Grape seed proanthocyanidin extract ameliorates murine autoimmune arthritis through regulation of TLR4/MyD88/NF-κB signaling pathway

Transcription

1 ORIGINAL ARTICLE Koren J Intern Me 218;33: Grpe see pronthoyniin extrt meliortes murine utoimmune rthritis through regultion of /MyD88/NF-κB signling pthwy Sng-Hyon Kim 1, Jihye Bng 2, Chng-Nm Son 1, Won-Ki Bek 3, n Ji-Min Kim 1 1 Division of Rheumtology, Deprtment of Internl Meiine, Keimyung University Dongsn Meil Center, Degu; 2 Keimyung University Shool of Meiine, Degu; 3 Deprtment of Miroiology, Keimyung University Shool of Meiine, Degu, Kore Reeive : Mrh 4, 216 Revise : April 8, 216 Aepte : April 27, 216 Corresponene to Ji-Min Kim, M.D. Division of Rheumtology, Deprtment of Internl Meiine, Keimyung University Dongsn Meil Center, 56 Dlseong-ro, Jung-gu, Degu 41931, Kore Tel: Fx: E-mil: okjimin@sm.or.kr Bkgroun/Aims: Grpe see pronthoyniin extrt (GSPE) hs een reporte to hve enefiil effet on regulting inf lmmtion. However, the nti-inflmmtory mehnism of GSPE remins unler. The im of this stuy ws to verify the influene of GSPE on the Toll-like reeptor 4 ()-meite signling pthwy in the regultion of murine utoimmune rthritis. Methos: Collgen-inue rthritis () ws inue in ilute rown non-gouti (DBA)/1J mie. The mie were trete with GSPE ( or 1 mg/kg) intrperitonelly. The severity of rthritis ws ssesse linilly, iohemilly, n histologilly. Immunostining for ws performe. The expressions of n ownstrem signling moleules were nlyze y Western lot. The effet of GSPE on lipopolyshrie (LPS)-inue tivtion ws lso evlute using RAW264.7 ells n firolst-like synovioytes (FLSs) from ptients with rheumtoi rthritis n from those with osteorthritis. Results: GSPE ttenute the linil severity of rthritis n erese histologil mge. GSPE tretment reue the numer of -stine ells in the synovium of mie with. GSPE lso ownregulte the expression of, myeloi ifferentition ftor 88 (MyD88) n phosphorylte IκBα synovil protein in mie. Conurrently, GSPE inhiite the nuler trnslotion of nuler ftor-κb (NF-κB) suunits (p65 n p5). LPS-inue tivtion ws suppresse y GSPE in humn FLS s well s in murine mrophges in vitro. Conlusions: Our results emonstrte tht GSPE meliorte y regulting the -MyD88-NF-κB signling pthwy. Keywors: Grpe see pronthoyniin extrt; Arthritis, experimentl; Tolllike reeptor 4; Arthritis, rheumtoi INTRODUCTION Rheumtoi rthritis (RA) is hroni utoimmune isese hrterize y persistent inflmmtion of the joint synovium n synovil hyperplsi. The errnt hnges, whih our in the synovium, le to joint mge n funtionl isility. Although the pthogenesis of RA hs not een lerly ientifie, it is ler tht sing network of pro-inflmmtory ytokines plys mjor role in the evelopment of RA [1]. Toll-like reeptor 4 () ontriutes to the inution of pro-inflmmtory ytokines y tivting innte Copyright 218 The Koren Assoition of Internl Meiine This is n Open Aess rtile istriute uner the terms of the Cretive Commons Attriution Non-Commeril Liense ( y-n/4./) whih permits unrestrite nonommeril use, istriution, n reproution in ny meium, provie the originl work is properly ite. pissn eissn

2 Kim SH, et l. New mehnism of tion of GSPE on immunity uner ertin irumstnes [2]. To te, severl stuies hve suggeste tht is implite in the pthogenesis of RA. is highly expresse in the inflme synovium of RA ptients [3]. The tivtion of the signling pthwy is involve in persistent inflmmtion n joint estrution in RA [4]. Assoitions etween single nuleotie polymorphisms n RA isese tivity hve een reporte [5]. Grpe see pronthoyniin extrt (GSPE) is stnrize wter-ethnol extrt erive from re grpe sees in whih vrious ntioxints inluing tehins n oligomeri pronthoyniins re umulte [6]. GSPE is me up of omintion of ingreients with 15% () tehin n () epitehin; 8% () epitehin 3-O-gllte, imers, trimers, tetrmers, n their glltes; n 5% pentmers, hexmers, heptmers, n their glltes [7]. Pronthoyniins re the most unnt phenoli ompouns in grpe sees. They hve een reporte to hve vrious io-protetive funtions suh s nti-teril, nti-virl, nti-llergi, nti-rinogeni, nti-thromoti, n vsoiltory tivities [8]. Pronthoyniins possess more potent nti-oxitive tivity thn vitmins C, E, n β-rotene [9]. Furthermore, the nti-inflmmtory properties of pronthoyniins on experimentl inflmmtion, inluing in niml moels of ulertive olitis, skin tumors, n ollgen-inue rthritis (), hve een shown in previous stuies [1-13]. However, little informtion is ville onerning how pronthoyniins regulte experimentl utoimmune rthritis. In this stuy, we hypothesize tht GSPE woul influene signling pthwys, whih ply ritil role in the pthogenesis of RA, in the evelopment of murine utoimmune rthritis. We lso exmine the immunologil effet of GSPE on firolst-like synovioytes (FLSs) in ptients with RA. METHODS Experimentl nimls Dilute rown non-gouti (DBA)/1J mie (6 to 8 weeks ol; SLC In., Hmmtsu, Jpn), were house in polyronte ges n fe with stnr mouse how (Rlston Purin, St. Louis, MO, USA) n wter liitum. All experimentl proeures were exmine n pprove y the Animl Reserh Ethis Committee of the Keimyung University (No. KM-29-6R). Inution of n GSPE tretment Mle DBA/1J mie were given n intrerml injetion of 1 μg of ovine type II ollgen emulsifie in omplete Freun s juvnt (1:1, w/v; Chonrex, Remon, WA, USA) into the se of the til. Three weeks fter primry immuniztion, ooster, whih onsiste of 1 μg of ovine type II ollgen emulsifie in inomplete Freun s juvnt (IFA; 1:1, v/v; Chonrex), ws injete intrermlly. Mie were ivie into three groups; ontrol,, n with GSPE tretment. To investigte the effets of GSPE on the evelopment of rthritis, mie were trete with GSPE (1 mg/kg oy weight) five times per week for 3 weeks intrperitonelly, fter the ooster injetion. GSPE ws kinly provie y Hnlim Phrmeutil Compny (Seoul, Kore). GSPE issolve in sline ws use for the experiments. Clinil rthritis severity sore Three oservers sore the severity of rthritis inepenently from y 24 fter primry immuniztion to y 42. Clinil rthritis sores of the mie were evlute s follows:, no eviene of swelling; 1, mil swelling onfine to the nkle or the mifoot (trsls); 2, moerte swelling extening from the nkle to the mifoot (trsls); n 3, severe swelling n erythem extening from the nkle to the mettrsl joints or nkylosis of the nkle joint. The sum of the sores of four lims represente the rthritis sore of eh mouse. Histologil nlysis Joint tissues were fixe with 4% prformlehye. They were then elifie for 1 week in hyrohlori i efore eing emee in prffin. Tissue setions (4 μm) were stine with H&E in orer to etermine the influx of inflmmtory ells. Inflmmtion ws sore using the following riteri:, no inflmmtion; 1, slight thikening of lining lyer or some infiltrting ells in sulining lyer; 2, slight thikening of lining lyer plus some infiltrting ells in sulining lyer; 3, thikening of lining lyer, influx of ells in sulining lyer n presene of ells in the synovil spe n synovium highly infiltrte with mny inflmmtory ells. The tissue setions were stine with sfrnin O to evlute rti

3 The Koren Journl of Internl Meiine Vol. 33, No. 3, My 218 lge egrtion uner light mirosopi exmintion. The extent of rtilge mge sore using the following riteri:, no estrution; 1, miniml erosion limite to single spots; 2, slight to moerte erosion in limite re; 3, more extensive erosion n estrution. Immunohistohemistry ws performe using the Vetstin ABC kit n 3,3 -iminoenziine s sustrte (oth from Vetor Lortories, Burlingme, CA, USA). Nonspeifi stining ws reue y protein loking with 2% norml got serum for 3 minutes, t room temperture. Joint tissues were inute with nti- ntioy (Snt Cruz Biotehnology, Dlls, TX, USA) overnight t 4ºC. Enzyme-linke immunosorent ssy The serum levels of nti-type II ollgen speifi immunogloulin G (IgG; Chonrex), tumor nerosis ftor α (TNF-α), interleukin 6 (IL-6), n IL-17 (R&D Systems, Minnepolis, MN, USA) were mesure oring to the mnufturer s protool using n enzyme-linke immunosorent ssy (ELISA) kit (R&D). For the etermintion of ytokine levels, loo smples from the mie were otine on y 42 n store t 7 C until use. Antioy onentrtions for eh serum smple were otine y referene to the stnr urve. The sorne vlues were etermine t 45 nm with n ELISA miroplte reer (Vetor3, Perkin Elmer, Wlthm, MA, USA). Protein extrtion n Western lot nlysis Ankles joints of mie were isolte y isrtiultion istl to tii. Synovil tissues from hin pws were ollete n frozen until experiments. The frozen synovil tissues were pulverize n synovil protein ws extrte s follows. Pulverize synovil tissues were lyse in ie-ol lysis uffer (ph 7.4, 5 mm Tris-HCl, 1% Noniet P-4,.25% soium eoxyholte, 15 mm NCl, 1 mm N 3 VO 4, n 1 mm NF) ontining protese inhiitors (2 mm phenylmethylsulfonyl fluorie, 1 μg/ml leupeptin, 1 μg/ml pepsttin, 1 μg/ml protinin, n 2 mm ethyleneiminetetreti i). Cytoplsmi n nuler extrts were prepre from joint tissues using the NE-PER nuler n ytoplsmi protein extrtion regent kit (Thermo Piere, Rokfor, IL, USA). Smples in eh group were sujete to 1% soium oeyl sulfte polyrylmie gel eletrophoresis on resolving gels ppropritely n trnsferre to nitroellulose memrne (Millipore, Billeri, MA, USA). Blots were inute overnight t 4 C with primry ntioies for, myeloi ifferentition ftor 88 (MyD88), Toll/IL-1 reeptor omin-ontining ptor inuing IFN-β (TRIF), IκBα, phosphorylte IκBα (p-iκbα), p65, p5 (ll from Snt Cruz Biotehnology), n β-tin (Sigm-Alrih, St. Louis, MO, USA). Anti-lminB1 (Snt Cruz Biotehnology) ws use s n internl ontrol of nuleus protein mrker. The memrne ws then wshe with mixture of Tris-uffere sline with Tween 2 (TBS-T) n inute with horserish peroxise-onjugte seonry ntioies (Snt Cruz Biotehnology). Protein ns were etete using enhne hemiluminesent regents (Amershm Phrmi Bioteh, Uppsl, Sween). Isoltion n ulture of FLS FLS were isolte y enzymti igestion of synovil tissue, provie y the Keimyung Humn Bio-Resoure Bnk, memer of the Ntionl Bionk of Kore. The tissue ws otine from ptients with osteorthritis (OA) n from those with RA who unerwent totl joint replement surgery or synovetomy t Keimyung University Dongsn Meil Center. All the ptients fulfille the Amerin College of Rheumtology riteri n gree to give their informe onsent (pprove y the Institutionl Review Bor [IRB] of Keimyung University Dongsn Meil Center [IRB No ]) [14]. The tissue smples were mine into 2 to 3 mm piees n trete for 4 hours with.5 mg/ml type II ollgense (Worthington Biohemil Corp., Lkewoo, NJ, USA) in Duleo s moifie Egle s meium (DMEM) t 37ºC in wter th. Synovioytes were grown in DMEM supplemente with 1% fetl ovine serum, 2 mm L-glutmine, 1 units/ml peniillin, n 1 ng/ml streptomyin. The ells were use for experiments from pssge 4 to 8, t whih time the popultion ws homogeneous. Sttistil nlysis The sttistil nlysis ws performe using the SPSS version 22 (IBM Co., Armonk, NY, USA). Stuent t tests n one-wy nlysis of vrine were rrie out. The epte level of signifine ws preset t p <.5. Eh experiment ws exeute t lest three times in uplite. Dt re presente s men ± SD

4 Kim SH, et l. New mehnism of tion of GSPE on RESULTS GSPE meliortes murine utoimmune rthritis We exmine the effet of GSPE on the evelopment of experimentl rthritis in vivo using mouse moel. DBA/1J mie were intrperitonelly injete with GSPE five times per week for 3 weeks fter the seon immuniztion with type II ollgen/ifa. The men rthritis sore of GSPE-trete mie ws signifintly lower thn tht of mie (Fig. 1A). Representtive histologi setions of hin pws of the mie re shown in Fig. 1B. The joints of mie revele the infiltrtion of inflmmtory ells, synovil prolifertion, n rtilge estrution (Fig. 1B). In mie trete with GSPE, there ws reution in infiltrte inflmmtory ells, synovil prolifertion, n rtilge mge (Fig. 1B). GSPE ereses serum levels of pro-inflmmtory ytokines in mie We investigte whether the therpeuti effet of GSPE in mie ffets the humorl immune response to type II ollgen n serum levels of pro-inflmmtory ytokines. The levels of nti-type II ollgen IgG, TNF-α, IL-6, n IL-17 in serum smples of mie were mesure using ELISA, 6 weeks fter primry immuniztion. The serum onentrtions of nti-type II ollgen IgG were signifintly reue in mie following GSPE tretment, when ompre with mie (Fig. 2A). There ws lso erese in serum levels of TNF-α (Fig. 2B), IL-6 (Fig. 2C), n IL-17 (Fig. 2D) in the GSPE-trete group. GSPE ministrtion reues expression in the synovium of mie hs een reporte to ply ritil role in the pthogenesis of [15,16]. Therefore, we investigte whether GSPE tretment influene expression in the synovil tissue of using immunohistohemil stining. To ssess the in vivo effet of GSPE on expression, histologil setions of the tiiotlr joints were stine in orer to visulize the presene of. As shown in Fig. 3A, less -positive ells were oserve in the joints of GSPE-trete mie thn in those of mie. We lso investigte the ef- H&E Sfrnin O Clinil rthritis sore A GSPE Synovil inflmmtion sore D25 D28 D32 D35 D39 D41 GSPE B GSPE Crtilge mge sore GSPE Figure 1. Grpe see pronthoyniin extrt (GSPE) tretment relieves the severity of ollgen-inue rthritis () in mie (n = 8 for eh group; ontrol,, GSPE-trete group). (A) For inution of, mle ilute rown non-gouti (DBA)/1J mie were given 1 μg of ovine type II ollgen emulsifie in omplete Freun s juvnt intrermlly into the se of the til. A ooster injetion ws ministere 3 weeks lter. GSPE (1 mg/kg) ws initite intrperitonelly fter the ooster injetion. The mie were trete with GSPE (1 mg/kg) five times per week for 3 weeks. The rthritis sore for eh mouse ws lulte s the sum of the sores of oth hin pws. Men vlues of the rthritis sores re represente on the grph. Error rs inite stnr evition of the men. (B) A hin lim of eh mouse ws stine with H&E (upper pnel) n sfrnin O (lower pnel). The joints from mie trete with GSPE emonstrte ttenute inflmmtion n rtilge estrution when ompre with those from mie ( 2). Error rs inite stnr evition of the men. p <.5, p <.1, ompre with the group, p <.1 ompre with the ontrol group

5 The Koren Journl of Internl Meiine Vol. 33, No. 3, My 218 A IL-6 (pg/ml) ollgen ntioy (pg/ml) GSPE B IL-17 (pg/ml) TNF-α (pg/ml) GSPE C GSPE GSPE Figure 2. Grpe see pronthoyniin extrt (GSPE) tretment suppresses immune response to type II ollgen n ereses serum levels of pro-inflmmtory ytokines. Serum onentrtions of (A) type II ollgen speifi totl immunogloulin G (IgG), (B) tumor nerosis ftor α (TNF-α), (C) interleukin 6 (IL-6), n (D) IL-17 were etermine using enzyme-linke immunosorent ssy in eh group of mie (n = 8 for eh group). Error rs represent stnr evition of the men. Eh experiment ws performe three times. p <.5, p <.1 ompre with the ollgen-inue rthritis () group, p <.5, p <.1 ompre with the ontrol group. D fet of GSPE on the intrellulr proteins involve in -meite signl trnsution in the synovium of, y using Western lotting. Synovil protein levels of, MyD88, n TRIF were nlyze. The expression levels of n MyD88 proteins were upregulte in the synovil extrts of mie when ompre with those of ontrol mie. n MyD88 expression levels were ownregulte in GSPE-trete mie when ompre with mie (Fig. 3B). The expression level of TRIF showe no inrese in mie when ompre to the ontrol mie. However, GSPE tretment ownregulte the expression level of TRIF in mie when ompre to mie (Fig. 3B). In orer to explore the effets of GSPE on the tivtion of nuler ftor-κb (NF-κB) signling, the levels of IκBα n p-iκbα were mesure. GSPE reue the level of p-iκbα protein in the synovil tissue of mie (Fig. 3C). The rtio of p-iκbα to totl IκBα ws lso lower in GSPE-trete mie thn in mie (Fig. 3C). We lso exmine whether GSPE ffete nuler trnslotion of the NF-κB suunits, p65 n p5. The nuler extrts n ytoplsmi lystes were purifie from the synovil tissue n sujete to Western lotting. The nuler loliztion of p65 n p5 NF-κB ws inhiite in the synovil extrts of GSPE-trete mie, while nuler loliztion of p65 n p5 NF-κB ws prominent in those of mie (Fig. 3D). GSPE suppresses lipopolyshrie-inue tivtion in RAW264.7 ells Our t emonstrtes tht GSPE ministrtion erese expression in the synovium of mie (Fig. 3A n 3B). We next investigte whether GSPE h n influene on expression in vitro y using murine mrophge ell line, RAW GSPE ownregulte the expression of, whih ws enhne y stimultion with lipopolyshrie (LPS) in RAW264.7 ells (Fig. 4A). We lso evlute the effet of GSPE on the expression of in FLS from ptients with OA n RA. As shown in Fig. 4B, LPS-inue upreg

6 Kim SH, et l. New mehnism of tion of GSPE on MyD88 GSPE TRIF A positive ell ount (ROI 3 3) f GSPE Protein expression/β-tin rtio β-atin GSPE GSPE p-iκbα IκBα B MyD88 TRIF β-atin Cytoplsm Nuleus GSPE p65 C p-iκbα/β-tin rtio p-iκbα/β-tin rtio p-iκbα GSPE IκBα GSPE p5 β-atin 1. p65, p5/β-tin rtio D GSPE Cytoplsm GSPE Nuleus p65 p5 p65 p5 e GSPE Figure 3. Grpe see pronthoyniin extrt (GSPE) ownregultes Toll-like reeptor 4 () expression n -meite signl trnsution proteins in the synovium of mie with ollgen-inue rthritis (; n = 8 for eh group). (A) expression ws ssesse y immunohistohemil stining. GSPE tretment le to reution in -positive ells in the synovium of mie, wheres the numer of -stine ells ws mrkely inrese in the synovium of mie, when ompre to tht of ontrol mie ( 2). (B) Western lot nlysis of synovil extrts from GSPE-trete mie showe erese protein level of, myeloi ifferentition ftor 88 (MyD88), n Toll/interleukin 1 reeptor omin-ontining ptor inuing IFN-β (TRIF), when ompre with mie. (C) The expression level of phosphorylte IκBα (p-iκbα) ws lso reue in GSPE-trete mie. (D) GSPE suppresse nuler trnslotion of nuler ftor-κb suunits (p65 n p5) in the synovium of mie. Error rs reflet stnr evition of the men. Eh experiment ws performe three times. ROI, region of interest. p <.5, p <.1, p <.1 ompre with the group, p <.5, e p <.1, f p <.1 ompre with the ontrol group

7 The Koren Journl of Internl Meiine Vol. 33, No. 3, My 218 OA-FLS A β-atin β-atin LPS GSPE B LPS GSPE /β-tin /β-tin /β-tin β-atin RA-FLS LPS GSPE Figure 4. Grpe see pronthoyniin extrt (GSPE) inhiits lipopolyshrie (LPS)-inue Toll-like reeptor 4 () expression in vitro. (A) RAW264.7 mrophges were o-inute with or without LPS (1 μg/ml) for 8 hours fter pretretment with or without GSPE (25 μg/ml) for 16 hours. LPS-inue expression ws ownregulte in RAW264.7 ells fter tretment with GSPE. (B) Firolst-like synovioyte (FLS) from ptients with osteorthritis (OA; n = 5) n rheumtoi rthritis (RA; n = 3) were inute in the sme onitions s pnel A. GSPE tretment reue LPS-inue expression in FLS from ptients with RA s well s in those from ptients with OA. Dt re presente s the men ± SD of three inepenent experiments. Eh experiment ws performe three times. p <.5, p <.1 ompre with the LPS only-stimulte group, p <.5, p <.1 ompre with the nil (ontrol onition). ultion ws suppresse y GSPE in FLS from ptients with RA n OA. DISCUSSION Pronthoyniins, whih re the min onstituents of GSPE, elong to the tegory of onense tnnins [17]. The soures of pronthoyniins inlue fruits, vegetles, nuts, sees, flowers, n rk [18]. Pronthoyniins re well-known s nturlly ourring nti-oxints. They hve een reporte to hve enefiil effets on moulting inflmmtion in humn ells, suh s ifferentite ipoytes n humn pulmonry epithelil ells in vitro, in ition to niml inflmmtory moels s mentione ove [19,2]. Some previous stuies hve shown tht GSPE hs n nti-inflmmtory effet in n niml moel of RA. Cho et l. [13] first reporte out the therpeuti effet of GSPE on n showe the effets of GSPE on oxitive stress n osteolstogenesis in vitro. Another stuy using murine moel of RA hs emonstrte the effet of GSPE fousing on inflmmtion-ssoite one estrution [21]. GSPE ttenute oth rthritis n oesity in oese mie [22]. However, little is known out the mehnism y whih GSPE regulte the inflmmtory response. Oxitive stress is reue y GSPE, n hs een reporte 618 to e involve in the pthogenesis of murine utoimmune rthritis. However, there hs een ontroversy s to whether oxitive stress instigtes, or suppresses the inflmmtion in moel of murine utoimmune rthritis [23-25]. One previous stuy reporte tht GSPE influenes murine utoimmune rthritis y regulting Foxp3 regultory n IL-17-prouing T ells, reiprol ontrol of whih is importnt in mnging utoimmune rthritis [26]. There ws nother report tht GSPE hs n nti-rthriti effet in juvnt-inue rthritis moel y moifying T ell susets [27]. In this stuy, we fouse on reveling the mehnism of tion of GSPE in suppressing murine utoimmune rthritis. Here, we emonstrte tht GSPE hs n nti-rthriti effet through the regultion of -meite signling for the first time. TLRs re innte reeptors, whih ply n essentil role in the innte immune system [28]. Among iverse TLRs, ws the first to e hrterize, n hs een reporte to ply signifint role in the pthogenesis of utoimmune iseses [29]. In prtiulr, lrge mount of reserh provies eviene tht is implite in the pthogenesis of RA. -efiient mie showe lower egree of severity n iniene of ompre to the wil-type mie [15]. LPS-inue tivtion ws gretly enhne in peripherl loo mononuler ells (PBMCs) from ptients with RA thn in those from

8 Kim SH, et l. New mehnism of tion of GSPE on ptients with OA or helthy ontrols [3]. tivtion y LPS inues the proution of pro-inflmmtory ytokines in humn FLS n PBMCs from ptients with RA [31,32]. upregultion inreses pro-inflmmtory tivity n inues egenertion in honroytes [33]. Although ontroversy exists regring the ssoition etween polymorphisms n RA, previous stuies hve reporte tht funtionl vrint of is ssoite with suseptiility to RA n tht non-missense geneti polymorphisms of onfer the risk of the evelopment of RA [34,35]. Our t emonstrte tht is highly expresse in the joints of mie. This oservtion supports previous stuy, whih onfirme tht tretment with ntgonist erese the severity of [16]. In this stuy, GSPE-trete mie h fewer -expressing ells in their synovium in omprison to those tht were not trete with GSPE. This suggests tht GSPE inhiits expression in the synovium in vivo. However, little is known out the effets of GSPE on the expression of in the synovium of mie. Therefore, further investigtion into the mehnism y whih GSPE suppresses the expression of in the synovium is require. is regre s n importnt reeptor in inflmmtory responses. ligtion les to the tivtion of ownstrem trnsription ftor, NF-κB, whih is linke y MyD88, key ptor protein in -NF-κB signling. The stimultion of reruits the MyD88 ptor protein, leing to IκB kinse tivtion n ulminting in IκBα phosphoryltion. IκBα phosphoryltion releses ytosoli sequestrtion of p65 n p5 NF-κB suunits n uses nuler repositioning of those proteins. In this stuy, we evlute the in vivo effet of GSPE on NF-κB n MyD88. We lso exmine the influene of GSPE on the vrious steps in NF-κB tivtion y performing ell frtiontion experiments. Our t showe tht GSPE tretment iminishes the phosphoryltion of IκBα n inhiits nuler trnslotion of p65 n p5 NF-κB suunits. The results inite tht GSPE inhiits NF-κB tivtion through the suppression of IκBα phosphoryltion. In ition to MyD88, TRIF is lso n ptor protein, whih respons to the tivtion of [36]. However, the t presente here show no signifint hnge in the expression of TRIF etween n ontrol mie. These results my imply tht the inflmmtory responses of utilize the -MyD88-epenent pthwy rther thn -TRIF-epenent pthwy, lthough further stuies re require to onfirm these tenttive implitions. Prior to in vitro experiments etile ove, we lso evlute the ytotoxi effet of GSPE on FLS from RA or OA ptients, n RAW264.7 ells using the 3-(4,5-imethylthizol-2-yl)-2,5-iphenylphenyltetrzolium romie (MTT) ssy. After 24-hour inution perio, GSPE, t onentrtions etween n 5 μg/ml showe no eviene of ytotoxiity to oth FLS n RAW264.7 ells (t not shown). FLS in the synovium ply ritil role in mintining inflmmtion n estroying rtilge y prouing pro-inflmmtory ytokines [37]. FLS hve een reporte to e le to express s well s vriety of hesion moleules [37,38]. Mrophges, whih re the min soure of pro-inflmmtory ytokines suh s TNF-α n IL-1, lso prtiipte in inuing inflmmtion n one erosion in ptients with RA [39]. The in vitro t from the present stuy emonstrte tht GSPE lso hs n influene on expression in humn ells in ition to murine ells. This stuy hs some limittion tht the stuy ws esigne to inlue only three groups; ontrol group, GSPE-untrete group, n GSPE-trete group. Inlusion of GSPE-trete ontrol group woul hve given more informtion out si effet of GSPE on ontrol DBA/1J mie. We hose one osge (1 mg/ kg) of GSPE in the stuy se on previous stuy s our stuy ws fouse on reveling the new mehnism of tion of GSPE [13]. However, ifferent osge group of GSPE woul hve me the stuy more informtive. group o-trete with GSPE n gonist woul hve lso provie more informtion tht inhiition y GSPE oul e reverse y gonist in mie. GSPE ws given intrperitonelly s we intene to minister more urte osge of GSPE to eh mouse in our stuy. However, orl ministrtion of GSPE might pproh to linil pplitions more losely. In onlusion, our stuy emonstrte tht GSPE ttenute utoimmune rthritis y regulting the / MyD88/NF-κB signling pthwy. GSPE effetively suppresse nti-type II ollgen IgG n pro-inflmmtory ytokines. A ruil innte reeptor for utoimmune iseses, expression in the synovil tissue ws signifintly influene y GSPE in vivo, s well s in vitro

9 The Koren Journl of Internl Meiine Vol. 33, No. 3, My 218 The results suggest tht GSPE oul e effetive in treting immunologil iseses suh s RA, in the pthogenesis of whih tivtion is involve. KEY MESSAGE 1. Our stuy emonstrte tht grpe see pronthoyniin extrt (GSPE) meliorte ollgen-inue rthritis y regulting the Tolllike reeptor 4 ()-MyD88-NF-κB signling pthwy. 2. GSPE lso regulte the expression of in humn firolst-like synovioytes. 3. Further stuies woul provie more eviene tht GSPE oul e effetive in treting immunologil iseses suh s rheumtoi rthritis, in the pthogenesis of whih tivtion is involve. Conflit of interest No potentil onflit of interest relevnt to this rtile ws reporte. Aknowlegments This work ws supporte y the reserh promoting grnt from the Keimyung University Dongsn Meil Center in 213. REFERENCES 1. Annunzito F, Cosmi L, Liott F, Mggi E, Romgnni S. Type 17 T helper ells-origins, fetures n possile roles in rheumti isese. Nt Rev Rheumtol 29;5: Psre C, Mezhitov R. Toll-like reeptors: linking innte n ptive immunity. Av Exp Me Biol 25;56: Rstke TR, Roelofs MF, Jenniskens YM, et l. Expression of toll-like reeptors 2 n 4 in rheumtoi synovil tissue n regultion y proinflmmtory ytokines interleukin-12 n interleukin-18 vi interferon-gmm. Arthritis Rheum 24;5: Ospelt C, Brentno F, Rengel Y, et l. Overexpression of toll-like reeptors 3 n 4 in synovil tissue from ptients with erly rheumtoi rthritis: toll-like reeptor expression in erly n longstning rthritis. Arthritis Rheum 28;58: Dvis ML, LeVn TD, Yu F, et l. Assoitions of toll-like reeptor (TLR)-4 single nuleotie polymorphisms n rheumtoi rthritis isese progression: n oservtionl ohort stuy. Int Immunophrmol 215;24: Bghi D, Bghi M, Stohs SJ, et l. Free rils n grpe see pronthoyniin extrt: importne in humn helth n isese prevention. Toxiology 2;148: Gett B, Fuzzti N, Griffini A, et l. Chrteriztion of pronthoyniins from grpe sees. Fitoterpi 2;71: Fine AM. Oligomeri pronthoyniin omplexes: history, struture, n phytophrmeutil pplitions. Altern Me Rev 2;5: Zhng XY, Li WG, Wu YJ, Bi DC, Liu NF. Pronthoyniin from grpe sees enhnes oxoruiin-inue ntitumor effet n reverses rug resistne in oxoruiin-resistnt K562/DOX ells. Cn J Physiol Phrmol 25;83: Li WG, Zhng XY, Wu YJ, Tin X. Anti-inflmmtory effet n mehnism of pronthoyniins from grpe sees. At Phrmol Sin 21;22: Li XL, Ci YQ, Qin H, Wu YJ. Therpeuti effet n mehnism of pronthoyniins from grpe sees in rts with TNBS-inue ulertive olitis. Cn J Physiol Phrmol 28;86: Meern SM, Vi M, Punthil T, Ktiyr SK. Dietry grpe see pronthoyniins inhiit 12-O-tetrenoyl phorol-13-ette-use skin tumor promotion in 7,12-imethylenz[]nthrene-initite mouse skin, whih is ssoite with the inhiition of inflmmtory responses. Crinogenesis 29;3: Cho ML, Heo YJ, Prk MK, et l. Grpe see pronthoyniin extrt (GSPE) ttenutes ollgen-inue rthritis. Immunol Lett 29;124: Aleth D, Neogi T, Silmn AJ, et l. 21 Rheumtoi rthritis lssifition riteri: n Amerin College of Rheumtology/Europen Legue Aginst Rheumtism ollortive inititive. Arthritis Rheum 21;62: Pierer M, Wgner U, Rossol M, Irhim S. Toll-like reeptor 4 is involve in inflmmtory n joint estrutive pthwys in ollgen-inue rthritis in DBA1J mie. PLoS One 211;6:e Aollhi-Roosz S, Joosten LA, Roelofs MF, et l. In- 62

10 Kim SH, et l. New mehnism of tion of GSPE on hiition of Toll-like reeptor 4 reks the inflmmtory loop in utoimmune estrutive rthritis. Arthritis Rheum 27;56: Brvo L. Polyphenols: hemistry, ietry soures, metolism, n nutritionl signifine. Nutr Rev 1998;56: Bghi D, Grg A, Krohn RL, Bghi M, Trn MX, Stohs SJ. Oxygen free ril svenging ilities of vitmins C n E, n grpe see pronthoyniin extrt in vitro. Res Commun Mol Pthol Phrmol 1997;95: Chon MR, Ceperuelo-Mllfre V, Mymo-Msip E, et l. Grpe-see proyniins moulte inflmmtion on humn ifferentite ipoytes in vitro. Cytokine 29;47: Kim H, Kim JY, Song HS, Prk KU, Mun KC, H E. Grpe see pronthoyniin extrt inhiits interleukin-17-inue interleukin-6 proution vi MAPK pthwy in humn pulmonry epithelil ells. Nunyn Shmieeergs Arh Phrmol 211;383: Prk JS, Prk MK, Oh HJ, et l. Grpe-see pronthoyniin extrt s suppressors of one estrution in inflmmtory utoimmune rthritis. PLoS One 212;7:e Jhun JY, Moon SJ, Yoon BY, et l. Grpe see pronthoyniin extrt-meite regultion of STAT3 proteins ontriutes to Treg ifferentition n ttenutes inflmmtion in murine moel of oesity-ssoite rthritis. PLoS One 213;8:e Choi EM. Oxitive sttus of DBA/1J mie with type II ollgen-inue rthritis. J Appl Toxiol 27;27: MCuin MD, Hou G, Arms GD, Dik R, Zhng Z, Brewer GJ. Tetrthiomolyte is effetive in mouse moel of rthritis. J Rheumtol 26;33: Gelermn KA, Hultqvist M, Pizzoll A, et l. Mrophges suppress T ell responses n rthritis evelopment in mie y prouing retive oxygen speies. J Clin Invest 27;117: Prk MK, Prk JS, Cho ML, et l. Grpe see pronthoyniin extrt (GSPE) ifferentilly regultes Foxp3() regultory n IL-17() pthogeni T ell in utoimmune rthritis. Immunol Lett 211;135: Ahm SF, Zoheir KM, Ael-Hmie HE, et l. Grpe see pronthoyniin extrt hs potent nti-rthriti effets on ollgen-inue rthritis y moifying the T ell lne. Int Immunophrmol 213;17: Mezhitov R, Preston-Hurlurt P, Jnewy CA Jr. A humn homologue of the Drosophil Toll protein signls tivtion of ptive immunity. Nture 1997;388: Sng A, Nkshim H, Akhoshi M, et l. Protetion ginst utoimmune nephritis in MyD88-efiient MRL/ lpr mie. Arthritis Rheum 27;56: Kowlski ML, Wolsk A, Grzegorzyk J, et l. Inrese responsiveness to toll-like reeptor 4 stimultion in peripherl loo mononuler ells from ptients with reent onset rheumtoi rthritis. Meitors Inflmm 28;28: Chovnov L, Vlek M, Krskov K, et l. Inrese proution of IL-6 n IL-17 in lipopolyshrie-stimulte peripherl mononulers from ptients with rheumtoi rthritis. Gen Physiol Biophys 213;32: Tng CH, Hsu CJ, Yng WH, Fong YC. Lipoteihoi i enhnes IL-6 proution in humn synovil firolsts vi TLR2 reeptor, PKCelt n -Sr epenent pthwys. Biohem Phrmol 21;79: Lorenz W, Buhrmnn C, Mosheri A, Lueers C, Shkiei M. Bteril lipopolyshries form proollgen-enotoxin omplexes tht trigger rtilge inflmmtion n egenertion: implitions for the evelopment of rheumtoi rthritis. Arthritis Res Ther 213;15:R Rstke TR, Frnke B, Hnssen S, et l. The Toll-like reeptor 4 Asp299Gly funtionl vrint is ssoite with erese rheumtoi rthritis isese suseptiility ut oes not influene isese severity n/or outome. Arthritis Rheum 24;5: Yng H, Wei C, Li Q, et l. Assoition of gene non-missense single nuleotie polymorphisms with rheumtoi rthritis in Chinese Hn popultion. Rheumtol Int 213;33: Plsson-MDermott EM, O Neill LA. Signl trnsution y the lipopolyshrie reeptor, Toll-like reeptor-4. Immunology 24;113: Brtok B, Firestein GS. Firolst-like synovioytes: key effetor ells in rheumtoi rthritis. Immunol Rev 21;233: Hung QQ, Pope RM. The role of toll-like reeptors in rheumtoi rthritis. Curr Rheumtol Rep 29;11: Dvignon JL, Hyer M, Bron M, et l. Trgeting monoytes/mrophges in the tretment of rheumtoi rthritis. Rheumtology (Oxfor) 213;52: