Aktivitas Inflamasi Dr Sambiloto

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 210736 16 pageshttpdxdoiorg1011552013210736

Research ArticleAP-1IRF-3 Targeted Anti-Inflammatory Activity ofAndrographolide Isolated from Andrographis paniculata

Ting Shen1 Woo Seok Yang1 Young-Su Yi1 Gi-Ho Sung2 Man Hee Rhee3

Haryoung Poo4 Mi-Yeon Kim5 Kyung-Woon Kim6 Jong Heon Kim7 and Jae Youl Cho1

1 Department of Genetic Engineering Sungkyunkwan University Suwon 440-746 Republic of Korea2Department of Herbal Crop Research National Institute of Horticultural amp Herbal ScienceRural Development Administration Suwon 441-707 Republic of Korea

3 College of Veterinary Medicine Kyungpook National University Daegu 702-701 Republic of Korea4 Laboratory of Chemical Genomics Korea Research Institute of Chemical Technology Daejeon 305-600 Republic of Korea5 School of Systems Biological Science Soongsil University Seoul 156-743 Republic of Korea6Animal Biotechnology Division National Institute of Animal Science RDA Suwon 441-706 Republic of Korea7 Cancer Cell andMolecular Biology Branch Research Institute National Cancer Center Goyang Gyeonggi 410-769 Republic of Korea

Correspondence should be addressed to Jong Heon Kim jhkimnccrekr and Jae Youl Cho jaechoskkuedu

Received 26 January 2013 Accepted 9 May 2013

Academic Editor Seung-Heon Hong

Copyright copy 2013 Ting Shen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Andrographolide (AG) is an abundant component of plants of the genus Andrographis and has a number of beneficial propertiesincluding neuroprotective anticancer anti-inflammatory and antidiabetic effects Despite numerous pharmacological studiesthe precise mechanism of AG is still ambiguous Thus in the present study we investigated the molecular mechanisms of AGand its target proteins as they pertain to anti-inflammatory responses AG suppressed the production of nitric oxide (NO) andprostaglandin E

2(PGE

2) as well as the mRNA abundance of inducible NO synthase (iNOS) tumor necrosis factor-alpha (TNF-120572)

cyclooxygenase (COX)-2 and interferon-beta (IFN-120573) in a dose-dependent manner in both lipopolysaccharide- (LPS-) activatedRAW2647 cells and peritoneal macrophages AG also substantially ameliorated the symptoms of LPS-induced hepatitis andEtOHHCl-induced gastritis in mice Based on the results of luciferase reporter gene assays kinase assays and measurement ofnuclear levels of transcription factors the anti-inflammatory effects of AG were found to be clearly mediated by inhibition ofboth (1) extracellular signal-regulated kinase (ERK)activator protein (AP)-1 and (2) I120581B kinase 120576 (IKK120576)interferon regulatoryfactor (IRF)-3 pathways In conclusion we detected a novel molecular signaling pathway by which AG can suppress inflammatoryresponses Thus AG is a promising anti-inflammatory drug with two pharmacological targets

1 Introduction

Acute and chronic inflammatory responses are both partof the natural defense mechanisms of the bodyrsquos innateimmune system and through such reactions the body isable to maintain immunological homeostasis Numerous ofreports continue to suggest that inflammatory responses areaccompanied by serious diseases including septic shockcancer atherosclerosis rheumatoid arthritis and diabetes [12] implying that inflammatory responses are a critical patron

to the pathophysiology of several diseases Thus manipu-lation of inflammatory responses may allow for preventionof serious diseases or symptoms Indeed significant efforthas been focused on exploring the molecular mechanismsof inflammatory responses to find therapeutic targets andin parallel on developing strong and safe anti-inflammatorydrugs to prevent or reverse many kinds of inflammation-related diseases Inflammatory events trigger macrophageactivation to produce nitric oxide (NO) prostaglandin

2 Evidence-Based Complementary and Alternative Medicine

O

O

CH

H2C

CH3

CH2

HCH2OH

HO

HO

H3C

Figure 1 Chemical structure of AG

E2(PGE2) tumor necrosis factor (TNF)-120572 or interleukin

(IL)-1120573 [3] Accordingly most studies have been performedusing macrophages and various toll-like receptor (TLR)ligands such as lipopolysaccharides (LPSs)

Although macrophage-mediated inflammatory respons-es are not fully understood several aspects have been charac-terized as follows (1) interactions between surface receptorssuch as TLR-4 or TLR-2 and their ligands are impor-tant [4] (2) subsequent activation occurs between adapter-inducing interferon-120573 (TRIF) and adaptor molecules includ-ing myeloid differentiation response gene 88 (MyD88)and TollInterleukin-1 receptor domain containing and(3) there is a significant cross-activation between MyD88transforming-growth-factor120573-activated kinase 1 (TAK1) andmitogen activated protein kinases (MAPKs) such as extra-cellular signal-regulated kinase (ERK) which is linked toactivation of nuclear factor-120581B (NF-120581B) and activator protein(AP)-1 [4 5] or TRIF TANK-binding kinase (TBK) andI120581B kinase 120576 (IKK120576) which are linked to the stimulationof interferon regulatory factor (IRF)-3-mediated interferon(IFN)-120572120573 production [6]

Recent findings include the critical function of Januskinase 2 (JAK2) and its counter-activated transcription fac-tormdashsignal transducer and activator of transcription (STAT-1)mdashin regulating the expression of proinflammatory genes[7] Although JAK2 activity linked to phosphorylation ofSTAT-1 or STAT-2 is increased by direct interaction withTLR4 on a time scale of a few minutes a major mechanismof activation of this enzyme during inflammatory responsesoccurs during late phase autologous stimulation with type IIFN-120572120573

AG (Figure 1) is a diterpenoid compound isolated fromAndrographis paniculata a medicinal plant ethnopharma-cologically prescribed in Korea China and Japan [8] AG

exhibits antioxidative anti-cancer anti-inflammatory antidi-abetes and antiaging properties [9 10] Although no AGderivatives have been tested in clinical trials to date sev-eral investigators have tried to mass-produce AG and itsderivatives due to its multiple inhibitory activities [11ndash14]The proposed anti-inflammatory effects of AG are basedon observations showing that it can suppress production ofinflammatory mediators including NO TNF-120572 IL-6 IL-12and PGE

2in activated macrophages [15 16] AG mediates

its anti-inflammatory effects by inhibiting the activities ofERK12 (MAPK pathway) and AKT (PI3 K pathway) in LPS-induced macrophages [17] In addition the ability of AG toeither activate protein phosphatase 2A and Akt or suppressAkt phosphorylation has been reported [18]

We performed a compound screen to identify anti-inflammatory drugs with high efficacy and low toxicityAmong several candidate compounds AG was chosenfor further study in macrophage-mediated inflammatoryresponses Although several studies have reported that AG isan anti-inflammatory drug the molecular anti-inflammatorymechanisms of AG were still vague Therefore in this studywe focused on exploring novel molecular mechanisms ofanti-inflammatory action of AG by studying TLR-mediatedinflammatory responses in macrophages Here we perceivethat AG exhibits anti-inflammatory effects in vitro and in vivovia novel pathways by inhibiting not only ERKAP-1 but alsoIKK120576IRF-3

2 Materials and Methods

21 Materials AG (MW 35046 Figure 1) indomethacin (3-45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromideMTT polyethylenimine (PEI) sodium carboxyl methylcell-ulose (Na CMC) pam3CSK and LPS (E coli 0111B4) werepurchased from Sigma-Aldrich Chemical Co (St Louis MOUSA) Poly(I C) U0126 (14-diamino-23-dicyano-14-bis [2-aminophenylthio] butadiene) SB203580 (4-[5-(4-fluorophe-nyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyri-dine) SP600125 (19-pyrazoloanthrone) and AG490 (2-cya-no-3-(34-dihydroxyphenyl)-N-(phenylmethyl)-2-propena-mide) were purchased from Calbiochem (La Jolla CAUSA) Rofecoxib (4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan- 2-one) was obtained from Amore Pacific RampD Center(Suwon Korea) Lipofectamine 2000was obtained from Invi-trogen (Carlsbad CA USA) Luciferase constructs contain-ing promoters for IRF-3 and AP-1 were a gift from ProfessorChung Hae Young (Pusan National University PusanKorea) and Addgene (Cambridge MA USA) An enzymeimmunoassay (EIA) kit for detecting PGE

2production was

purchased from Amersham (Little Chalfont Buckingha-mshire UK) The ERK kinase assay kit was purchased fromMillipore (Billerica MA USA) Fetal bovine serum (FBS)and RPMI1640 were obtained from Gibco (Grand IslandNY USA) RAW2647 and human embryonic kidney 293(HEK293) cells were purchased from ATCC (Rockville MDUSA) All other chemicals were of analytical grade Antibod-ies specific for either the total or phosphorylated forms ofc-Fos c-Jun ATF2 CEBP Fra-1 STAT-1 IRF-3 TBK1 IKK120576tyrosine threonine serine Janus kinase (JAK)2 extracellular

Evidence-Based Complementary and Alternative Medicine 3

signal-related kinase (ERK) c-Jun N-terminal kinase (JNK)p38 Akt I120581B120572 120574-tubulin and 120573-actin were obtained fromeither Cell Signaling Technologies (Beverly MA USA) orSanta Cruz Biotechnology (Santa Cruz CA USA)

22 Mice Six-week-old male ICR BalbC and C57BL6mice were purchased from Daehan Biolink (ChungbukKorea) Mice were given pellets (Samyang Daejeon Korea)and water ad libitum under a 12 h lightdark cycle Studieswere performed in accordance with guidelines established bythe Kangwon National University Institutional Animal Careand Use Committee

23 Preparation ofMouse PeritonealMacrophages andHumanTonsil-Derived Macrophages Peritoneal exudates wereobtained fromC57BL6 male mice (7-8 weeks old 17ndash21 g) bylavage 4 days after intraperitoneal injection of 1mL of sterile4 thioglycollate broth (Difco Laboratories Detroit MIUSA) as described previously [19] After washing with RPMI1640 medium containing 2 FBS peritoneal macrophages(1 times 106 cellsmL) were plated in 100mm tissue culture dishesfor 4 h at 37∘C in a 5 CO

2humidified atmosphere To

prepare human tonsil-derived macrophages [20] surgicallyremoved tonsils were cut into 2-3mm thick pieces anddigested for 15min at 37∘C with collagenase (BoehringerMannheim Germany) After rinsing with RPMI 1640 bycentrifugation at 400timesg for 7min cells were filtered througha 70120583mnylonmesh Next red blood cells were depleted withGeyrsquos solution cells were washed with RPMI 1640 mediumcontaining 2FBS and the resulting tonsil-derived cells wereplated in Petri dishes After four hours adherent cells werecollected using a cell scraper and used as macrophages Afterwashing the cells again human tonsil-derived macrophages(2times106 cellsmL) were plated to evaluate the PGE

2inhibitory

activity of AGThe use of human tissues was approved by theInstitutional Review Board of Yonsei University College ofMedicine in Korea

24 Cell Culture RAW2647 cells a murine macrophage cellline and HEK293 cells were maintained in RPMI 1640or DMEM supplemented with 100UmL of penicillin100 120583gmL of streptomycin and 10 FBS Cells were grownat 37∘C and 5 CO

2in humidified air

25 Determination of NO and PGE2Production RAW2647

cells and peritoneal macrophages (1 times 106 cellsmL) werepreincubated for 18 h after which cells were treated with AG(0 to 100 120583M) for 30min and then further incubated withLPS (1120583gmL) for 24 h NO production was determined byGriess assay and the production of PGE

2was determined by

an enzyme immunoassay kit as described previously [21]

26 Cell Viability Assay RAW2647 cells (1 times 106 cellsmL)were preincubated 18 h after which AG (0 to 100 120583M)was added to the cell suspensions and incubated for 24 hCytotoxic effects of AG were evaluated by conventionalMTT assay as described previously [22] Briefly 3 h prior toculture termination 10 120583L of an MTT solution (10mgmL in

phosphate buffered saline pH 74) was added and cells werereturned to the incubator After 3 h the MTT reaction wasquenched by the addition of 15 sodium dodecyl sulfate intoeach well which solubilized formazan produced by cells [23]Absorbance at 570ndash630 nm (OD

570minus630) was measured with a

Spectramax 250 microplate reader

27 LPS-Induced Hepatitis Mouse Model A model of exper-imental liver inflammation was induced by LPS injectionaccording to a publishedmethod [24] Briefly fastedC57BL6mice were treated orally with AG (40mgkg) once a dayfor 6 days One hour after the final administration ofAG LPS (10mgkg) or LPS (5 120583gkg) and D-galactosamine(800mgkg) were administered intraperitoneally Each ani-mal was anesthetized with an overdose of urethane 1 h afteradministration of hepatitis inducers and blood was drawnfrom the portal vein The livers were then excised and gentlyrinsed under running tap water Serum was obtained bycentrifugation of blood at 3000 rpm for 15min The levels ofserum alanine aminotransferase (ALT) and aspartate amino-transferase (AST) were measured with a Roche Modularspectrophotometric autoanalyzer

28 EtOHHCl-Induced Gastritis Mouse Model Experimen-tal stomach inflammation was induced with EtOHHClaccording to a published method [25] Fasted ICR mice weretreated orally with AG (40mgkg) or ranitidine (40mgkg)twice per day for 3 days the in vivo dosage of AG wasdetermined based on previous in vivo tests in which 5 to30mgkg was administered tomice [26]Thirty minutes afterthe final injection of AG 400120583L of 60 ethanol in 150mMHCl was administered orally Each animal was anesthetizedwith an overdose of urethane 1 h after the administrationof necrotizing agents The stomachs were then excised andgently rinsed under running tap water After opening thestomach along the greater curvature and spreading it outon a board the area (mm2) of mucosal erosive lesions weremeasured using a pixel counter under blinded conditions

29mRNAAnalysis by Semiquantitative Reverse Transcriptase(RT) and Real-Time Polymerase Chain Reaction To deter-mine the levels of mRNA expression of inflammatory medi-ators total RNA was extracted from LPS-treated RAW2647cells with TRIzol Reagent (Gibco Grand Island NY USA)according to the manufacturerrsquos instructions Total RNA wasstored at minus70∘C until use Semi-quantitative RT reactionswere conducted as described previously [27 28] Quan-tification of mRNA was performed by real-time RT-PCRwith SYBR Premix Ex Taq according to the manufacturerrsquosinstructions (Takara Bio Inc Shiga Japan) using a real-timethermal cycler (Bio-Rad Hercules CA USA) as describedpreviously [29] The primers used (Bioneer Daejeon Korea)are listed in Table 1

210 Transfection and Luciferase Reporter Gene AssaysHEK293 or RAW2647 cells (1 times 106 cellsmL) were trans-fected with 1120583g of plasmids containing TRIF TBK1 AP-1-Luc IRF-3-Luc iNOS-Luc COX-2-Luc and 120573-galactosidase


Table 1 PCR primers used in this study

Name Sequence (51015840 to 31015840)Real-time PCR

iNOS F GGAGCCTTTAGACCTCAACAGAR TGAACGAGGAGGGTGGTG

IFN-120573 F TCCAAGAAAGGACGAACATTCGR GAGGCCATTTGGGAACTTCT

COX-2 F CACTACATCCTGACCCACTTR ATGCTCCTGCTTGAGTATGT

IRF3 F GGCTGACTTTGGCATCTTR TTCCTCTTCCAGGTTGACA

GAPDH F CAATGAATACGGCTACAGCAACR AGGGAGATGCTCAGTGTTGG

SemiquantitativePCR

iNOS F CCCTTCCGAAGTTTCTGGCAGCAGR GGCTGTCAGAGCCTCGTGGCTTTGG

TNF-120572 F TTGACCTCAGCGCTGAGTTGR CCTGTAGCCCACGTCGTAGC

IFN-120573 F CAGGATGAGGACATGAGCACCR CTCTGCAGACTCAAACTCCAC


GAPDH F CACTCACGGCAAATTCAACGGCAR GACTCCACGACATACTCAGCAC

in the absence or presence of PMAionomycin or LPS usingPEI or lipofectamine 2000 in a 12-well plate as describedpreviously [30] or according to manufacturerrsquos instructionCells were incubated for 48 h after transfection and thenused for assays Luciferase assays were performed using theLuciferase Assay System (Promega Madison WI USA) aspreviously reported [31]

211 Preparation of Cell Lysates Nuclear Fractionation andAnalysis by Immunoblotting and Immunoprecipitation Liversfrom LPS-treated mice RAW2647 cells or HEK293 cells(5times106 cellsmL) were washed 3 times in cold PBSwith 1mMsodium orthovanadate and lysed in lysis buffer (20mM Tris-HCl pH 74 2mM EDTA 2mM ethyleneglycotetraaceticacid 50mM 120573-glycerophosphate 1mM sodium orthovana-date 1mM dithiothreitol 1 Triton X-100 10 glycerol10 120583gmL aprotinin 10 120583gmL pepstatin 1mM benzimideand 2mM PMSF) for 30min under rotation at 4∘C Lysateswere clarified by centrifugation at 16000timesg for 10min at 4∘Cand stored at minus20∘C until use Nuclear lysates were preparedin a three-step procedure [32] After treatment cells were firstcollected with a rubber policeman washed with 1times PBS andlysed in 500120583L of lysis buffer (50mM KCl 05 Nonidet P-40 25mMHEPES (pH 78) 1mM phenylmethylsulfonyl flu-oride 10 120583gmL leupeptin 20120583gmL aprotinin and 100120583M14-dithiothreitol (DTT)) on ice for 4min Next cell lysateswere centrifuged at 19326timesg for 1min in a microcentrifuge

In the second step the pellet (nuclear fraction) was washedonce with wash buffer which had the same composition aslysis buffer lackingNonidet P-40 In the final step nuclei weretreated with an extraction buffer containing 500mM KCl10 glycerol and several other reagents mentioned abovefor the lysis buffer The nucleiextraction buffer mixture wasfrozen at minus80∘C and then thawed on ice and centrifuged at19326timesg for 5minThe resulting supernatantwas collected asthe nuclear extract Soluble cell lysates were immunoblottedand protein levels were quantified as described previously[33] For immunoprecipitation cell lysates containing equalamounts of protein (500120583g) from RAW2647 cells (1 times 107cellsmL) were precleared with 10120583L of protein A-coupledSepharose beads (50 vv) (Amersham Little ChalfontBuckinghamshire UK) for 1 h at 4∘C Precleared sampleswere then incubated with 5 120583L of antibody for IKK120576 IRF-3phospho-ERK or TBK1 overnight at 4∘C Immune complexeswere mixed with 10 120583L of protein A-coupled Sepharose beads(50 vv) and incubated for 3 h at 4∘C

212 ERK and IKK120576 Enzyme Assay To determine theinhibitory effects of AG or U0126 on LPS-activated ERK orIKK120576 enzyme activities immunoprecipitated phospho-ERKor IKK120576 prepared from RAW2647 cells (5 times 106 cellsmL)treated with LPS (1 120583gmL) for 30 (ERK) or 10 (IKK120576) minwere incubated with AG or U0126 in the presence orabsence of substrate protein [myelin basic protein (MBP)]or immunoprecipitated IRF-3 from unstimulated RAW2647cells according to the manufacturerrsquos instructions TheERK or IKK120576 kinase activities were determined with anti-phospho-MBP or anti-phospho-IRF-3 antibodies afterimmunoblotting analysis

213 Statistical Analysis Data are presented as themeanplusmn SDof experiments performed with 6 biological replicates (119899 = 6)per treatment and subjected to statistical analysis Similarexperimental datawere observed in three independent exper-iments For statistical comparisons results were analyzedusing ANOVA with Scheffersquos post hoc test or Kruskal-WallisMann-Whitney test Differences with values of 119875 lt005 were considered statistically significant All statisticaltests performed using SPSS (SPSS Inc Chicago IL)

3 Results

31 AG Suppresses the Production of Inflammatory Mediatorsin Macrophages AG inhibited release of NO and PGE

2in

macrophages activated by different TLR stimulators includ-ing LPS poly(I C) and pam3CSK AG suppressed LPS-induced production of NO and PGE

2in both RAW2647

cells and peritoneal macrophages which had a purity of983 as determined by flow cytometry (data not shown)in a dose-dependent manner (Figure 2(a)) with IC

50values

ranging from 64 to 367 120583M (Table 2) AG also suppressedthe production of NO and PGE

2in RAW2647 cells and

peritoneal macrophages activated by poly(I C) or pam3CSKin a dose-dependent manner with IC

50values ranging from

131 to 395 120583M and from 163 120583M to 437 120583M respectively


0 625 125 25 500

20

40

60

80

100

120

NO

and

PGE 2

prod

uctio

n (

of c

ontro

l)

AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

NO

0 625 125 25 500

20

40

60

80

100

120

NO

and

PGE 2

prod

uctio

n (

of c

ontro

l)

AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

NOPGE2 PGE2

RAW2647 cells LPS (1120583gmL) Peritoneal macrophages LPS (1120583gmL)

(a)

0

20

40

60

80

100

120 Human tonsil-derived macrophages

AG (120583M)LPS (1120583gmL) minus

minusminus

++

50

lowast

PGE 2

prod

uctio

n (

of c

ontro

l)

(b)

0

20

40

60

80

100

120

RAW2647 cellsPeritoneal macrophages

Cel

l via

bilit

y (

of c

ontro

l)

AG (120583M)0 625 125 25 50

(c)

Figure 2 The effect of AG on production of inflammatory mediators and cell viability (a) RAW2647 cells (1 times 106 cellsmL) (left panels)peritoneal macrophages (2times106 cellsmL) (right panels) or (b) human tonsil-derivedmacrophages (2times106 cellsmL) were treated with AG inthe absence or presence of LPS (1120583gmL) for 24 h Supernatants were collected and the concentration of NO and PGE

2in the supernatant was

determined by Griess assay and EIA as described in Section 2 (c) Viability of RAW2647 cells (1 times 106 cellsmL) and peritoneal macrophages(2times106 cellsmL) treated with AGwere evaluated byMTT assay Data are presented as themean plusmn SD of an experiment done with 6 biologicalreplicates (119899 = 6) per treatment lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control group

(Table 2) Moreover PGE2production in LPS-treated human

tonsil-derived macrophages was also remarkably suppressedupon treatment with AG (50120583M) (Figure 2(b)) The viabilityof RAW2647 cells and peritoneal macrophages was notaffected by exposure to AG for either 6 or 12 h and treatmentfor 24 h produced only weak cytotoxicity (less than 20) atthe highest AG concentration tested (50120583M) (Figure 2(c))Similarly the AG derivative dehydro-AG also strongly inhib-ited NO production in LPS-treated RAW2647 cells at 50 120583Mwith no cytotoxicity (data not shown)

32 AG Suppresses the mRNA Expression of InflammatoryGenes in Macrophages Semi-quantitative RT-PCR(Figure 3(a)) and real-time PCR (Figure 3(b)) revealedthat AG (50120583M) strongly suppressed the mRNA expressionof inflammatory genes (iNOS COX-2 and TNF-120572) inRAW2647 cells To confirm the suppressive effects of AGtowards these inflammatory mediators at the transcriptionallevel reporter gene assays were conducted using constructscontaining either the COX-2 or iNOS promoter regionlinked to a luciferase gene In accordance with our PCR


Table 2 Inhibitory potency of AG on the production of inflamma-tory mediators in RAW2647 cells and peritoneal macrophages

Cells Ligand IC50 value (120583M)NO PGE2

RAW2647cells

LPS 144 plusmn 03 367 plusmn 06

Poly (I C) 131 plusmn 06 289 plusmn 05

Pam3CSK 163 plusmn 07 212 plusmn 05

Peritonealmacrophages




RAW2647 cells (1 times 106 cellsmL) or peritoneal macrophages (2 times106 cellsmL) were treated with LPS (1120583gmL) poly(I C) (200120583gmL) orpam3CSK (10120583gmL) in the presence or absence ofAG for 24 h Supernatantswere collected and the concentrations of NO and PGE2 in the supernatantswere determined by Griess assay and EIA respectively as described inSection 2

results AG strongly suppressed PMAionomycin enhancedpromoter activity of iNOS and COX-2 in a dose-dependentmanner (Figure 3(c))

33 AG Inhibits Activation of AP-1 and STAT-1 in Macro-phages The nuclear translocation patterns of p65 (NF-120581B)members of the AP-1 family (c-Fos c-Jun ATF-2 Fra-1 andCEBP) and STAT-1 are shown in Figures 4(a) and 4(b) andAG suppressed nuclear localization of c-Fos phospho-ATF-2 and phospho-STAT-1 However unlike previous reports[34] AG did not block the LPS stimulated increase in nucleartranslocation of p65 levels after 15min (data not shown)Together these findings suggest that upstream signalingevents for activation of the AP-1 family and STAT-1 are majortargets of AG in LPS-mediated inflammatory responses

34 ERK is a Primary Target of AG in Inhibition of AP-1 Acti-vation Reporter gene assays confirmed that AG significantlysuppressed AP-1 activation triggered by LPS in RAW2647cells in a dose-dependentmanner (Figure 5(a))However AGdid not suppress NF-120581B activation induced by either TNF-120572treatment or by cotransfection with MyD88 TRIF or TBK1(data not shown) This finding differs from that of previousreports [34] however we did find that TRAM-induced NF-120581B activation was diminished by AG in a dose-dependentmanner (data not shown) suggesting that NF-120581B inhibitionby AG appears to be signal dependent

Because AP-1 translocation is mainly regulated byMAPKs we investigated the possible involvement of ERKp38 and JNK in AG inhibition No inhibition of phospho-MAPK levels by AG was observed up to 60min of incuba-tion (Figure 5(b)) suggesting that AG does not inhibit theactivities of upstream kinases responsible for MAPK phos-phorylationHowever the possibility remained thatAGcoulddirectly suppress the activities of ERK p38 or JNK Usinga reporter gene assay we next investigated the contributionof MAPK to AP-1 activation As shown in Figure 5(c) (leftpanel) the ERK inhibitor U0126 but not other inhibitors

strongly attenuated AP-1-mediated luciferase activity sug-gesting that ERK may be the major pathway involved in AP-1 translocation To confirm whether AG is capable of abro-gating the enhanced enzyme activity of ERK a direct kinaseassay was performed As shown in Figure 5(c) (right panel)AG suppressed the phosphorylation of the ERK substrateMBP although its inhibitory activity was not stronger thanthat of U0126 This result strongly suggests that AG is able todirectly modulate the enzyme activity of ERK

35 IKK120576 is an Additional Target of AG in Inhibiting IRF-3 and STAT-1 Activation Because the levels of phospho-STAT-1 but not total STAT-1 were decreased by AG in thenuclear fraction we next examined the inhibitory mecha-nism of AG against STAT activation (Figure 4(b)) First thepossibility of JAK2 involvement an essential requirement forthe phosphorylation of STAT-1 [35] was tested As shown inFigure 6(a) AG diminished the phosphorylation of JAK2 at75min up to 55 but not at significantly earlier time points(2 to 5min) suggesting that AG-mediated inhibition of JAK2activation is not a directearly event Because late phaseactivation of JAK2 is known to be autologously activatedby type I interferons such as IFN-120573 we examined whetherAG suppresses IFN-120573 production As expected AG clearlyinhibited the upregulation of luciferase activity mediated byIRF-3 binding to the IFN-120573-promoter region after cotrans-fection with TRIF or TBK1 (Figure 6(b)) Further mRNAlevels of IFN-120573 evaluated by RT-PCR (Figure 6(c) left panel)and real-time PCR (Figure 6(c) right panel) were decreasedby AG In agreement with these results AG also suppressednuclear levels of phospho-IRF-3 induced by LPS in a dose-dependent manner (Figure 6(d)) To examine whether AGinhibits the activities of upstream kinases such as TBK1 andIKK120576 [36 37] which induce IRF-3 phosphorylation [36]we analyzed the activities of both TBK1 and IKK120576 by invitro kinase assay Specifically we looked at the formationof molecular complexes between these enzymes As shownin Figure 6(e) while AG strongly blocked the kinase activityof IKK120576 responsible for IRF-3 phosphorylation the kinaseactivity of TBK1 was not blocked by AG (data not shown)Similarly complex formation between IKK120576 and TBK1 acritical step for the activation of IKK120576 was suppressed by AGup to 60 (Figure 6(f))

36 AG Ameliorates LPS-Induced Hepatitis and EtOHHCl-Induced Gastritis in Animal Disease Models To examinewhether AG has anti-inflammatory effects in animal modelsof inflammatory disease we employed LPS-induced hepatitisand EtOHHCl-induced gastritis in mice Orally adminis-tered AG (40mgkg) significantly attenuated LPS-inducedliver damage (Figure 7(a) right panel) and decreased elevatedserum levels of ALT and AST derived from damaged hepa-tocytes (Figure 7(a) left panel) ConverselyD-galactosamineboosted LPS-induced hepatic damage leading to a dramaticincrease of serum parameters (ALT and AST) up to 1527and 1950ULWe also examined whether AG could suppressenhanced phospho-ATF-2 level in livers from LPS-treatedmice In agreement with in vitro effect (Figure 4(a) left


LPS (1120583gmL)AG (50120583M) minusminus

minus

+

+ +

iNOS

COX-2

TNF-120572

GAPDH

6h

(a)

0

20

40

60

80

100

120

140

NorLPSLPS + AG (50120583M)

iNOS COX-2 TNF-120572

lowastlowast

lowastlowastlowastlowast

mRN

A le

vel (

o

f con

trol)

(b)

0

2

4

6

8

10

12

iNOS-LucCOX-2-Luc

Luci

fera

se ac

tivity

(fol

d in

crea

se)

Normal minus 25 50

lowastlowast

lowastlowast

lowast

lowast

AG (120583M)

PMA (100nM) + ionomycin (1120583M)

(c)

Figure 3The effect of AG onmRNA expression of iNOS COX-2 and TNF-120572 genes in LPS-treated RAW2647 cells RAW2647 cells (5times106cellsmL) were incubated with AG in the absence or presence of LPS (1120583gmL) for 6 h mRNA levels for iNOS COX-2 and TNF-120572 weredetermined by (a) semi-quantitative PCR and (b) by real-time PCR GAPDH was used as an internal control (c) The promoter activitiesof iNOS and COX-2 were determined by luciferase reporter gene assay in HEK293 cells as described in Section 2 Data ((b) and (c)) arepresented as the mean plusmn SD of an experiment done with 6 biological replicates (119899 = 6) per treatment (a) is a representative image of threedifferent gels with similar results lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control group

panel) AG strongly suppressed ATF-2 phosphorylation inthe LPS-induced hepatitis mouse model (Figure 7(b)) In thecase of the EtOHHCl-induced gastritis model AG stronglyreduced the area of the gastric lesions similar to ranitidine(40mgkg) (Figure 7(c)) and rofecoxib (10mgkg) a selectiveCOX-2 inhibitor (data not shown) suggesting that AG is anorally available anti-inflammatory drugwith effects similar tothose of known anti-inflammatory remedies [38]

4 Discussion

AG was originally identified as a component of the medic-inal plant Andrographis paniculata known in Korea asCheonshimryeon and in China as Chuan Xin Lian and hasbeen traditionally prescribed for numerous inflammatorydiseases including infection inflammation cold fever anddiarrhea in Korea China and Japan [39 40] A significant


00

02

04

06

08

10

12

c-Fos

minusminus

minus ++++

125 25 50

LPS (1120583gmL)AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Rela

tive i

nten

sity

c-Fos25 50

15min

c-Jun

p-CEBP

CEBP

LaminAC

minusminus

++++minusLPS (1120583gmL)AG (120583M) 125

p-ATF-2

p-ATF-2

p-Fra-1Fra-1

p-Fra-1

(a)

STAT-1

p-ST

AT-1

(rel

ativ

e int

ensit

y)

00

02

04

06

08

10

12

LPS (1120583gmL)AG (50120583M) minus +minus

minus + +

120min

lowastlowast

120574-tubulin

p-STAT-1

(b)

Figure 4 The effect of AG on nuclear translocation of total or phosphoforms of transcription factors ((a) (b)) RAW2647 cells (5 times 106cellsmL) were incubated with AG in the absence or presence of LPS (1120583gmL) for the indicated time After preparing nuclear fractionslevels of total or phospho-forms of nuclear localized transcription factors namely (a) c-Fos c-Jun p-ATF-2 p-CEBP p-Fra-1 CEBP andFra-1 and (b) p-STAT-1 and STAT-1 were identified by immunoblotting Relative intensities were calculated with a DNR Bioimaging system(GelQuant software version 27) Data ((a) right panel and (b) lower panel) are presented as the mean plusmn SD of relative intensity values of 3blots (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control group


0

2

4

6

8

10

LPS (1120583gmL)AG(120583M)

minus

minusminus

+++

25 50

RAW2647 cells

lowastlowast

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

(a)

LPS (1120583gmL)minus

minus

minusminusminus + + +

++++++

p-ERK

ERK

p-p38

p38

p-JNK

120573-actin

5min 30min 60min

JNK

AG (50120583M)

(b)

PMA (100nM)U0126 (20120583M)SB203580 (10120583M)SP600125 (10120583M)AG490 (25120583M)

HEK293 cells

0

5

10

15

20

25

30

35

minusminus

minus

minus

minus

minus

minus

minus

minus

minus minus

minusminus minus

minus

minus

minus

minus

minus

minus

minus

+ + ++

+

+

+

+ +

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

00

02

04

06

08

10

12

p-MBP

lgG heavy chain

lowast

lowastlowast

LPS (1120583gmL)AG (50120583M)

minus

minus

minus

minus

minus minus

minus

+

++

+

+

30min

Rela

tive i

nten

sity

U0126(20 120583M)

(c)

Figure 5 The effect of AG on AP-1 activation (a) RAW2647 cells cotransfected with AP-1-luciferase and 120573-gal constructs (as a transfectioncontrol) were treated with AG in the absence or presence of LPS (1120583gmL) for 12 h Luciferase activity was determined by luminescence asdescribed in Section 2 (b) RAW2647 cells (5 times 106 cellsmL) were incubated with AG in the absence or presence of LPS (1120583gmL) for theindicated times After preparing whole cell lysates levels of total or phospho-forms of upstream signaling enzymes (ERK p38 and JNK) wereidentified by immunoblotting ((c) left panel) HEK293 cells co-transfectedwithAP-1-luciferase and120573-gal constructs were treatedwithMAPKinhibitors (U0126 SB203580 and SP600125) or the JAK2 inhibitor AG490 in the absence or presence of PMA (100 nM) for 12 h Luciferaseactivities were determined by luminescence ((c) right panel) A direct ERK enzyme assay was performed with immunoprecipitated phospho-ERK prepared fromRAW2647 cells (5times106 cellsmL) incubated with LPS (1120583gmL) for 30min in the presence or absence of AG orU0126 for30min using an ERK kinase assay kit Relative intensities of phospho-MBP level were calculated with a DNR Bioimaging system (Gelquantsoftware version 27) Data are presented as the mean plusmn SD of an experiment done with 6 biological replicates (119899 = 6) per treatment ((a) and(c) left panels) or 3 different blots ((c) right lower panel) Other data (b) are representative images of three different experiments that hadsimilar results lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control group

body of scientific evidence supports the ethnopharmacolog-ical significance of Andrographis paniculata and its com-ponents with respect to their therapeutic efficacy againstinflammatory diseases Specifically the ethyl acetate fractionof this plant strongly attenuates LPS-induced lung inflam-mation [41] Likewise carrageenan-induced inflammatorysymptoms were also reported to be suppressed bymethanolic

extracts of Andrographis paniculata [42] AG was identifiedas a major active component of Andrographis paniculata andhas been demonstrated to have anti-inflammatory activityas assessed by the carrageenan-induced inflammation model[43] an ovalbumin-A-induced mouse asthma model [34]and an allergic-lung inflammation model [16] Our exper-iments also clearly supported the anti-inflammatory effects


p-JAK2

JAK2

p-JAK2

JAK2

2min 25min 5min

AG (50120583M)

AG (50120583M)

minus

minus minus minus minus

+

+

+ +++

+

+

+

minus

minus

minus minusminus

+ +

+

+ +

+

+ +

+

45min 60min 75min

01 03 1 12 13 16 07 RI

LPS (1120583gmL)

LPS (1120583gmL)

(a)

0

5

10

15

20

25

TRIFTBK1

minus minus 125 25 50

AG (120583M)TRIF or TBK1

IRF-

3-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

lowast

lowast

lowastlowastlowastlowastlowastlowast

HEK293 cells

(b)

0

20

40

60

80

100

120

LPS (1120583gmL)AG (120583M)

minus

minus minus

+++

25 50

IFN

-120573m

RNA

leve

l (

of c

ontro

l)

lowastlowast

lowastlowast

GAPDH

IFN-120573

COX-2

iNOS

LPS (1120583gmL)AG (50120583M)

1hminus

minus minus

+

+

+

(c)

Rela

tive i

nten

sity

02

04

06

08

12

120574-tubulin

p-IRF-3

minusminus

minus ++ ++

125 25 50

5min

lowastlowast

LPS (1120583gmL)AG (120583M)

1

0

(d)

Figure 6 Continued


IP IKK120576 or IRF-3

Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min

LPS (1120583gmL) minus

minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)

Figure 6 The effect of AG on JAK2STAT-1 signaling (a) RAW2647 cells (5 times 106 cellsmL) were incubated with AG in the absence orpresence of LPS (1120583gmL) for the indicated times After preparing whole cell lysates levels of total or phospho-forms of JAK2 were examinedby immunoblotting (b) HEK293 cells cotransfected with IRF-3-luciferase and 120573-gal constructs (as a transfection control) were treated withAG in the absence or presence (cotransfection) of inducer protein constructs (TRIF or TBK1) for 12 h Luciferase activity was determined byluminescence (c) RAW2647 cells (5 times 106 cellsmL) were incubated with AG in the absence or presence of LPS (1120583gmL) for 1 h Levelsof mRNA for IFN-120573 iNOS and COX-2 were determined by semi-quantitative RT-PCR (left panel) or real-time PCR (right panel) (d)RAW2647 cells (5 times 106 cellsmL) were incubated with AG in absence or the presence of LPS (1120583gmL) for 5min After preparing nuclearfractions levels of total or phospho-forms of IRF-3 were examined by immunoprecipitation and immunoblotting (e) IKK120576 kinase activitywith AGwas determined by a conventional kinase assay with immunoprecipitated IKK120576 fromRAW2647 cells treated with LPS for 10min andimmunoprecipitated IRF-3 from normal RAW2647 cells (f) Molecular complex formation between TBK1 and IKK120576 from LPS (1 120583gmL)-treated RAW2647 cells was examined by immunoblotting Relative intensities were calculated with the DNR Bio-imaging system (Gelquantsoftware version 27) Data are presented as the mean plusmn SD of an experiment done with 6 biological replicates (119899 = 6) per treatment (b) and(c) right panel) or 3 biological replicates (119899 = 3) per treatment ((d) lower panel and (e) lower panel) The other data ((a) (c) left panel (d)upper panel and (f)) are representative images of three different experiments that showed similar results RI Relative intensity lowast119875 lt 005and lowastlowast119875 lt 001 compared to the control group

of AG on inflammatory responses both in vitro and in vivoAG suppressed the production of major inflammatory medi-ators such as NO and PGE

2in a dose-dependent manner

regardless of macrophage type (primary or cancer cells)(Figure 2) as well as inflammatory stimuli [LPS pam3CSKand poly(IC)] (data not shown) The mild-conditionedhepatitis symptoms in LPS-treated mice (Figure 7(a)) as wellas severe inflammatory stomach lesions in EtOHHCl-treatedmice (Figure 7(b)) were also robustly suppressed by orallyadministered AG (40mgkg)

The anti-inflammatory effects of AG appear to be due toits ability to inhibit various inflammatory mediators at thetranscriptional level Our data indicated that AG-mediatedinhibition was strongly linked to suppression of mRNAexpression of inflammatory genes such as iNOS COX-2 andTNF-120572 (Figure 3) In addition to our data other researchgroups have shown that AG can strongly suppress transcrip-tional upregulation of proinflammatory cytokines (TNF-120572IL-1120573 IL-4 IL-5 IL-6 and IL-13) cytokine receptors (IL-1120573Rand TNFR) adhesion molecules (E-selectin and VCAM-1)


Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT

AG (40mgkg) minusminus

minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)

HClEtOH +AG (40mgkg)HClEtOHNormal

HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)

Figure 7 In vivo anti-inflammatory effect of AG on LPS-induced hepatitis and HClEtOH-induced gastritis animal models Mice wereadministered AG orally for 6 days and treated intraperitoneally with LPS or LPSD-galactosamine to induce hepatitis After 1 h ((a) leftpanel) livers were resected and photos were taken ((a) right panel) after which serum was prepared to measure biochemical parameters(AST and ALT) (b) The inhibitory activity of AG on the phosphorylation of transcription factor ATF2 in the livers from LPS-treated micewas examined by immunoblottingThe levels of total or phospho-forms of ATF2 were measured from whole liver lysates prepared from LPS-treated mice (c) Mice were orally administered with AG or ranitidine for 3 days and treated orally with EtOHHCl to induce gastritis After1 h ((c) left panel) stomachs were resected and photos were taken of the gastric lesions in the stomach The areas of gastric lesions ((c) rightpanel) were measured Data ((a) right panel and (c) right panel) are presented as the mean plusmn SD of an experiment done with 6 mice (119899 = 6)lowast

119875 lt 005 compared to the control group

and chemokines (CCL8 and CXCL11) in macrophagesvascular endothelial cells and eosinophils [34 44 45]

In the present study the COX-2 inhibitor rofecoxibstrongly ameliorated HClEtOH-induced stomach inflam-mation (data not shown) Given that COX-1 inhibitors(aspirin indomethacin and other NSAIDs) are known toinduce gastritis [46] and potent NOS inhibitors (eg NG-Nitro-L-argininemethyl ester) exacerbate the EtOH-inducedgastric ulcer index by up to 2-fold [47] we considered thepossibility that COX-2 activity or COX-2-mediated levels ofPGE2could play a pathophysiological role in the generation

of EtOHHCl-induced stomach inflammation Thus AG-mediated inhibition of COX-2 expression was considered tobe one of themajor contributing factors in its gastroprotectiveactivity towards NO and TNF-120572

The ability of AG to inhibit transcriptional activationhas been mostly reported via its ability to suppress NF-120581B activation [48] Regarding the inhibitory mechanism ofAG it has been demonstrated that AG binds to reducedcysteine 62 of the NF-120581B p50 subunit following thiolationand AG fails to inhibit binding of p50 in a C62S mutant [48]However under our conditions when NF-120581B activation was


TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B

c-Fos c-JunI120581B120572

STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase

AG-targeted pathways

ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3

Figure 8 Schematic diagram of the proposed molecular anti-inflammatory mechanism of AG

induced by LPS and TNF-120572 or cotransfection with adaptormolecules such as MyD88 and TRIF we did not observeAG-mediated inhibition of p65 translocation and NF-120581Bactivation although TRAM-induced NF-120581B activation in areporter gene assay and LPS-induced I120581B120572 phosphorylationwere both dose-dependently suppressed by AG (data notshown) On the contrary there is a report that AG blocks p65nuclear translocation and DNA-binding activity in nuclearextracts from lung tissues of OVA-challenged mice [34]Likewise a report that AG can increase protein phosphatase2A activity and subsequent dephosphorylation of p65 at S536[18] appears to support our results According to our dataAP-1 and IRF-3 are critical transcription factor targets ofAG pharmacology in LPS-treated macrophages SpecificallyAP-1-induced luciferase activity in LPS-activated RAW2647cells was suppressed by AG (Figure 5(a)) which also clearlydecreased levels of both nuclear and whole cell c-Fos andphospho-ATF-2 in LPS-treated RAW2647 cells (Figure 4(a))andmouse livers (Figure 7(b))Moreover AG decreased IRF-3-mediated luciferase activity in HEK293 cells transfectedwith TRIF and TBK1 (Figure 6(b)) as well as the abundanceof nuclear phospho-IRF-3 (Figure 6(d)) Together these datasuggest that the inflammatory pathways of the AP-1 familyand IRF-3 could be major inhibitory targets of AG in TLR4-activated macrophages Similar findings that support thispossibility were previously observed in the downregulationof MMP-7 via the inactivation and decrease of nuclear AP-1in A549 cells [49 50] although there was nomention of IRF-3as a target of AG action in these studies

In spite of numerous studies the direct molecular tar-gets of AG have not yet been fully elucidated Previousstudies have been limited mostly to simple identificationof certain biochemical phenomena including the measure-ment of phosphorylated forms of enzymes found in apoptotic

and inflammatory signaling cascades For example AG hasbeen shown to reduce protein levels of activated Akt inan HRas-transformed rat kidney epithelial (RK3E) cell line[51] phosphorylation of ERK in the invasion of CT26 cells[52] enzyme activity of 120572-glucosidase [53] phosphorylationlevels of ERK12 and AKT in LPS-treated macrophages[17] and phosphorylation of ERK15 induced by anti-CD3antibody or PMAionomycin in T cells However none ofthese studies tested whether AG directly targets a specifickinase activity Interestingly ERK is an AP-1 upstreamenzyme that was directly targeted by AG under our kinaseassay conditions (Figure 5(c) right panel) unlike a previousreport indicating that AG suppresses ERK phosphorylation[17 54] Nonetheless our data and accumulated evidencein other reports strongly suggest that ERK is a primarytarget of AG pharmacology essential for the suppression ofdownstream transcription factor AP-1 with respect to both itsanti-inflammatory and anti-cancer activities

The JAK2STAT-1 pathway inhibitor AG490 was previ-ously shown to exhibit strong anti-inflammatory activity andthus suppression of phospho-STAT-1 levels by AG is regardedas another critical event for its pharmacological action [55]However our data suggested that AG does not act as adirect inhibitor of JAK23 since early phosphorylation ofthe enzyme triggered by autophosphorylation [56] was notblocked by AG (Figure 6(a)) and JAK3 kinase activity wasnot reduced (data not shown) Rather upstream signalingrequired for late phase JAK2STAT-1 activation might be apotential target of AG Interestingly AGblocked early expres-sion of IFN-120573 but not iNOS and COX-2 during LPS stimula-tion (Figure 6(c)) and reduced luciferase activity triggered bycotransfection of an IFN-120573 promoter-containing a reportergene construct with IRF-3 binding sequences and TRIF orTBK1 (Figure 6(b)) Ultimately our kinase assays led us to


conclude that the kinase activity of IKK120576 but not TBK1 canbe directly suppressed byAG (Figure 6(e)) and thatmolecularcomplex formation between TBK1 and IKK120576 (Figure 6(f))and phosphorylation of IRF-3 were subsequently inhibited(Figure 6(d)) Therefore while our data strongly suggest thatAG has dual independent inhibitory targets (ERK and IKK120576)in TLR-activated inflammatory signaling further studies areneeded to determine how AG can simultaneously inhibit twodifferent enzymes namely ERK and IKK120576 Considering thatAG is capable of binding to cysteine residues of target proteins(p50) [48] thiolation of specific cysteine residues in theseenzymes which is also seen in other chemically reactive com-pounds such as benzene metabolites [57] and sesquiterpenelactones [21 58] could be used to explain its biochemicalmode of action The possibility of AG-induced thiolation ofboth ERK and IKK120576 will be verified in our next study

5 Conclusions

We demonstrated that AG strongly suppresses both invitro and in vivo inflammatory responses in TLR-activatedmacrophages such as the production of NO and PGE

2 and

the expression of proinflammatory genes such as iNOSCOX-2 and TNF-120572 AG also ameliorated the symptoms ofboth LPS-triggered hepatitis and EtOHHCl-induced gastri-tis in mice In particular AG was shown to act as a dualinhibitor of ERK and IKK120576 which are particularly importantin TLR4-mediated inflammation As a consequence AP-1(c-fosATF-2) and IRF-3JAK2STAT-1 pathways were sup-pressed by AG as summarized in Figure 8 AG-containingAndrographis paniculata has been ethnopharmacologicallyused for a long time and is orally available [59 60] aswell as easily detected in serum [61] Furthermore severalcarrier systems have been developed to improve the oraladsorption of AG [62] and chemical derivatization of AGhas already been performed by many scientists [43 63]Thus together with the existing literature our data stronglysuggest that AG andor its derivatives could be used as novelanti-inflammatory agents To support this possibility we willuse additional in vivo inflammatory models such as acute(septic shock and carrageenan-induced arthritis) and chronic(collagen- or adjuvant-induced arthritis) disease conditionsfor future preclinical trials

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Ting Shen Woo Seok Yang and Young-Su Yi contributedequally to this work

Acknowledgments

This research was supported by the Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) funded by the Ministry of Education Science and

Technology (no 0004975) Jong Heon Kim was supported bythe National Cancer Center Grant (1110110) of Korea

References

[1] M Ferencık V Stvrtinova I Hulın and M Novak ldquoInflam-mationmdasha lifelong companion Attempt at a non-analyticalholistic viewrdquo Folia Microbiologica vol 52 no 2 pp 159ndash1732007

[2] N Hogg ldquoFree radicals in diseaserdquo Seminars in ReproductiveEndocrinology vol 16 no 4 pp 241ndash248 1998

[3] Y G Lee W M Lee J Y Kim et al ldquoSrc kinase-targeted anti-inflammatory activity of davallialactone from Inonotus xeran-ticus in lipopolysaccharide-activated RAW2647 cellsrdquo BritishJournal of Pharmacology vol 154 no 4 pp 852ndash863 2008

[4] O Takeuchi and S Akira ldquoToll-like receptors their physiolo-gical role and signal transduction systemrdquo International Immu-nopharmacology vol 1 no 4 pp 625ndash635 2001

[5] M Yamamoto and S Akira ldquoLipid a receptor TLR4-mediatedsignaling pathwaysrdquo Advances in Experimental Medicine andBiology vol 667 pp 59ndash68 2009

[6] T Kawai and S Akira ldquoToll-like receptor downstream signal-ingrdquoArthritis Research andTherapy vol 7 no 1 pp 12ndash19 2005

[7] O S Kim E J Park E H Joe and I Jou ldquoJAK-STAT signal-ing mediates gangliosides-induced inflammatory responses inbrain microglial cellsrdquo Journal of Biological Chemistry vol 277no 43 pp 40594ndash40601 2002

[8] RM Kunwar K P Shrestha and RW Bussmann ldquoTraditionalherbal medicine in far-west Nepal a pharmacological appra-isalrdquo Journal of Ethnobiology and Ethnomedicine vol 6 article35 2010

[9] N P Trivedi U M Rawal and B P Patel ldquoHepatoprotectiveeffect of andrographolide against hexachlorocyclohexane-indu-ced oxidative injuryrdquo Integrative Cancer Therapies vol 6 no 3pp 271ndash280 2007

[10] H P Chao C D Kuo J H Chiu and S L Fu ldquoAndrographolideexhibits anti-invasive activity against colon cancer cells viainhibition of MMP2 activityrdquo Planta Medica vol 76 no 16 pp1827ndash1833 2010

[11] J Li W Huang H Zhang XWang and H Zhou ldquoSynthesis ofandrographolide derivatives and their TNF-120572 and IL-6 expres-sion inhibitory activitiesrdquo Bioorganic and Medicinal ChemistryLetters vol 17 no 24 pp 6891ndash6894 2007

[12] S R Jada G S Subur C Matthews et al ldquoSemisynthesis andin vitro anticancer activities of andrographolide analoguesrdquoPhytochemistry vol 68 no 6 pp 904ndash912 2007

[13] SNanduri V KNyavanandi S S RaoThunuguntla et al ldquoSyn-thesis and structure-activity relationships of andrographolideanalogues as novel cytotoxic agentsrdquo Bioorganic and MedicinalChemistry Letters vol 14 no 18 pp 4711ndash4717 2004

[14] Z Wang P Yu G Zhang et al ldquoDesign synthesis and antibac-terial activity of novel andrographolide derivativesrdquo Bioorganicand Medicinal Chemistry vol 18 no 12 pp 4269ndash4274 2010

[15] W F Chiou C F Chen and J J Lin ldquoMechanisms of sup-pression of inducible nitric oxide synthase (iNOS) expressionin RAW 2647 cells by andrographoliderdquo British Journal ofPharmacology vol 129 no 8 pp 1553ndash1560 2000

[16] A A Abu-Ghefreh H Canatan and C I Ezeamuzie ldquoIn vitroand in vivo anti-inflammatory effects of andrographoliderdquo Inter-national Immunopharmacology vol 9 no 3 pp 313ndash318 2009


[17] WWang J Wang S F Dong et al ldquoImmunomodulatory activ-ity of andrographolide on macrophage activation and specificantibody responserdquo Acta Pharmacologica Sinica vol 31 no 2pp 191ndash201 2010

[18] C Y Hsieh M J Hsu G Hsiao et al ldquoAndrographolide enha-nces nuclear factor-120581B subunit p65 Ser 536 dephosphorylationthrough activation of protein phosphatase 2A in vascularsmooth muscle cellsrdquo Journal of Biological Chemistry vol 286no 8 pp 5942ndash5955 2011

[19] T J Kang J S Moon S Lee and D Yim ldquoPolyacetylene com-pound from Cirsium japonicum var ussuriense inhibits theLPS-induced inflammatory reaction via suppression of NF-120581Bactivity in RAW 2647 cellsrdquo Biomolecules andTherapeutics vol19 no 1 pp 97ndash101 2011

[20] O R Lee G Sathiyaraj Y J Kim et al ldquoDefense genes inducedby pathogens and abiotic stresses in Panax ginseng C AMeyerrdquoJournal of Ginseng Research vol 35 no 1 pp 1ndash11 2011

[21] J Y Cho K U Baik J H Jung and M H Park ldquoIn vitro anti-inflammatory effects of cynaropicrin a sesquiterpene lactonefrom Saussurea lappardquo European Journal of Pharmacology vol398 no 3 pp 399ndash407 2000

[22] Y S Kim Y H Kim J R Noh E S Cho J H Park and H YSon ldquoProtective effect of Korean red ginseng against aflatoxinB1-induced hepatotoxicity in ratrdquo Journal of Ginseng Researchvol 35 no 2 pp 243ndash249 2011

[23] S K Jo J Y Hong H J Park and S K Lee ldquoAnticancer activityof novel daphnane diterpenoids from Daphne genkwa throughcell-cycle arrest and suppression of AktSTATSrc signalings inhuman lung cancer cellsrdquo Biomolecules amp Therapeutics vol 20no 6 pp 513ndash519 2012

[24] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

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

[26] A A Abu-Ghefreh H Canatan and C I Ezeamuzie ldquoIn vitroand in vivo anti-inflammatory effects of andrographoliderdquoInternational Immunopharmacology vol 9 no 3 pp 313ndash3182009

[27] YO Kim and SW Lee ldquoMicroarray analysis of gene expressionby ginseng water extracts in a mouse adrenal cortex afterimmobilization stressrdquo Journal of Ginseng Research vol 35 no1 pp 111ndash123 2011

[28] Y G Choi S Yeo S H Kim and S Lim ldquoAnti-inflammatorychanges of gene expression by Artemisia iwayomogi in theLPS-stimulated human gingival fibroblastmicroarray analysisrdquoArchives of Pharmacal Research vol 35 no 3 pp 549ndash563 2012

[29] S Kim S Shim D S Choi J H Kim Y B Kwon and J KwonldquoModulation of LPS-stimulated astroglial activation by ginsengtotal saponinsrdquo Journal of Ginseng Research vol 35 no 1 pp80ndash85 2011

[30] G Devitt MThomas A M Klibanov T Pfeiffer and V BoschldquoOptimized protocol for the large scale production of HIVpseudovirions by transient transfection of HEK293T cells withlinear fully deacylated polyethyleniminerdquo Journal of VirologicalMethods vol 146 no 1-2 pp 298ndash304 2007

[31] K K Jung H S Lee J Y Cho et al ldquoInhibitory effect of cur-cumin on nitric oxide production from lipopolysaccharide-activated primary microgliardquo Life Sciences vol 79 no 21 pp2022ndash2031 2006

[32] S E Byeon Y G Lee B H Kim et al ldquoSurfactin blocks NOproduction in lipopolysaccharide-activated macrophages byinhibiting NF-120581B activationrdquo Journal of Microbiology andBiotechnology vol 18 no 12 pp 1984ndash1989 2008

[33] J A Lee M Y Lee I S Shin C S Seo H Ha and H K ShinldquoAnti-inflammatory effects of Amomum compactum on RAW264 7 cells via induction of heme oxygenase-1rdquo Archives ofPharmacal Research vol 35 no 4 pp 739ndash746 2012

[34] Z Bao S Guan C Cheng et al ldquoA novel antiinflammatory rolefor andrographolide in asthma via inhibition of the nuclearfactor-120581b pathwayrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 657ndash665 2009

[35] W H Choi K A Ji S B Jeon et al ldquoAnti-inflammatory roles ofretinoic acid in rat brain astrocytes suppression of interferon-120574-induced JAKSTAT phosphorylationrdquo Biochemical and Bio-physical Research Communications vol 329 no 1 pp 125ndash1312005

[36] F Ikeda C M Hecker A Rozenknop et al ldquoInvolvement ofthe ubiquitin-like domain of TBK1IKK-i kinases in regulationof IFN-inducible genesrdquoThe EMBO Journal vol 26 no 14 pp3451ndash3462 2007

[37] M Solis R Romieu-Mourez D Goubau et al ldquoInvolvement ofTBK1 and IKK120576 in lipopolysaccharide-induced activation of theinterferon response in primary humanmacrophagesrdquo EuropeanJournal of Immunology vol 37 no 2 pp 528ndash539 2007

[38] S E Byeon J Y Chung Y G Lee BH Kim KH Kim and J YCho ldquoIn vitro and in vivo anti-inflammatory effects of taheeboa water extract from the inner bark of Tabebuia avellanedaerdquoJournal of Ethnopharmacology vol 119 no 1 pp 145ndash152 2008

[39] R A Burgos J L Hancke J C Bertoglio et al ldquoEfficacy of anAndrographis paniculata composition for the relief of rheuma-toid arthritis symptoms a prospective randomized placebo-controlled trialrdquo Clinical Rheumatology vol 28 no 8 pp 931ndash946 2009

[40] C V Chandrasekaran A Gupta and A Agarwal ldquoEffect of anextract of Andrographis paniculata leaves on inflammatory andallergic mediators in vitrordquo Journal of Ethnopharmacology vol129 no 2 pp 203ndash207 2010

[41] W W Chao Y H Kuo S L Hsieh and B F Lin ldquoInhibitoryeffects of ethyl acetate extract of Andrographis paniculata onNF-120581B trans-activation activity and LPS-induced acute inflam-mation in micerdquo Evidence-based Complementary and Alterna-tive Medicine vol 2011 Article ID 254531 9 pages 2011

[42] K Sheeja P K Shihab and G Kuttan ldquoAntioxidant and anti-inflammatory activities of the plant Andrographis paniculataneesrdquo Immunopharmacology and Immunotoxicology vol 28 no1 pp 129ndash140 2006

[43] C Aromdee N Sriubolmas S Wiyakrutta S Suebsasna andW Khunkitti ldquoEffect of the derivatives of andrographolide onthe morphology of Bacillus subtilisrdquo Archives of Pharmacal Re-search vol 34 no 1 pp 71ndash77 2011

[44] W Parichatikanond C Suthisisang P Dhepakson and A Her-unsalee ldquoStudy of anti-inflammatory activities of the purecompounds from Andrographis paniculata (burmf) Nees andtheir effects on gene expressionrdquo International Immunopharma-cology vol 10 no 11 pp 1361ndash1373 2010

[45] Y J Wang J T Wang Q X Fan and J G Geng ldquoAndrogra-pholide inhibits NF-120581B activation and attenuates neointimal


hyperplasia in arterial restenosisrdquo Cell Research vol 17 no 11pp 933ndash941 2007

[46] Y A Masannat M Harron and G Harinath ldquoNonsteroidalanti-inflammatory drugs-associated colopathyrdquo ANZ Journal ofSurgery vol 80 no 1-2 pp 96ndash99 2010

[47] H Kushima C A Hiruma-Lima M A Santos E Viana MCoelho-Ferreira and A R M Souza Brito ldquoGastroprotectiveactivity of Pradosia huberi on experimentally induced gastriclesions in rodents role of endogenous sulphydryls and nitricoxiderdquo Journal of Ethnopharmacology vol 101 no 1ndash3 pp 61ndash672005

[48] Y F Xia B Q Ye Y D Li et al ldquoAndrographolide attenuatesinflammation by inhibition of NF-120581B activation through cova-lent modification of reduced cysteine 62 of p 50rdquo Journal ofImmunology vol 173 no 6 pp 4207ndash4217 2004

[49] MD Shi HH Lin T A Chiang et al ldquoAndrographolide couldinhibit human colorectal carcinoma Lovo cells migration andinvasion via down-regulation of MMP-7 expressionrdquo Chemico-Biological Interactions vol 180 no 3 pp 344ndash352 2009

[50] Y C Lee H H Lin C H Hsu C J Wang T A Chiang andJ H Chen ldquoInhibitory effects of andrographolide on migrationand invasion in human non-small cell lung cancer A549 cellsvia down-regulation of PI3KAkt signaling pathwayrdquo EuropeanJournal of Pharmacology vol 632 no 1ndash3 pp 23ndash32 2010

[51] S K Hung L C Hung C D Kuo et al ldquoAndrographolidesensitizes Ras-transformed cells to radiation in vitro and invivordquo International Journal of Radiation Oncology BiologyPhysics vol 77 no 4 pp 1232ndash1239 2010


[53] J Xu S Huang H Luo et al ldquoQSAR studies on andrograph-olide derivatives as 120572-glucosidase inhibitorsrdquo InternationalJournal of Molecular Sciences vol 11 no 3 pp 880ndash895 2010

[54] H R Tsai L M Yang W J Tsai and W F Chiou ldquoAndro-grapholide acts through inhibition of ERK12 andAkt phospho-rylation to suppress chemotactic migrationrdquo European Journalof Pharmacology vol 498 no 1ndash3 pp 45ndash52 2004

[55] L Li J Zhang B Jin E R Block and J M Patel ldquoNitric oxideupregulation of caspase-8mRNAexpression in lung endothelialcells role of JAK2STAT-1 signalingrdquo Molecular and CellularBiochemistry vol 305 no 1-2 pp 71ndash77 2007

[56] S A Litherland T X Xie K M Grebe et al ldquoSignal transduc-tion activator of transcription 5 (STAT5) dysfunction in autoim-mune monocytes and macrophagesrdquo Journal of Autoimmunityvol 24 no 4 pp 297ndash310 2005

[57] T Y Lee K C Lee and H H Chang ldquoModulation of thecannabinoid receptors by andrographolide attenuates hepaticapoptosis following bile duct ligation in rats with fibrosisrdquoApoptosis vol 15 no 8 pp 904ndash914 2010

[58] A J Garcıa-Pineres V Castro G Mora et al ldquoCysteine 38in p65NF-120581B plays a crucial role in DNA binding inhibitionby sesquiterpene lactonesrdquo Journal of Biological Chemistry vol276 no 43 pp 39713ndash39720 2001

[59] K H Hessling R W Schlick R Luckey K Gratz S A AQaiyumi and E P Allhoff ldquoThe therapeutic value of the ambu-lant extracorporeal shock wave lithotripsy of salivary stonesResults of a prospective studyrdquo Laryngo-Rhino-Otologie vol 72no 3 pp 109ndash115 1993

[60] A Panossian A Hovhannisyan G Mamikonyan et al ldquoPhar-macokinetic and oral bioavailability of andrographolide from

Andrographis paniculata fixed combinationKan Jang in rats andhumanrdquo Phytomedicine vol 7 no 5 pp 351ndash364 2000

[61] S S Handa and A Sharma ldquoHepatoprotective activity of And-rographolide from Andrographis paniculata against carbonte-trachloriderdquo Indian Journal of Medical Research vol 92 pp276ndash283 1990

[62] K Ren Z Zhang Y Li et al ldquoPhysicochemical characteristicsand oral bioavailability of andrographolide complexed withhydroxypropyl-120573-cyclodextrinrdquo Pharmazie vol 64 no 8 pp515ndash520 2009

[63] G F Dai H W Xu J F Wang F W Liu and H M LiuldquoStudies on the novel 120572-glucosidase inhibitory activity andstructure-activity relationships for andrographolide analoguesrdquoBioorganic and Medicinal Chemistry Letters vol 16 no 10 pp2710ndash2713 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


O

O

CH

H2C

CH3

CH2

HCH2OH

HO

HO

H3C

Figure 1 Chemical structure of AG

E2(PGE2) tumor necrosis factor (TNF)-120572 or interleukin

(IL)-1120573 [3] Accordingly most studies have been performedusing macrophages and various toll-like receptor (TLR)ligands such as lipopolysaccharides (LPSs)

Although macrophage-mediated inflammatory respons-es are not fully understood several aspects have been charac-terized as follows (1) interactions between surface receptorssuch as TLR-4 or TLR-2 and their ligands are impor-tant [4] (2) subsequent activation occurs between adapter-inducing interferon-120573 (TRIF) and adaptor molecules includ-ing myeloid differentiation response gene 88 (MyD88)and TollInterleukin-1 receptor domain containing and(3) there is a significant cross-activation between MyD88transforming-growth-factor120573-activated kinase 1 (TAK1) andmitogen activated protein kinases (MAPKs) such as extra-cellular signal-regulated kinase (ERK) which is linked toactivation of nuclear factor-120581B (NF-120581B) and activator protein(AP)-1 [4 5] or TRIF TANK-binding kinase (TBK) andI120581B kinase 120576 (IKK120576) which are linked to the stimulationof interferon regulatory factor (IRF)-3-mediated interferon(IFN)-120572120573 production [6]

Recent findings include the critical function of Januskinase 2 (JAK2) and its counter-activated transcription fac-tormdashsignal transducer and activator of transcription (STAT-1)mdashin regulating the expression of proinflammatory genes[7] Although JAK2 activity linked to phosphorylation ofSTAT-1 or STAT-2 is increased by direct interaction withTLR4 on a time scale of a few minutes a major mechanismof activation of this enzyme during inflammatory responsesoccurs during late phase autologous stimulation with type IIFN-120572120573

AG (Figure 1) is a diterpenoid compound isolated fromAndrographis paniculata a medicinal plant ethnopharma-cologically prescribed in Korea China and Japan [8] AG

exhibits antioxidative anti-cancer anti-inflammatory antidi-abetes and antiaging properties [9 10] Although no AGderivatives have been tested in clinical trials to date sev-eral investigators have tried to mass-produce AG and itsderivatives due to its multiple inhibitory activities [11ndash14]The proposed anti-inflammatory effects of AG are basedon observations showing that it can suppress production ofinflammatory mediators including NO TNF-120572 IL-6 IL-12and PGE

2in activated macrophages [15 16] AG mediates

its anti-inflammatory effects by inhibiting the activities ofERK12 (MAPK pathway) and AKT (PI3 K pathway) in LPS-induced macrophages [17] In addition the ability of AG toeither activate protein phosphatase 2A and Akt or suppressAkt phosphorylation has been reported [18]

We performed a compound screen to identify anti-inflammatory drugs with high efficacy and low toxicityAmong several candidate compounds AG was chosenfor further study in macrophage-mediated inflammatoryresponses Although several studies have reported that AG isan anti-inflammatory drug the molecular anti-inflammatorymechanisms of AG were still vague Therefore in this studywe focused on exploring novel molecular mechanisms ofanti-inflammatory action of AG by studying TLR-mediatedinflammatory responses in macrophages Here we perceivethat AG exhibits anti-inflammatory effects in vitro and in vivovia novel pathways by inhibiting not only ERKAP-1 but alsoIKK120576IRF-3

2 Materials and Methods

21 Materials AG (MW 35046 Figure 1) indomethacin (3-45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromideMTT polyethylenimine (PEI) sodium carboxyl methylcell-ulose (Na CMC) pam3CSK and LPS (E coli 0111B4) werepurchased from Sigma-Aldrich Chemical Co (St Louis MOUSA) Poly(I C) U0126 (14-diamino-23-dicyano-14-bis [2-aminophenylthio] butadiene) SB203580 (4-[5-(4-fluorophe-nyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyri-dine) SP600125 (19-pyrazoloanthrone) and AG490 (2-cya-no-3-(34-dihydroxyphenyl)-N-(phenylmethyl)-2-propena-mide) were purchased from Calbiochem (La Jolla CAUSA) Rofecoxib (4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan- 2-one) was obtained from Amore Pacific RampD Center(Suwon Korea) Lipofectamine 2000was obtained from Invi-trogen (Carlsbad CA USA) Luciferase constructs contain-ing promoters for IRF-3 and AP-1 were a gift from ProfessorChung Hae Young (Pusan National University PusanKorea) and Addgene (Cambridge MA USA) An enzymeimmunoassay (EIA) kit for detecting PGE

2production was

purchased from Amersham (Little Chalfont Buckingha-mshire UK) The ERK kinase assay kit was purchased fromMillipore (Billerica MA USA) Fetal bovine serum (FBS)and RPMI1640 were obtained from Gibco (Grand IslandNY USA) RAW2647 and human embryonic kidney 293(HEK293) cells were purchased from ATCC (Rockville MDUSA) All other chemicals were of analytical grade Antibod-ies specific for either the total or phosphorylated forms ofc-Fos c-Jun ATF2 CEBP Fra-1 STAT-1 IRF-3 TBK1 IKK120576tyrosine threonine serine Janus kinase (JAK)2 extracellular







2inhibitory



2in humidified air



2was determined by


















SemiquantitativePCR











3 Results


2in


2in both RAW2647


50values







0 625 125 25 500

20

40

60

80

100

120

NO

and

PGE 2

prod

uctio

n (

of c

ontro

l)

AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

NO

0 625 125 25 500

20

40

60

80

100

120

NO

and

PGE 2

prod

uctio

n (

of c

ontro

l)

AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

NOPGE2 PGE2


(a)

0

20

40

60

80

100



minusminus

++

50

lowast

PGE 2

prod

uctio

n (

of c

ontro

l)

(b)

0

20

40

60

80

100

120


Cel

l via

bilit

y (

of c

ontro

l)

AG (120583M)0 625 125 25 50

(c)










RAW2647cells


















minus

+

+ +

iNOS

COX-2

TNF-120572

GAPDH

6h

(a)

0

20

40

60

80

100

120

140



lowastlowast


mRN

A le

vel (

o

f con

trol)

(b)

0

2

4

6

8

10

12

iNOS-LucCOX-2-Luc

Luci

fera

se ac

tivity

(fol

d in

crea

se)

Normal minus 25 50

lowastlowast

lowastlowast

lowast

lowast

AG (120583M)


(c)



4 Discussion



00

02

04

06

08

10

12

c-Fos

minusminus

minus ++++

125 25 50

LPS (1120583gmL)AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Rela

tive i

nten

sity

c-Fos25 50

15min

c-Jun

p-CEBP

CEBP

LaminAC

minusminus

++++minusLPS (1120583gmL)AG (120583M) 125

p-ATF-2

p-ATF-2

p-Fra-1Fra-1

p-Fra-1

(a)

STAT-1

p-ST

AT-1

(rel

ativ

e int

ensit

y)

00

02

04

06

08

10

12


minus + +

120min

lowastlowast

120574-tubulin

p-STAT-1

(b)



0

2

4

6

8

10

LPS (1120583gmL)AG(120583M)

minus

minusminus

+++

25 50

RAW2647 cells

lowastlowast

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

(a)


minus


++++++

p-ERK

ERK

p-p38

p38

p-JNK

120573-actin

5min 30min 60min

JNK

AG (50120583M)

(b)


HEK293 cells

0

5

10

15

20

25

30

35

minusminus

minus

minus

minus

minus

minus

minus

minus

minus minus

minusminus minus

minus

minus

minus

minus

minus

minus

minus

+ + ++

+

+

+

+ +

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

00

02

04

06

08

10

12

p-MBP

lgG heavy chain

lowast

lowastlowast

LPS (1120583gmL)AG (50120583M)

minus

minus

minus

minus

minus minus

minus

+

++

+

+

30min

Rela

tive i

nten

sity

U0126(20 120583M)

(c)





p-JAK2

JAK2

p-JAK2

JAK2

2min 25min 5min

AG (50120583M)

AG (50120583M)

minus


+

+

+ +++

+

+

+

minus

minus

minus minusminus

+ +

+

+ +

+

+ +

+

45min 60min 75min

01 03 1 12 13 16 07 RI

LPS (1120583gmL)

LPS (1120583gmL)

(a)

0

5

10

15

20

25

TRIFTBK1



IRF-

3-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

lowast

lowast


HEK293 cells

(b)

0

20

40

60

80

100

120

LPS (1120583gmL)AG (120583M)

minus

minus minus

+++

25 50

IFN

-120573m

RNA

leve

l (

of c

ontro

l)

lowastlowast

lowastlowast

GAPDH

IFN-120573

COX-2

iNOS

LPS (1120583gmL)AG (50120583M)

1hminus

minus minus

+

+

+

(c)

Rela

tive i

nten

sity

02

04

06

08

12

120574-tubulin

p-IRF-3

minusminus

minus ++ ++

125 25 50

5min

lowastlowast

LPS (1120583gmL)AG (120583M)

1

0

(d)

Figure 6 Continued



Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min


minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)







Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2inhibitory



2in humidified air



2was determined by


















SemiquantitativePCR











3 Results


2in


2in both RAW2647


50values







0 625 125 25 500

20

40

60

80

100

120

NO

and

PGE 2

prod

uctio

n (

of c

ontro

l)

AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

NO

0 625 125 25 500

20

40

60

80

100

120

NO

and

PGE 2

prod

uctio

n (

of c

ontro

l)

AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

NOPGE2 PGE2


(a)

0

20

40

60

80

100



minusminus

++

50

lowast

PGE 2

prod

uctio

n (

of c

ontro

l)

(b)

0

20

40

60

80

100

120


Cel

l via

bilit

y (

of c

ontro

l)

AG (120583M)0 625 125 25 50

(c)










RAW2647cells


















minus

+

+ +

iNOS

COX-2

TNF-120572

GAPDH

6h

(a)

0

20

40

60

80

100

120

140



lowastlowast


mRN

A le

vel (

o

f con

trol)

(b)

0

2

4

6

8

10

12

iNOS-LucCOX-2-Luc

Luci

fera

se ac

tivity

(fol

d in

crea

se)

Normal minus 25 50

lowastlowast

lowastlowast

lowast

lowast

AG (120583M)


(c)



4 Discussion



00

02

04

06

08

10

12

c-Fos

minusminus

minus ++++

125 25 50

LPS (1120583gmL)AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Rela

tive i

nten

sity

c-Fos25 50

15min

c-Jun

p-CEBP

CEBP

LaminAC

minusminus

++++minusLPS (1120583gmL)AG (120583M) 125

p-ATF-2

p-ATF-2

p-Fra-1Fra-1

p-Fra-1

(a)

STAT-1

p-ST

AT-1

(rel

ativ

e int

ensit

y)

00

02

04

06

08

10

12


minus + +

120min

lowastlowast

120574-tubulin

p-STAT-1

(b)



0

2

4

6

8

10

LPS (1120583gmL)AG(120583M)

minus

minusminus

+++

25 50

RAW2647 cells

lowastlowast

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

(a)


minus


++++++

p-ERK

ERK

p-p38

p38

p-JNK

120573-actin

5min 30min 60min

JNK

AG (50120583M)

(b)


HEK293 cells

0

5

10

15

20

25

30

35

minusminus

minus

minus

minus

minus

minus

minus

minus

minus minus

minusminus minus

minus

minus

minus

minus

minus

minus

minus

+ + ++

+

+

+

+ +

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

00

02

04

06

08

10

12

p-MBP

lgG heavy chain

lowast

lowastlowast

LPS (1120583gmL)AG (50120583M)

minus

minus

minus

minus

minus minus

minus

+

++

+

+

30min

Rela

tive i

nten

sity

U0126(20 120583M)

(c)





p-JAK2

JAK2

p-JAK2

JAK2

2min 25min 5min

AG (50120583M)

AG (50120583M)

minus


+

+

+ +++

+

+

+

minus

minus

minus minusminus

+ +

+

+ +

+

+ +

+

45min 60min 75min

01 03 1 12 13 16 07 RI

LPS (1120583gmL)

LPS (1120583gmL)

(a)

0

5

10

15

20

25

TRIFTBK1



IRF-

3-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

lowast

lowast


HEK293 cells

(b)

0

20

40

60

80

100

120

LPS (1120583gmL)AG (120583M)

minus

minus minus

+++

25 50

IFN

-120573m

RNA

leve

l (

of c

ontro

l)

lowastlowast

lowastlowast

GAPDH

IFN-120573

COX-2

iNOS

LPS (1120583gmL)AG (50120583M)

1hminus

minus minus

+

+

+

(c)

Rela

tive i

nten

sity

02

04

06

08

12

120574-tubulin

p-IRF-3

minusminus

minus ++ ++

125 25 50

5min

lowastlowast

LPS (1120583gmL)AG (120583M)

1

0

(d)

Figure 6 Continued



Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min


minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)







Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























SemiquantitativePCR











3 Results


2in


2in both RAW2647


50values







0 625 125 25 500

20

40

60

80

100

120

NO

and

PGE 2

prod

uctio

n (

of c

ontro

l)

AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

NO

0 625 125 25 500

20

40

60

80

100

120

NO

and

PGE 2

prod

uctio

n (

of c

ontro

l)

AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

NOPGE2 PGE2


(a)

0

20

40

60

80

100



minusminus

++

50

lowast

PGE 2

prod

uctio

n (

of c

ontro

l)

(b)

0

20

40

60

80

100

120


Cel

l via

bilit

y (

of c

ontro

l)

AG (120583M)0 625 125 25 50

(c)










RAW2647cells


















minus

+

+ +

iNOS

COX-2

TNF-120572

GAPDH

6h

(a)

0

20

40

60

80

100

120

140



lowastlowast


mRN

A le

vel (

o

f con

trol)

(b)

0

2

4

6

8

10

12

iNOS-LucCOX-2-Luc

Luci

fera

se ac

tivity

(fol

d in

crea

se)

Normal minus 25 50

lowastlowast

lowastlowast

lowast

lowast

AG (120583M)


(c)



4 Discussion



00

02

04

06

08

10

12

c-Fos

minusminus

minus ++++

125 25 50

LPS (1120583gmL)AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Rela

tive i

nten

sity

c-Fos25 50

15min

c-Jun

p-CEBP

CEBP

LaminAC

minusminus

++++minusLPS (1120583gmL)AG (120583M) 125

p-ATF-2

p-ATF-2

p-Fra-1Fra-1

p-Fra-1

(a)

STAT-1

p-ST

AT-1

(rel

ativ

e int

ensit

y)

00

02

04

06

08

10

12


minus + +

120min

lowastlowast

120574-tubulin

p-STAT-1

(b)



0

2

4

6

8

10

LPS (1120583gmL)AG(120583M)

minus

minusminus

+++

25 50

RAW2647 cells

lowastlowast

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

(a)


minus


++++++

p-ERK

ERK

p-p38

p38

p-JNK

120573-actin

5min 30min 60min

JNK

AG (50120583M)

(b)


HEK293 cells

0

5

10

15

20

25

30

35

minusminus

minus

minus

minus

minus

minus

minus

minus

minus minus

minusminus minus

minus

minus

minus

minus

minus

minus

minus

+ + ++

+

+

+

+ +

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

00

02

04

06

08

10

12

p-MBP

lgG heavy chain

lowast

lowastlowast

LPS (1120583gmL)AG (50120583M)

minus

minus

minus

minus

minus minus

minus

+

++

+

+

30min

Rela

tive i

nten

sity

U0126(20 120583M)

(c)





p-JAK2

JAK2

p-JAK2

JAK2

2min 25min 5min

AG (50120583M)

AG (50120583M)

minus


+

+

+ +++

+

+

+

minus

minus

minus minusminus

+ +

+

+ +

+

+ +

+

45min 60min 75min

01 03 1 12 13 16 07 RI

LPS (1120583gmL)

LPS (1120583gmL)

(a)

0

5

10

15

20

25

TRIFTBK1



IRF-

3-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

lowast

lowast


HEK293 cells

(b)

0

20

40

60

80

100

120

LPS (1120583gmL)AG (120583M)

minus

minus minus

+++

25 50

IFN

-120573m

RNA

leve

l (

of c

ontro

l)

lowastlowast

lowastlowast

GAPDH

IFN-120573

COX-2

iNOS

LPS (1120583gmL)AG (50120583M)

1hminus

minus minus

+

+

+

(c)

Rela

tive i

nten

sity

02

04

06

08

12

120574-tubulin

p-IRF-3

minusminus

minus ++ ++

125 25 50

5min

lowastlowast

LPS (1120583gmL)AG (120583M)

1

0

(d)

Figure 6 Continued



Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min


minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)







Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 625 125 25 500

20

40

60

80

100

120

NO

and

PGE 2

prod

uctio

n (

of c

ontro

l)

AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

NO

0 625 125 25 500

20

40

60

80

100

120

NO

and

PGE 2

prod

uctio

n (

of c

ontro

l)

AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

NOPGE2 PGE2


(a)

0

20

40

60

80

100



minusminus

++

50

lowast

PGE 2

prod

uctio

n (

of c

ontro

l)

(b)

0

20

40

60

80

100

120


Cel

l via

bilit

y (

of c

ontro

l)

AG (120583M)0 625 125 25 50

(c)










RAW2647cells


















minus

+

+ +

iNOS

COX-2

TNF-120572

GAPDH

6h

(a)

0

20

40

60

80

100

120

140



lowastlowast


mRN

A le

vel (

o

f con

trol)

(b)

0

2

4

6

8

10

12

iNOS-LucCOX-2-Luc

Luci

fera

se ac

tivity

(fol

d in

crea

se)

Normal minus 25 50

lowastlowast

lowastlowast

lowast

lowast

AG (120583M)


(c)



4 Discussion



00

02

04

06

08

10

12

c-Fos

minusminus

minus ++++

125 25 50

LPS (1120583gmL)AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Rela

tive i

nten

sity

c-Fos25 50

15min

c-Jun

p-CEBP

CEBP

LaminAC

minusminus

++++minusLPS (1120583gmL)AG (120583M) 125

p-ATF-2

p-ATF-2

p-Fra-1Fra-1

p-Fra-1

(a)

STAT-1

p-ST

AT-1

(rel

ativ

e int

ensit

y)

00

02

04

06

08

10

12


minus + +

120min

lowastlowast

120574-tubulin

p-STAT-1

(b)



0

2

4

6

8

10

LPS (1120583gmL)AG(120583M)

minus

minusminus

+++

25 50

RAW2647 cells

lowastlowast

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

(a)


minus


++++++

p-ERK

ERK

p-p38

p38

p-JNK

120573-actin

5min 30min 60min

JNK

AG (50120583M)

(b)


HEK293 cells

0

5

10

15

20

25

30

35

minusminus

minus

minus

minus

minus

minus

minus

minus

minus minus

minusminus minus

minus

minus

minus

minus

minus

minus

minus

+ + ++

+

+

+

+ +

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

00

02

04

06

08

10

12

p-MBP

lgG heavy chain

lowast

lowastlowast

LPS (1120583gmL)AG (50120583M)

minus

minus

minus

minus

minus minus

minus

+

++

+

+

30min

Rela

tive i

nten

sity

U0126(20 120583M)

(c)





p-JAK2

JAK2

p-JAK2

JAK2

2min 25min 5min

AG (50120583M)

AG (50120583M)

minus


+

+

+ +++

+

+

+

minus

minus

minus minusminus

+ +

+

+ +

+

+ +

+

45min 60min 75min

01 03 1 12 13 16 07 RI

LPS (1120583gmL)

LPS (1120583gmL)

(a)

0

5

10

15

20

25

TRIFTBK1



IRF-

3-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

lowast

lowast


HEK293 cells

(b)

0

20

40

60

80

100

120

LPS (1120583gmL)AG (120583M)

minus

minus minus

+++

25 50

IFN

-120573m

RNA

leve

l (

of c

ontro

l)

lowastlowast

lowastlowast

GAPDH

IFN-120573

COX-2

iNOS

LPS (1120583gmL)AG (50120583M)

1hminus

minus minus

+

+

+

(c)

Rela

tive i

nten

sity

02

04

06

08

12

120574-tubulin

p-IRF-3

minusminus

minus ++ ++

125 25 50

5min

lowastlowast

LPS (1120583gmL)AG (120583M)

1

0

(d)

Figure 6 Continued



Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min


minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)







Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















RAW2647cells


















minus

+

+ +

iNOS

COX-2

TNF-120572

GAPDH

6h

(a)

0

20

40

60

80

100

120

140



lowastlowast


mRN

A le

vel (

o

f con

trol)

(b)

0

2

4

6

8

10

12

iNOS-LucCOX-2-Luc

Luci

fera

se ac

tivity

(fol

d in

crea

se)

Normal minus 25 50

lowastlowast

lowastlowast

lowast

lowast

AG (120583M)


(c)



4 Discussion



00

02

04

06

08

10

12

c-Fos

minusminus

minus ++++

125 25 50

LPS (1120583gmL)AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Rela

tive i

nten

sity

c-Fos25 50

15min

c-Jun

p-CEBP

CEBP

LaminAC

minusminus

++++minusLPS (1120583gmL)AG (120583M) 125

p-ATF-2

p-ATF-2

p-Fra-1Fra-1

p-Fra-1

(a)

STAT-1

p-ST

AT-1

(rel

ativ

e int

ensit

y)

00

02

04

06

08

10

12


minus + +

120min

lowastlowast

120574-tubulin

p-STAT-1

(b)



0

2

4

6

8

10

LPS (1120583gmL)AG(120583M)

minus

minusminus

+++

25 50

RAW2647 cells

lowastlowast

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

(a)


minus


++++++

p-ERK

ERK

p-p38

p38

p-JNK

120573-actin

5min 30min 60min

JNK

AG (50120583M)

(b)


HEK293 cells

0

5

10

15

20

25

30

35

minusminus

minus

minus

minus

minus

minus

minus

minus

minus minus

minusminus minus

minus

minus

minus

minus

minus

minus

minus

+ + ++

+

+

+

+ +

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

00

02

04

06

08

10

12

p-MBP

lgG heavy chain

lowast

lowastlowast

LPS (1120583gmL)AG (50120583M)

minus

minus

minus

minus

minus minus

minus

+

++

+

+

30min

Rela

tive i

nten

sity

U0126(20 120583M)

(c)





p-JAK2

JAK2

p-JAK2

JAK2

2min 25min 5min

AG (50120583M)

AG (50120583M)

minus


+

+

+ +++

+

+

+

minus

minus

minus minusminus

+ +

+

+ +

+

+ +

+

45min 60min 75min

01 03 1 12 13 16 07 RI

LPS (1120583gmL)

LPS (1120583gmL)

(a)

0

5

10

15

20

25

TRIFTBK1



IRF-

3-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

lowast

lowast


HEK293 cells

(b)

0

20

40

60

80

100

120

LPS (1120583gmL)AG (120583M)

minus

minus minus

+++

25 50

IFN

-120573m

RNA

leve

l (

of c

ontro

l)

lowastlowast

lowastlowast

GAPDH

IFN-120573

COX-2

iNOS

LPS (1120583gmL)AG (50120583M)

1hminus

minus minus

+

+

+

(c)

Rela

tive i

nten

sity

02

04

06

08

12

120574-tubulin

p-IRF-3

minusminus

minus ++ ++

125 25 50

5min

lowastlowast

LPS (1120583gmL)AG (120583M)

1

0

(d)

Figure 6 Continued



Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min


minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)







Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















minus

+

+ +

iNOS

COX-2

TNF-120572

GAPDH

6h

(a)

0

20

40

60

80

100

120

140



lowastlowast


mRN

A le

vel (

o

f con

trol)

(b)

0

2

4

6

8

10

12

iNOS-LucCOX-2-Luc

Luci

fera

se ac

tivity

(fol

d in

crea

se)

Normal minus 25 50

lowastlowast

lowastlowast

lowast

lowast

AG (120583M)


(c)



4 Discussion



00

02

04

06

08

10

12

c-Fos

minusminus

minus ++++

125 25 50

LPS (1120583gmL)AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Rela

tive i

nten

sity

c-Fos25 50

15min

c-Jun

p-CEBP

CEBP

LaminAC

minusminus

++++minusLPS (1120583gmL)AG (120583M) 125

p-ATF-2

p-ATF-2

p-Fra-1Fra-1

p-Fra-1

(a)

STAT-1

p-ST

AT-1

(rel

ativ

e int

ensit

y)

00

02

04

06

08

10

12


minus + +

120min

lowastlowast

120574-tubulin

p-STAT-1

(b)



0

2

4

6

8

10

LPS (1120583gmL)AG(120583M)

minus

minusminus

+++

25 50

RAW2647 cells

lowastlowast

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

(a)


minus


++++++

p-ERK

ERK

p-p38

p38

p-JNK

120573-actin

5min 30min 60min

JNK

AG (50120583M)

(b)


HEK293 cells

0

5

10

15

20

25

30

35

minusminus

minus

minus

minus

minus

minus

minus

minus

minus minus

minusminus minus

minus

minus

minus

minus

minus

minus

minus

+ + ++

+

+

+

+ +

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

00

02

04

06

08

10

12

p-MBP

lgG heavy chain

lowast

lowastlowast

LPS (1120583gmL)AG (50120583M)

minus

minus

minus

minus

minus minus

minus

+

++

+

+

30min

Rela

tive i

nten

sity

U0126(20 120583M)

(c)





p-JAK2

JAK2

p-JAK2

JAK2

2min 25min 5min

AG (50120583M)

AG (50120583M)

minus


+

+

+ +++

+

+

+

minus

minus

minus minusminus

+ +

+

+ +

+

+ +

+

45min 60min 75min

01 03 1 12 13 16 07 RI

LPS (1120583gmL)

LPS (1120583gmL)

(a)

0

5

10

15

20

25

TRIFTBK1



IRF-

3-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

lowast

lowast


HEK293 cells

(b)

0

20

40

60

80

100

120

LPS (1120583gmL)AG (120583M)

minus

minus minus

+++

25 50

IFN

-120573m

RNA

leve

l (

of c

ontro

l)

lowastlowast

lowastlowast

GAPDH

IFN-120573

COX-2

iNOS

LPS (1120583gmL)AG (50120583M)

1hminus

minus minus

+

+

+

(c)

Rela

tive i

nten

sity

02

04

06

08

12

120574-tubulin

p-IRF-3

minusminus

minus ++ ++

125 25 50

5min

lowastlowast

LPS (1120583gmL)AG (120583M)

1

0

(d)

Figure 6 Continued



Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min


minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)







Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

02

04

06

08

10

12

c-Fos

minusminus

minus ++++

125 25 50

LPS (1120583gmL)AG (120583M)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Rela

tive i

nten

sity

c-Fos25 50

15min

c-Jun

p-CEBP

CEBP

LaminAC

minusminus

++++minusLPS (1120583gmL)AG (120583M) 125

p-ATF-2

p-ATF-2

p-Fra-1Fra-1

p-Fra-1

(a)

STAT-1

p-ST

AT-1

(rel

ativ

e int

ensit

y)

00

02

04

06

08

10

12


minus + +

120min

lowastlowast

120574-tubulin

p-STAT-1

(b)



0

2

4

6

8

10

LPS (1120583gmL)AG(120583M)

minus

minusminus

+++

25 50

RAW2647 cells

lowastlowast

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

(a)


minus


++++++

p-ERK

ERK

p-p38

p38

p-JNK

120573-actin

5min 30min 60min

JNK

AG (50120583M)

(b)


HEK293 cells

0

5

10

15

20

25

30

35

minusminus

minus

minus

minus

minus

minus

minus

minus

minus minus

minusminus minus

minus

minus

minus

minus

minus

minus

minus

+ + ++

+

+

+

+ +

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

00

02

04

06

08

10

12

p-MBP

lgG heavy chain

lowast

lowastlowast

LPS (1120583gmL)AG (50120583M)

minus

minus

minus

minus

minus minus

minus

+

++

+

+

30min

Rela

tive i

nten

sity

U0126(20 120583M)

(c)





p-JAK2

JAK2

p-JAK2

JAK2

2min 25min 5min

AG (50120583M)

AG (50120583M)

minus


+

+

+ +++

+

+

+

minus

minus

minus minusminus

+ +

+

+ +

+

+ +

+

45min 60min 75min

01 03 1 12 13 16 07 RI

LPS (1120583gmL)

LPS (1120583gmL)

(a)

0

5

10

15

20

25

TRIFTBK1



IRF-

3-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

lowast

lowast


HEK293 cells

(b)

0

20

40

60

80

100

120

LPS (1120583gmL)AG (120583M)

minus

minus minus

+++

25 50

IFN

-120573m

RNA

leve

l (

of c

ontro

l)

lowastlowast

lowastlowast

GAPDH

IFN-120573

COX-2

iNOS

LPS (1120583gmL)AG (50120583M)

1hminus

minus minus

+

+

+

(c)

Rela

tive i

nten

sity

02

04

06

08

12

120574-tubulin

p-IRF-3

minusminus

minus ++ ++

125 25 50

5min

lowastlowast

LPS (1120583gmL)AG (120583M)

1

0

(d)

Figure 6 Continued



Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min


minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)







Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

LPS (1120583gmL)AG(120583M)

minus

minusminus

+++

25 50

RAW2647 cells

lowastlowast

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

(a)


minus


++++++

p-ERK

ERK

p-p38

p38

p-JNK

120573-actin

5min 30min 60min

JNK

AG (50120583M)

(b)


HEK293 cells

0

5

10

15

20

25

30

35

minusminus

minus

minus

minus

minus

minus

minus

minus

minus minus

minusminus minus

minus

minus

minus

minus

minus

minus

minus

+ + ++

+

+

+

+ +

lowastlowast

AP-

1-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

00

02

04

06

08

10

12

p-MBP

lgG heavy chain

lowast

lowastlowast

LPS (1120583gmL)AG (50120583M)

minus

minus

minus

minus

minus minus

minus

+

++

+

+

30min

Rela

tive i

nten

sity

U0126(20 120583M)

(c)





p-JAK2

JAK2

p-JAK2

JAK2

2min 25min 5min

AG (50120583M)

AG (50120583M)

minus


+

+

+ +++

+

+

+

minus

minus

minus minusminus

+ +

+

+ +

+

+ +

+

45min 60min 75min

01 03 1 12 13 16 07 RI

LPS (1120583gmL)

LPS (1120583gmL)

(a)

0

5

10

15

20

25

TRIFTBK1



IRF-

3-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

lowast

lowast


HEK293 cells

(b)

0

20

40

60

80

100

120

LPS (1120583gmL)AG (120583M)

minus

minus minus

+++

25 50

IFN

-120573m

RNA

leve

l (

of c

ontro

l)

lowastlowast

lowastlowast

GAPDH

IFN-120573

COX-2

iNOS

LPS (1120583gmL)AG (50120583M)

1hminus

minus minus

+

+

+

(c)

Rela

tive i

nten

sity

02

04

06

08

12

120574-tubulin

p-IRF-3

minusminus

minus ++ ++

125 25 50

5min

lowastlowast

LPS (1120583gmL)AG (120583M)

1

0

(d)

Figure 6 Continued



Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min


minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)







Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p-JAK2

JAK2

p-JAK2

JAK2

2min 25min 5min

AG (50120583M)

AG (50120583M)

minus


+

+

+ +++

+

+

+

minus

minus

minus minusminus

+ +

+

+ +

+

+ +

+

45min 60min 75min

01 03 1 12 13 16 07 RI

LPS (1120583gmL)

LPS (1120583gmL)

(a)

0

5

10

15

20

25

TRIFTBK1



IRF-

3-m

edia

ted

luci

fera

se ac

tivity

(fold

incr

ease

)

lowast

lowast


HEK293 cells

(b)

0

20

40

60

80

100

120

LPS (1120583gmL)AG (120583M)

minus

minus minus

+++

25 50

IFN

-120573m

RNA

leve

l (

of c

ontro

l)

lowastlowast

lowastlowast

GAPDH

IFN-120573

COX-2

iNOS

LPS (1120583gmL)AG (50120583M)

1hminus

minus minus

+

+

+

(c)

Rela

tive i

nten

sity

02

04

06

08

12

120574-tubulin

p-IRF-3

minusminus

minus ++ ++

125 25 50

5min

lowastlowast

LPS (1120583gmL)AG (120583M)

1

0

(d)

Figure 6 Continued



Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min


minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)







Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Rela

tive i

nten

sity

02

04

06

08

12

lowast

IKK120576

IRF-3

p-IRF-3

AG (50120583M)

WB

minus +

1

0

(e)

IP TBK1

15min


minus minus +

++

IKK120576

TBK1

WB 02 1 04 RI

AG (50120583M)

(f)







Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Normal LPSLPS +

AG (40mgkg)LPS +

D-GalN (800mgkg)

0

50

100

150

200

250

300

ASTALT


minus + +

+

AST

and

ALT

leve

ls (U

L)

lowast

lowast

LPS (10mgkg)

(a)

ATF-2

AG (40mgkg)

p-ATF-2

LPS (10mgkg) minus

minus minus

+ +

+

(b)


HCl

EtO

H-in

duce

d ga

stric

lesio

ns(

of c

ontro

l)

AG (40mgkg)

CMC (05)0

20

40

60

80

100

120

lowast

lowast

minus

minus

minus

+

minus

++

+

+

Ranitidine (40mgkg)

HClEtOH +

Ranitidine (40mgkg)

(c)








TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TLR4

Pam3CSK LPS

ERK p38 JNK

MyD88

TBK1

AP-1

NO

IFN-120573

PGE2JAK2

TLR3

NF-120581B


STAT-1 COX-2iNOS

IFN-120573 IFN-R

TRAF6TLR2TRIF

MAPK kinase


ATF-2

Pro-TNF-120572

Macrophages

IKK120576

IKK

TNF-120572

Poly (I C)

IRF-3








5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions


2 and






Acknowledgments



References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Aktivitas Inflamasi Dr Sambiloto