Research Article Blockade of Airway Inflammation by Kaempferol via Disturbing Tyk-STAT Signaling in Airway Epithelial Cells and in Asthmatic Mice

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1 Hinwi Pulishing Corportion Eviene-Bse Complementry n Alterntive Meiine Volume, Artile ID 7, pges Reserh Artile Bloke of Airwy Inflmmtion y Kempferol vi Disturing Tyk-STAT Signling in Airwy Epithelil Cells n in Asthmti Mie Ju-Hyun Gong, Dekeun Shin, Seon-Young Hn, Sin-Hye Prk, Min-Kyung Kng, Jung-Lye Kim, n Young-Hee Kng Deprtment of Foo n Nutrition, Hllym University, Chunheon, Gngwon-o -7, Repuli of Kore Corresponene shoul e resse to Young-Hee Kng; yhkng@hllym..kr Reeive Deemer ; Revise 8 Mrh ; Aepte April Aemi Eitor: Seung-Heon Hong Copyright Ju-Hyun Gong et l. This is n open ess rtile istriute uner the Cretive Commons Attriution Liense, whih permits unrestrite use, istriution, n reproution in ny meium, provie the originl work is properly ite. Asthm is hrterize y ronhil inflmmtion using inrese irwy hyperresponsiveness n eosinophili. The intertion etween irwy epithelium n inflmmtory meitors plys key role in the sthmti pthogenesis. The in vitro stuy eluite inhiitory effets of kempferol, flvonoi foun in pples n mny erries, on inflmmtion in humn irwy epithelil BEAS-B ells. Nontoxi kempferol t μm suppresse the LPS-inue IL-8 proution through the TLR tivtion, inhiiting eotxin- inution. The in vivo stuy explore the emoting effets of kempferol on sthmti inflmmtion in BALB/ mie sensitize with ovlumin (OVA). Mouse mrophge inflmmtory protein- proution n CXCR expression were upregulte in OVA-hllenge mie, whih ws ttenute y orl ministrtion of mg/kg kempferol. Kempferol llye the irwy tissue levels of eotxin- n eotxin reeptor CCR enhne y OVA hllenge. This stuy further explore the loke of Tyk-STAT signling y kempferol in oth LPS-stimulte BEAS-B ells n OVA-hllenge mie. LPS tivte Tyk responsile for eotxin- inution, while kempferol ose-epenently inhiite LPS- or IL-8-inflme Tyk tivtion. Similr inhiition of Tyk tivtion y kempferol ws oserve in OVA-inue mie. Aitionlly, LPS stimulte the tivtion of STAT/ signling onomitnt with ownregulte expression of Tyk-inhiiting SOCS. In ontrst, kempferol enumere STAT/ signling with restortion of SOCS expression. Consistently, orl ministrtion of kempferol loke STAT trnstivtion elevte y OVA hllenge. These results emonstrte tht kempferol llevite irwy inflmmtion through moulting Tyk-STAT/ signling responsive to IL-8 in enotoxin-expose irwy epithelium n in sthmti mie. Therefore, kempferol my e therpeuti gent trgeting sthmti iseses.. Introution Allergi sthm is hrterize y the infiltrtion of eosinophils, mst ells, n T-lymphoytes into irwy epithelium [, ]. This infiltrtion usully les to ronhil epithelil lyer esqumtion, golet ell hyperplsi, n sumuos thikening []. The interply etween irwy epithelil ells n the immune ells plys n importnt role in the pthogenesis of n llergi sthm ttk []. Aoringly, the irwy epithelium is oth trget of inflmmtory n physil insults n n effeter of ongoing irwy inflmmtion. In sthmti proess, ntigen-sensitize T helper (Th) ells proue speifi ytokines, whih use severl key fetures of llergi ronhil sthm []. Both IL- n IL- my stimulte epithelil ells to proue hemokines suh s eotxin n growth ftors [6]. The eosinophil tthment n infiltrtion into the irwy epithelium entil ining of eotxin to C-C hemokine reeptor type (CCR) expresse on eosinophils []. Proinflmmtory IL-8 is serete y mrophges n lung epithelil ell into lung flui n reruits neutrophils n eosinophils to the sites of inflmmtion [7]. Aoringly, the IL-8 overexpression in humn ronhil epithelil ells my ply pivotl role in the eosinophil infiltrtion into inflme irwys [8]. Exposure to lipopolyshrie (LPS) inreses the severity of sthm, whih tivtes Toll-like reeptor (TLR) signling in the regultion of Th-riven lung inflmmtion [9]. Severl stuies hve shown tht the TLR tivtion y LPS

2 Eviene-Bse Complementry n Alterntive Meiine promotes inflmmtory mehnisms inluing nuler ftor (NF)-κB n Jnus-tivte kinse (JAK)/signl trnsuers n tivtors of trnsription (STAT) pthwys []. Cytokine stimultion tivtes the STAT pthwy vi phosphoryltion of tyrosine resiues y reeptor-ssoite JAK fmily memers []. Thus, the regultion of IL-8 response in irwy epithelil ells through the inflmmtory signling pthwy prevents explosive inflmmtory retions. The suppressors of ytokine signling (SOCS) hve emerge s the physiologil or pthologil regultors of ytokine responses in the inflmmtory systems []. The SOCS proteins hve importnt mehnism for the negtive regultion of the ytokine-stat pthwy []. STAT6 is importnt in the regultion of lung inflmmtion in response to llergens n viruses in murine moels with sthm []. However, muh less is known out the role of STAT/ in meiting llergi responses in sthm. Kempferol is nturl flvonol-type flvonoi tht hs een isolte from plnt soures. Kempferol effetively suppresses the evelopment of IgE-meite llergi inflmmtion of intestinl ell moels y inhiiting the seretion of llergi meitors []. The flvonol fisetin meliortes sthmti phenotypes, whih is ssoite with reution of Th responses s well s suppression of NF-κB nits ownstrem hemokines [6]. Queretin n kempferol inhiite IgE-meite relese of proinflmmtory meitors from humn mst ells, whih my e ue to inhiition of intrellulr lium influx n PKCθ signling [7]. Reently, we hve emonstrte tht kempferol suppresses eosinophil infiltrtion n irwy inflmmtion in llergi sthm [8]. It ws lso foun tht kempferol ttenute irwy llergi responses through isturing NF-κBsignling n eotxin- seretion. However, the moleulr mehnisms unerlying the ntillergi tions of kempferol shoul e fully lrifie. Bse on the literture eviene tht kempferol possesses nti-inflmmtory n ntillergi tivities, this stuy investigte whether kempferol inhiite inflmmtion in LPS-inue irwy epithelil BEAS-B ells through loking TLR tivtion. It ws teste tht IL-8 ws responsile for LPS-stimulte eotxin- inution in epithelil ells. Furthermore, the suppressive effets of kempferol on irwy inflmmtion were evlute in OVA-hllenge BALB/ mie y mesuring mrophge inflmmtory protein (MIP)-, CCR, n eotxin-. This stuy eluite whether kempferol enumere Tyk-STAT signling pthwy responsive to LPS n OVA in irwy inflmmtion n eosinophili.. Mterils n Methos.. Chemils. M99, humn epierml growth ftor (EGF), hyroortisone, geltin, humn insulin, potrnsferrin, LPS, n lumin from hiken egg white were otine from the Sigm-Alrih Chemil (St. Louis, MO, USA), s were ll other regents, unless speifilly stte elsewhere. Fetl ovine serum (FBS), peniillin-streptomyin, n trypsin-edta were purhse from the Lonz (Wlkersville, MD, USA). Humn ronhil irwy epithelil ell line, BEAS-B, ws provie y the Amerin Type Culture Colletion (ATCC, Mnsss, VA, USA). Imjet Alum (queous solution of mg/ml luminum hyroxie n mg/ml mgnesium hyroxie plus intive stilizers) ws purhse from Thermo Fisher Sientifi (Rokfor, IL, USA). For western lot nlysis n immunohistohemil ssy, ntioies ginst humn phospho-tyk, humn phospho- STAT/, STAT, n mouse phospho-stat were otine from Cell Signling Tehnology (Beverly, MA, USA). Antihumn eotxin- n ntihumn IL-8 were purhse from R&D Systems (Minnepolis, MN, USA). Antihumn TLR, ntimouse CCR, n ntihumn SOCS were purhse from the Snt Cruz Biotehnology (Snt Cruz, CA, USA). Humn Tyk inhiitor ws provie y Cliohem (Drmstt, Germny). Horserish peroxise-onjugte got ntirit IgG, onkey ntigot IgG, n got ntimouse IgG were quire from Jkson Immunoreserh Lortories (West Grove, PA, USA). Alumin from ovine serum (essentilly ftty i free) n skim milk were quire from Beton Dikinson Compny (Sprks, MD, USA). Enzymelinke immunosorent ssy (ELISA) kits of humn IL-8, mouse MIP-, n mouse eotxin- were purhse from R&D Systems... BEAS-B Cell Culture n Viility. BEAS-B ells were ulture in mm HEPES-uffere M99 ontining % FBS, mm glutmine, U/mL peniillin, μg/ml streptomyin supplemente with. μg/ml insulin,.6 μg/ml hyroortisone,. μg/ml potrnsferrin, n ng/ml humn EGF. The 9 9% onfluene of BEAS-B ells ws sustine t 7 C in n tmosphere of % CO uring ell experiments. Kempferol t μm ws pretrete overnight, n then LPS or IL-8 pplie to BEAS-B ells to inue eotxin-, phospho-stat, n phospho-stat. A pek expression of eotxin- ws ttine when LPS ws e to BEAS-B ells for 8 h [8]. The ytotoxiity of μm kempferol ws etermine fter 8 h ulture of BEAS-B ells using n MTT (-(,- imethylthizol-yl)-iphenyl tetrzolium romie, Duhef Biohemie, Hrlem, The Netherlns) ssy. Briefly, ells were mintine in fresh meium inluing mg/ml MTT t 7 Cforh.Gentleshkingwsonutetoissolve purple formzn prout in. ml isopropnol, n the sorne of formzn ws etermine t λ = 7nm using miroplte reer (Bio-R Moel, Herules, CA, USA)... Inution of Airwy Inflmmtion in Murine Moel. Six-week-ol mle BALB/ mie (Hllym University Breeing Center for Lortory Animls) were kept on h light/ h rk yle t ± Cwith± % reltive humiity uner speifi pthogen-free onitions. Mie were fe nonpurifie iet (RoFeeTM, DBL, Umsung, Kore) n were provie with wter liitum t the Animl Fility of Hllym University. The nonpurifie iet omposition ws s follows: not less thn (NLT).% rue protein, NLT.% rue ft, not more thn (NMT) 8.% rue fier,

3 Eviene-Bse Complementry n Alterntive Meiine NMT 8.% rue sh, NLT.% lium, n NLT.% phosphorus.miewerellowetolimtizeforweek efore eginning the experiments. Mie were ivie into four sugroups (n = 6 for eh sugroup). Mie were sensitize with μg OVAissolveinsolutionofμL PBS n μlimjetalumysuutneousinjetiontwie on y n y. Kempferol solution (. ml, or mg/kg BW) ws orlly ministrte to OVA-sensitize mie h efore OVA hllenge. On y 8, y 9, n y, the % OVA inhltion to mie ws performe for min in plsti hmer linke to n ultrsoni neulizer (Clenny Aerosol, Meel, S. Polo i Torrile, Itly). ControlmieweresensitizenhllengewithPBSs the OVA vehile. All mie were srifie with n nestheti ( μl/kg Rompun n 8 μl/kg Zoletil, intrperitonel injetion) h fter the lst hllenge (y ). The right lungs were ollete, frozen to liqui nitrogen, n kept t 8 C for the extrtion, n the left lungs were preserve n fixe in % prformlehye n then use for the stining. All experiments were pprove y the Committee on Animl Experimenttion of Hllym University n performe in ompline with the University s Guielines for the Cre n Use of Lortory Animls (Hllym -66). No mie were e, n no pprent signs of exhustion were oserve uring the experimentl perio... Western Blot Anlysis. Whole BEAS-B ell lystes or BALB/ lung tissue extrts were prepre in M Tris-HCl (ph 6.8) lysis uffer ontining % SDS, % glyerophosphte,. M N VO,. M NF, n protese inhiitor oktil. Equl volumes of ell ulture superntnts n equl mounts of ell lystes or tissue extrts proteins were eletrophorese on 8 % SDS-PAGE gel n trnsferre onto nitroellulose memrne. Bloking of nonspeifi ining ws performe in TBS-T uffer ( mm Tris- HCl (ph 7.), mm NCl, n.% Tween ) ontining either % ovine serum lumin or % nonft ry milk for h. The memrne ws inute overnight t Cwithspeifiprimryntioy.Aftertriplewshing with TBS-T uffer, the memrne ws then pplie to got ntirit IgG, onkey ntigot IgG, or got ntimouse IgG onjugte to horserish peroxise for h. Following nother triple wshing, trget protein ws etermine using the SuperSignl West Pio Chemiluminesene etetion regents (Piere Biotehnology, Rokfor, IL, USA) n the Agf meil X-ry film lue (Agf HelthCre NV, Mortsel, Belgium). Antihumn βtin inution ws omplishe for the omprtive ontrol... Reverse Trnsriptse-Polymerse Chin Retion (RT- PCR) Anlysis. Followingultureprotools,totlRNAws isolte from LPS-trete BEAS-B ells using ommerilly ville Trizol regent kit (Invitrogen, Crls, CA, USA). The RNA ( μg) ws reversily trnsrie with units of reverse trnsriptse n. mg/ml oligo-(t) primer (Bioneer, Dejeon, Repuli of Kore). The expressionsofthemrnatrnsriptsoftlr(forwrprimer -CAT TGG TGT GTC GGT CCT CA-, reverse primer -ACT GCC AGG TCT GAG CAA TC-,77p)nβtin (forwr primer -GAC TAC CTC ATG AAG ATC-, reverse primer -GAT CCA CAT CTG CTG GAA-,p) were evlute y RT-PCR. The PCR ws performe in μl uffer ( mm Tris-HCl (ph 9.), mm MgCl,mM NTP,unitsofTqDNApolymerse,nμM of eh primer) n terminte y heting t 9 Cformin.After thermoyling n eletrophoresis of μl PCR prouts on.% grose-formlehye gel, the ns were visulize using TFX- M moel-uv trnsillumintor (Viler- Lourmt, Mrne-l-Vllée, Frne), n gel photogrphs were otine. The sene of ontminnts ws routinely heke y the RT-PCR ssy of negtive ontrol smples without primer ition..6. ELISA. Cell-free ulture mei were ollete from BEAS-B ells n store t C. IL-8 seretion n tissue levels of MIP- n eotxin- were exmine in ulture mei or BALB/ lung tissue extrts y using eh ELISA kit (Miroplte Reer, Moleulr Devies, Sunnyvle, CA, USA)..7. Lung Immunohistohemistry. Immunohistohemil nlysis ws rrie out y using ntioies ginst mouse CXCR, mouse phospho-tyk, n phospho-stat. All lung tissue setions were sujete to series of immunohistohemil proeures inluing the Ag retrievl followe y quenhing of enogenous peroxise tivity. For the mesurement of tissue levels of CXCR n phospho-tyk, n immunofluoresent histohemil nlysis ws onute using ntimouse CXCR or ntimouse phospho-tyk n Cy- or FITC-onjugte ntigot IgG. Nuler stining ws one with,6-imiino--phenylinole (DAPI). For the etetion of phospho-stat,, -iminoenziine hromogeni sustrte etetion kit (Dko, Crpinteri, CA, USA) ws use. Counter stining ws onute with hemtoxylin. Eh slie ws mounte in VetMount mounting meium (Vetor Lortories, Burlingme, CA, USA). Imges of eh slie were tken using n optil mirosope system (Axiomger, Zeiss, Germny). Protein levels of CXCR, phospho-tyk, n phospho-stat were quntifie y the imge nlysis progrm of the mirosope system..8. Sttistil Anlysis. The t re presente s men ± SEM for eh tretment group in the in vivo n in vitro experiments. Sttistil nlyses were onute using Sttistil Anlysis Systems progrm (SAS Institute, Cry, NC, USA). One-wy ANOVA ws use to etermine inhiitory effets of kempferol on irwy inflmmtion n llergi responses in epithelil ells n sensitize mie. Differenes mong tretment groups were nlyze with Dunn s multiple rnge test n were onsiere to e signifint t P<..

4 Eviene-Bse Complementry n Alterntive Meiine. Result.. Suppression of LPS-Promote TLR Inution n IL-8 Proution y Kempferol. Mmmlin TLR is the signltrnsuing reeptor tivte y the teril LPS n lipotehoi i [, 9]. Western lot nlysis showe tht TLR serve s n epithelil reeptor to LPS for the irwy inflmmtory proess. Humn BEAS-B ells were inute with μg/ml LPS in the sene n presene of μm kempferol for 8 h. The expression of TLR ws gretly elevte in LPS-stimulte BEAS-B ells (Figure ()). This stuy investigte whether μm kempferol inhiite the inution of TLR triggere y LPS. When BEAS-B ells were inute with μm kempferol for h, there ws no notle ytotoxiity oserve [8]. When nontoxi kempferol ws e, the TLR inution ws inhiite in ose-epenent mnner. Aitionlly, kempferol suppresse the expression of TLR mrna (Figure ()). This stuy eluite tht LPS inue ellulr expression of IL-8 through stimulting TLR signling n tht kempferol enumere IL-8 inution. LPS enhne ellulr seretion of IL-8, whih ws mpene y the nontoxi TLR inhiitor OxPAPC t μg/ml (Figure ()). Similr inhiition ws oserve with μm kempferol. In ition, LPS inrese the IL-8 seretion of BEAS-B ells (Figure ()). However, the tretment of LPS-expose ells with μm kempferol mrkely ttenute suh seretion. The urrent stuy ttempte to prove tht IL-8 is one of the pivotl ftors responsile for sthmti irwy inflmmtion. Chemokines with protein sequene homology to humnil-8hvenoteenientifieinmie[]. The CXC hemokines KC n MIP- (lso known s CXCL) re funtionl homologs of humn IL-8 in mie. Aoringly, the MIP- levels in mouse lung tissue were mesure. OVA hllenge inrese MIP- proution in mouse lung tissue (Figure ()). However, kempferol supplemente to OVAhllenge mie mrkely iminishe MIP- proution. Furthermore, this stuy exmine the inution of CXCR, the reeptor to IL-8, in lung tissues of OVA-hllenge mie. There ws lk of ytoplsmi stining in the negtive ontrol mie (Figure ()). However, strong ytoplsmi reish stining ws oserve in OVA-hllenge mie. In ontrst, the CXCR inution ws ose epenently ttenute in mie supplemente with kempferol (Figure ())... Attenution of LPS-Inue Eotxin- Expression y Kempferol. This stuy investigte whether IL-8 ws involve in the eosinophil infiltrtion y inuing eotxin- protein in enotoxin-experiene irwy epithelil ells. Eotxin- expression ws gretly enhne in IL-8-stimulte BEAS-B ells, whih ws reverse y treting μm kempferol (Figure ()). Aoringly, the suppression of IL- 8 proution y kempferol my e ssoite with its loke of erly irwy inflmmtion. Aitionlly, μg/ml OxPAPC olishe the inution of eotxin- protein in LPS-expose BEAS-B ells (Figure ()), initing tht its inution y LPS ws meite vi the TLR signling enumere y kempferol. The role of eotxin- in the irwy inflmmtion ws verifie in lung tissues of OVA-hllenge mie. CCR n serve s reeptor for severl ifferent hemokines suh s mrophge inflmmtory proteins, monoyte hemottrtnt proteins, n eotxins. Most of the ligns to CCR re ssoite with sthm, n CCR hs eome n ppeling possiility in sthm tretment or therpy []. The lung tissue level of CCR ws enhne in OVA-expose mie (Figure ()). In ontrst, the supplementtion of kempferol rogte the CCR protein level t the kempferol-given osges of n mg/kg. Aitionlly, this stuy etermine the eotxin- proution in lung tissues of OVA-hllenge mie. OVA elevte the eotxin- protein level in mouse lung tissues (Figure ()). However, in OVA-experiene mie supplemente with kempferol, the eotxin- proution ws ose epenently iminishe... Inhiitory Effet of Kempferol on Tyk-STAT Ativtion. Ativtion of TLR y LPS les to promotion of the inflmmtory mehnisms inluing JNK/SAPK, NF-κB, n JAK/STAT pthwys []. This stuy eluite whether Tyk ownstrem signling ws responsile for irwy inflmmtion inue y LPS. BEAS-B ells were inute with μg/ml LPS, n Tyk tivtion ws etermine se on 6 h intervl up to h. Tyk phosphoryltion ws grully elevte up to 8 h n therefter iminishe (Figure ()). When kempferol ws e to LPS-expose BEAS-B ells, the Tyk tivtion ws suppresse in ose-epenent mnner (Figure ()). Similr effets on Tyk tivtion were oserve with IL-8 (Figure()). This stuy further teste whether the eotxin- inution through TLR signling y oth LPS n IL-8 entile Tyk tivtion. The Tyk inhiitor t μm suppressethe eotxin- inution in IL-8-stimulte BEAS-B ells in similr mnnerto μm kempferol (Figure ()). Likewise, phosphorylte Tyk ws notly oserve in perironhil regions of OVA-expose mouse lung tissues, eviene y immunofluoresent FITC tissue stining (Figure()). However, the FITC green fluoresene isppere in lung tissues y supplying kempferol to OVA-hllenge mie t the osges of n mg/kg (Figure ()), initing tht kempferol iminishe irwy inflmmtion y eterring the Tyk tivtion... Disturne of STAT Trnstivtion y Kempferol. Next, this stuy exmine whether the phosphoryltion of STAT n STAT, the Tyk ownstrem effetors, ws promote y LPS in BEAS-B ells. The phosphoryltion of oth STAT n STAT peke t 8 h n stye up in LPS-expose BEAS-B ells (Figure 6()). When BEAS-B ells were tivte y μg/ml LPS, μm kempferol signifintly suppresse the phosphoryltion of STAT n STAT, resulting in n inrese in unphosphorylte STAT (Figure 6()). Thus, kempferol my e n ntgonist to this inution of STAT/ signling in response to LPS in BEAS- B ells. This implies tht LPS promote Tyk tivtion n

5 Eviene-Bse Complementry n Alterntive Meiine Untrete ontrol μg/ml LPS Kempferol (μm) Mrker (p) Untrete μg/ml LPS ontrol 6 6 (min) TLR 89 kd β-tin kd β-tin ( p) TLR (77 p) TLR (fol of ontrol), Mrker Untrete (p) ontrol μg/ml LPS Kempferol (μm) β-tin ( p) TLR (77 p) () () μg/ml LPS Untrete ontrol μg/ml OxPAPC μm Kempferol IL-8 8 kd IL-8 (fol of ontrol) IL-8 seretion (ng/ml).... Untrete ontrol Kempferol (μm) μg/ml LPS () () Figure : Inhiitory effets of kempferol on expression levels () n trnsription () of TLR n loke of IL-8 seretion y TLR inhiition () n IL-8 seretion () in LPS-stimulte BEAS-B ells. After ulturing ells with μg/ml LPS in the sene n presene of μm kempferol or μg/ml OxPAPC for 8 h, ell extrts were sujete to 8% SDS-PAGE n western lot nlysis with primry ntioy ginst TLR n IL-8. Representtive lot t were otine from experiments, n β-tin protein ws use s n internl ontrol. The mrna levels of TLR were nlyze y using RT-PCR (). β-tin ws use s housekeeping gene for the omplifition with TLR. The r grphs (men ± SEM) in the ottom pnels represent quntittive results of lots. Cell ulture mei were ollete for the mesurement of IL-8 seretion y using n ELISA kit (). Vlues not shring ommon letter re signifintly ifferent t P <.. sequentilly tivte STAT/ signling leing to irwy inflmmtion. SOCS fmily memers re ytokine-inuile negtive regultors of ytokine signling. The expression of SOCS, the protein ining to Tyk/JAK n inhiiting their tivity, ws mpene in LPS-experiene BEAS-B ells (Figure 6()). This result prove tht LPS positively regulte STAT signling pthwy, while kempferol isture this pthwy y restoring the SOCS expression in n opposite fshion. Aoringly, kempferol my lunt IL-8 signling y enhning the inhiitory funtion of SOCS trgeting Tyk tivity.

6 6 Eviene-Bse Complementry n Alterntive Meiine 9 8 MIP- proution (ng/mg tissue protein) 7 6 Control mie OVA-hllenge mie + Kempferol (mg/kg) % OVA sensitiztion 9 () OVA-hllenge mg/kg kempferol-trete mie OVA-hllenge mg/kg kempferol-trete mie 8 7 CXCR tissue level (fol of negtive ontrol) 6 () + Kempferol (mg/kg) % OVA sensitiztion () Figure : Inhiition of MIP- proution () n CXCR expression ( n ) y kempferol. Inhiition of MIP- proution n CXCR expression y kempferol in OVA-hllenge mouse lung tissues. Tissue extrts were ollete for the mesurement of MIP- proution y using n ELISA kit (). Immunofluoresene nlysis showing inhiition of CXCR expression in OVA-hllenge mouse lung tissues y kempferol (). CXCR loliztion ws visulize with Cy-onjugte seonry ntioy. Nuler stining ws one with DAPI. CXCR ws ientifie s re stining n quntifie y using n optil mirosope system (). Eh photogrph ws representtive of four mie. Mgnifition: -fol. Vlues in the r grphs (men ± SEM) not shring ommon letter re signifintly ifferent t P <.. Consistent with LPS-inue STAT tivtion in irwy epithelil ells, the OVA hllenge inrese nuler trnslotion of STAT, mnifeste s rown nuler stining. There ws signifint inrese in the numers of rk rown-stine nuler STAT oserve (Figure 7()), emonstrting tht the OVA initement inflme nuler tivtion of STAT. In ontrst, the OVA-promote STAT trnstivtion iminishe in kempferol-elivere mie (Figure 7()). Therefore, the speifi loke of Tyk-STAT in the irwy/lung of sensitize mie y orl ministrtion of kempferol my e useful nti-inflmmtory strtegy to onfer sthmti protetion.. Disussion Inflmmtory n llergi sthm is hrterize y the infiltrtion of eosinophils, mst ells, n T lymphoytes into irwy epithelium [, ]. The interply etween these

7 Eviene-Bse Complementry n Alterntive Meiine 7 ng/ml IL-8 μg/ml LPS Eotxin- Untrete ontrol Kempferol (μm) 8. kd Eotxin- Untrete ontrol μg/ml OxPAPC μm Kempferol 8. kd β-tin kd β-tin kd Eotxin- (fols of ontrol) 7 6 Eotxin- (fol of ontrol) () () % OVA sensitiztion Kempferol (mg/kg) + CCR kd 6 CCR (fol of negtive ontrol) Eotxin- proution (ng/mg tissue protein) + Kempferol (mg/kg) () () % OVA sensitiztion Figure : Western lot t showing inhiition of eotxin- expression y kempferol or OxPAPC in LPS- or IL-8-stimulte in BEAS-B ells ( n ). After ulturing ells with μg/ml LPS in the sene n presene of μm kempferol or μg/ml OxPAPC for 8 h, ell extrts were sujete to % SDS-PAGE n western lot nlysis with primry ntioy ginst eotxin-. β-tin protein ws use s n internl ontrol. Inhiition of CCR expression n eotxin- seretion y kempferol in OVA-hllenge mouse lung tissues ( n ). Tissue extrts were sujete to western lot nlysis with primry ntioy ginst murine CCR (). The r grphs (men ± SEM, n=) in the ottom pnels represent quntittive results. Eotxin- proution ws mesure in OVA-hllenge mouse lung tissues y using n ELISA kit (). Vlues in r grphs not shring letter inite signifint ifferene t P <..

8 8 Eviene-Bse Complementry n Alterntive Meiine Phospho-Tyk β-tin μg/ml LPS (h) kd kd () Phospho-Tyk Untrete ontrol μg/ml LPS Kempferol (μm) kd Phospho-Tyk Untrete ontrol ng/ml IL-8 Kempferol (μm) kd β-tin Phospho-Tyk (fol of ontrol) kd β-tin Phospho-Tyk (fol of ontrol) kd () () Figure : Temporl tivtion of Tyk () n its loke y kempferol in LPS-expose epithelil ells. BEAS-B ells were ulture with μg/ml LPS () or ng/ml IL-8 () in the sene n presene of μm kempferol for 8 h. Totl ell protein extrts were sujete to western lot nlysis with primry ntioy ginst phosphorylte Tyk. β-tin ws use s n internl ontrol.the r grphs(men ± SEM, n = ) in the ottom pnels represent quntittive ensitometri results. Mens without ommon letter iffer, P <.. ells n irwy epithelil ells plys n importnt role in the pthogenesis of n sthmti episoe []. The speifi ytokines suh s IL-, IL-, n IL- use severl key fetures of llergi ronhil sthm []. In ition, the eosinophil tthment n infiltrtion into the irwy epithelium entil the ining of eotxin proteins to CCR expresse on eosinophils, sophils, n Th ells []. The CXC hemokine IL-8 is proinflmmtory meitor ssoite with the hemotxis n egrnultion of neutrophils, T ells, sophils,n eosinophils [7]. It is proue y mrophges n lung epithelil ells into lung flui n reruits neutrophils n eosinophils to the sites of inflmmtion. Aoringly, the overexpression of IL-8 in humn ronhil epithelil ells plys pivotl role in the reruitment n infiltrtion of eosinophils into inflme irwys, whih le to irwy wll remoeling through tivte intrellulr signling pthwys. This stuy showe the enhne seretion of IL- 8 n eotxin- from enotoxin-expose irwy epithelil ells n the inrese inution of MIP- n CXCR in lung tissues of OVA-hllenge mie. MIP- is funtionl homolog of humn IL-8 in mie. CXCR is the reeptor to IL-8 tht is powerful neutrophil hemotti ftor. Also, kempferol mpene epithelil seretion of IL-8 n eotxin- n the inution of lung tissue CXCR. Aitionlly, kempferol inhiite the CCR inution n eotxin- seretion enhne y OVA hllenge, initing tht kempferol my inhiit inflmmtory ell infiltrtion into the lesion sites of sthmti inflmmtion. It shoul e note tht the eotxin- seretion my e seonry to IL-8 inution. Severl polyphenols re effetive in llying llergi inflmmtion, resulting in symptom relief with the use of llopthi meiines [ ]. Soy isoflvones suppress irwy inflmmtion, hyperresponsiveness, n irwy remoeling in murine moel of llergi sthm []. Chrysin inhiits mst ell-erive llergi inflmmtory retions y loking proution of histmine relese n proinflmmtory ytokines []. Although iverse unetermine moleulr trgets hve een eviene, the moleulr mehnisms unerlying ntillergi tions of polyphenols remin to e lrifie [, 6]. Our reent stuy emonstrte tht kempferol suppresse eosinophil infiltrtion n irwy inflmmtion in llergi sthm through isturing NF-κB signling n eotxin- seretion [8]. Similrly, fisetin meliortes sthmti phenotypes onomitnt with suppression of NF-κB n its ownstrem

9 Eviene-Bse Complementry n Alterntive Meiine 9 ng/ml IL-8 Untrete ontrol μm μm Tyk inhiitor Kempferol Eotxin- 8. kd β-tin kd Control mie OVA-hllenge mie Eotxin- (fol of ontrol) 6 () OVA-hllenge mg/kg kempferol-trete mie OVA-hllenge mg/kg kempferol-trete mie Phospho-Tyk tissue level (fol of negtive ontrol) () + Kempferol (mg/kg) % OVA sensitiztion () Figure:EffetofTykinhiitiononeotxin-expressioninIL-8-stimulteBEAS-Bells().BEAS-BellsweretretewithμM Tyk inhiitor n μm kempferol expose to ng/ml IL-8 for 8 h. Cell extrts were sujete to western lot nlysis with primry ntioy ginst eotxin- ( seprte experiments, ()). β-tin ws uses n internl ontrol. The r grphs (men± SEM) in theottom pnel represent ensitometri results. Immunofluoresene nlysis showing inhiition of Tyk tivtion in OVA-hllenge mouse lung tissues y kempferol ( n ). Cytoplsmi Tyk ws visulize with n FITC-onjugte seonry ntioy. Nuler stining ws one with DAPI. Tyk ws ientifie s green stining n quntifie y using n optil mirosope system (). Eh photogrph is representtive of four mie. Mgnifition: -fol. Mens without ommon letter iffer, P <.. hemokines [6]. In ition, stiin inhiitstheeosinophil migrtion n tivity of hemokines n hesion moleules involve in the inflmmtory proess of sthm y suppressing the NF-κB pthwy[7]. Queretin inhiits IgE-meite relese of proinflmmtory meitors from humn mst ells, possily ue to inhiition of intrellulr lium influx n PKCθ signling [7]. However, the possile tion mehnism(s) of kempferol ntgonizing the inution of inflmmtory meitors responsile for irwy llergi inflmmtion re not still efine. Exposure to LPS inreses the severity of sthm, whih tivtes TLR signling in regultion of Th-riven irwy isese [7]. In this stuy, the epithelil inution of IL- 8 vi TLR pthwy stimulte eotxin- expression ssoite with sthmti inflmmtion. Consistently in OVAhllenge irwy tissues MIP-, CXCR, n CCR were simultneously inue, initive of possile irwy tivtion of eotxin- y IL-8. Most of ligns to CCR re ssoite with sthm, n CCR hs eome n ppeling possiility in sthm tretment or therpy [, 8]. Kempferol

10 Eviene-Bse Complementry n Alterntive Meiine Phospho-STAT (Tyr 7) β-tin μg/ml LPS (h) 79 kd kd () μg/ml LPS Phospho-STAT (ser 77) Untrete ontrol Kempferol (μm) 9 kd Phospho-STAT (Tyr 7) Totl STAT 79 kd 86 kd SOCS- Untrete ontrol μg/ml LPS Kempferol (μm) kd Phospho-STAT/ (fol of ontrol) β-tin.... e kd SOCS- (fol of ontrol) β-tin kd Phospho-STAT Phospho-STAT () Figure 6: Time ourse response of STAT (), suppression of STAT/ phosphoryltion (), n elevtion of SOCS- expression () y kempferol in LPS-stimulte BEAS-B ells. BEAS-B ells were trete with μm kempferol in the sene n presene of μg/ml LPS for 8 h. For western lot nlysis, totl ell protein extrts were immunolotte with primry ntioy ginst phosphorylte STAT, phosphorylte STAT, totl STAT, SOCS-, or β-tin s n internl ontrol. The r grphs (men ± SEM, n=) in the ottom pnels represent quntittive ensitometri results. Mens without ommon letter iffer, P <.. () suppresse the inution of CXCR n CCR enhne y OVA hllenge. Ativte TLR les to the promotion of the inflmmtory mehnisms inluing severl ownstrem pthwys of mitogen-tivte protein kinsen, NF-κB, n JAK/STAT []. The urrent stuy investigte Tyk-STATresponsive mehnism y whih kempferol isle the IL- 8 responses in lung/irwy epithelil ells through inflmmtory TLR signling pthwy. The ownregultion of IL- 8responseykempferolinirwyepithelilellsthrough isturing signling pthwys of Tyk-STAT/ prevente explosive sthmti retions ue to eotxin- tivtion. The STAT proteins, ytokine-inuile trnsription ftors, re ruil for ytokine signling n the ute phse responses []. However, their role in meiting llergi responses in sthm is not well efine. One investigtion showe tht irwy epithelil STAT ws responsile for llergi inflmmtion y moulting Th ell reruitment n effetor funtion in murine moel of hroni sthm [9]. OurstuyfounthtSTATnSTATmighteinvolve in enotoxin-inue irwy epithelil IL-8 signling n susequent eotxin- tivtion. Likewise, the inhiition of STAT n STAT meliorte experimentl sthm y moulting lung CD(+) enriti ells phenotype n funtion []. Thus, trgeting STAT n STAT my provie the sis for novel therpy for sthmti inflmmtion []. In the urrent stuy, kempferol ttenute the STAT tivtion through loking the IL-8-Tyk pthwy linke to epithelil TLR signling inflme y LPS. Consistently, kempferol iminishe the levels of STAT tivte in OVA-hllenge mouse irwy/lung tissues. The polyphenol hesperiin- -O-methylether inhiits irwy hyperresponsiveness in murine moel of sthm y eresing the numer of inflmmtory ells n OVA-speifi IgE levels in serum n BALF []. In ition, hlorogeni i

11 Eviene-Bse Complementry n Alterntive Meiine Control mie OVA-hllenge mie 6 OVA-hllenge mg/kg kempferol-trete mie OVA-hllenge mg/kg kempferol-trete mie Phospho-STAT tissue level (fol of negtive ontrol) + Kempferol (mg/kg) % OVA sensitiztion () () Figure 7: Immunohistohemil stining of phosphorylte STAT in lung tissues otine from mie supplemente with mg/kg kempferol. Lung tissue setions were immunostine with speifi primry ntioy ginst phosphorylte STAT n, - iminoenziine-onjugte IgG. Counter stining ws onute with hemtoxylin. Phosphorylte STAT ws ientifie s rown nuler stining () n quntifie y using n optil mirosope system (). Eh photogrph is representtive of four mie. Mgnifition: -fol. Mens without ommon letter iffer, P <.. suppresses pulmonry eosinophili, IgE proution, n Th-type ytokine proution in n OVA-inue llergi sthm through inhiiting tivtion of STAT6 n JNK []. On the other hn, SOCS proteins hve n importnt mehnismforthenegtiveregultionoftheytokine-stat pthwy. Thus, SOCS proteins hve een explore s trgets for therpeuti strtegies in llergi sthm []. In this stuy, the SOCS expression ws reue in LPS-expose irwy epithelil ells, whih ws reverse y the kempferol tretment. Colletively, kempferol ooste the inhiition of the Tyk-STAT/ pthwy responsile for the ytokine signling of IL-8 n eventully regulte llergi sthm phenotype. In summry, this stuy investigte the potentil of kempferol s trgets for therpeuti strtegies in enotoxinor ytokine-ssoite irwy inflmmtion. Nontoxi kempferol suppresse LPS-inue proution of IL-8 n susequent inution of eotxin-. The IL-8 inution of eotxin- entile the meition of the TLR signling pthwy tht ws loke y kempferol. Aitionlly, this ompoun isture the IL-8-Tyk-STAT/ signling with upregultion of SOCS in enotoxin-expose irwy epithelil ells. In the in vivo BALB/ mouse stuy, kempferol ministrtion loke the inution of MIP-, CXCR, nccrinirwy/lungtissueselevteyovahllenge. Moreover, kempferol enumere the OVA hllengeinflme Tyk-STAT tivtion. Therefore, kempferol ws effetive in meliorting llergi n inflmmtory irwy iseses through isturing Tyk-STAT-responsive signling pthwy instigte y IL-8 vent in ellulr or niml moels of llergi sthm. Arevitions CCR: C-C hemokine reeptor type CXCR: C-X-C hemokine reeptor type JAK: Jnus kinse IL: Interleukin LPS: Lipopolyshrie NF-κB: Nuler ftor-κb OVA: Ovlumin PKC: Protein kinse SOCS: Suppressors of ytokine signling STAT: Signl trnsuers n tivtors of trnsription Th: T helper TLR: Toll-like reeptor Tyk: Tyrosine kinse.

12 Eviene-Bse Complementry n Alterntive Meiine Funing This stuy ws supporte y the Ntionl Reserh Fountion of Kore (96) n y the Ministry of Eution n Siene Tehnology n Ntionl Reserh Fountion of Kore through the Humn Resoure Trining Projet for Regionl Innovtion (--A----). Conflit of Interests J.H.Gong,D.Shin,S.Y.Hn,S.H.Prk,M.K.Kng,J. L. Kim, n Y. H. Kng elre tht there is no onflit of interests. Referenes [] S. P. Hogn, Reent vnes in eosinophil iology, Interntionl Arhives of Allergy n Immunology, vol., supplement, pp., 7. [] J. E. Pese, Asthm, llergy n hemokines, Current Drug Trgets,vol.7,no.,pp.,6. [] S. T. Holgte, The sentinel role of the irwy epithelium in sthm pthogenesis, Immunologil Reviews, vol.,no., pp. 9,. [] H. Kuipers n B. N. Lmreht, The interply of enriti ells, Th ells n regultory T ells in sthm, Current Opinion in Immunology,vol.6,no.6,pp.7 78,. [] C. J. Oliphnt, J. L. Brlow, n A. N. MKenzie, Insights into the initition of type immune responses, Immunology, vol., pp. 78 8,. 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13 Eviene-Bse Complementry n Alterntive Meiine murine moel of sthm, Journl of Immunology, vol. 78, no., pp , 7. [] M. Husing, M. Tepe, C. Uel et l., Inution of tolerogeni lung CD+ T ells y lol tretment with pstat- n pstat- inhiitor meliorte experimentl llergi sthm, Interntionl Immunophrmology, vol., pp.,. [] M. Profit, A. Sl, A. Bonnno et l., Cysteinyl leukotriene- reeptor tivtion in humn ronhil epithelil ell line les to signl trnsuer n tivtor of trnsription - meite eosinophil hesion, Journl of Phrmology n Experimentl Therpeutis,vol.,no.,pp.,8. [] Y.L.Yng,H.T.Hsu,K.H.Wng,C.S.Wng,C.M.Chen,n W. C. Ko, Hesperiin- -o-methylether is more potent thn hesperiin in phosphoiesterse inhiition n suppression of ovlumin-inue irwy hyperresponsiveness, Eviene- Bse Complementry n Alterntive Meiine, vol., Artile ID 986, pges,. [] H. R. Kim, D. M. Lee, S. H. Lee et l., Chlorogeni i suppresses pulmonry eosinophili, IgE proution, n Th-type ytokine proution in n ovlumin-inue llergi sthm: tivtion of STAT-6 n JNK is inhiite y hlorogeni i, Interntionl Immunophrmology, vol.,no.,pp. 8,.

14 MEDIATORS of INFLAMMATION The Sientifi Worl Journl Hinwi Pulishing Corportion Gstroenterology Reserh n Prtie Hinwi Pulishing Corportion Journl of Hinwi Pulishing Corportion Dietes Reserh Hinwi Pulishing Corportion Hinwi Pulishing Corportion Interntionl Journl of Journl of Enorinology Immunology Reserh Hinwi Pulishing Corportion Disese Mrkers Hinwi Pulishing Corportion Sumit your mnusripts t BioMe Reserh Interntionl PPAR Reserh Hinwi Pulishing Corportion Hinwi Pulishing Corportion Journl of Oesity Journl of Ophthlmology Hinwi Pulishing Corportion Eviene-Bse Complementry n Alterntive Meiine Stem Cells Interntionl Hinwi Pulishing Corportion Hinwi Pulishing Corportion Journl of Onology Hinwi Pulishing Corportion Hinwi Pulishing Corportion Prkinson s Disese Computtionl n Mthemtil Methos in Meiine Hinwi Pulishing Corportion AIDS Behviourl Neurology Hinwi Pulishing Corportion Reserh n Tretment Hinwi Pulishing Corportion Hinwi Pulishing Corportion Oxitive Meiine n Cellulr Longevity Hinwi Pulishing Corportion