Flavonoid Apigenin Inhibits Lipopolysaccharide-Induced Inflammatory Response through Multiple ...

19 Pages·2015·2.63 MB·English

Virginia Commonwealth University
VCU Scholars Compass
Microbiology and Immunology Publications Dept. of Microbiology and Immunology
2014
Flavonoid Apigenin Inhibits Lipopolysaccharide-
Induced Inflammatory Response through Multiple
Mechanisms in Macrophages
Xiaoxuan Zhang
China Pharmaceutical University, Virginia Commonwealth University
Guangji Wang
China Pharmaceutical University
Emily C. Gurley
Virginia Commonwealth University
Huiping Zhou
Virginia Commonwealth University, Wenzhou Medical University
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Flavonoid Apigenin Inhibits Lipopolysaccharide-Induced
Inflammatory Response through Multiple Mechanisms in
Macrophages
Xiaoxuan Zhang1,2, Guangji Wang1*, Emily C. Gurley2, Huiping Zhou2,3,4*
1Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, P. R. China, 2Department of Microbiology & Immunology, Virginia
CommonwealthUniversity,Richmond,Virginia,UnitedStatesofAmerica,3DepartmentofInternalMedicine/GastroenterologyandMcGuireVeteransAffairsMedical
Center,Richmond,Virginia,UnitedStatesofAmerica,4SchoolofPharmacy,WenzhouMedicalUniversity,Wenzhou,P.R.China
Abstract
Background:Apigeninisanon-toxicnaturalflavonoidthatisabundantlypresentincommonfruitsandvegetables.Ithas
beenreportedthatapigeninhasvariousbeneficialhealtheffectssuchasanti-inflammationandchemoprevention.Multiple
studieshaveshownthatinflammationisanimportantriskfactorforatherosclerosis,diabetes,sepsis,variousliverdiseases,
and other metabolic diseases. Although it has been long realized that apigenin has anti-inflammatory activities, the
underlyingfunctional mechanisms are stillnotfully understood.
MethodologyandPrincipalFindings: In the present study, we examined the effect of apigenin on LPS-induced
inflammatoryresponseandfurtherelucidatedthepotentialunderlyingmechanismsinhumanTHP-1-inducedmacrophages
andmouseJ774A.1macrophages.ByusingthePrimePCRarray,wewereabletoidentifythemajortargetgenesregulated
by apigenin in LPS-mediated immune response. The results indicated that apigenin significantly inhibited LPS-induced
productionofpro-inflammatorycytokines,suchasIL-6,IL-1b,andTNF-athroughmodulatingmultipleintracellularsignaling
pathwaysinmacrophages.ApigenininhibitedLPS-inducedIL-1bproductionbyinhibitingcaspase-1activationthroughthe
disruption of the NLRP3 inflammasome assembly. Apigenin also prevented LPS-induced IL-6 and IL-1b production by
reducingthemRNAstabilityviainhibitingERK1/2activation.Inaddition,apigeninsignificantlyinhibitedTNF-aandIL-1b-
inducedactivation ofNF-kB.
ConclusionandSignificance:Apigenin Inhibits LPS-induced Inflammatory Response through multiple mechanisms in
macrophages. These results provided important scientific evidences for the potential application of apigenin as a
therapeuticagent forinflammatory diseases.
Citation: Zhang X, Wang G, Gurley EC, Zhou H (2014) Flavonoid Apigenin Inhibits Lipopolysaccharide-Induced Inflammatory Response through Multiple
MechanismsinMacrophages.PLoSONE9(9):e107072.doi:10.1371/journal.pone.0107072
Editor:MengweiZang,BostonUniversitySchoolofMedicine,UnitedStatesofAmerica
ReceivedJune27,2014;AcceptedAugust12,2014;PublishedSeptember5,2014
Thisisanopen-accessarticle,freeofallcopyright,andmaybefreelyreproduced,distributed,transmitted,modified,builtupon,orotherwiseusedbyanyonefor
anylawfulpurpose.TheworkismadeavailableundertheCreativeCommonsCC0publicdomaindedication.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Allrelevantdataarewithinthepaper.
Funding:ThisworkwassupportedinpartbyNIHgrantsR01DK-057543andR01AT004148,VAMeritAward1I01BX001390,NationalScienceFoundationofChina
Grants(81070245,81270489,81273589,and81202591),Jiangsuprovincepromotionfoundationforthekeylabofdrugmetabolismandpharmacokinetics
(NM2012012),andProjectforinternationalS&TcooperationprogramofChina(2011DFG333380).Thefundershadnoroleinstudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*Email:[email protected](HZ);[email protected](GW)
Introduction and macrophage chemoattractant protein-1 (MCP-1). Persistent
production of these proinflammatory cytokines can cause severe
Lipopolysaccharide(LPS),amajorcomponentofgram-negative tissue destruction and eventually organ failure. The traditional
bacteria cell membrane, is a well-characterized inducer of the steroidal anti-inflammatory drugs (SAIDs) and nonsteroidal anti-
inflammatoryresponse.Theinitialacuteinnateimmuneresponse inflammatory drugs (NSAIDs) are the commonly used to treat
to LPS primes the adaptive immune system against further acuteinflammatorydisorders.However,theseconventionaldrugs
infection [1]. Macrophages are the major players in both innate havenotbeensuccessfulintreatingchronicinflammatorydiseases
andadaptiveinflammatoryresponses.Ithasbeenwell-recognized duetoseveresideeffects[4].Theneedforthedevelopmentofnew
that the prolonged activation of the inflammatory response anti-inflammatorydrugswithhigherpotencyandlowertoxicityis
contributes to a wide variety of chronic human diseases such as urgent tocombat various complexinflammatory diseases.
arteriosclerosis, sepsis, obesity, diabetes, various liver diseases, Flavonoids are a family of polyphenolic compounds, that are
inflammatory bowel disease, autoimmune diseases, allergy and widely distributed in the plant kingdom and consumed in
cancer [2,3]. Activation of macrophages by LPS leads to the significant amounts as part of thehuman diet [5]. The beneficial
increased secretion of a large set of proinflammatory cytokines, effects of these flavonoids to human health have been well-
suchastumornecrosisfactor(TNF)-alpha,interleukin(IL)-1,IL-6, documented [5–12]. Epidemiological studies have shown that
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Anti-InflammatoryEffectofApigenin
Figure1.Thechemicalstructureofapigenin.
doi:10.1371/journal.pone.0107072.g001
flavonoids in a healthy diet have potentially beneficial effects in lar/molecular mechanisms by which apigenin modulates LPS-
inflammatory diseases and can reduce the risk of various cancers induced inflammatory response in macrophages have not been
[13]. Apigenin (49, 5, 7,-trihydroxyflavone) (Fig.1) is a non-toxic fully revealed.
andnon-mutagenicdietaryflavonoid,whichisabundantlypresent In the present study, we examined the effect of apigenin on
in common fruits and vegetables, such as oranges, grapefruits, LPS-induced inflammatory response in macrophages and further
parsley, onions, chamomile, wheat sprouts, and some seasonings exploredthepotentialcellular/molecularmechanismsinvolvedin
[13,14]. During last decade, apigenin has garnered increased itsanti-inflammatory effects.
interest as a health promoting agent because of its low intrinsic
toxicity and high chemopreventiveefficiency [13,14]. It has been Methods
shown that apigenin induces human pancreatic cancer cell death
via inhibition of the glycogen synthase kinase-3b/nuclear factor Materials
kappaB(NF-kB)signalingpathway[15].Inaddition,apigeninhas Apigenin,LPS,phorbol12-myristate13-acetate(PMA),actino-
been reported to have anti-inflammatory activities. It protects mycinD,andhygromycinBwerepurchasedfromSigma-Aldrich
endothelial cells from LPS-induced inflammation and inhibits (St.Louis,MO).CellCountingKit-8(CCK-8)wasfromDojindo
allergen-induced airwayinflammation [16,17].Severalintracellu- Molecular Technologies (Kumamoto, Japan). Antibodies against
lar signaling pathways have been suggested to be involved in IL-1b,p-ERK1/2,ERK1,ERK2andASCwerefromSantaCruz
apigenin-mediated anti-inflammatory effects, such as NF-kB, Biotechnology (Santa Cruz, CA). Antibody against caspase-1 was
MAPK/ERK, and JNK pathways [18,19]. However, the cellu- from Millipore Corporation (Billerica, MA). Mouse monoclonal
Table1. Real-timePCRprimers.
Gene ForwardPrimer(59-39) ReversePrimer(59-39)
hGAPDH ACATCATCCCTGCCTCTACTGG TCCGACGCCTGCTTCACC
hIL-1b TGGCTTATTACAGTGGCAATG GTGGTGGTCGGAGATTCG
hIL-6 CAGATTTGAGAGTAGTGAGGAAC CGCAGAATGAGATGAGTTGTC
TNF-a CGAGTCTGGGCAGGTCTAC GGGAGGCGTTTGGGAAGG
hCaspase-1 CCACATCCTCAGGCTCAGAAG GCGGCTTGACTTGTCCATTATTG
hNLRP3 AGGAAGATGATGTTGGACTGG GTGGATGGGTGGGTTTGG
mGAPDH GTCGTGGATCTGACGTGCC GATGCCTGCTTCACCACCTT
mIL-1b AATCTCACAGCAGCACATC AGCAGGTTATCATCATCATCC
mIL-6 GAGGATACCACTCCCAACAGACC AAGTGCATCATCGTTGTTCATACA
mTNF-a GCCTCCCTCTCATCAGTTC ACTTGGTGGTTTGCTACG
doi:10.1371/journal.pone.0107072.t001
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Anti-InflammatoryEffectofApigenin
Figure2.TheclustergramofPrimePCRassayinhumanTHP-1-
derived macrophages. Cells were pretreated with apigenin (A,
25mM) or DMSO for 2h, and then treated with LPS (100ng/mL) or
vehicle control for 24h. Total cellular RNA was isolated and reverse
transcribed.ThemRNAlevelsof91genesinvolvedintheinflammatory
immuneresponseweredetectedusingapre-designedPrimePCRassay
following the manufacturer’s instructions. The results were analyzed
usingBio-RadCFXmanagersoftware.Theclustergramofallthetested
genesisshown.
doi:10.1371/journal.pone.0107072.g002
antibody against b-actin and NLRP3 polyclonal antibody were
from Thermo Scientific (Wilmington, DE). IRDye secondary
antibodies were purchased from LI-COR Biosciences (Lincoln,
NE). Recombinant human/mouse IL-1b, IL-6 and TNF-a, anti-
human/mouse IL-1b, IL-6 and TNF-a antibodies, biotinylated
anti-human/mouse IL-1b, IL-6 and TNF-a antibodies, and
avidin-HRP were from eBioscience (San Diego, CA). Immune
Response Tier 1 H96 Plate, Bio-Rad protein assay reagent,
Precision Plus Kaledoscope Standards, iQTM SYBR Green
Supermix were obtained from Bio-Rad (Hercules, CA). QIAzol
Lysis Reagent was obtained from QIAGEN Sciences (German-
town,MD).HighCapacitycDNAReverseTranscriptionKitwas
from Life Technologies (Grand Island, NY). FuGene HD
transfectionReagent,pGL4.32[luc2P/NF-kB-RE/Hygro]vector
and pGL4.76 [hRluc/Hygro] vector were from Promega (Mad-
sion, WI).
Cell culture and treatment
Human THP-1 monotypic cells (from ATCC, Cat# TIB-
202TM)wereculturedinRPMI1640mediumsupplementedwith
10%FBS,100U/mLmLpenicillin,and100mg/mLstreptomycin
at37uCwith5%CO2.THP-1monocytesweretreatedwithPMA
(100 ng/mL)for5daystoinducedifferentiationintomacrophag-
es. MouseJ774A.1 macrophages (from ATCC, Cat# TIB-67TM)
weremaintainedinDMEMsupplementedwith10%FBS,100U/
mL penicillin, and 100mg/mL streptomycin at 37uC with 5%
CO2. Cells from passages 12 to 15 were used in these studies.
HEK293 cells were cultured in DMEM supplemented with 10%
FBS, 0.1mM non-essential amino acid and 1% Penicillin-
Streptomycin. Apigenin was dissolved in dimethyl sulfoxide
(DMSO) and directly added into the culture medium. Based on
the preliminary toxicity test and previous published studies
[20,21], the concentrations of apigenin used in this study were
6.25, 12.5 and 25mM. For each result, a minimum of three
independent experiments were performed.
Immune response PrimerPCR assay
Human THP-1-derived macrophages were pretreated with
apigenin(25 mM)for2 handthentreatedwithLPS(100 ng/mL)
for 24h. Total cellular RNA was isolated using QIAzol Lysis
Reagentandreversetranscribedintothefirst-strandcDNAusing
aHigh-CapacitycDNATranscriptionKitasdescribedpreviously
[22].The20ngofcDNAofcontrolorLPS-orLPS+apigenin-
treated samples were loaded into the Immune Response Tier 1
H96 plate. The mRNA levels of 91 genes involved in the
inflammatory immune response were detected at the same time
using iQTM SYBR Green Supermix on Bio-Red CFX96 real-
timePCRsystem.ThedatawereanalyzedbyusingBio-RadCFX
manager software.
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Anti-InflammatoryEffectofApigenin
Figure3.TheclustergramofthemajortargetgenesofapigenininhumanTHP-1-derivedmacrophages.Cellswerepretreatedwith
apigenin(A,25mM)orDMSOfor2h,andthentreatedwithLPS(100ng/mL)orvehiclecontrolfor24h.TotalcellularRNAwasisolatedandreverse
transcribed. The mRNA levels of 91 genes involved in an inflammatory immune response were detected using a pre-designed PrimePCR assay
followingthemanufacturer’sinstructions.TheresultswereanalyzedusingBio-RadCFXmanagersoftware.Theclustergramofmajortargetgenesis
shown.
doi:10.1371/journal.pone.0107072.g003
RNA isolation and quantitative real-time RT-PCR transcribed into first-strand cDNA using a High-Capacity cDNA
Cells were pretreated with apigenin for 2 h and then treated Transcription Kit. The mRNA levels of IL-1b, IL-6, TNF-a,
withLPS(100 ng/mL)orvehiclecontrolfor24h.TotalCellular caspase-1,ASC,NLRP3andGAPDHwerequantifiedusinggene-
RNA was isolated using QIAzol Lysis Reagent and reverse specific primers (Table1). iQTM SYBR Green Supermix was
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Anti-InflammatoryEffectofApigenin
Figure4.EffectofapigeninonLPS-inducedproinflammatorycytokinesmRNAexpressioninhumanTHP-1-derivedmacrophages.
Cellswerepretreatedwithdifferentconcentrationsofapigenin(A,6.25,12.5,25mM)for2h,thentreatedwithLPS(100ng/mL)for24hours.Total
cellularRNAwasisolatedandreversetranscribed.TherelativemRNAlevelsofIL-1b,IL-6,andTNF-aweredetectedbyreal-timeRT-PCRasdescribed
under‘‘Methods’’.Valuesaremean6S.E.ofthreeindependentexperiments.Statisticalsignificancerelativetovehiclecontrol,##p,0.01;###p,
0.001;StatisticalsignificancerelativetoLPSgroup,*p,0.05,**p,0.01,***p,0.001.A.IL-1b;B.IL-6;C.TNF-a.
doi:10.1371/journal.pone.0107072.g004
usedasafluorescentdyetodetectthepresenceofdouble-stranded copies of a NF-kB response element (NF-kB-RE) that drives the
DNA.ThemRNAlevelsoftargetgeneswerequantifiedwithreal- transcription of the luciferase reporter gene luc2P (Photinus
time RT-PCRas describedpreviously [22]. pyralis). Briefly, 293cells were transfected with pGL4.32[luc2P/
NF-kB-RE/Hygro] or pGL4.76 [hRluc/Hygro] using FuGene
Enzyme-Linked Immunosorbent Assay (ELISA) HDtransfectionreagent.Thestablytransfectedcellswereselected
Cells were pretreated with apigenin for 2 h and then treated by Hygromycin B(100 mg/mL) for two weeks. To determine the
with LPS (100ng/mL) or vehicle control for 24h. At the end of effectofapigeninoninflammatorycytokine-mediatedactivationof
thetreatment,cellculturemediawerecollectedandcentrifugedat NF-kB, 293 cells expressing NF-kB-RE were pretreated with
14,0006rpmfor1 min.TheproteinlevelsofTNF-a,IL-1b,and apigeninfor2 hours,andthentreatedwithhumanIL-1b(10 ng/
IL-6 in the media were determined by ELISA as described mL) or TNF-a (10ng/mL), or vehicle control for 4hours. The
previously[22].Thetotalproteinconcentrationsoftheviablecells luciferase activity was detected on a Glomax Multi-functional
weredeterminedusingBio-Radproteinassayreagent.Theprotein Plate Reader (Promega) using the Promega Luciferase Assay
levelsoftheTNF-a,IL-1b,andIL-6inmediawerenormalizedto System kit according to manufacturer’s instructions. The relative
the total protein amount of the viable cells and expressed as pg/ luciferaseactivityofeachgroupwascomparedtocontrolvehicle.
mg proteins.
Statistical analysis
Assessment of IL-1b and IL-6 mRNA stability Alloftheexperimentswererepeatedatleastthreetimesandthe
data were expressed as mean 6 SD. One-way ANOVA was
Cells were pretreated with apigenin for 2 h and then treated
employed to analyze the differences between sets of data. To
withLPS(100 ng/mL)orvehiclecontrolfor2 hbeforeadditionof
confirm the differences occurred between groups, post hoc tests
actinomycinD(5.0 mg/mL,time0).Thecellswereharvestedfor
were used for follow-up test. Statistics were performed using
isolationoftotalcellularRNAat0.5,1,2,4and6 hafteraddition
GraphPadPrism5.0(GraphPad,SanDiego,CA).Avalueofp,
of actinomycin D. The mRNA stability of IL-1b and IL-6 were
0.05 wasconsideredstatistically significant.
quantified with real-timePCR as describedpreviously [23].
Results
Western blot analysis
Total cell lysates were prepared as previously described [24]. Identification of the target genes regulated by apigenin
TheproteinconcentrationwasdeterminedusingBio-Radprotein
in LPS-mediated immune response in macrophages
assay reagent. A total of 70mg of proteins was resolved on 10%
Althoughapigeninhasbeenreportedtohaveanti-inflammatory
Bis–Tris gels and transferred to nitrocellulose membranes. The
activities and is able to inhibit the expression of several
proteinlevelsoftargetgenesweredetectedusingspecificprimary
proinflammatory cytokines such as TNF-a and IL-6, there is no
antibodies and IRDye secondary antibodies on Odyssey Fluores-
information available regarding the effect of apigenin on LPS-
cenceImagingSystem(LI-CORBiosciences,USA).Thedensityof
induced immune response. In order to determine the effect of
the immunoblots was analyzed using Odyssey V3.0 software and
apigeninonLPS-inducedinflammatoryresponseandidentifythe
normalized with b-Actin.
specifictargetgenes,wedidthequantitativereal-timePrimePCR
array using the Bio-Rad predesigned assay specifically for
Immunofluorescence staining of ASC
inflammatory immune response. The differentiated THP-1
HumanTHP-1-derivedmacrophageswereculturedon22622- macrophages were pretreated with apigenin (25mM) for 2 h and
mm glass coverslips in 6-well plates. Cells were pretreated with then treated with LPS (100 ng/mL) or vehicle control for 24h.
apigenin (25mM)for2 h andthentreatedwithLPS (100 ng/ml) TotalcellularRNAwasisolatedandreversetranscribedintofirst-
or vehicle control for 24h. Cells were fixed with 3.7% strand cDNA. A total of 20ng of cDNA was used to run a real-
formaldehyde in PBS for 30min, permeabilized with 0.1% timePrimePCRarrayaccordingtotheprotocolrecommendedby
Triton-X-100for3 min,andblockedwith3%normalgoatserum the manufacturer. The results indicated that in LPS-stimulated
for 1 h. Rabbit anti-ASC antibody was incubated for 4 h at RT. macrophages, more than two dozen genes were significantly up-
The negative control was incubated with the same amount of regulated including IL-6, IL-8, IL1b, IL12b, NFKBIA (nuclear
normal rabbit IgG. TheAlexa Fluor 488-labeled goatanti-rabbit factorofkappalightpolypeptidegeneenhancerinB-cellsinhibitor
antibody was incubated for 1h at RT. The coverslips were alpha), and NFKB1. But IL-10 and TLR4 were significantly
mounted with fluorescence mounting medium with DAPI. The down-regulated (Fig.2).
intracellular ASC speck formation was visualized under an Cytokinesareimportantimmunomodulationagentsinregulat-
Olympus 1671 fluorescence microscope with a 60x oil objective ing host responses to infection, inflammation, sepsis, and cancer
using a dual-filter set for FITC and DAPI. The images were [25]. As shown in Fig.3, apigenin not only significantly inhibited
captured usingIPLab 4.0software. LPS-inducedup-regulationofpro-inflammatorycytokines,suchas
IL-1b,IL-6,andIL-12b,butalsoreducedLPS-inducedincreaseof
Measurement of NF-kB activity inflammatorychemokineCCL5andadhesionmolecules(ICAM1
NF-kB activity was detected using a NF-kB-luciferase reporter andVCAM1).Inaddition,LPS-induceddown-regulationofIL-10
vector, pGL4.32 [luc2P/NF-kB-RE/Hygro]. It contains five wasreversedbyapigenin.Theseresultssuggestthatapigeninmay
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Anti-InflammatoryEffectofApigenin
Figure5.EffectofapigeninonLPS-induced proinflammatorycytokinesmRNAexpressioninmouseJ774A.1macrophages.Cells
werepretreatedwithdifferentconcentrationsofapigenin(A,6.25,12.5,25mM)for2handthentreatedwithLPS(100ng/mL)for24hours.Total
cellularRNAwasisolatedandreversetranscribed.TherelativemRNAlevelsofIL-1b,IL-6,andTNF-aweredetectedbyreal-timeRT-PCRasdescribed
under‘‘Methods’’.Valuesaremean6S.E.ofthreeindependentexperiments.Statisticalsignificancerelativetovehiclecontrol,##p,0.01;###p,
0.001;StatisticalsignificancerelativetoLPSgroup,**p,0.01,***p,0.001.A.IL-1b;B.IL-6;C.TNF-a.
doi:10.1371/journal.pone.0107072.g005
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Figure6.EffectofapigeninonLPS-inducedIL-6andTNF-aproteinexpressioninhumanTHP-1-derivedmacrophages.Cellswere
pretreatedwithdifferentconcentrationsofapigenin(A,6.25,12.5,25mM)for2handthentreatedwithLPS(100ng/mL)for24h.Attheendof
treatment,eachcellculturemediumwascollected.TheproteinlevelsofIL-6andTNF-aweredeterminedbyELISAasdescribedunder‘‘Methods’’.
Valuesaremean6S.E.ofthreeindependentexperiments.Statisticalsignificancerelativetovehiclecontrol,###p,0.001;Statisticalsignificance
relativetoLPSgroup,**p,0.01,***p,0.001.A.IL-6;B.TNF-a.
doi:10.1371/journal.pone.0107072.g006
modulate LPS-induced inflammatory response through multiple TNF-a in human THP-1-derived macrophages, which were
mechanisms. completely inhibited by apigenin in a dose-dependent manner.
Similarly, apigenin also markedly inhibited LPS-induced expres-
Effect of apigenin on LPS-induced expression of pro- sion of IL-1b, IL-6, and TNF-a in a dose-dependent manner in
inflammatory cytokines in macrophages mouse J774A.1 macrophages(Fig.5).
In order to verify the results of PrimePCR Array and confirm
the anti-inflammatory effect of apigenin in macrophages, we EffectofapigeninonLPS-inducedsecretionofIL-6,TNF-a
measured the mRNA levels of several key pro-inflammatory and IL-1b protein in macrophages
cytokines involved in inflammatory response including IL-1b, In order to determine whether the inhibition of the mRNA
TNF-a, and IL-6 using real-time RT-PCR [22,26,27]. As shown expressionofpro-inflammatorycytokinesbyapigeniniscorrelated
inFig.4,LPSmarkedlyincreasedmRNAlevelsofIL-1b,IL-6and tothereductionofproteinlevels,wemeasuredtheTNF-aandIL-
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Figure 7. Effect of apigenin on LPS-induced IL-6 and TNF-a protein expression in in mouse J774A.1 macrophages. Cells were
pretreatedwithdifferentconcentrationsofapigenin(A,6.25,12.5,25mM)for2handthentreatedwithLPS(100ng/mL)for24h.Attheendof
treatment,eachcellculturemediumwascollected.TheproteinlevelsofIL-6andTNF-aweredeterminedbyELISAasdescribedunder‘‘Methods’’.
Valuesaremean6S.E.ofthreeindependentexperiments.Statisticalsignificancerelativetovehiclecontrol,###p,0.001;Statisticalsignificance
relativetoLPSgroup,**p,0.01,***p,0.001.A.IL-6;B.TNF-a.
doi:10.1371/journal.pone.0107072.g007
6secretedintocellculturemediausingELISA.AsshowninFig.6, and IL-1b maturation, we measured the intracellular pro-IL-1b
apigenin significantly reduced LPS-induced secretion of IL-6 in proteinlevelsbyWesternblotanalysisandthesecretedmatureIL-
humanTHP-1-derivedmacrophages.However,apigeninwasless 1b protein levels in culture media by ELISA. As shown in
potent in regulating TNFa expression. Similar results were Fig.8AandB,LPSsignificantlyincreasedpro-IL-1bproteinlevels,
obtained usingmouse J774A.1 macrophages(Fig.7). but apigenin had no inhibitory effect on pro-IL-1b protein
The mature IL-1b production is rigorously controlled by expression.However,theLPS-inducedsecretionofmatureIL-1b
expression, maturation and secretion. The pro-inflammatory was dose-dependently inhibited by apigenin in human THP-1-
stimuli induces expression of the inactive IL-1b precursor (pro- derivedmacrophages(Fig.8C).Theseresultssuggestthatapigenin
IL-1b),whichlacksaclassicsignalpeptideandisfurtherprocessed may regulate the maturation of IL-1b by targeting intracellular
into mature active IL-b by an intracellular cysteine protease, caspase-1.
caspase-1, and secreted from the cell [28,29]. To determine
whether apigenin had anyeffect on pro-IL-1b protein expression
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