Preventive effects of dietary hydroxytyrosol acetate, an extra virgin olive oil polyphenol in murine collagen-induced arthritis

Depósi o de in es igación de la Uni e sidad de Se illa h ps://idus.us.es/ “This is an Accep ed Manusc ip o an a icle published in [ MOLECULAR NUTRITION & FOOD RESEARCH] on [18 Sep embe 2015], a ailable a : h ps://doi.o g/[h ps://doi.o g/10.1002/mn .201500304].” Fo Pee Re iew P e en i e e ec s o die a y hyd ox y y osol ace a e, an ex a i gin oli e oil polyphenol in mu ine collagen- induced a h i is Jou nal: Molecula Nu i ion and Food Resea ch Manusc ip ID: mn .201500304.R2 Wiley - Manusc ip ype: Resea ch A icle Da e Submi ed by he Au ho : n/a Comple e Lis o Au ho s: Rosillo, Ma ia Angeles; Uni e si y o Se ille, Pha macology Sánchez-Hidalgo, Ma ina; Uni e si y o Se ille, Pha macology González-Benjumea, Alejand o; Uni e si y o Se ille, O ganic Chemis y Fe nández-Bolaños, José G.; Uni e si y o Se ille, O ganic Chemis y Lubbe s, E ik; E asmus Medical Cen e , Rheuma ology Ala cón-de-la-Las a, Ca alina; Uni e si y o Se ille, Pha macology Keywo ds: CIA, EVOO, Hyd oxy y osol ace a e, In lamma ion, Rheuma oid a h i is Wiley-VCH Molecula Nu i ion and Food Resea ch Fo Pee Re iew P e en i e e ec s o die a y hyd oxy y osol ace a e, an ex a i gin oli e oil polyphenol in mu ine collagen-induced a h i is Ma ía Angeles Rosillo 1 , Ma ina Sánchez-Hidalgo 1 , Alejand o González- Benjumea 2 , José G. Fe nández-Bolaños 2 , E ik Lubbe s 3 , Ca alina Ala cón-de-la- Las a 1, * 1 Depa men o Pha macology, Facul y o Pha macy, Uni e si y o Se ille, Spain 2 Depa men o O ganic Chemis y, Facul y o Chemis y, Uni e si y o Se ille, Spain 3 Depa men o Rheuma ology, E asmus MC, Uni e si y Medical Cen e , Ro e dam, The Ne he lands * Co esponding au ho : D . Ca alina Ala cón de la Las a Depa men o Pha macology. Facul y o Pha macy. Uni e si y o Se ille, Spain. P o eso Ga cía González, 2. 41012 Se ille Telephone: +34 954 559877 Fax: + 34 954 55 6074 [email p o ec ed]s Abb e ia ions (COMP) ca ilage olime ic ma ix p o ein, (COX-2) cyclooxygenase-2, (ERK) ex acellula signal- egula ed kinases, (EVOO) ex a i gin oli e oil, (H&E) hema oxylin and eosin, (HO-1) heme oxygenase-1, (HTy) hyd oxy y osol, (HTy-Ac) Hyd oxy y osol ace a e, (JAK/STAT) Janus kinase- signal ansduce and ac i a o o ansc ip ion, (JNK) c-Jun N- e minal kinases, (MAPKs) mi ogen-ac i a ed p o ein kinases, (MMPs) me allop o einases, (MMP-3) me allop o einase-3, (mPGES-1) p os aglandin E syn hase-1, (NF-κB) nuclea ansc ip ion ac o -kappa B, (N 2) nuclea ac o E2- ela ed ac o 2, (RA) heuma oid a h i is, (SD) s anda d die , (STAT-3) signal ansduce and ac i a o o ansc ip ion (TNF-α) umo nec osis ac o α Keywo ds CIA, EVOO, Hyd oxy y osol ace a e, In lamma ion, Rheuma oid a h i is Page 1 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Fo Pee Re iew Abs ac Scope: Hyd oxy y osol ace a e (HTy-Ac), an ex a i gin oli e oil (EVOO) polyphenol, has ecen ly exhibi ed an ioxidan and an i-in lamma o y e ec s on LPS-s imula ed mac ophages and ulce a i e coli is. This s udy was designed o e alua e die a y HTy-Ac supplemen a ion e ec s on collagen-induced a h i is (CIA) in mice. Me hods and esul s: DBA-1/J mice we e ed om weaning wi h 0.05% HTy-Ac. A e 6 weeks, a h i is was induced by ype II collagen. Mice we e sac i iced 42 days a e i s immuniza ion. Blood was ecollec ed and paws we e his ological and biochemically p ocessed. HTy-Ac die signi ican ly p e en ed a h i is de elopmen and dec eased se um IgG1 and IgG2a, ca ilage olime ic ma ix p o ein (COMP) and me allop o einase-3 (MMP-3) le els, as well as, p o-in lamma o y cy okines le els (TNF-α, IFN-γ, IL-1β, IL-6 and IL-17A). The ac i a ion o Janus kinase-signal ansduce and ac i a o o ansc ip ion (JAK/STAT), mi ogen-ac i a ed p o ein kinases (MAPKs) and nuclea ansc ip ion ac o -kappa B (NF-κB) pa hways we e d as ically amelio a ed whe eas nuclea ac o E2- ela ed ac o 2 (N 2) and heme oxygenase-1 (HO-1) p o ein exp essions we e signi ican ly up- egula ed in hose mice ed wi h HTy-Ac. Conclusion: HTy-Ac imp o ed he oxida i e e en s and e u ned p o-in lamma o y p o eins exp ession o basal le els p obably h ough JAK/STAT, MAPKs and NF-κB pa hways. HTy-Ac supplemen migh p o ide a basis o de eloping a new die a y s a egy o he p e en ion o heuma oid a h i is. Page 2 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Fo Pee Re iew 1. In oduc ion Rheuma oid a h i is (RA) is a ch onic sys emic in lamma o y disease cha ac e ized by in lamma ion o mul iple join s and des uc ion o ca ilage and bone. The ea lies e en is he de elopmen o sys emic au oimmuni y by exogenous and au ologous an igens and hus au o- an ibodies and la e high le els o cy okines can be de ec ed yea s be o e clinical symp om [1, 2]. A a join le el, he e a e syno ial hype plasia and massi e in il a ion o immune cells, including CD4 + T-cells, B-cells, na u al kille cells, mac ophages, dend i ic cells, neu ophils and mas cells. Al hough he cause o RA emains unclea , inc easing e idences indica e ha p o- in lamma o y cy okines such as umo nec osis ac o α (TNFα), IL-1β, IL-6, IFN-γ and me allop o einases (MMPs) p oduced om RA syno ium play an impo an ole in he e osion o a icula ca ilage and subchond al bones [3, 4]. The p ocess o gene exp ession o hese p o-in lamma o y media o s in ol es mul iple signal ansduc ion pa hways including mi ogen-ac i a ed p o ein kinases (MAPKs) which comp ises ex acellula signal- egula ed kinases (ERK1/2 o p42/p44), c-Jun N- e minal kinases (JNK)1/2/3 and p38, and he nuclea ansc ip ion ac o -kappa B (NF-κB) which a e ac i a ed in he syno ium o pa ien s wi h RA [5, 6]. In pa icula , NF-κB plays an impo an ole in MMPs induc ion and also egula es a wide ange o genes ha con ibu e o in lamma ion, such IL-1β, TNFα, IL-6, chemokines and mic osomal p os aglandin E syn hase-1 (mPGES-1), an e icien downs eam enzyme co-localized and unc ionally coupled wi h he inducible enzymes cyclooxygenase-2 (COX-2). Bo h, COX-2 and mPGES-1, a e up- egula ed and esponsible o he o e p oduc ion o p os aglandin E 2 (PGE 2 ) which may a ec join in eg i y h ough EP 4 ecep o ac i a ion [7]. The signal ansduce and ac i a o o ansc ip ion (STAT)-3 is ano he c i ical ansc ip ion ac o in lamma o y pa hway ac i a ed in esponse o cy okines in ol ed in he pa hogenesis o RA by s ee ing he abno mal ac i a ion, au oma ici y, and p olonged su i al o syno ial cells. Specially, o e exp ession o STAT-3 has been epo ed in syno ial memb anes om RA pa ien s co ela ing wi h pai ed se um IL-6 [8]. Likewise, nuclea ac o E2- ela ed ac o 2 (N 2) is a key ansc ip ion ac o o ches a o o he induc ion o se e al an ioxidan enzymes, such as heme oxygenase (HO)-1. The ac i a ion o HO-1 in in lamma o y condi ions could be pa o an adap i e mechanism o limi cy o oxici y. In ac , i has been epo ed ha HO-1 de iciency in mice esul s in a ch onic in lamma o y s a e [9]. Despi e signi ican ad ances in he apies o he ea men o many au oimmune diseases such as RA, a pe sis en unme need s ill exis s o mo e e ec i e, du able, and con enien ea men op ions. In his sense, he in e es by die a y supplemen s and nu aceu icals wi hou undesi able e ec s ha accompany he classical pha maco he apy is g owing. Cu en epidemiological and expe imen al s udies suppo a bene icial ole o die a y polyphenols in se e al in lamma o y diseases, including RA. In his ega d, we ha e p e iously demons a ed ha o al adminis a ion o a phenolic ex ac om ex a i gin oli e oil (EVOO) was able o down- egula e he a h i ic p ocess in he collagen-induced a h i is (CIA) model o RA [10]. EVOO is ich in a a ie y o phenolic compounds, mainly cons i u ed by secoi idoid de i a i es o 2-(3,4-dihyd oxyphenyl)e hanol (hyd oxy y osol, HTy) and o 2-(4-hyd oxyphenyl)e hanol ( y osol), and hyd oxy y osyl ace a e (HTy-Ac), along wi h mino amoun s o ee HTy [11]. To da e, HTy-Ac has shown p o ec ion e ec s agains oxida i e DNA damage in blood cells [12], as well as agains i on-induced oxida i e s ess in human ce ical cells (HeLa) [13]. In addi ion, a s udy om González-Co ea e al., [14] showed a neu op o ec i e e ec o HTy-Ac in a model o hypoxia– eoxygena ion in a b ain slices, bo h in i o and a e o al adminis a ion. A g ea e an ipla ele agg ega ing ac i i y han HTy has also been demons a ed [15]. Mo e ecen ly, we ha e showed ha HTy-Ac, exe ed an an i-in lamma o y e ec on acu e ulce a i e coli is. This e ec in ol es a dec ease in COX-2 and iNOS p o ein exp ession p obably ough JNK MAPK and NF-κB signaling pa hways [16]. In addi ion HTy-Ac was able o modula e in lamma o y esponse in mu ine pe i oneal mac ophages [17]. Page 3 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Fo Pee Re iew Taken his backg ound in o accoun , he p esen s udy was designed o e alua e he e ec s o HTy-Ac die a y supplemen a ion, in he a h i is model o CIA in mice. In addi ion o mac oscopic and his ological analyses, we ha e de e mined he e ec s o HTy-Ac on he p oduc ion o in lamma o y media o s. In o de o gain a be e insigh in o mechanisms o ac ion, signaling pa hways we e also explo ed. 2. Ma e ial and Me hods. To e alua e he bene icial e ec s o HTy-Ac on CIA model, DBA 1J mice we e used. Mice we e andomized in ou expe imen al g oups (10 animals pe g oup): (1) naï e g oup, (2) Con ol g oup (CIA), (3) HTy die g oup (CIA-HTy) and (4) HTy-AC die g oup (CIA-HTy-Ac). A e 6 weeks, a h i is was induced by ype II collagen. Mice we e sac i iced 42 days a e i s immuniza ion. Blood was ecollec ed and paws we e his ological and biochemically p ocessed. ELISA, his opa hological analysis and immunoblo ing we e pe o med as indica ed in Suppo ing In o ma ion Ma e ial and Me hods. 3. Resul s 3.1. E ec s o die a y HTy-Ac on CIA-induced AR model. The de elopmen o a h i is was moni o ed un il day 42. The ime-cou se o a h i ic sco e indica es ha con ol CIA mice de eloped a p og essi e de elopmen o a h i is obse ed om day 34 (Fig. 1A). Howe e , mice ed wi h HTy-Ac die showed a signi ican delayed onse (p<0.05 and p<0.01 s. CIA) and dec eased he disease se e i y o CIA educing disease incidence, numbe o in ol ed paws, oo pad hickness and clinical index om days 37 o 42. Su p isingly, HTy die a y did no modi y he de elopmen o a h i is. These esul s sugges ed ha die a y en iched wi h HTy-Ac no only could e a d he de elopmen bu i also may ha e a he apeu ic e ec on ongoing in lamma o y a h i is. Rep esen a i e pho og aphs o hind paws om he di e en expe imen al animal g oups a e shown in igu e 1B. In addi ion, H&E s aining e ealed ha his ological ea u es o he join om naï e animals we e ypical o no mal s uc u e wi h syno ial memb ane composed o syno ial cells and collagen and a clea syno ial space (Fig. 1C, a). On he con a y, join om con ol CIA mice exhibi ed his ological changes indica i e o se e e a h i is, cha ac e ized by an ex ensi e in il a ion o in lamma o y cells in o a icula issues, exuda ion in o he syno ial space, syno ial hype plasia and ca ilage e osion (Fig. 1C, b). These his ological ea u es we e less e iden in CIA -HTy-Ac g oup (Fig. 1C, d). 3.2. HTy-Ac die educed le els o CII-speci ic an ibodies. To de e mine he e ec o HTy-Ac die a y on au oan ibody p oduc ion, se um was collec ed a day 42 and an i-CII speci ic IgG an ibodies we e measu ed. As shown in Figu e 2A, bo ine IgG1 and IgG2a le els we e signi ican ly lowe in die a y HTy-Ac eed g oup in compa ison wi h CIA con ol (p< 0.01 s. CIA) and HTy eed g oup (p<0.01 and p<0.05 s. HTy g oup). Simila ly, mouse IgG1 and IgG2a le els we e also signi ican ly amelio a ed in mice ed wi h HTy-Ac die (p<0.01 and p<0.05 s. CIA and p<0.05 s. HTy g oup) 3.3. HTy-Ac die dec eased se um MMP-3 and COMP le els in CIA-induced RA. In he p esen s udy we de e mined se um MMP-3 le els, as syno ial in lamma o y bioma ke , in CIA mice o in es iga e he po en ial bene icial e ec s o ou nu i ional he apeu ic s a egy. Ci cula ing MMP-3 le els we e ma kedly inc eased (p<0.01 s. naï e) in se um om a h i ic CIA con ol g oup (Fig. 2C). By con as , a educ ion in MMP-3 se um le els was obse ed in hose a h i ic animals eeding wi h HTy-Ac (p<0.05 s. CIA). In addi ion, se um le els o COMP we e signi ican ly ele a ed in CIA animals when compa ed o naï e con ols (p<0.001 s. naï e). On he con a y, CIA-HTy-Ac animal g oup signi ican ly dec eased COMP le els in compa ison wi h CIA g oup (p<0.001 s. CIA) eaching le els compa able o hose desc ibed in naï e animals (Fig. 2C); whe eas die a y HTy ea men was ine ec i e. 3.4. E ec s o die a y HTy-Ac on join media o s. I has been epo ed ha TNF-α, IL-1β, IL-6, IFN-γ and IL-17A a e c i ical cy okines in ol ed in he pa hogenesis o RA [1]. To explo e whe he cy okines join le els we e pa alleled o he disease se e i y o CIA, concen a ion o hese cy okines we e examined in paw homogena es Page 4 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Fo Pee Re iew by ELISA. As shown in Figu e 3, TNF-α, IL-1β, IL-6, IFN-γ and IL-17A le els we e signi ican ly inc eased in paw homogena es om CIA animals when compa ed wi h naï e mice (p<0.01 and p<0.01 s. naï e) sugges ing i s ela ionship wi h he syno ial issue in lamma ion. Con e sely, ou esul s indica e ha animals ed wi h HTy-Ac die showed a signi ican educ ion in all hose p o-in lamma o y cy okines p oduc ion in compa ison wi h CIA g oup (TNF-α: p<0.05, IL- 1β: p<0.001, IL-6: p<0.01, IFN-γ: p<0.01 and IL-17A: p<0.001 s. CIA). 3.5. E ec o die a y HTy-Ac on COX-2 and mPGES-1 exp ession. COX-2 and mPGES1 exp essions we e de e mined by wes e n blo in paw homogena e (Fig. 4A). A h i ic con ol animal g oup showed an o e exp ession o bo h hese p o-in lamma o y enzymes (COX-2: p<0.01 and mPGES1: p<0.001 s. naï e) whe eas HTy-Ac die was able o educe he p o ein exp ession le els o bo h hem (p<0.01 s. CIA) (Fig. 4B). By con as , die a y HTy ea men ailed o educe signi ican ly he p o ein exp ession le els o bo h COX-2 and mPGES1 in CIA mice. On he o he hand, le els o PGE 2 we e measu ed on he paw homogena e, HTy-Ac was capable o educe he le els o hese eicosanoid. (Fig. 4B) 3.6. E ec o die a y HTy-Ac on p-STAT-3 p o ein exp ession. STAT-3 has been desc ibed as a c i ical ansc ip ion ac o in ol ed in he pa hogenesis o RA by s ee ing he abno mal ac i a ion, au oma ici y, and p olonged su i al o syno ial cells. I has been also desc ibed ha IL-6 ac i e he JAK/STAT pa hway and mainly culmina es in he ac i a ion o he STAT-3 ansc ip ion ac o [18]. We e alua ed p-STAT-3 p o ein exp ession by wes e n blo om hind paw homogena es. S a is ical analysis e ealed a signi ican p-STAT- 3 o e exp ession in CIA g oup when compa ed o he naï e con ol g oups (p<0.001 s. naï e) whe eas nu i ional he apy wi h HTy-Ac signi ican ly supp essed STAT3 phospho yla ion in a h i ic CIA mice (p<0.01 s. CIA), his supp ession was also signi ican in compa ison wi h HTy-CIA g oup (p<0.01 s. CIA -HTy) (Fig. 5A). These esul s a e in acco dance wi h hose ob ained in he measu emen o p o-in lamma o y cy okines le els sugges ing ha die a y HTy-Ac may ep ess STAT-3 ac i a ion educing IL-6 le els in CIA mice. 3.7. Die a y HTy-Ac induces N 2/HO-1 an ioxidan pa hway ac i a ion. The exp ession o he p o eins HO-1 and N 2 we e also e alua ed in paw homogena es by wes e n blo ing. Ou da a show ha HO-1 was signi ican ly down egula ed in CIA mice du ing he main enance o ch onic in lamma ion (p<0.001 s. naï e); howe e , die a y HTy-Ac ea men induced a HO-1 o e exp ession in compa ison wi h CIA a h i ic con ol g oup (p<0.05 s. CIA) (Fig. 5B). N 2 ac i a ion has been epo ed o play an impo an ole in HO-1 exp ession. Acco ding o ou esul s, N 2 exp ession was simila ly educed in hose a h i ic animals ed wi h SD die (p<0.001 s. naï e) whe eas a signi ican inc ease in N 2 p o ein le els was obse ed in CIA-HTy-Ac animals (p<0.05 s. CIA and p<0.05 s. CIA-HTy) (Fig.5B). 3.8. E ec o die a y HTy-Ac on MAPKs signaling pa hway. MAPKs play a key ole inducing he p o-in lamma o y gene exp ession which ini ia es in lamma o y esponses. We in es iga ed he e ec o die a y HTy-Ac on MAPKs (JNK and p38) signaling pa hway ac i a ion in CIA mice. In he p esen s udy, phospho yla ion o JNK, p38 and ERK (1/2) p o eins inc eased signi ican ly in cy osolic ex ac s om CIA mice paw homogena es. None heless, he p o eins exp ession o all phospho yla ed MAPKs p o eins, p- JNK, p-p38 and p-ERK (1/2) was signi ican ly amelio a ed a e die a y HTy-Ac ea men (p<0.05 and p<0.01 s. CIA) (Fig. 6A). Again, he HTy die showed i s ine ec i eness along he expe imen al pe iod. 3.9. E ec s o die a y HTy-Ac on NF-κB signaling pa hway. We also in es iga ed he e ec s o HTy-Ac on IkB-α deg ada ion in cy oplasmic ex ac s om mice join s. As shown in igu e 6B, IκB-α exp ession was signi ican ly educed in CIA-induced a h i is mice when compa ed wi h naï e mice (p < 0.001 s. naï e). The IκB-α exp ession obse ed in CIA mice was consis en wi h an inc ease in IκB-α deg ada ion hus allowing NF-κB ansloca ion in o he nucleus o bind speci ic DNA sequences leading o p o-in lamma o y genes ansc ip ion. Acco ding o he esul s ob ained, he e ec obse ed in a h i ic con ol g oup on IκB-α p o ein deg ada ion was p e en ed by die a y HTy-Ac ea men . On he Page 5 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Fo Pee Re iew con a y, he nuclea p65 p o ein le els we e signi ican ly inc eased in CIA g oup (p<0.001 s. naï e) whe eas die a y HTy-Ac ea men p e en ed he CIA-induced nuclea ansloca ion le el o p65 in paw homogena e in compa ison wi h hose a h i ic animals ed wi h s anda d die (p<0.01 s. CIA) (Fig. 6B) a oiding he NF-kB-media ed ansc ip ional ac i a ion. HTy die ailed o modi y IκB-α deg ada ion and nuclea p65 ansloca ion in CIA mice. 4. Discussion Ou indings, ha e shown, o he i s ime, ha die a y HTy-Ac en ichmen was able o p e en and down- egula e he a h i ic p ocess in he CIA model o RA. This model is commonly used o in es iga e ele an pa hogenic mechanisms o RA as well as new an ia h i ic ea men s [19]. HTy-Ac was desc ibed o he i s ime in oli e oil by B enes e al.[20] and is ound in mos Spanish i gin oli e oils. Mo eo e , ecen ly, i was epo ed by Ma eos e al.[21] ha HTy-Ac is mo e soluble in he lipophilic phases han HTy, due o he p esence o he es e g oup, which was demons a ed in a Caco-2 cell model. Thus, his inc eased lipophilici y means ha HTy-Ac is be e abso bed ac oss in es inal epi helial cell monolaye s han ee HTy [22]. CIA induc ion esul ed in he de elopmen o a p onounced syno i is associa ed wi h ca ilage deg ada ion and bone e osion [23]. Howe e , die a y HTy- Ac supplemen a ion could imp o e he a h i is sco e which was co ela ed wi h a mino mig a ion o in lamma o y cells in o a icula issues in addi ion o a ma ked educ ion o join edema, syno ial hype plasia and ca ilage e osion in compa ison wi h hose animals ed wi h SD and HTy en iched die s. A ole o humo al immuni y in he pa hogenesis o au oimmune a h i is is sugges ed by e idence de i ed om animal models as well as pa ien s wi h RA. An ibody/an igen complexes a e abundan ly ound in he join s o RA pa ien s and a e belie ed o play a ole in igge ing he join in lamma ion [24]. CIA pa hogenesis is cha ac e ized by he gene a ion o an i-CII an i-bodies. All iso ypes (IgG1, IgG2a and IgG2b) o an i-CII an ibodies we e a h i ogenic, wi h he IgG1 and IgG2b iso ypes as he domina ing a h i ogenic an ibodies [25]. In he p esen s udy, die a y HTy-Ac supplemen a ion signi ican ly dec eased se um le els o IgG1-and IgG2a- an i-CII an ibodies in CIA mice. These esul s sugges a po en ial ole o HTy-Ac in egula ing B cell esponses, which may be in pa , esponsible o i s an i-a h i ic e ec s. The de elopmen and p og ession o RA is closely ela ed o an imbalance o cy okine ne wo k. In RA syno ium, ele a ed le els o p o-in lamma o y cy okines such as TNF-α, IL-1β, IL-6, IL-17 and INF-γ a e p oduced by mac ophages and syno ial ib oblas s. These p o-in lamma o y cy okines bo h di ec ly and indi ec ly exe hei e ec s h ough he p oduc ion o addi ional p o-in lamma o y cy okines, chemokines and MMPs a he pannus ca ilage junc ion leading o ca ilage deg ada ion [26-28]. In addi ion, TNF-α s imula es os eoclas ogenesis [29], supp esses he ec ui men o os eoblas s and inhibi he exp ession o ma ix genes [30], whe eas IL-6 inc eases os eoclas numbe s in abecula bone [31]. Besides, IL-17 is a T cell- de i ed cy okine able o induce he elease o IL-8 and IL-6, and plays a conside able ole in he addi i e/syne gis ic e ec s induced by TNF-α and IL-1β [32]. The p esen s udy showed ha a h i ic mice ed wi h HTy-Ac en iched die had dec eased IL- 1β, IL-6, IFN-γ, IL-17 and TNF-α le els in paw homogena e in compa ison wi h CIA mice ed wi h SD and HTy en iched die . These esul s indica e ha die a y HTy-Ac exe s an i- in lamma o y ac i i ies by he blockage o IL-1β, IL-6, IFN-γ, IL-17 and TNF-α p oduc ion. COMP, a p ominen non-collagenous componen o ca ilage, accoun s o app oxima ely 1% o he we weigh o a icula issue and shows g ea po en ial as a biological ma ke o ca ilage me abolism in a h i is [33]. Inc eased agmen s o COMP ha e been epo ed in pa ien s wi h os eoa h i is, RA, and join inju y, hus and he moni o ing o COMP le els in syno ial luid o se um has been sugges ed o be a help ul me hod o assessing he p esence and p og ession o a h i is. In ac , se um COMP is educed in RA pa ien s in emission[33]. In addi ion, COMP is a pu a i e subs a e o MMPs. In pa icula , MMP-3, is a p o einase sec e ed by syno ial ib oblas s and chond ocy es. I s syn hesis and ac i a ion is induced by a ious ac o s, including p o-in lamma o y cy okines and Toll-like ecep o ligand. I s ac i i y Page 6 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Fo Pee Re iew is associa ed wi h highe join damage in RA [34] and CIA [4] and esul s in deg ada ion o agg ecan co e p o ein, ca ilage link p o ein, ib onec in, and collagen ypes IV, VII, IX, and XI. Likewise, se um MMP-3 le el is sugges ed as a p edic o o join des uc ion in ea ly RA o es ablished RA and should be used in associa ion wi h usual in lamma o y ma ke s o ollow he apy e iciency [35]. Ou da a a e in ag eemen wi h abo e s udies and showed ha high se um COMP and MMP-3 le els we e associa ed wi h disease ac i i y and join p og ession in CIA mice, by con as he p oduc ion o bo h ca ilage and syno ial bioma ke s we e signi ican ly inhibi ed by he HTy-Ac en iched die in CIA mice. COX-2 and mPGES-1, enzymes esponsible o he o e p oduc ion o PGE 2 in in lamma ion, a e up- egula ed [36] con ibu ing o he p og ession o RA h ough EP 4 ecep o ac i a ion [7]. We ha e shown ha die a y HTy-Ac supplemen a ion educed PGE2 le els which could be possibly due o dec eased exp ession o bo h COX-2 and mPGES-1 in he join . The e o e, egula ion o hese p o-in lamma o y bioma ke s by HTy-Ac could ep esen a po en ial molecula a ge suscep ible o HTy-Ac modula ion, which has no been demons a ed p e iously. Signal ansduc ion pa hways closely in ol ed in in lamma ion include he MAPKs, JAK-STAT and NF-κB pa hways [37, 38]. In ac , NF-κB nuclea ansc ip ion ac o plays a pi o al ole in he de elopmen and ac i a ion o Th-1 esponses [39] and is esponsible in addi ion o MAPKs o COX-2 up- egula ion [40]. We in es iga ed whe he he ac i a ion o NF-κB signaling pa hway was inhibi ed by HTy-Ac die . Ou da a a e in ag eemen wi h Sanchez-Fidalgo e al. ha showed ha HTy-Ac inc eased he inhibi o y p o ein IkB-α and educed p65 ansloca ion, indica ing ha die a y HTy-Ac inhibi ed he NF-κB ac i a ion by blocking IκB-α deg ada ion in dex an sul a e sodium -induced coli is in mice [16]. MAPK amily membe s, including p38 kinases, ERKs 1 and 2 and JNKs, a e in ol ed in many impo an cell p ocesses, mainly he egula ion o he syn hesis o chemokines, cy okines, adhesion molecules and PGs in ol ed in RA [6, 41]. Besides JNK MAPK modula es MMPs p oduc ion by syno ial ib oblas s and d i es os eoclas di e en ia ion in RA [42]. In pa icula , p38 MAPK egula es MMP-3 induc ion in ib oblas s [43] and os eoclas di e en ia ion [44]. Mo eo e , MAPKs phospho yla e he JAK-STAT impo an in p o-in lamma o y cy okine- media ed signaling pa hways such as Th17 cell di e en ia ion, leading o STAT-3 ac i a ion by phospho yla ion on y osine esidues esul ing in he o ma ion o STAT dime s ha ansloca e in o he nucleus o bind speci ic DNA sequences [45]. In his line, STAT-3 o e exp ession has been also de ec ed in syno ial memb anes om RA pa ien s and i has been epo ed o con ibu e o he ch onici y o CIA induced a h i is model [4, 46]. Ou indings a e in conco dance wi h abo e epo s and showed ha p38 and JNK MAPKs phospho yla ion we e inc eased in CIA con ol mice. Simila ly, STAT-3 o e exp ession was also e idenced in RA syno ium o con ol CIA mice and was posi i ely ela ed o he se e i y o syno i is, whe eas die a y HTy-Ac supplemen a ion educed signi ican ly bo h MAPKs and STAT-3 ac i a ion a ansc ip ional le el. Collec i ely, ou da a sugges ha die a y HTy-Ac may ep ess IL-17 p oduc ion in e e ing nega i ely wi h JNK, p-38, p-ERK MAPKs and STAT-3 signaling pa hways. N 2, is a edox-sensi i e ansc ip ion ac o and binds o an ioxidan esponse elemen s (ARE) loca ed in he p omo e egions o many de oxi ying/an ioxidan genes, including HO-1 [47]. In in lamma o y condi ions, HO-1 exp ession p o ein could be pa o an adap i e mechanism o limi cy o oxici y ia se e al mechanisms including sca enging o eac i e oxygen o ni ogen species, egula ion o cell p oli e a ion and p e en ion o apop osis. Likewise, i has been epo ed ha HO-1 de iciency in mice esul s in a ch onic in lamma o y s a e [9]. Thus, N 2 egula es edox s a us and plays key oles in cellula de ense by enhancing he emo al o eac i e oxygen species [48]. Fu he mo e, i has been documen ed ha de iciency o N 2 accele a es he e ec o phase o a h i is and agg a a es join disease [49]. In he p esen s udy, in conco dance wi h Ka a as e al. [50], exp essions o N 2 and HO-1 we e dec eased in he a h i is g oup, by con as die a y HTy-Ac could es o e N 2 and HO-1 exp essions Page 7 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Fo Pee Re iew 254x190mm (96 x 96 DPI) Page 14 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Fo Pee Re iew 254x190mm (96 x 96 DPI) Page 15 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Fo Pee Re iew 254x190mm (96 x 96 DPI) Page 16 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Fo Pee Re iew 254x190mm (96 x 96 DPI) Page 17 o 17 Wiley-VCH Molecula Nu i ion and Food Resea ch 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60