research vol 37 no 3 pp293ndash299 2013


[24] C K Youn S J Park M Y Lee et al ldquoSilibinin inhibits LPS-inducedmacrophage activation by blocking p38MAPK inRAW2647 cellsrdquo Biomolecules and Therapeutics vol 21 no 4 pp258ndash263 2013

[25] T Shen J LeeMH Park et al ldquoGinsenosideRp1 a ginsenoside

derivative blocks promoter activation of iNOS and Cox-2genes by suppression of an IKK120573-mediated NF-120581B pathway inHEK293 cellsrdquo Journal of Ginseng Research vol 35 no 2 pp200ndash208 2011

[26] M H Kim Y J Son S Y Lee et al ldquoJAK2-targeted anti-inflammatory effect of a resveratrol derivative 24-dihydroxy-N-(4-hydroxyphenyl)benzamiderdquo Biochemical Pharmacologyvol 86 no 12 pp 1747ndash1761 2013

[27] J K Yoo H Y Jung C Kim W S Son and J K Kim ldquoMiR-7641 modulates the expression of CXCL1 during endothelialdifferentiation derived from human embryonic stem cellsrdquoArchives of Pharmacal Research vol 36 no 3 pp 353ndash358 2013

[28] S E Byeon T Yu Y Yang et al ldquoHydroquinone regulateshemeoxygenase-1 expression via modulation of Src kinaseactivity through thiolation of cysteine residuesrdquo Free RadicalBiology and Medicine vol 57 pp 105ndash118 2013

[29] D Shin KWKimH Y Chung S Yoon and JMoon ldquoEffect ofsinapic acid against carbon tetrachloride-induced acute hepaticinjury in ratsrdquoArchives of Pharmacal Research vol 36 no 5 pp626ndash633 2013

[30] S Okabe H Miyake and Y Awane ldquoCytoprotective effectsof NC-1300 and omeprazole on HClsdotethanol-induced gastriclesions in ratsrdquo Japanese Journal of Pharmacology vol 42 no1 pp 123ndash133 1986

[31] W S YangD JeongGNamet al ldquoAP-1 pathway-targeted inhi-bition of inflammatory responses in LPS-treated macrophagesand EtOHHCl-treated stomach by Archidendron clypeariamethanol extractrdquo Journal of Ethnopharmacology vol 146 no2 pp 637ndash644 2013

[32] T Yu Y Yi Y Yang J Oh D Jeong and J Y Cho ldquoThe piv-otal role of TBK1 in inflammatory responses mediated bymacrophagesrdquoMediators of Inflammation vol 2012 Article ID979105 8 pages 2012

[33] S E Byeon Y S Yi J Oh B C Yoo S Hong and J Y CholdquoThe role of Src kinase in macrophage-mediated inflammatoryresponsesrdquo Mediators of Inflammation vol 2012 Article ID512926 18 pages 2012

[34] E S Han J Y Oh and H Park ldquoCordyceps militaris extractsuppresses dextran sodium sulfate-induced acute colitis inmiceand production of inflammatory mediators from macrophagesand mast cellsrdquo Journal of Ethnopharmacology vol 134 no 3pp 703ndash710 2011

[35] T Yu J Shim Y Yang et al ldquo3-(4-(tert-Octyl)phenox-y)propane-12-diol suppresses inflammatory responses via inhi-bition of multiple kinasesrdquo Biochemical Pharmacology vol 83no 11 pp 1540ndash1551 2012

[36] S E Byeon J Lee B C Yoo et al ldquoP38-Targeted inhibitionof interleukin-12 expression by ethanol extract from Cordy-ceps bassiana in lipopolysaccharide-activated macrophagesrdquoImmunopharmacology and Immunotoxicology vol 33 no 1 pp90ndash96 2011

[37] D K Park and H Park ldquoEthanol extract of cordyceps militarisgrown on germinated soybeans attenuates dextran-sodium-sulfate-(DSS-) induced colitis by suppressing the expressionof matrix metalloproteinases and inflammatory mediatorsrdquoBioMed Research International vol 2013 Article ID 102918 10pages 2013

[38] L Lu ldquoStudy on effect of Cordyceps sinensis and artemisininin preventing recurrence of lupus nephritisrdquo Chinese Journal ofModern Developments in Traditional Medicine vol 22 no 3 pp169ndash171 2002

[39] H G Kim B Shrestha S Y Lim et al ldquoCordycepin inhibitslipopolysaccharide-induced inflammation by the suppressionof NF-120581B through Akt and p38 inhibition in RAW 2647macrophage cellsrdquo European Journal of Pharmacology vol 545no 2-3 pp 192ndash199 2006

[40] J O Lee B Shrestha G H Sung S K Han T W Kim and JM Sung ldquoCultural characteristics and fruiting body productionin Cordyceps bassianardquoMycobiology vol 38 no 2 pp 118ndash1212010

[41] GWu L Li G H Sung et al ldquoInhibition of 24-dinitrofluorob-enzene-induced atopic dermatitis by topical application of thebutanol extract of Cordyceps bassiana inNCNgamicerdquo Journalof Ethnopharmacology vol 134 no 2 pp 504ndash509 2011

[42] T J Oh S H Hyun S G Lee Y J Chun G H Sung and HK Choi ldquoNMR and GC-MS based metabolic profiling andfree-radical scavenging activities of Cordyceps pruinosamyceliacultivated under different media and light conditionsrdquo PLoSONE vol 9 no 3 Article ID e90823 2014

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


will then generate intracellular signalling events throughnonreceptor type protein tyrosine kinases including Syk Srcphosphoinositide-3-kinase (PI3K) and mitogen activatedprotein kinase (MAPK) upstream of extracellular signal-related kinase (ERK) p38 and c-Jun N-terminal kinase(JNK) Ultimately all of these intracellular responses reachactivation of transcription factors including CREB nuclearfactor-120581B (NF-120581B) and activator protein-1 (AP-1) [1 2] Theoutcomes of such activation are linked to the expression ofvarious proinflammatory genes and inflammatory mediators[3ndash5] In addition recent findings have raised the possibilitythat sustained inflammatory responses can cause seriousdiseases such as septic shock cancer diabetes gastritis andatherosclerosis[5ndash7]Therefore development of stronger andsafer anti-inflammatory remedies may contribute to morepromising strategies to effectively treat inflammation-deriveddiseases

The genus Cordyceps which includes Cordyceps sinen-sis Cordyceps militaris Cordyceps pruinosa and Cordycepsbassiana comprises ethnopharmacologically valuable mush-rooms in Korea China and Japan [8] The genus Cordycepshas been traditionally used for the treatment of variousinflammatory and infectious diseases such as eczema skindiseases chronic bronchitis asthma and tuberculosis orused as a tonic for longevity endurance and vitality [8 9]ThemushroomCordycepswas also described in ancient tradi-tional Chinese medicine literatures as a medicinal ingredientprescribed for relieving chronic bronchitis chronic obstruc-tive pulmonary disease and tuberculosis [10 11] Additionalstudies have found that these mushrooms have numerousbiological activities based on antioxidative anticancer antidi-abetes antibacterial antifungal and antifatigue properties[12 13] Fromphytochemical approaches it has been reportedthat cordycepin ergosterol peroxide and polysaccharides arethe major components responsible for the various pharma-cological activities of Cordyceps [14] However in spite oftheir excellent pharmaceutical and nutraceutical potential itis difficult to mass produce these mushrooms To overcomethe limitations of mass production of Cordyceps species ourgroup has developed an artificial culturing condition witha grain rice-enriched medium that is able to achieve massproduction of fruit bodies of Cordyceps militaris Cordycepspruinosa and Cordyceps bassiana under defined cultureconditions

Among the mushrooms that we demonstrated to havegrown in a mass production setting we focused especiallyon Cordyceps pruinosa (Figure 1(a)) as only a few papershave reported its pharmacological activities towards cancerand inflammation [15 16] and the cultivation rate of its fruitbody is much higher than other species Although the anti-inflammatory activity of the methanol extract of Cordycepspruinosa has been suggested previously by determination ofIL-1120573 TNF-120572 NO and PGE

2levels [15] the exact molec-

ular mechanism for these effects has not been fully elu-cidated Therefore in this studywe aimed to explore anti-inflammatory mechanism of Cordyceps pruinosa butanolfraction by using kinase assays luciferase reporter assaysmolecular binding assays and overexpression strategies aswell as a mouse model of gastritis

Table 1 PCR primers used in this study

Name Sequence (51015840 to 31015840)

iNOS F CCCTTCCGAAGTTTCTGGCAGCAGR GGCTGTCAGAGCCTCGTGGCTTTGG

TNF-120572 F TTGACCTCAGCGCTGAGTTGR CCTGTAGCCCACGTCGTAGC

IL-6 F GTACTCCAGAAGACCAGAGGR TGCTGGTGACAACCACGGCC

GAPDH F CACTCACGGCAAATTCAACGGCAR GACTCCACGACATACTCAGCAC

2 Materials and Methods

21 Materials Artificially cultivated fresh fruiting bodiesof Cordyceps pruinosa were obtained from Mushtech Co(Chuncheon Korea) andwere authenticated byDr JM Sung(Mushtech Chuncheon Korea) A voucher specimen(EFCC 11968) was deposited in the EntomopathogenicFungal Culture Collection Kangwon National UniversityKorea Forskolin cordycepin (3-45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) H

2DCFDA

fluorescein isothiocyanate (FITC)-dextran phorbol 12-myristate 13-acetate (PMA) and LPS from Escherichia coli0111B4 were purchased from Sigma-Aldrich (St Louis MO)BAY 11-7082 was obtained from Calbiochem (La Jolla CA)Luciferase constructs containing binding promoters for NF-120581B CREB and AP-1 were gifts from Professor Chung HaeYoung (Pusan National University Pusan Korea) and ManHee Rhee (Kyungpook National University Daegu Korea)Enzyme immunosorbent assay (ELISA) kits for determininglevels of TNF-120572 were purchased from Amersham (LittleChalfont Buckinghamshire UK) Foetal bovine serum(FBS) and RPMI1640 were obtained from GIBCO (GrandIsland NY) RAW2647 and HEK293 cells were purchasedfrom ATCC (Rockville MD) All other chemicals were ofSigma-Aldrich grade Phosphospecific and total antibodiesfor transcription factors (p65 p50 and c-Jun) MAPK (ERKp38 and JNK) I120581B120572 IKK120573 AKT lamin AC and 120573-actinwere obtained from Cell Signalling Technology (BeverlyMA) Primers (Table 1) were designed in our laboratory andwere synthesized by Bioneer (Daejeon Korea)

22 Preparation and Characteristics of Cp-BF After dryingthe fruiting bodies of Cordyceps pruinosa at 50∘C an ethanolextract was prepared and several solvent fractions weresubsequently prepared with n-hexane n-butanol and ethylacetate After evaporation of the fractions under reducedpressure each fraction was dried using a freeze-dryer to givesolid subfractions Among the various fractions the butanolfraction (Cp-BF) gave a 7 yieldThe phytochemical charac-teristics of Cp-BF were identified by high performance liquidchromatography (HPLC) analysis as reported previously [17]the system was equipped with KNAUER (Wellchrom HPLC-pump K-1001 Wellchrom fast scanning spectrophotometerK-2600 and 4 channel degasser K-500) The elution solventsconsisted of distilled water and acetonitrileThe gradient step


(a)


Residue

n-Hexane fr Residue

EtOAc fr


(476 g)

(167 g) EtOAc (800mL) X3

n-BuOH (800mL) X3


(75 g)

H2O

H2O

H2OCH2Cl2 fr

CH2Cl2 (800mL) X3

(b)

Cordycepin

Rt (min) [area]

Cordycepin

Cp-BF

148min [127]

153min [112]

Abso

rban

ce at

260

nm

100

50

0

0 10 20 30 40

Rt (min) [area]

Abso

rban

ce at

260

nm

100

50

0

0 10 20 30 40

Rt (min) [area]

Abso

rban

ce at

260

nm

100

50

0

0 10 20 30 40

(c)





















570)













3 Results








NO

pro

duct

ion

( o

f con

trol)

Cp-BF (120583gmL)+ +

50+

100+

200+25

0

20

40

60

80

100

120


minus minus

lowastlowast


(a)

TNF-120572

pro

duct

ion

( o

f con

trol)

+ +100

+200

0

20

40

60

80

100

120


minus minus

lowastlowast

(b)

0

20

40

60

80

100

120

140

Phag

ocyt

ic u

ptak

e (

of c

ontro

l)

50 10025125Cp-BF (120583gmL)

minus minus

lowast


(c)

0

200

400

600

800

ROS

gene

ratio

n (M

FI)


Cp-BF (120583gmL)LPS (1120583gmL)


H2DCFDA (50120583M)

(d)

0

20

40

60

80

100

120

140


Cel

l via

bilit

y (

of c

ontro

l)

0 50 100 20025Cp-BF (120583gmL)

(e)





IL-6

+50

+100

+200

+

GAPDH

iNOS


LPS (1120583gmL)6h

TNF-120572

(a)

0

2

4

6

8

10

12

14

16

AP-1CREB

Stimulus

Luci

fera

se ac

tivity

(fol

d in

crea

se)

+ +50

+100

+200

+25


NF-120581B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

p65

+ + + + + ++ + +

p50

Lamin ACc-Jun


15min 30min 60min

(c)


4 Discussion






+ + + ++ + + + + + + +

p-ERKERK

p-JNK

p-p38p38

JNK



p-I120581B120572I120581B120572

120573-Actin

+ + + + + ++ + +

p-Akt

Akt



120573-Actin

IKK120572120573

p-IKK120572120573

2min 3min 5min

(a)

0

20

40

60

80

100

120

140

Cp-BF

Kina

se ac

tivity

( o

f con

trol)

+ +(200120583gmL)


lowast

(b)

0

50

100

150

200

250

300

(fold

incr

ease

)

0+

200+

100+

50+

minus

minus

Cp-BF (120583gmL)

p-I120581B120572

120573-Actin

NF-120581

B lu

cife

rase

activ

ity

IKK120573 (05 120583g)


(c)

p-p65

+ +100

p-p50

HEK293 cells

p-I120581B120572120573-Actin


minus minus

(d)

WB

+ ++

+IgG Input

+

LC


I120581B120572Cp-BF (100120583gkg)


(e)

0

20

40

60

80

100

120

NO

pro

duct

ion

and

phag

ocyt

ic

upta

ke (

of c

ontro

l)

0 10 15 0 10 15


BAY11-7082 (120583M)


lowastlowast

lowastlowast

(f)




EtOHHCl

(200mgkg) (40mgkg)

(a)

0

20

40

60

80

100

120


Size

of b

lood

spot

s (

of c

ontro

l)

(200mgkg) (40mgkg)

lowastlowast

lowastlowast

(b)

EtOHHCl + ++

Stomach

minusminusminus

p-I120581B120572

120573-Actin

Cp-BF (200mgkg)

(c)







LPS


SrcSyk

p85PI3K

PDK1AKT

AP-1 CREB

MKK36 MKK47

ERK p38

c-fos c-Jun

MEK12

iNOS

NO

HClEtOH

Cp-BF

IL-6Pro-TNF-120572

NF-120581B

TNF-120572

I120581B120572

IKK120572120573

JNK







Abbreviations





reaction





Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)


Residue

n-Hexane fr Residue

EtOAc fr


(476 g)

(167 g) EtOAc (800mL) X3

n-BuOH (800mL) X3


(75 g)

H2O

H2O

H2OCH2Cl2 fr

CH2Cl2 (800mL) X3

(b)

Cordycepin

Rt (min) [area]

Cordycepin

Cp-BF

148min [127]

153min [112]

Abso

rban

ce at

260

nm

100

50

0

0 10 20 30 40

Rt (min) [area]

Abso

rban

ce at

260

nm

100

50

0

0 10 20 30 40

Rt (min) [area]

Abso

rban

ce at

260

nm

100

50

0

0 10 20 30 40

(c)





















570)













3 Results








NO

pro

duct

ion

( o

f con

trol)

Cp-BF (120583gmL)+ +

50+

100+

200+25

0

20

40

60

80

100

120


minus minus

lowastlowast


(a)

TNF-120572

pro

duct

ion

( o

f con

trol)

+ +100

+200

0

20

40

60

80

100

120


minus minus

lowastlowast

(b)

0

20

40

60

80

100

120

140

Phag

ocyt

ic u

ptak

e (

of c

ontro

l)

50 10025125Cp-BF (120583gmL)

minus minus

lowast


(c)

0

200

400

600

800

ROS

gene

ratio

n (M

FI)


Cp-BF (120583gmL)LPS (1120583gmL)


H2DCFDA (50120583M)

(d)

0

20

40

60

80

100

120

140


Cel

l via

bilit

y (

of c

ontro

l)

0 50 100 20025Cp-BF (120583gmL)

(e)





IL-6

+50

+100

+200

+

GAPDH

iNOS


LPS (1120583gmL)6h

TNF-120572

(a)

0

2

4

6

8

10

12

14

16

AP-1CREB

Stimulus

Luci

fera

se ac

tivity

(fol

d in

crea

se)

+ +50

+100

+200

+25


NF-120581B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

p65

+ + + + + ++ + +

p50

Lamin ACc-Jun


15min 30min 60min

(c)


4 Discussion






+ + + ++ + + + + + + +

p-ERKERK

p-JNK

p-p38p38

JNK



p-I120581B120572I120581B120572

120573-Actin

+ + + + + ++ + +

p-Akt

Akt



120573-Actin

IKK120572120573

p-IKK120572120573

2min 3min 5min

(a)

0

20

40

60

80

100

120

140

Cp-BF

Kina

se ac

tivity

( o

f con

trol)

+ +(200120583gmL)


lowast

(b)

0

50

100

150

200

250

300

(fold

incr

ease

)

0+

200+

100+

50+

minus

minus

Cp-BF (120583gmL)

p-I120581B120572

120573-Actin

NF-120581

B lu

cife

rase

activ

ity

IKK120573 (05 120583g)


(c)

p-p65

+ +100

p-p50

HEK293 cells

p-I120581B120572120573-Actin


minus minus

(d)

WB

+ ++

+IgG Input

+

LC


I120581B120572Cp-BF (100120583gkg)


(e)

0

20

40

60

80

100

120

NO

pro

duct

ion

and

phag

ocyt

ic

upta

ke (

of c

ontro

l)

0 10 15 0 10 15


BAY11-7082 (120583M)


lowastlowast

lowastlowast

(f)




EtOHHCl

(200mgkg) (40mgkg)

(a)

0

20

40

60

80

100

120


Size

of b

lood

spot

s (

of c

ontro

l)

(200mgkg) (40mgkg)

lowastlowast

lowastlowast

(b)

EtOHHCl + ++

Stomach

minusminusminus

p-I120581B120572

120573-Actin

Cp-BF (200mgkg)

(c)







LPS


SrcSyk

p85PI3K

PDK1AKT

AP-1 CREB

MKK36 MKK47

ERK p38

c-fos c-Jun

MEK12

iNOS

NO

HClEtOH

Cp-BF

IL-6Pro-TNF-120572

NF-120581B

TNF-120572

I120581B120572

IKK120572120573

JNK







Abbreviations





reaction





Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























570)













3 Results








NO

pro

duct

ion

( o

f con

trol)

Cp-BF (120583gmL)+ +

50+

100+

200+25

0

20

40

60

80

100

120


minus minus

lowastlowast


(a)

TNF-120572

pro

duct

ion

( o

f con

trol)

+ +100

+200

0

20

40

60

80

100

120


minus minus

lowastlowast

(b)

0

20

40

60

80

100

120

140

Phag

ocyt

ic u

ptak

e (

of c

ontro

l)

50 10025125Cp-BF (120583gmL)

minus minus

lowast


(c)

0

200

400

600

800

ROS

gene

ratio

n (M

FI)


Cp-BF (120583gmL)LPS (1120583gmL)


H2DCFDA (50120583M)

(d)

0

20

40

60

80

100

120

140


Cel

l via

bilit

y (

of c

ontro

l)

0 50 100 20025Cp-BF (120583gmL)

(e)





IL-6

+50

+100

+200

+

GAPDH

iNOS


LPS (1120583gmL)6h

TNF-120572

(a)

0

2

4

6

8

10

12

14

16

AP-1CREB

Stimulus

Luci

fera

se ac

tivity

(fol

d in

crea

se)

+ +50

+100

+200

+25


NF-120581B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

p65

+ + + + + ++ + +

p50

Lamin ACc-Jun


15min 30min 60min

(c)


4 Discussion






+ + + ++ + + + + + + +

p-ERKERK

p-JNK

p-p38p38

JNK



p-I120581B120572I120581B120572

120573-Actin

+ + + + + ++ + +

p-Akt

Akt



120573-Actin

IKK120572120573

p-IKK120572120573

2min 3min 5min

(a)

0

20

40

60

80

100

120

140

Cp-BF

Kina

se ac

tivity

( o

f con

trol)

+ +(200120583gmL)


lowast

(b)

0

50

100

150

200

250

300

(fold

incr

ease

)

0+

200+

100+

50+

minus

minus

Cp-BF (120583gmL)

p-I120581B120572

120573-Actin

NF-120581

B lu

cife

rase

activ

ity

IKK120573 (05 120583g)


(c)

p-p65

+ +100

p-p50

HEK293 cells

p-I120581B120572120573-Actin


minus minus

(d)

WB

+ ++

+IgG Input

+

LC


I120581B120572Cp-BF (100120583gkg)


(e)

0

20

40

60

80

100

120

NO

pro

duct

ion

and

phag

ocyt

ic

upta

ke (

of c

ontro

l)

0 10 15 0 10 15


BAY11-7082 (120583M)


lowastlowast

lowastlowast

(f)




EtOHHCl

(200mgkg) (40mgkg)

(a)

0

20

40

60

80

100

120


Size

of b

lood

spot

s (

of c

ontro

l)

(200mgkg) (40mgkg)

lowastlowast

lowastlowast

(b)

EtOHHCl + ++

Stomach

minusminusminus

p-I120581B120572

120573-Actin

Cp-BF (200mgkg)

(c)







LPS


SrcSyk

p85PI3K

PDK1AKT

AP-1 CREB

MKK36 MKK47

ERK p38

c-fos c-Jun

MEK12

iNOS

NO

HClEtOH

Cp-BF

IL-6Pro-TNF-120572

NF-120581B

TNF-120572

I120581B120572

IKK120572120573

JNK







Abbreviations





reaction





Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results








NO

pro

duct

ion

( o

f con

trol)

Cp-BF (120583gmL)+ +

50+

100+

200+25

0

20

40

60

80

100

120


minus minus

lowastlowast


(a)

TNF-120572

pro

duct

ion

( o

f con

trol)

+ +100

+200

0

20

40

60

80

100

120


minus minus

lowastlowast

(b)

0

20

40

60

80

100

120

140

Phag

ocyt

ic u

ptak

e (

of c

ontro

l)

50 10025125Cp-BF (120583gmL)

minus minus

lowast


(c)

0

200

400

600

800

ROS

gene

ratio

n (M

FI)


Cp-BF (120583gmL)LPS (1120583gmL)


H2DCFDA (50120583M)

(d)

0

20

40

60

80

100

120

140


Cel

l via

bilit

y (

of c

ontro

l)

0 50 100 20025Cp-BF (120583gmL)

(e)





IL-6

+50

+100

+200

+

GAPDH

iNOS


LPS (1120583gmL)6h

TNF-120572

(a)

0

2

4

6

8

10

12

14

16

AP-1CREB

Stimulus

Luci

fera

se ac

tivity

(fol

d in

crea

se)

+ +50

+100

+200

+25


NF-120581B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

p65

+ + + + + ++ + +

p50

Lamin ACc-Jun


15min 30min 60min

(c)


4 Discussion






+ + + ++ + + + + + + +

p-ERKERK

p-JNK

p-p38p38

JNK



p-I120581B120572I120581B120572

120573-Actin

+ + + + + ++ + +

p-Akt

Akt



120573-Actin

IKK120572120573

p-IKK120572120573

2min 3min 5min

(a)

0

20

40

60

80

100

120

140

Cp-BF

Kina

se ac

tivity

( o

f con

trol)

+ +(200120583gmL)


lowast

(b)

0

50

100

150

200

250

300

(fold

incr

ease

)

0+

200+

100+

50+

minus

minus

Cp-BF (120583gmL)

p-I120581B120572

120573-Actin

NF-120581

B lu

cife

rase

activ

ity

IKK120573 (05 120583g)


(c)

p-p65

+ +100

p-p50

HEK293 cells

p-I120581B120572120573-Actin


minus minus

(d)

WB

+ ++

+IgG Input

+

LC


I120581B120572Cp-BF (100120583gkg)


(e)

0

20

40

60

80

100

120

NO

pro

duct

ion

and

phag

ocyt

ic

upta

ke (

of c

ontro

l)

0 10 15 0 10 15


BAY11-7082 (120583M)


lowastlowast

lowastlowast

(f)




EtOHHCl

(200mgkg) (40mgkg)

(a)

0

20

40

60

80

100

120


Size

of b

lood

spot

s (

of c

ontro

l)

(200mgkg) (40mgkg)

lowastlowast

lowastlowast

(b)

EtOHHCl + ++

Stomach

minusminusminus

p-I120581B120572

120573-Actin

Cp-BF (200mgkg)

(c)







LPS


SrcSyk

p85PI3K

PDK1AKT

AP-1 CREB

MKK36 MKK47

ERK p38

c-fos c-Jun

MEK12

iNOS

NO

HClEtOH

Cp-BF

IL-6Pro-TNF-120572

NF-120581B

TNF-120572

I120581B120572

IKK120572120573

JNK







Abbreviations





reaction





Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NO

pro

duct

ion

( o

f con

trol)

Cp-BF (120583gmL)+ +

50+

100+

200+25

0

20

40

60

80

100

120


minus minus

lowastlowast


(a)

TNF-120572

pro

duct

ion

( o

f con

trol)

+ +100

+200

0

20

40

60

80

100

120


minus minus

lowastlowast

(b)

0

20

40

60

80

100

120

140

Phag

ocyt

ic u

ptak

e (

of c

ontro

l)

50 10025125Cp-BF (120583gmL)

minus minus

lowast


(c)

0

200

400

600

800

ROS

gene

ratio

n (M

FI)


Cp-BF (120583gmL)LPS (1120583gmL)


H2DCFDA (50120583M)

(d)

0

20

40

60

80

100

120

140


Cel

l via

bilit

y (

of c

ontro

l)

0 50 100 20025Cp-BF (120583gmL)

(e)





IL-6

+50

+100

+200

+

GAPDH

iNOS


LPS (1120583gmL)6h

TNF-120572

(a)

0

2

4

6

8

10

12

14

16

AP-1CREB

Stimulus

Luci

fera

se ac

tivity

(fol

d in

crea

se)

+ +50

+100

+200

+25


NF-120581B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

p65

+ + + + + ++ + +

p50

Lamin ACc-Jun


15min 30min 60min

(c)


4 Discussion






+ + + ++ + + + + + + +

p-ERKERK

p-JNK

p-p38p38

JNK



p-I120581B120572I120581B120572

120573-Actin

+ + + + + ++ + +

p-Akt

Akt



120573-Actin

IKK120572120573

p-IKK120572120573

2min 3min 5min

(a)

0

20

40

60

80

100

120

140

Cp-BF

Kina

se ac

tivity

( o

f con

trol)

+ +(200120583gmL)


lowast

(b)

0

50

100

150

200

250

300

(fold

incr

ease

)

0+

200+

100+

50+

minus

minus

Cp-BF (120583gmL)

p-I120581B120572

120573-Actin

NF-120581

B lu

cife

rase

activ

ity

IKK120573 (05 120583g)


(c)

p-p65

+ +100

p-p50

HEK293 cells

p-I120581B120572120573-Actin


minus minus

(d)

WB

+ ++

+IgG Input

+

LC


I120581B120572Cp-BF (100120583gkg)


(e)

0

20

40

60

80

100

120

NO

pro

duct

ion

and

phag

ocyt

ic

upta

ke (

of c

ontro

l)

0 10 15 0 10 15


BAY11-7082 (120583M)


lowastlowast

lowastlowast

(f)




EtOHHCl

(200mgkg) (40mgkg)

(a)

0

20

40

60

80

100

120


Size

of b

lood

spot

s (

of c

ontro

l)

(200mgkg) (40mgkg)

lowastlowast

lowastlowast

(b)

EtOHHCl + ++

Stomach

minusminusminus

p-I120581B120572

120573-Actin

Cp-BF (200mgkg)

(c)







LPS


SrcSyk

p85PI3K

PDK1AKT

AP-1 CREB

MKK36 MKK47

ERK p38

c-fos c-Jun

MEK12

iNOS

NO

HClEtOH

Cp-BF

IL-6Pro-TNF-120572

NF-120581B

TNF-120572

I120581B120572

IKK120572120573

JNK







Abbreviations





reaction





Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-6

+50

+100

+200

+

GAPDH

iNOS


LPS (1120583gmL)6h

TNF-120572

(a)

0

2

4

6

8

10

12

14

16

AP-1CREB

Stimulus

Luci

fera

se ac

tivity

(fol

d in

crea

se)

+ +50

+100

+200

+25


NF-120581B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

p65

+ + + + + ++ + +

p50

Lamin ACc-Jun


15min 30min 60min

(c)


4 Discussion






+ + + ++ + + + + + + +

p-ERKERK

p-JNK

p-p38p38

JNK



p-I120581B120572I120581B120572

120573-Actin

+ + + + + ++ + +

p-Akt

Akt



120573-Actin

IKK120572120573

p-IKK120572120573

2min 3min 5min

(a)

0

20

40

60

80

100

120

140

Cp-BF

Kina

se ac

tivity

( o

f con

trol)

+ +(200120583gmL)


lowast

(b)

0

50

100

150

200

250

300

(fold

incr

ease

)

0+

200+

100+

50+

minus

minus

Cp-BF (120583gmL)

p-I120581B120572

120573-Actin

NF-120581

B lu

cife

rase

activ

ity

IKK120573 (05 120583g)


(c)

p-p65

+ +100

p-p50

HEK293 cells

p-I120581B120572120573-Actin


minus minus

(d)

WB

+ ++

+IgG Input

+

LC


I120581B120572Cp-BF (100120583gkg)


(e)

0

20

40

60

80

100

120

NO

pro

duct

ion

and

phag

ocyt

ic

upta

ke (

of c

ontro

l)

0 10 15 0 10 15


BAY11-7082 (120583M)


lowastlowast

lowastlowast

(f)




EtOHHCl

(200mgkg) (40mgkg)

(a)

0

20

40

60

80

100

120


Size

of b

lood

spot

s (

of c

ontro

l)

(200mgkg) (40mgkg)

lowastlowast

lowastlowast

(b)

EtOHHCl + ++

Stomach

minusminusminus

p-I120581B120572

120573-Actin

Cp-BF (200mgkg)

(c)







LPS


SrcSyk

p85PI3K

PDK1AKT

AP-1 CREB

MKK36 MKK47

ERK p38

c-fos c-Jun

MEK12

iNOS

NO

HClEtOH

Cp-BF

IL-6Pro-TNF-120572

NF-120581B

TNF-120572

I120581B120572

IKK120572120573

JNK







Abbreviations





reaction





Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















+ + + ++ + + + + + + +

p-ERKERK

p-JNK

p-p38p38

JNK



p-I120581B120572I120581B120572

120573-Actin

+ + + + + ++ + +

p-Akt

Akt



120573-Actin

IKK120572120573

p-IKK120572120573

2min 3min 5min

(a)

0

20

40

60

80

100

120

140

Cp-BF

Kina

se ac

tivity

( o

f con

trol)

+ +(200120583gmL)


lowast

(b)

0

50

100

150

200

250

300

(fold

incr

ease

)

0+

200+

100+

50+

minus

minus

Cp-BF (120583gmL)

p-I120581B120572

120573-Actin

NF-120581

B lu

cife

rase

activ

ity

IKK120573 (05 120583g)


(c)

p-p65

+ +100

p-p50

HEK293 cells

p-I120581B120572120573-Actin


minus minus

(d)

WB

+ ++

+IgG Input

+

LC


I120581B120572Cp-BF (100120583gkg)


(e)

0

20

40

60

80

100

120

NO

pro

duct

ion

and

phag

ocyt

ic

upta

ke (

of c

ontro

l)

0 10 15 0 10 15


BAY11-7082 (120583M)


lowastlowast

lowastlowast

(f)




EtOHHCl

(200mgkg) (40mgkg)

(a)

0

20

40

60

80

100

120


Size

of b

lood

spot

s (

of c

ontro

l)

(200mgkg) (40mgkg)

lowastlowast

lowastlowast

(b)

EtOHHCl + ++

Stomach

minusminusminus

p-I120581B120572

120573-Actin

Cp-BF (200mgkg)

(c)







LPS


SrcSyk

p85PI3K

PDK1AKT

AP-1 CREB

MKK36 MKK47

ERK p38

c-fos c-Jun

MEK12

iNOS

NO

HClEtOH

Cp-BF

IL-6Pro-TNF-120572

NF-120581B

TNF-120572

I120581B120572

IKK120572120573

JNK







Abbreviations





reaction





Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















EtOHHCl

(200mgkg) (40mgkg)

(a)

0

20

40

60

80

100

120


Size

of b

lood

spot

s (

of c

ontro

l)

(200mgkg) (40mgkg)

lowastlowast

lowastlowast

(b)

EtOHHCl + ++

Stomach

minusminusminus

p-I120581B120572

120573-Actin

Cp-BF (200mgkg)

(c)







LPS


SrcSyk

p85PI3K

PDK1AKT

AP-1 CREB

MKK36 MKK47

ERK p38

c-fos c-Jun

MEK12

iNOS

NO

HClEtOH

Cp-BF

IL-6Pro-TNF-120572

NF-120581B

TNF-120572

I120581B120572

IKK120572120573

JNK







Abbreviations





reaction





Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LPS


SrcSyk

p85PI3K

PDK1AKT

AP-1 CREB

MKK36 MKK47

ERK p38

c-fos c-Jun

MEK12

iNOS

NO

HClEtOH

Cp-BF

IL-6Pro-TNF-120572

NF-120581B

TNF-120572

I120581B120572

IKK120572120573

JNK







Abbreviations





reaction





Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















reaction





Acknowledgment


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 ikk -targeted anti-inflammatory activities of a

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