Erucin Exerts Anti-Inflammatory Properties in Murine Macrophages and Mouse Skin: Possible Mediation through the Inhibition of NFκB Signaling

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1 Int. J. Mol. Si. 213, 14, ; doi:1.339/ijms Artile OPEN ACCESS Interntionl Journl of Moleulr Sienes ISSN Eruin Exerts Anti-Inflmmtory Properties in Murine Mrophges nd Mouse Skin: Possile Medition through the Inhiition of NFκB Signling Hn Jin Cho 1,2, Ki Won Lee 2,3,4 nd Jung Hn Yoon Prk 1,3, * Deprtment of Food nd Nutrition, Hllym University, Chunheon 2-72, Kore; E-Mil: hnjini@hllym..kr Institute of Green Bio Siene & Tehnology Seoul Ntionl University, Pyeonghng , Kore; E-Mil: kiwon@snu..kr Advned Institutes of Convergene Tehnology, Seoul Ntionl University, Suwon, Gyonggi-do , Kore WCU Biomodultion Mjor, Deprtment of Agriulturl Biotehnology nd Center for Food nd Bioonvergene, Seoul Ntionl University, Seoul , Kore * Author to whom orrespondene should e ddressed; E-Mil: jyoon@hllym..kr; Tel.: ; Fx: Reeived: 9 Septemer 213; in revised form: 25 Septemer 213 / Aepted: 3 Septemer 213 / Pulished: 15 Otoer 213 Astrt: Eruin, n isothioynte, is hydrolysis produt of gluoeruin found in rugul nd hs reently een reported to hve nti-ner properties in vrious ner ells. In this study, we ssessed the nti-inflmmtory effets of eruin nd the underlying mehnisms, using lipopolyshride ()-stimulted RAW murine mrophges nd 12-O-tetrdenoylphorol-13-ette-treted mouse skin. In RAW ells, eruin (2.5, 5 μmol/l) inhiited -indued prodution of nitri oxide nd prostglndin E 2. Eruin inhiited -indued degrdtion of the inhiitor of κbα nd trnslotion of p65 to the nuleus nd, susequently, redued -indued nuler ftor κb (NFκB) DNA inding tivities, s well s the trnsriptionl tivity of NFκB, leding to the deresed expression of NFκB-trget genes, inluding tumor nerosis ftor-α, interleukin (IL)-6, IL-1β, induile nitri oxide synthse (inos) nd ylooxygense (COX)-2, s well s trnsriptionl tivity of inos nd COX-2. In mie, eruin (1, 3 nmoles) tretment signifintly inhiited phorol ester-indued formtion of er edem nd expression of inos nd COX-2 proteins. These results indite tht eruin exerts potent

2 Int. J. Mol. Si. 213, nti-inflmmtory tivity y inhiiting the pro-inflmmtory enzymes nd ytokines, whih my e medited, t lest in prt, vi the inhiition of NFκB signling. Keywords: eruin; inflmmtion; mouse skin; murine mrophges 1. Introdution Inflmmtion is the primry response of living tissues to injury nd infetion nd plys n importnt role in oth innte nd dptive immunity. However, side from these positive roles of inflmmtion, unresolved nd reurrent inflmmtion is ssoited with vriety of diseses, inluding ner, type 2 dietes, oesity, rdiovsulr diseses nd neurodegenertive diseses [1]. Sine inflmmtory ells produe inflmmtory meditors (retive oxygen/nitrogen speies nd ytokines) whih n indue muttion in DNA [2,3], exessive inflmmtory response is use of ner. Therefore, prevention nd/or inhiition of inflmmtion re importnt for the prevention of these hroni degenertive diseses, nd it is importnt to identify nti-inflmmtory dietry ompounds nd to explore their underlying mehnisms of tions. Consumption of ruiferous vegetles is ssoited with severl helth enefits ginst ner, hert helth, dietes, sthm nd Alzheimer s disese [4,5]. These enefits of ruiferous vegetles hve een ttriuted to their ontining vrious iotive ompounds (sulfur-ontining ompounds, indole derivtives nd phenoli ompounds). Among these ompounds, isothioyntes (sulfur-ontining ompounds nd produed y enzymti onversion of gluosinoltes) hve een shown to exert vrious iologil effets, inluding nti-inflmmtory properties [6]. For exmple, enzyl isothioynte (BITC), phenethyl isothioynte (PITC) nd sulforphne (SFN) hve een found to possess nti-inflmmtory tivities [7 1]. Eruin, 4-(methylthio)utyl isothioynte (struture in Figure 1A), is hydrolysis produt of gluoeruin, whih is found in rugul, nd n in vivo redution produt of SFN. Eruin hs een reported to exert hemopreventive effets y vrious mehnisms, inluding ltertion of phse I, II nd III enzymes, regultion of ell prolifertion y indution of ell yle rrest nd poptosis nd downregultion of ndrogen reeptor signling [11]. The inflmmtory responses re medited y vrious types of ells, inluding lymphoytes, mrophges, dendriti ells nd proliferting firolsts of the onnetive tissue [12]. During inflmmtion, irulting monoytes migrte into inflmed tissues nd differentite into mrophges. The tivtion of mrophges results in the relese of pro-inflmmtory ytokines, suh s tumor nerosis ftor-α (TNF-α), interleukin (IL)-6 nd IL-1β nd the prodution of nitri oxide (NO) y induile nitri oxide synthse (inos) [13]. Prostglndin E 2 (PGE 2 ), key meditor of immunopthology, is lso produed y ylooxygense-2 (COX-2) in tivted mrophges [14]. Nuler ftor κb (NFκB), trnsription ftor, plys ritil role in multiple iologil proesses, inluding inflmmtion, ell prolifertion nd poptosis. It is well known tht NFκB regultes the trnsription of multiple genes ssoited with inflmmtory responses, suh s inos, COX-2, TNF-α, IL-1β nd IL-6 [15]. Therefore, the inhiition of the NFκB pthwy ould e good strtegy to prevent nd/or ure inflmmtion-ssoited diseses, nd numerous inhiitors of NFκB, inluding nturl produts, hve een reported through wide vriety of studies [16,17].

3 Nitrite (μmol/l) PGE 2 (ng/l) Cell viility (sorne t 57 nm) Int. J. Mol. Si. 213, In this study, we explored the nti-inflmmtory effets of eruin nd the underlying mehnisms using lipopolyshride ()-stimulted mrophges. Our results indite tht eruin dereses the expression of inflmmtory meditors (inos nd COX-2) nd pro-inflmmtory ytokines (TNF-α, IL-1β nd IL-6) through the inhiition of NFκB signling. We lso demonstrte the potent in vivo nti-inflmmtory effets of eruin in 12-O-tetrdenoylphorol-13-ette (TPA)-treted mouse skin. 2. Results nd Disussion 2.1. Eruin Dereses -Indued Prodution of NO nd PGE 2 in RAW Cells In order to determine non-ytotoxi onentrtions of eruin for RAW ells, we first treted the ells with vrious onentrtions of eruin for 24 h. Cell viility ws not ffeted y eruin up to the onentrtion of 1 μmol/l (Figure 1B). In order to evlute the nti-inflmmtory effets of eruin in -stimulted ells, 24-h-onditioned medi were olleted nd ssyed for NO y the Griess regent system nd for PGE 2 y ELISA. Eruin tretment deresed -indued prodution of NO nd PGE 2 in dose-dependent mnner (Figure 1C,D). Figure 1. Eruin dereses lipopolyshride ()-indued NO nd prostglndin E 2 (PGE 2 ) prodution in RAW ells. (A) Struture of eruin; (B D) RAW ells were treted with vrious onentrtions of eruin in the presene of. Vile ell numers were estimted y the MTT Assy (B); The 24-h-onditioned medi were olleted for the estimtion of NO (C) nd PGE 2 (D) onentrtions. Eh r represents the men ± SEM (n = 4). Mens without ommon letter differ (p <.5). A B 2 S Eruin N C S C D 5, 4, 3, d 2, 1, d

4 inos mrna (reltive onentrtion) COX-2 mrna (reltive onentrtion) Int. J. Mol. Si. 213, We next investigted whether eruin inhiits the expression of inos nd COX-2. The results of Western lot nlysis reveled tht inresed the protein expression of inos nd COX-2, whih ws deresed y eruin tretment (Figure 2A). The levels of inos nd COX-2 mrnas were hnged in prllel with those of their orresponding proteins (Figure 2B,C). Eruin tretment signifintly inhiited -indued inos nd COX-2 trnsriptionl tivity (Figure 2D,E). These results indite tht eruin dereses -indued NO nd PGE 2 prodution through the downregultion of inos nd COX-2 expression. Figure 2. Eruin dereses -indued expression of induile nitri oxide synthse (inos) nd ylooxygense (COX)-2 in RAW ells. RAW ells were treted with vrious onentrtions of eruin in the presene of. (A) Cell lystes were sujeted to Western lotting with their relevnt ntiodies. The reltive undne of eh nd to their own β-tin ws quntified, nd the ontrol levels were set t 1%. The djusted men ± SEM (n = 3) of eh nd is shown ove eh lot; (B,C) Totl RNA ws isolted, nd rel-time RT-PCR ws performed; (D,E) RAW ells were trnsfeted with the murine inos or COX-2 reporter gene onstrut. The trnsfeted ells were treted with vrious onentrtions of eruin in the presene of. Cell lystes were prepred to determine luiferse tivity. Eh r represents the men ± SEM (n = 4). Mens without ommon letter differ (p <.5). ND: not deteted. A ND ND inos COX-2 -tin B 8 C 1, , 5

5 inos trnsriptionl tivity (% of ontrol) COX-2 trnsriptionl tivity (% of ontrol) Int. J. Mol. Si. 213, Figure 2. Cont. D E 2,5 2, 1,5 1, Eruin Dereses -Indued Prodution of TNF-α, IL-6 nd IL-1β in RAW Cells We next exmined whether eruin dereses the expression of pro-inflmmtory ytokines. The mounts of TNF-α, IL-6 nd IL-1β relesed into onditioned medi were inresed y tretment, nd the inreses were inhiited y eruin tretment (Figure 3A). Additionlly, results of rel-time RT-PCR reveled tht eruin inhiited -indued mrna expression of TNF-α, IL-6 nd IL-1β (Figure 3B) Eruin Inhiits -Indued Ativtion of NFκB Signling in RAW Cells As NFκB regultes the expression of genes enoding inos, COX-2, TNF-α, IL-6 nd IL-1β [18,19], we next determined whether eruin inhiits NFκB signling. Upon stimultion, inhiitors of NFκB (IκB)-α re phosphorylted nd degrded, nd the NFκB proteins in the ytoplsm re lierted from IκB nd trnsloted to the nuleus, where they ind to the promoter regions of NFκB-responsive genes [15]. tretment redued the levels of IκB-α, nd eruin suppressed the -indued redution in IκB-α (Figure 4A). The levels of ytosoli p65 protein were redued y tretment, whih ws suppressed y eruin, wheres the levels of nuler p65 were mrkedly inresed y, nd this inrese ws suppressed y eruin tretment. Additionlly, NFκB DNA inding ws mrkedly inresed y, whih ws suppressed y eruin pre-tretment (Figure 4B). Furthermore, eruin inhiited the trnsriptionl tivity of NFκB in -stimulted ells (Figure 4C).

6 IL-1β (ng/l) IL-1β mrna (reltive onentrtion) IL-6 (ng/l) IL-6 mrna (reltive onentrtion) TNF-α (ng/l) TNF-α mrna (reltive onentrtion) Int. J. Mol. Si. 213, Figure 3. Eruin dereses -indued TNF-α, IL-6 nd IL-1β prodution in RAW ells. RAW ells were treted with vrious onentrtions of eruin in the presene of. (A) The 24-h-onditioned medi were olleted for the estimtion of TNF-α, IL-6 nd IL-1β onentrtions; (B) Totl RNA ws isolted nd rel-time RT-PCR performed. Eh r represents the men ± SEM (n = 4). Mens without ommon letter differ (p <.5). A 25, 2, 15, 1, B , d 1 8, 6, 4, 1,2 1, 8 6 2, d , 1, , 5

7 NF B reporter tivity (Aritrry Unit) Int. J. Mol. Si. 213, Figure 4. Eruin inhiits -indued tivtion of NFκB signling in RAW ells. (A,B) RAW ells were treted with vrious onentrtions of eruin for 3 min. ws then dded nd inuted for nother 2 min. (A) Totl ell lystes were sujeted to Western lotting with their relevnt ntiodies; (B) Cytosoli frtions nd nuler extrts were prepred for Western lotting with n nti-p65 ntiody nd Eletrophoreti Moility Shift Assy (EMSA). The reltive undne of eh nd ws quntified, nd the ontrol levels were set t 1%. The djusted men ± SEM (n = 3) of eh nd is shown ove eh lot; (C) RAW ells were trnsfeted with NFκB-luiferse reporter plsmid. The trnsfeted ells were treted with vrious onentrtions of eruin in the presene of. Cell lystes were prepred to determine luiferse tivity. Eh r represents the men ± SEM (n = 4). Mens without ommon letter differ (p <.5). B p65 (Cytosol) A d I B p65 (Nuler) -tin NF B DNA inding tivity C Eruin Dereses TPA-Indued Edem Formtion in Mouse Inflmmtion Model We next tested the in vivo nti-inflmmtory effet of eruin using the TPA-indued mouse er edem model. Femle ICR mie were treted (in the left er) with vrious doses of eruin 3 min efore the pplition of TPA. The pplition of eruin to mouse er signifintly inhiited TPA-indued edem formtion (Figure 5A). Immunofluoresent stining reveled tht eruin pre-tretment inhiited TPA-indued inos expression in er epidermis (Figure 5B,C). Using similr skin inflmmtion

8 Stining intensity (Aritrry Unit) COX-2 inos Weight of edem (mg) Int. J. Mol. Si. 213, model, we hve previously reported tht TPA tretment inreses the infiltrtion of inflmmtory ells in mouse skin [2]. TPA tretment indued the expression of COX-2 in infiltrting inflmmtory ells, nd this expression ws suppressed y eruin pre-tretment (Figure 5B,C). Figure 5. Eruin dereses 12-O-tetrdenoylphorol-13-ette (TPA)-indued edem formtion in mouse inflmmtion model. Eruin ws topilly pplied to the mouse er 3 min prior to the topil pplition of TPA. (A) The weights of 6-mm dimeter er punh smples were mesured 4 h fter TPA tretment. Eh r represents the men ± SEM (n = 4); (B) Er setions were stined with their relevnt ntiodies, s desried in the Experimentl setion. Photogrphs of immunofluoresent stining (sle r = 5 μm), whih re representtive of four different nimls, re shown; (C) Quntifition ws performed y lulting the stining intensities using ImgeJ. Eh r represents the men ± SEM (n = 4). Mens without ommon letter differ (p <.5). DEXA: dexmethsone. A TPA (5 nmoles) 1 3 Dex Eruin (nmoles) B TPA (5 nmoles) 1 3 DEXA C 5.E+6 4.E+6 3.E+6 Eruin (nmoles) nmoles Eruin TPA + nmoles Eruin TPA + 1 nmoles Eruin TPA + 3 nmoles Eruin TPA + DEXA 2.E+6 1.E+6.E+ inos COX-2

9 Int. J. Mol. Si. 213, Disussion Cruiferous vegetles, inluding ge, rooli, mustrd nd rugul, re rih soures of gluosinoltes, sulfur-ontining ompounds. Reently, gluosinolte metolites hve generted gret del of interest, due to their iologil effets, inluding nti-inflmmtory effets [7 1], s well s nti-ner tivity [4,5,21]. For exmple, BITC (derived from gluotropeolin) nd PITC (derived from gluonsturtiin) hve een found to possess nti-ner [22,23] nd nti-inflmmtory tivities [7,8]. Additionlly, SFN, n isothioynte derived from hydrolysis of gluorphnin, is well-known hemopreventive ompound [23] nd nti-inflmmtory gent [9,1]. As the inflmmtion meditors nd ellulr effetors re importnt onstituents of the tumor miroenvironments, inflmmtion hs een inriminted in the development nd progression of vriety of humn ners [24]. Thus, it n e postulted tht the inhiition of inflmmtion is n importnt strtegy for the inhiition of ner development nd progression y these ompounds. Eruin, the sulfide nlog of SFN, hs een shown to exhiit nti-ner tivities [11]. However, to dte, the nti-inflmmtory effets of eruin hve not een studied in detil, exept in the reent study y Yehud nd ollegues, whih reported tht eruin deresed the trnsription of pro-inflmmtory moleules (TNF-α, IL-1β nd IL-12) in THP-1 humn ute monoyti leukemi ells, whih were treted with [25]. In the present study, we demonstrte tht eruin exerts potent nti-inflmmtory properties in -stimulted mrophges, whih is medited through the inhiition of NFκB signling. We lso show tht eruin t very low doses (1 nd 3 nmoles) effetively inhiits inflmmtory responses in mouse skin, suggesting tht this ompound hs the potentil to e used s n nti-inflmmtory gent. In the present study, we hve demonstrted tht μmol/l nd 1 3 nmol of eruin exert nti-inflmmtory effets in mrophges nd in mie, respetively. In niml studies, Aoui nd ollegues reently showed tht orl dministrtion of eruin (7.375 μmol/25 g mie) effetively deresed tumor growth in xenogrft model of ldder ner [26]. Eruin is hydrolysis produt of gluoeruin found in rugul nd rooli sprouts. It hs een reported tht when humn sujets onsumed 4 g of rooli sprouts (ontining 71 μmol gluoeruin), plsm eruin peks were rehed 3 h fter the onsumption, nd the plsm onentrtions of eruin were pproximtely 1 μmol/l [27], inditing tht gluoeruin in food is rpidly metolized nd eruin is quikly sored into the lood. Together, these results indite tht the iovilility of eruin is high nd eruin is effetive t low dose in humns nd mie. Additionlly, eruin my e generted in the ody from the onsumption of SFN preursors, euse there is evidene for interonversion etween SFN nd eruin in humns [27,28]. For exmple, eruin metolites were found in urine smples of sujets onsuming rooli nd red nd white ge (not ontin gluoeruin), nd the rtio of gluoeruin/gluorphnin in the rooli sprouts nd tht of eruin/sfn metolites in the plsm of sujets were different. However, future studies re needed to determine the effiieny of interonversion etween SFN nd eruin. NFκB, n induile trnsription ftor, regultes the expression of lrge numer of genes tht re involved in the regultion of inflmmtion [12]. For exmple, induile enzymes (inos nd COX-2) nd pro-inflmmtory ytokines (inluding TNF-α, IL-6 nd IL-1β) were regulted y NFκB tivtion [16,18,19]. In the present study, we oserved tht eruin: (1) inhiits the degrdtion of IκB-α nd the trnsriptionl tivity of NFκB; (2) dereses NO nd PGE 2 prodution through

10 Int. J. Mol. Si. 213, inhiition of inos nd COX-2 expression; nd (3) dereses the seretion nd mrna expression of TNF-α, IL-1β nd IL-6 in -stimulted mrophges. Furthermore, tretment with very low doses (1 nd 3 nmoles) of eruin dereses the formtion of edems nd the expression of inos nd COX-2 proteins in TPA-treted mouse skin (Figure 5). These results indite tht eruin inhiits the expression of pro-inflmmtory enzymes nd the seretion of ytokines, whih is medited vi the inhiition of NFκB signling. The NFκB fmily onsists of five memers, p65 (RelA), p5, RelB, p52 nd -Rel. NFκB is expressed in ll ell types, inluding mrophges, dendriti ells nd firolsts, s the min sites of inflmmtion. The different NFκB pthwys (nonil nd non-nonil) re distinguished y how they re stimulted nd regulted y meditors nd hve different funtions [12]. In the nonil NFκB pthwy, the NFκB proteins (p65 nd p5) re lolized in the ytoplsm y IκB. Upon tivtion y stimuli, suh s, IκB kinse phosphoryltes IκB protein, therey leding to the degrdtion of IκB y the 26S protesome. The lierted NFκB proteins re then trnsloted to the nuleus, where they ind to the promoter regions of NFκB-responsive genes, resulting in inresed gene expression involved in the pro-inflmmtory response [12,15]. In the present study, using murine mrophges, we lerly demonstrte tht eruin inhiits -indued degrdtion of IκB-α, trnslotion of p65 from the ytosol to nuleus nd NFκB DNA iding tivity nd NFκB reporter tivity (Figure 4). Results from our lortory, s well s those from other lortories indite tht the inhiition of NFκB signling is one of the mehnisms y whih other isothioyntes (BITC, PITC nd SFN) exert nti-inflmmtory properties [7 1]. Together, these results indite tht eruin exerts nti-inflmmtory properties y mehnisms similr to those of BITC, PITC nd SFN. Future study is needed to explore whether mehnisms other thn the inhiition of NFκB signling re involved in the nti-inflmmtory effet of eruin. 3. Experimentl Setion 3.1. Mterils The regents were quired from the following suppliers: eruin, LKT Lortories (Sint Pul, MN, USA); 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrzolium romide (MTT),, TPA nd nti-β-tin ntiody, Sigm (St Louis, MO, USA); ntiodies ginst inos nd COX-2 for Western lot nlysis, BD Trnsdution Lortories (Plo Alto, CA, USA); nti-nfκb p65 ntiody, Snt Cruz Biotehnology (Snt Cruz, CA, USA); nd nti-iκb-α ntiody, Cell Signling Tehnology (Beverly, MA, USA) Cell Culture nd MTT Assy The RAW ell line ws purhsed from the Amerin Type Culture Colletion (Mnsss, VA, USA) nd ws mintined in Duleo s Modified Egle Medium (DMEM) ontining 1% fetl ovine serum (FBS), 1 ku/l peniillin nd 1 mg/l streptomyin. To exmine the effets of eruin on ell viility, the ells were plted in 24-well pltes t density of 5, ells/well with DMEM ontining 1% FBS. Cells were serum-deprived for 24 h in DMEM ontining 1% FBS nd were

11 Int. J. Mol. Si. 213, then treted with vrious onentrtions of eruin in the presene of (1 mg/l) for 24 h. Vile ell numers were then estimted vi MTT Assy NO, PGE 2 nd Cytokine Assys The RAW ells were treted with eruin nd s desried ove, nd the 24-h-onditioned medi were olleted. The onentrtions of NO nd PGE 2 were mesured using the Griess regent system (Promeg, Mdison, WI, USA) nd PGE 2 ssy kit (R & D Systems, Minnepolis, MN, USA), respetively. The onentrtions of TNF-α, IL-6 nd IL-1β were mesured using ELISA kits (ebiosiene, Sn Diego, CA, USA) Western Blot Anlysis nd Eletrophoreti Moility Shift Assy (EMSA) Totl ell lystes, ytosoli frtions nd nuler extrts were prepred, nd Western lot nlyses were performed s desried previously [29]. For the Eletrophoreti Moility Shift Assy (EMSA), nuler extrts were inuted with [ 32 P]-leled NFκB oligonuleotide proes (Promeg) for 3 min [29]. Protein-DNA omplexes were resolved y 5% non-denturing gel, nd the gels were then visulized y utordiogrphy. The intensity of the nds otined from Western lot nlysis nd EMSA results ws quntified y using ImgeJ softwre (NIH, Bethesd, MD, USA). The ontrol levels were set t 1%, nd the djusted men ± SEM (n = 3) of eh nd is shown ove eh lot Rel-Time RT-PCR Totl RNA ws isolted using n RNesy Plus Mini Kit (Qigen, Vleni, CA, USA), nd DNA ws synthesized using Mxime RT PreMix (intron Biotehnology, Seongnm, Kore). Rel-time RT-PCR ws onduted s previously desried [29] Luiferse Reporter Gene Assy For reporter ssys, RAW ells were otrnsfeted with pgl-minos-1588 [3], pgl-mcox [31] or NFκB-luiferse reporter plsmid (Tkr Bio, Otsu, Shig, Jpn) together with ontrol reporter plsmid prl-tk (Promeg) using Nuleofetor-II (Amx, Githersurg, MD, USA). The trnsfeted ells were plted nd treted with vrious onentrtion of eruin in the presene of for 6 h. Luiferse ssys were performed using the Dul-Luiferse Reporter Assy, system ording the mnufturer s instrutions (Promeg). The firefly luiferse tivity ws normlized to the Renill luiferse tivity Mouse Er Edem Femle ICR mie (4 weeks of ge) were purhsed from Orient Bio In. (Gpyung, Kore) nd were limtized to lortory onditions t the niml reserh fility of Hllym University (Chunheon, Kore). All niml experimentl protools were pproved y the Animl Cre nd Use Committee of Hllym University (Hllym21-7-1). To evlute the nti-inflmmtory effet of eruin on er edem formtion, the mie were treted on the left er with vrious doses (, 1 nd 3 nmoles) of eruin for 3 min. Edem ws indued in the

12 Int. J. Mol. Si. 213, left er vi the topil pplition of 5 nmoles of TPA. Eruin nd TPA were dissolved in 2 μl of dimethyl sulfoxide/etone (15:85, v/v). Four hours fter TPA tretment, iopsies of the left nd right ers were performed using 6-mm punh, nd the individul iopsy ws weighed. The mount of edem formtion ws lulted y sutrting the weight of the right er (vehile treted) from tht of the left er (tretment). Dexmethsone (5 μg) ws used s positive ontrol. For immunofluoresent stining, er iopsies were fixed in 4% prformldehyde nd were emedded in prffin wx. Prffin-emedded setions (5 μm) were hydrted through xylene nd grded lohol. Setions were then permeilized with ie-old methnol nd inuted with nti-inos ntiody or nti-cox-2 ntiody (Cymn Chemils, Ann Aror, MI, USA). Setions were susequently wshed 4 times in TBS with.1% Tween 2 nd were inuted with Alex Fluor 488 got nti-rit-igg ntiody (Invitrogen, Crlsd, CA, USA). Nulei were ounterstined with 4',6-dimidino-2-phenylindole. Fluoresent imges were otined using Crl Zeiss AxioImger mirosope (Crl Zeiss, Jen, Germny), nd the fluoresene intensity ws lulted using ImgeJ softwre Sttistil Anlysis Dt were expressed s mens ± SEM nd nlyzed y ANOVA. Differenes etween tretment groups were nlyzed using Dunn s multiple rnge test, utilizing SAS sttistil softwre, version 9.2 (SAS Institute, Cry, NC, USA). Differenes were onsidered signifint t p < Conlusions In summry, using RAW murine mrophges, we demonstrte tht eruin inhiits the -indued prodution of NO nd PGE 2, y modulting the expression of inos nd COX-2 proteins, s well s the seretion of pro-inflmmtory ytokines (TNF-α, IL-6 nd IL-1β). Additionlly, we demonstrte tht eruin inhiits the trnsriptionl tivity of inos nd COX-2 nd mrna expression of inos, COX-2 TNF-α, IL-6 nd IL-1β, s well s NFκB signling. Furthermore, eruin suppresses inflmmtory responses nd inhiits the expression of inos nd COX-2 in TPA-treted mouse skin. Reurrent nd persistent inflmmtion is ssoited with rod vriety of diseses. The present results suggest tht eruin n e used s nturl nti-inflmmtory gent, whih n help to prevent or relieve hroni inflmmtion-relted diseses. Aknowledgments This reserh ws supported y the Bsi Siene Reserh Progrm through the Ntionl Reserh Foundtion of Kore (NRF), funded y the Ministry of Edution (NRF-212R1A1A2667) nd y n NRF grnt funded y the Koren government (MSIP) (NRF-213R1A2A2A54533). The uthors would like to thnk Ji Hee Kim for her tehnil ssistne. Conflits of Interest The uthors delre tht they hve no onflit of interest.

13 Int. J. Mol. Si. 213, Referenes 1. Aller, M.A.; Aris, N.; Fuentes-Julin, S.; Blzquez-Mrtinez, A.; Argudo, S.; Miguel, M.P.; Aris, J.L.; Aris, J. Coupling inflmmtion with evo-devo. Med. Hypotheses 212, 78, Lin, W.W.; Krin, M. A ytokine-medited link etween innte immunity, inflmmtion, nd ner. J. Clin. Invest. 27, 117, Lu, H.; Ouyng, W.; Hung, C. Inflmmtion, key event in ner development. Mol. Cner Res. 26, 4, Mnhli, S.; Chidmr Murthy, K.N.; Ptil, B.S. Cruil fts out helth enefits of populr ruiferous vegetles. J. Funt. Foods 212, 4, Higdon, J.V.; Delge, B.; Willims, D.E.; Dshwood, R.H. Cruiferous vegetles nd humn ner risk: Epidemiologi evidene nd mehnisti sis. Phrmol. Res. 27, 55, Brown, K.K.; Hmpton, M.B. Biologil trgets of isothioyntes. Biohim. Biophys. At 211, 181, Lee, Y.M.; Seon, M.R.; Cho, H.J.; Kim, J.S.; Prk, J.H. Benzyl isothioynte exhiits nti-inflmmtory effets in murine mrophges nd in mouse skin. J. Mol. Med. 29, 87, Lee, Y.M.; Cho, H.J.; Ponnurj, S.P.; Kim, J.; Kim, J.S.; Kim, S.G.; Prk, J.H. Phenethyl isothioynte inhiits 12-O-tetrdenoylphorol-13-ette-indued inflmmtory responses in mouse skin. J. Med. Food 211, 14, Woo, K.J.; Kwon, T.K. Sulforphne suppresses lipopolyshride-indued ylooxygense-2 (COX-2) expression through the modultion of multiple trgets in COX-2 gene promoter. Int. Immunophrmol. 27, 7, Heiss, E.; Herhus, C.; Klimo, K.; Brtsh, H.; Gerhuser, C. Nuler ftor kpp B is moleulr trget for sulforphne-medited nti-inflmmtory mehnisms. J. Biol. Chem. 21, 276, Melhini, A.; Trk, M.H. Biologil profile of eruin: A new promising ntiner gent from ruiferous vegetles. Toxins 21, 2, Gsprini, C.; Feldmnn, M. NF-kppB s trget for modulting inflmmtory responses. Curr. Phrm. Des. 212, 18, Vlledor, A.F.; Comld, M.; Sntmri-Bi, L.F.; Lloers, J.; Celd, A. Mrophge proinflmmtory tivtion nd detivtion: A question of lne. Adv. Immunol. 21, 18, Klinski, P. Regultion of immune responses y prostglndin E2. J. Immunol. 212, 188, Ymmoto, Y.; Gynor, R.B. IkppB kinses: Key regultors of the NF-kppB pthwy. Trends Biohem. Si. 24, 29, Gupt, S.C.; Sundrm, C.; Reuter, S.; Aggrwl, B.B. Inhiiting NF-kppB tivtion y smll moleules s therpeuti strtegy. Biohim. Biophys. At 21, 1799, Ymmoto, Y.; Gynor, R.B. Therpeuti potentil of inhiition of the NF-kppB pthwy in the tretment of inflmmtion nd ner. J. Clin. Invest. 21, 17, Lwrene, T. The nuler ftor NF-kppB pthwy in inflmmtion. Cold Spring Hr. Perspet. Biol. 29, 1, 1651.

14 Int. J. Mol. Si. 213, Bud, V.; Krin, M. Is NF-kppB good trget for ner therpy? Hopes nd pitflls. Nt. Rev. Drug Disov. 29, 8, Cho, H.J.; Lim, S.S.; Lee, Y.S.; Kim, J.S.; Lee, C.H.; Kwon, D.Y.; Prk, J.H. Hexne/ethnol extrt of Glyyrrhiz urlensis liorie exerts potent nti-inflmmtory effets in murine mrophges nd in mouse skin. Food Chem. 21, 121, Hyes, J.D.; Kelleher, M.O.; Eggleston, I.M. The ner hemopreventive tions of phytohemils derived from gluosinoltes. Eur. J. Nutr. 28, 47, Ro, C.V. Benzyl isothioynte: Doule troule for rest ner ells. Cner Prev. Res. 213, 6, Cheung, K.L.; Kong, A.N. Moleulr trgets of dietry phenethyl isothioynte nd sulforphne for ner hemoprevention. AAPS J. 21, 12, Mntovni, A.; Allven, P.; Si, A.; Blkwill, F. Cner-relted inflmmtion. Nture 28, 454, Yehud, H.; Sorok, Y.; Zlotkin-Frusi, M.; Gilhr, A.; Milner, Y.; Tmir, S. Isothioyntes inhiit psorisis-relted proinflmmtory ftors in humn skin. Inflmm. Res. 212, 61, Aoui, B.; Riedl, K.M.; Rlston, R.A.; Thoms-Ahner, J.M.; Shwrtz, S.J.; Clinton, S.K.; Mortzvi, A. Inhiition of ldder ner y rooli isothioyntes sulforphne nd eruin: Chrteriztion, metolism, nd interonversion. Mol. Nutr. Food Res. 212, 56, Clrke, J.D.; Hsu, A.; Riedl, K.; Bell, D.; Shwrtz, S.J.; Stevens, J.F.; Ho, E. Biovilility nd inter-onversion of sulforphne nd eruin in humn sujets onsuming rooli sprouts or rooli supplement in ross-over study design. Phrmol. Res. 211, 64, Vermeulen, M.; Vn den Berg, R.; Freidig, A.P.; vn Blderen, P.J.; Ves, W.H. Assoition etween onsumption of ruiferous vegetles nd ondiments nd exretion in urine of isothioynte merpturi ids. J. Agri. Food Chem. 26, 54, Cho, H.; Seon, M.; Lee, Y.; Kim, J.; Kim, J.; Kim, S.; Prk, J. 3,3'-Diindolylmethne suppresses the inflmmtory response to lipopolyshride in murine mrophges. J. Nutr. 28, 138, Cho, I.J.; Lee, A.K.; Lee, S.J.; Lee, M.G.; Kim, S.G. Repression y oxidtive stress of inos nd ytokine gene indution in mrophges results from AP-1 nd NF-kppB inhiition medited y B ell trnslotion gene-1 tivtion. Free Rdi. Biol. Med. 25, 39, Ki, S.H.; Choi, M.J.; Lee, C.H.; Kim, S.G. Glph12 speifilly regultes COX-2 indution y sphingosine 1-phosphte. Role for JNK-dependent uiquitintion nd degrdtion of IkppBlph. J. Biol. Chem. 27, 282, y the uthors; liensee MDPI, Bsel, Switzerlnd. This rtile is n open ess rtile distriuted under the terms nd onditions of the Cretive Commons Attriution liense (