The Effect of Curcumin on T Helper 1/T Helper 17 Balance in Rat Collagen-Induced Arthritis Model

Transcription

1 Glol Journl of Phrmology 9 (1): 87-96, 2015 ISSN IDOSI Pulitions, 2015 DOI: /idosi.gjp The Effet of Curumin on T Helper 1/T Helper 17 Blne in Rt Collgen-Indued Arthritis Model Amny El-Wkkd, Aeer M. Bdr, Hi Siii, Elshim Mohsen nd Somy El-Dee 1 Deprtment of Medil Physiology, Ntionl Reserh Centre, Ciro, Egypt 2 Deprtment of Zoology, Fulty of Siene, Ciro University, Giz 12613, Egypt Astrt: Immunomodultory imlne is the hrteristi feture of utoimmune diseses like rheumtoid rthritis (RA). T helper (Th) 17 plys ritil role in the pthogenesis of RA. The lne of Th1 nd Th17 is importnt for norml immune response. In RA therpy, methotrexte (MTX) hs n importnt use depending on its nti-inflmmtory effets, however, is limited due to its side effets. Curumin is nturl plnt produt used s n immunomodultor. This study imed to investigte the impt of urumin nd MTX dministrtion singly or omined on rt model with ollgen-indued rthritis. The results reveled tht the level of serum interleukin (IL)-17 showed positive orreltion with IL-23, interferon-gmm (IFN- ) nd trnsforming growth ftor-et (TGF- ) nd negtive orreltion with IL-27 t the end of the experiment. Tretment with urumin, MTX nd their omintions lso used signifint (P<0.05) redution in the pro-inflmmtory ytokines (IL-17, IL-23, IFN-, nd TGF- ) nd improvement of the histopthologil pperne of the hind pw s ompred to rthriti rts t the end. However, single tretment with urumin dded more meliortion y indution of hondroytes in the histopthology of the hind pw. Therey, the omined tretment of urumin nd MTX is ssoited with suppression of Th1/Th17 ytokines nd showed signifint prodution of IL-27 s protetive ytokine. Key words: T Helper 17 Cytokines Curumin Methotroxte Collgen-Indued Arthritis Interleukin- 27 Trnsforming Growth Ftor-Bet Interferon-Gmm, Interleukin-23 INTRODUCTION prodution, leding to hroni inflmmtion tht hrterizes RA [10]. Rheumtoid rthritis (RA) is mjor humn The inrese of IL-17 levels ws found to promote the utoimmune disese whih is hroni progressive prodution of other ytokines tht my medite rtilge disese ut with unknown etiology [1,2]. RA generlly destrution suh s IL-1, tumor nerosis ftor-lph ffets vrious ody joints eing hroni systemi (TNF- ), IL-6 nd reeptor tivtor of nuler ftor inflmmtory disorder [3,4]. Mny previous studies kpp-et lignd (RANKL) s well s hemokines suh doumented tht T ells, B ells nd ytokines ply s mrophge inflmmtory protein nd IL-8 [11]. pivotl role in the pthogenesis of RA [5, 6]. These pro-inflmmtory ytokines involved in The mjor ell type in the synovil infiltrtion in progression of RA vi n upregultion of ylooxygense the joints is T ells [7-9]. Attention ws drwn to the nd metlloproteinses leding to tivtion of joint pro-inflmmtory ytokines tht my led to the initition inflmmtion nd destrution of synovil joints. IL-27 is nd ugmenttion of hroni inflmmtion in RA. Thus, heterodimeri ytokine, produed y mny ells using of niml models to investigte ytokine- inluding B ells, monoytes, mrophges nd dendriti dependeny in eh phse of rthritis progression ws ells. It elongs to the IL-6/IL-12 ytokine fmily. It hs tken into onsidertion [10]. An interreltionship een mentioned to e implited in the pthogenesis of etween synovil firolsts nd T helper (Th)-17 utoimmune diseses, with pivotl role s oth proimmunity ws found in niml models of RA,, s they nd nti-inflmmtory ytokine. The immunosuppressive promote the migrtion of Th17 ells to the ffeted joints effets of IL-27 hve een ssoited with inhiiting the using profound inrese in interleukin (IL)-17 development of Th17 ells nd induing IL-10 prodution. Corresponding Author: Aeer Mhmoud Bdr, Deprtment, Fulty of Siene, Ciro University, Giz 12613, Egypt. Tel: , Fx: , E-mil: eerdr@gmil.om. 87

2 Glol J. Phrmol., 9 (1): 87-96, 2015 IL-27 hs n influene on vrious B ell susets nd guidelines nd pproved y the Institutionl Animl suppression of ntiody prodution [12]. Ethis Committee. Methotrexte (MTX) is the most used RA therpies s n immunosuppressive gent [13]. MTX is the first line Drugs nd Chemils: Complete Freund's djuvnt therpy for RA [14], s it ts on tively proliferting (CFA), dimethyl sulfoxide (DMSO) nd urumin were ells inhiiting the synthesis of DNA, RNA thymidine purhsed from Sigm, St. Louis, Mo, USA. Lyophilized nd proteins. In ddition, MTX ws shown to e relted ovine type II ollgen ws purhsed from BioCol to the derese of mrophges, T ells nd plsm GmH, Mihendorf, Germny nd MTX ws purhsed ells numer. It lso my inhiit osteolstogenesis y from Eewe, Austri. Interferon-gmm (IFN- ), deresing the prodution of TNF- nd IL-6 or y trnsforming growth ftor-et (TGF- ), IL-17, IL-23 nd reduing RANTL seretions y synovil firolsts or IL-27 were purhsed from WKEA MED Supplies Corp, mrophges [15]. In RA therpy, the used drugs NY, USA. Curumin ws dissolved in DMSO t dose of expeted to modulte the inflmmtion proesses, 100 mg/kg ody weight (.w) nd dministered suppress the joint destrution nd re sfe from side intrperitonelly (i.p) thrie week [21]. MTX ws effets. Unfortuntely, the mjority of these drugs dissolved in phosphte uffered sline t dose of typilly re ompnied y severe side effets inluding 1.0 mg/kg (.w) one week, dministered i.p [21,22]. gstrointestinl leeding, inresed lood pressure, DMSO ws dministered i.p t dose of 0.2 ml/100 elerted osteoporosis, myelosuppression, mg.w thrie week [23]. heptotoxiity, oulr toxiity, hypersensitivity nd llergi retions, s well s inresed risk of infetions Indution of Collgen-Indued Arthritis: Lyophilized [16,17]. Therefore, there is growing diretion of using ovine type II ollgen ws dissolved t 4 mg/ml 0.05M nturl produts from plnt extrts s therpeuti gents eti id y gentle stirring overnight t 4 C. In n ie for mny diseses. th, CFA nd ollgen solutions were mixed y using Sientists re heding to study the impt of homogenizer with smll lde (5mm in dimeter). One omplementry nd lterntive mediine for tretment volume of ollgen solution ws dded to n equl trying to void the side effets nd high ost of volume of CFA drop-wise while mixing t low speed. onventionlly used nti-inflmmtory drugs. Curumin, Mixing ws ontinued until stiff emulsion resulted t hydrophoi polyphenol (n tive onstituent of mximum speed (pproximtely 30,000 r.p.m. for 2-3 min). turmeri) proved to hve supposed effet on the The emulsion ws ooled on ie th prior mixing. regultion of multiple moleulr trgets [18]. It hs The emulsion ws trnsferred to Hmilton glss 1 ml nti-oxidnt, nti-inflmmtory nd pro-poptoti syringe. 0.2 ml (200 µg ollgen) of the emulsion ws properties [19, 20]. The present study imed to indue rt injeted suutneously t the se of the til. A ooster ollgen-indued rthritis model (CIA) whih is somewht injetion (0.1 ml of the emulsion) ws dministered on dy more nlogues to humn RA. To ssess the influene of 7 fter initil immuniztion. All tretments were initited urumin nd/or MTX dministrtion on Th17 nd Th1 from the 20th dy nd ontinued till dy 69. relted ytokines during the pthogenesis of the disese. To ddress the reltionship etween IL-17 nd Experimentl Design: Animls were divided into 7 IL-27. To evlute the effet of urumin nd/or MTX groups, 10 rts eh: Group N: norml rts were left dministrtion on the histology of hind pw. without ny tretment s ontrol. Group D: (vehile ontrol) injeted with 0.2 ml/100 g.w DMSO, i.p route MATERIALS AND METHODS thrie week for 7 weeks. Group A: (rthritis ontrol) rts were immunized with lyophilized ovine type II ollgen Animls: Mle lino rts of Wistr strin, weighing in CFA t the se of the til to indue rthritis nd then g, were otined from the niml house of were injeted with ooster dose on dy 7. Group C: Ntionl Reserh Center, Egypt. Rts were llowed to (urumin+rthritis+urumin) rts were injeted with limte for one week prior to initition of urumin y dose of 100 mg/kg.w thrie week for experimenttion. The environmentl onditions were 7 weeks y i.p route prior to indution of rthritis nd s properly stndrdized with 12-h drk/light yle, tretment fter indution for nother 7 weeks. Group C: onstnt temperture of 20 C nd reltive humidity of (rthritis+urumin) rthriti rts were injeted with 50-55%. Rts were fed on stndrd rodent diet urumin y dose of 100 mg/kg.w thrie week for (Formul diet 5008; L diet, St Louis, MO) with wter 7 weeks y i.p route. Group M: (rthritis+mtx) rthriti d liitum. The nimls were treted nd red ording rts were injeted with 1 mg/kg.w MTX y i.p route to the Ntionl Helth nd Medil Reserh Counil one week for 7 weeks. Group CM: 88

3 Glol J. Phrmol., 9 (1): 87-96, 2015 (rthritis+mtx+urumin) rthriti rts were injeted with rts t different time points following urumin nd/or 1 mg/ Kg.w MTX y i.p route one week for 7 weeks. MTX tretment inluding the kinetis of Th17 nd Th1 After 30 min of MTX tretment, the sme rts were responses. The levels of different ytokines were injeted with 100 mg/kg.w urumin thrie week y i.p monitored in the vrious groups utilized in the study. route for 7 weeks. Blood smples were olleted on dys 0, 20, 41, 48, 61 nd 69 nd olleted ser were used for Interleukin-17: As depited in Tle (1), there ws ssying different ytokines. profound signifint inrese in the level of IL-17 in group A ompred to groups N nd D t ll the time points. However, the IL-17 levels dropped signifintly in groups C, M nd CM s ompred to group A from dy 41 until dy 69. In group C, the IL-17 level grdully deresed fter dy 20 ompred to groups N, D nd A. Histologil Exmintion of Hind Pws: Rts were euthnized on the 69th dy, the hind pws were hrvested nd fixed for 3-7 dys with 10% phosphte-uffered formlin (Fisher Sientifi, Fir Lwn, NJ), wshed in slowly running tp wter for 30 min Therefter, pws were delified in HCI/Formi id (1:1) working solution (Fisher Sientifi), the solution ws hnged eh dy until delifition ws omplete [1]. One delifition ws omplete, the pws were rinsed in wter riefly nd trnsferred to mmoni solution nd left for 30 min to neutrlize ids left in the speimens, wshed in running tp wter thoroughly up to 24 h, emedded in prffn. Trnsverse nd longitudinl seril setions of 6 to 12 µm were stined in lum hemtoxylin nd eosin (H&E) (Sigm-Aldrih), exmined under the mirosope for morphologil nd ellulr infiltrtions ssessment in the joints. Cytokine Assys: The onentrtions of IFN-, TGF-, IL-17, IL-23 nd IL-27 in serum smples were determined y sndwih Enzyme Linked Immunosoront Assy (ELISA). The ssys were performed s suggested y the mnufturer s instrutions (WKEA MED Supplies Corp, NY, USA). The levels of ytokines were expressed s pg/ml. Sttistil Anlysis: Dt were represented s men ± stndrd error of men. The present dt were nlyzed using one-wy ANOVA to demonstrte the effet of different tretments on the onentrtion of ytokines. In ddition, post-hoc ws pplied to ompre the signifint differene mong the exmined groups t = Correltion oeffiient ws used to fit the reltionship etween vrious ytokines using SPSS version RESULTS The onset of rthritis ourred etween dys 10-15, it ws hrterized y redness nd swelling of the hind pw nd ws onfirmed y histologil exmintion. Cytokines Assys: We investigted the immunologil hnges ssoited with the severity of CIA in Wistr 89 Interleukin-23: Dt presented in Tle (2) demonstrted tht the level of IL-23 in group A ws signifintly higher thn groups N nd D long the period of experiment exept t dy 41. At dy 20, the level of IL-23 in group C ws signifintly higher thn group N nd signifintly lower thn group A (Tle 2). At dy 41, groups CM nd C exhiited signifint elevtion regrding ll other groups, while the lowest level ws found in group M. At dy 48, the men level of IL-23 ws signifintly elevted in group C in omprison to ll other groups. On the other hnd, the other groups showed no signifint hnges s the level rnged etween (16.85±0.23) nd (17.48±1.59). At dys 61 nd 69, IL-23 level ws signifintly elevted in group A ompred to most other groups, wheres the lowest levels were found in groups M nd CM. Interferon-Gmm: At dy 20, level of IFN- ws signifintly elevted in group C ompred to groups N nd D (Tle 3). In ontrst, groups CM nd M showed no signifint hnges with restortion of norml levels from dy 41 till the end of the experiment. Interleukin-27: At dy 20, the serum level of IL-27 ws signifintly inresed in group A ompred to groups N nd C (Tle 4). At dy 41, the IL-27 level in group A dropped profoundly ompred to its level in dy 20 nd ontinued to derese till the end of the experiment. However, the level of IL-27 ws signifintly inresed (5.25 ± 0.23) in group M t dy 41 in omprison to other groups under the study, ut t the end of the experiment (dy 69) the level dropped (2.83 ± 0.22) profoundly. The lest level of IL-27 ppered in group CM nd exhiited restortion s ompred to group N. At dy 48, the men level of IL-27 ws elevted in groups A, C, M nd CM regrding groups N nd D. At dy 61 the serum level of IL-27 ws signifintly inresed in group CM, followed y group M, wheres the other groups showed no signifint hnges ompred to groups N nd D. While t dy 69, the level of IL-27 in group CM followed y C

4 Glol J. Phrmol., 9 (1): 87-96, 2015 Tle 1: Interleukin -17 level in norml nd rthriti rts efore nd fter tretment with urumin nd methotrexte singly or omined Group Dy 0 Dy 20 Dy 41 Dy 48 Dy 61 Dy 69 N ± ± ± ± ± ± 0.02 D ± ± ± ± 0.04 C ± ± ± ± ± ± 0.82 A ± ± ± ± ± 0.08 C ± ± ± ± 0.44 M ± ± ± ± 0.69 CM ± ± ± ± 0.52 Dt re represented s men ± stndrd error of men. In olumns: Mens with the sme supersript letters re similr (insignifint, P > 0.05), wheres others re signifint (P < 0.05) t =0.05. N: norml, D: rts injeted with DMSO, C: rts injeted with urumin efore indution of rthritis then treted with urumin fter indution, A: rthriti nontreted rts, C: rthriti rts treted with urumin, M: rthriti rts treted with MTX nd CM: rthriti rts injeted with oth urumin nd MTX Tle 2: Interleukin-23 level in norml nd rthriti rts efore nd fter tretment with urumin nd methotrexte singly or omined Group Dy 0 Dy 20 Dy 41 Dy 48 Dy 61 Dy 69 N ± ± ± ± ± ± 0.41 D ± ± ± ± 0.62 C ± ± ± ± ± ± 1.79 A ± ± ± ± ± 0.48 C ± ± ± ± 0.83 M ± ± ± ± 0.99 CM ± ± ± 1.77 I ± 1.46 Dt re represented s men ± stndrd error of men. In olumns: Mens with the sme supersript letters re similr (insignifint, P > 0.05), wheres others re signifint (P < 0.05) t =0.05. N: norml, D: rts injeted with DMSO, C: rts injeted with urumin efore indution of rthritis then treted with urumin fter indution, A: rthriti nontreted rts, C: rthriti rts treted with urumin, M: rthriti rts treted with MTX nd CM: rthriti rts injeted with oth urumin nd MTX Tle 3: Interferon-gmm level in norml nd rthriti rts efore nd fter tretment with urumin nd methotrexte singly or omined Goup Dy 0 Dy 20 Dy 41 Dy 48 Dy 61 Dy 69 N ± ± 0.82 d ± ± 1.4 d ± ± 0.50 D d ± ± 2.75 d ± ± 1.0 C ± ± ± ± ± ± 4.25 A ± ± ± ± ± 4.68 C ± ± ± ± 3.01 M d ± ± 6.33 d ± ± 7.25 CM d ± ± 3.5 d ± ± 6.79 Dt re represented s men ± stndrd error of men. In olumns: Mens with the sme supersript letters re similr (insignifint, P > 0.05), wheres others re signifint (P < 0.05) t =0.05. N: norml, D: rts injeted with DMSO, C: rts injeted with urumin efore indution of rthritis then treted with urumin fter indution, A: rthriti nontreted rts, C: rthriti rts treted with urumin, M: rthriti rts treted with MTX nd CM: rthriti rts injeted with oth urumin nd MTX Tle 4: Interleukin -27 level in norml nd rthriti rts efore nd fter tretment with urumin nd methotrexte singly or omined Group Dy 0 Dy 20 Dy 41 Dy 48 Dy 61 Dy 69 N 2.61 ± ± ± ± ± ± 0.31 D 3.21 ± ± ± ± 0.06 C 3.38 ± ± ± ± ± ± 0.66 A 4.15 ± ± ± ± ± 0.2 C 3.04 ± ± ± ± 0.27 M 5.25 ± ± ± ± 0.22 CM 2.83 ± ± ± ± 0.13 Dt re represented s men ± stndrd error of men. In olumns: Mens with the sme supersript letters re similr (insignifint, P > 0.05), wheres others re signifint (P < 0.05) t =0.05. N: norml, D: rts injeted with DMSO, C: rts injeted with urumin efore indution of rthritis then treted with urumin fter indution, A: rthriti nontreted rts, C: rthriti rts treted with urumin, M: rthriti rts treted with MTX nd CM: rthriti rts injeted with oth urumin nd MTX 90

5 Glol J. Phrmol., 9 (1): 87-96, 2015 Tle 5: Trnsforming growth ftor- level in norml nd rthriti rts efore nd fter tretment with urumin nd methotrexte singly or omined Effet of urumin nd methotrexte tretment on TGF- levels in norml nd rthriti rts Group Dy 0 Dy 20 Dy 41 Dy 48 Dy 61 Dy 69 N ± ± ± ± ± ± 0.27 D ± ± ± ± 0.70 C ± ± ± ± ± ± 0.51 A 27.1 ± ± ± ± ± 0.50 C ± ± ± ± 1.03 M ± ± ± ± 1.61 CM ± ± ± ± 1.52 Dt re represented s men ± stndrd error of men. In olumns: Mens with the sme supersript letters re similr (insignifint, P > 0.05), wheres others re signifint (P < 0.05) t =0.05. N: norml, D: rts injeted with DMSO, C: rts injeted with urumin efore indution of rthritis then treted with urumin fter indution, A: rthriti nontreted rts, C: rthriti rts treted with urumin, M: rthriti rts treted with MTX nd CM: rthriti rts injeted with oth urumin nd MTX Tle 6: Correltion etween of the level of IL-17 nd levels of IL-23, Il-27, IFN- nd TGF- t dy 69 in ll groups of the study Group IL-23 IL-27 IFN- TGF- C A C M CM C: rts injeted with urumin efore indution of rthritis then treted with urumin fter indution, A: rthriti non treted rts, C: rthriti rts treted with urumin, M: rthriti rts treted with MTX nd CM: rthriti rts injeted with oth urumin nd MTX Fig. 1: Histologil fetures of hind pw mong vrious studied groups t the end of the experiment. (A) Hind pw setions from norml group (group N) showed norml histopthologil rhiteture. (B) Hind pw setions from DEMSO treted group (group D) showed norml hispthology. (C, D, E) Hind pw setions from group A (rthritis rts) showed; mirofrtures of rtiulr rtilge (smll rrow) ssoited with firolsts prolifertion (lrge rrow) (C), nerosis of rtiulr rtilge (smll rrow) nd fol hemrrhge (rrow hed) (D), leukoyti ells infiltrtion (rrows) (E). (F) Group C (urumin 100 mg/kg +CIA) showed no hispthologil hnges. (G) Group C (rthritis+urumin 100 mg/kg) showed prolifertion of hondroytes (rrows). (H, I) Group M (rthritis+methotrexte) nd group CM (rthritis+methotrexte +urumin) showed norml histologil pperne. All tissues stined with hemtoxylin nd eosin H&E. (Figs. A, C, D, I, J X200; Figs. B, E, F, G X400) 91

6 Glol J. Phrmol., 9 (1): 87-96, 2015 nd C exhiited higher signifint levels ompred to groups N nd D. In group CM, the IL-27 level ws elevted throughout the experiment from dys 48 to 69 in omprison to group N. Trnsforming Growth Ftor- : The level of TGFthroughout the experimentl intervls is illustrted in Tle 5. At dy 20, TGF- level ws signifintly inresed in groups A nd C ompred to groups N nd D. In group A, the serum level of TGF- ws signifintly higher thn groups N nd D t ll the time points. From dy 41 to dy 69, the level of TGF- in groups C, M nd CM ws not signifintly hnged from group N nd group D. Correltion Anlysis Between IL-17 nd the Other Cytokines: Reltion etween the level of IL-17 nd other ytokines in the experimentl groups t dy 69 is represented in Tle 6. Serum level of IL-17 showed positive orreltion with the levels of IL-23, IFN- nd TGF-. However, serum level of IL-17 showed negtive orreltion with IL-27 level inluding orreltion oeffiients rnging etween -0.41, , -0.94, in groups A, C, C, M nd CM respetively. Additionlly, group CM reveled the highest negtive orreltion vlue thn ll other groups. Histology of Hind Pws: Histologil exmintion of hind pws t the end of the experiment ws demonstrted in Fig. 1. Hind pws from group N showed norml rhiteture with no inflmmtory ells infiltrtions (Fig. 1A). Also, most setions from group D reveled no histopthologil hnges (Fig. 1B). Pws from group A showed widening of synovil vity, thikening of the synovil memrne, disruption of the rtilginous tissue nd evidene of one dmge s ompred to norml joint struture. Setions reveled mirofrtures of rtiulr rtilge ssoited firolsts prolifertion (Fig. 1C), nerosis of rtiulr rtilge ssoited with firosis nd fol hemorrhge (Fig. 1D) nd leukoyti ells infiltrtion (Fig. 1E). Norml histologil struture ws notied in most setions from groups C, M nd CM (Figs. 1F, 1H nd 1I). Group C reveled prolifertion of hondroytes in the pw of some setions (Fig. 1G). DISCUSSION The ellulr mehnism of one nd rtilge destrution in RA still remins unler [17]. Synovil tissues of RA joints produe vrious inflmmtory ytokines, suh s IL-l nd TNF-, whih re elieved to ply importnt roles in joint destrution. One key property of IL-I7A is its orhestrl role in mediting the migrtion of inflmmtory ells, whih tkes entrl ple in RA pthogenesis [24]. It is thought tht Il-23 plys n importnt role in the survivl nd expnsion of pthologil Th17 ells [25-27]. IL-23 is involved in the differentition of Th17 ells in pro-inflmmtory omplex, espeilly in the presene of TGF- nd IL-6 [25]. Notly, the inrese in IL-17 nd IL-23 ppers to e speifi for RA [26]. MTX is the entrl drug in the mngement of RA, known to suppress joint destrution in RA nd other immune medited inflmmtory diseses. However, MTX indues signifint dverse events in onsiderle numer of ptients [28]. Considering these toxiologil spets, omintion therpy of urumin nd MTX my provide enefiil therpeuti strtegy with redued side effets in RA. In the present study, we ssessed the level of IL-17 s the signture ytokine of Th17 nd other ytokines during the ourse of CIA model nd to investigte the effet of MTX singly or omined with urumin. Curumin omprises 2-8% of most turmeri preprtions nd is generlly regrded s its most tive omponent, hving potent ntioxidnt, nti-inflmmtory nd nti-rinogeni properties [29]. Here, the immunologi pttern of rthriti rts (group A) t the onset of the disese t dy 20 hrterized with signifint lower level of IL-23 nd higher levels of IL-17, IL-27, IFN- nd TGF- ompred to group N. However, pre-tretment with urumin in group C ltered this pttern, using rised levels of IFN- etween dys 20 nd 61 nd redued levels of IL-17 etween dys 41 n 69 ompred to group A. The oserved inrese in IFNlevel ws mthed with the redution in the level of IL-17. It hs een reported y other investigtors tht IFN- n downregulte the IL-17 response [30,31]. These results explin its ility to regulte differentition of CD4+ T ells to Th17 nd Th1 in norml stte. The present work showed tht tretment with urumin for seven weeks fter rthritis indution (group C) indued signifint redution in the levels of IL-17, IL-23, IFN- nd TGF- ompred to non-treted rthriti rts (group A). The suppression in the levels of these inflmmtory ytokines within urumin treted rthriti (group C) rts suggested n nti-inflmmtory effet of urumin. The elevted expression of the levels of IL-17 nd IFN- in experimentl utoimmune enephlomyelitis model ws deresed following in vivo 92

7 Glol J. Phrmol., 9 (1): 87-96, 2015 tretment with urumin [32]. Moreover, Arthriti rts omprison to group A. Thus, IL-27 plys n importnt treted with urumin showed signifint inhiition of role s protetive ytokine in the pthogenesis nd serum proinflmmtory ytokine (IL-1 ) when ompred proposed s potentil tretment for rthritis [40,41]. with non-treted rthriti [33]. RA is systemi disese tht ffets multitude of It ws stted tht IL-17 upregultes the prodution of orgns. Previously, it hs een shown tht RA indution pro-inflmmtory ytokines espeilly IFN- [34] whih used drsti histopthologil hnges in thymus, liver, indited the positive orreltion etween IL-17 nd lung nd kidney setions [42]. Additionlly, the IFN-. Th17 ells re developed from nïve CD4+T ells destrutive effets ompnied with MTX tretment on under the influene of network of inflmmtory these seondry orgns were minimized nd meliorted ytokines inluding IL-1, IL-6 nd TGF-, whih support y omined tretment of oth urumin nd MTX [43]. the ommitment to this linege [26]. Thus, this reltion Our dt illustrted tht the indution of rthritis used ould explin the positive orreltion etween oth IL-17 severe nd drsti dmge in the hind pws s oserved nd TGF- in the present study. Additionlly, we in the exmined setions from group A. MTX nd/or demonstrted negtive orreltion etween IL-17 nd IL- urumin tretment suppressed nd meliorted the 27. This negtive orreltion ould e used y the drsti dmge used y the indution of rthritis. MTX inhiitory effets of IL-27 on Th1, Th2 nd Th17 susets hs powerful nti-inflmmtory effet in vivo nd of T ells s well s the expnsion of induile inhiits humn synovil firolst RANKL prodution regultory T ells [35-37]. It lso inhiits the nd osteolstogenesis in dose-dependent mnner [15]. development of proinflmmtory Th17 ells y Also, in se of group C urumin tretment used the suppressing the expression of Th17 mster trnsription prolifertion of hondroytes. These results re in ftor ROR- t, therey preventing the prodution of IL- greement with previous studies whih demonstrted tht 17A nd IL-17F in nive T ells nd up-regultion of IL- urumin is promising therpeuti gent for the l0 expression [38]. In ontext of the ove ytokine tretment of RA s it hs nti-inflmmtory nd ntilne in the pthogenesis of utoimmune rthritis, the poptoti effets in hondroytes [44]. Curumin hs potentil nti-rthriti tivity of urumin might skew the tivities similr to the nti-tnf drugs, ut without their ytokine milieu towrds reduing the pthogeni Th1 serious side-effets [3]. (IFN- )/Th17 (IL-17) response. These findings highlight tht urumin differentilly regultes CD4+ Th ell CONCLUSION responses in CIA. In the urrent work, post-tretment with MTX (group The findings of the study indite tht urumin hs M) meliortes CIA rts, using redution in the levels n meliortive effet on CIA rts, involving downwrd of IL-17, IL-23, IFN- nd TGF- signifintly ompred to trend in pro-inflmmtory ytokines, inluding IL-17, ILnon-treted rthriti rts (group A) throughout the 23, IFN- nd TGF-. Notly, urumin tretment experiment. However, IL-27 level ws not signifintly indued the prolifertion of hondroytes. An inverse hnged from dys 48 to 69 in omprison to group A. intertion is onsidered etween IL-17 nd IL-27. This redution in the serum levels of the mesured Therefore, the omined tretment with oth urumin ytokines n e ttriuted to the nti-inflmmtory nd MTX ould modulte the ytokines prodution properties nd n dditionl nti-prolifertive tivity of through upregultion of the protetive ytokine MTX. Also, the immunosuppressive tivity of MTX, represented y IL-27, used the restortion of the norml medited y poptosis of seletive tivted, ut not struture of the hind pw. resting, T ells of peripherl lood in the S/G2 phse of the ell yle, even fter short-term exposure to MTX [39]. REFERENCES The present dt demonstrted tht omined tretment of MTX nd urumin hs the ility to 1. Yng, Y.H., R. Rjih, D.Y. Lee, Z. M, H. Yu, meliorte the linil severity of CIA rts t the end of H.H. Fong, L. Lo, B.M. Bermn nd K.D. Moudgil, the experiment, using signifint derese in the levels Suppression of ongoing experimentl rthritis of IL-17, IL-23, IFN- nd TGF- nd n inrese in the y hinese herl formul (huo-luo-xio-ling dn) level of IL-27 ompred to group A. Compring with involves hnges in ntigen-indued immunologil urumin or MTX single used groups, omined nd iohemil meditors of inflmmtion. Evidene tretment of urumin nd MTX showed signifint Bsed Complementry nd Alterntive Mediine, inrese in the level IL-27 etween dys 61 nd 69 in 2011:

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9 Glol J. Phrmol., 9 (1): 87-96, Hemeid, R.A. nd O.M. Mohfez, Curumin 32. Knksi, S., E. Cslini, C.C. Wlline, C. Mo, ttenutes methotrxte-indued hepti oxidtive W. Cherwe nd J.J. Bright, Differentil dmge in rts. Journl of the Egyptin Ntionl regultion of CD4(+) T helper ell responses y Cner Institute, 20: urumin in experimentl utoimmune 24. Zrioul, S., M.L. Toh, A. Tourndre, Y. Zhou, enephlomyelitis. Journl of Nutritionl M.A. Czlis, A. Phot, V. Miosse nd P. Miosse, Biohemistry, 23: IL-17RA nd IL-17RC reeptors re 33. Kunh, M., V.G. Nidu, B.D. Shu, S.G. Gdeplli essentil for IL-17A-indued ELR+ CXC hemokine nd R. Sistl, Curumin potentites the expression in synovioytes nd re overexpressed nti-rthriti effet of prednisolone in Freund's in rheumtoid lood. Journl of Immunology, omplete djuvnt-indued rthriti rts. Journl 180: of Phrmology nd Phrmotherpeutis, 25. Duvllet, E., L. Semerno, E. Assier, G. Flgrone 66: nd M.C. Boissier, Interleukin-23: key 34. Truhetet, M.E., M.D. Mosslyi nd K. Bonife, ytokine in inflmmtory diseses. Annls of IL-17 in the rheumtologist's line of sight. Mediine, 43: BioMed Reserh Interntionl, 2013: Qu, N.L., M. Xu, I. Mizoguhi, J. Furusw, 35. Jnkowski, M., P. Kopiñski nd A. Go, K. Kneko, K. Wtne, J. Mizuguhi, M. Itoh, Interleukin-27: iologil properties nd linil Y. Kwkmi nd T. Yoshimoto, Pivotl roles of pplition. Arhivum Immunologie et Therpie T-helper 17-relted ytokines, IL-17, IL-22 nd IL-23 Experimentlis (Wrsz), 58: in inflmmtory diseses. Clinil nd Developmentl 36. Hirhr, K., K. Ghoreshi, X.P. Yng, H. Tkhshi, Immunology, 2013: A. Lurene, G. Vhedi, G. Siumè, A.O. Hll, 27. Gffen, S.L., R. Jin, A.V. Grg nd D.J. Cu, C.D. Dupont, L.M. Frniso, Q. Chen, M. Tnk, The IL-23-IL-17 immune xis: from mehnisms to Y. Knno, H.W. Sun, A.H. Shrpe, C.A. Hunter nd therpeuti testing. Nture Review Immunology, J.J. O'She, Interleukin-27 priming of T ells 14: ontrols IL-17 prodution in trns vi indution of 28. Romão, V.C., H. Cnhão nd J.E. Fonse, Old the lignd PD-L1. Immunity, 36: drugs, old prolems: where do we stnd in predition 37. Mddur, M.S., P. Miosse, S.V. Kveri nd J. Byry, of rheumtoid rthritis responsiveness to Th17 ells: iology, pthogenesis of methotrexte nd other syntheti DMARDs?. BMC utoimmune nd inflmmtory diseses nd Mediine, 11: 17. therpeuti strtegies. Amerin Journl of 29. Soetikno, V., F.R. Sri, P.T. Veerveedu, Pthology, 181: R.A. Thndvryn, M. Hrim, V. Sukumrn, 38. Diegelmnn, J., T. Olszk, B. Göke, R.S. Blumerg nd A.P. Lkshmnn, K. Suzuki, H. Kwhi nd S. Brnd, A novel role for interleukin-27 (IL-27) K. Wtne, Curumin meliortes s meditor of intestinl epithelil rrier protetion mrophge infiltrtion y inhiiting NF- B medited vi differentil signl trnsduer nd tivtion nd proinflmmtory ytokines in tivtor of trnsription (STAT) protein signling streptozotoin indued-dieti nephropthy. nd indution of ntiteril nd nti-inflmmtory Nutrition nd Metolism (Lond), 8: 35. proteins. J. Biologil Chemistry, 287: Prk, H., Z. Li, X.O. Yng, S.H. Chng, R. Nuriev, 39. Esprz, L., J. De Hro, S. Bled nd F. Ain, Y.H. Wng, Y. Wng, L. Hood, Z. Zhu, Q. Tin nd Non-Fs (CD95/APO1)-medited poptosis of C. Dong, A distint linege of CD4 T ells tivted T ells inhiits the development of regultes tissue inflmmtion y produing theroslerosis. Intertive CrdioVsulr nd interleukin 17. Nture Immunology, 6: Thori Surgery, 15: Chu, C.Q., D. Swrt, D. Alorn, J. Toker nd 40. Klliolis, G.D., R.A. Gordon nd L.B. Ivshkiv, K.B. Elkon, Interferon-gmm regultes Suppression of TNF- nd IL-1 signling identifies suseptiility to ollgen-indued rthritis through mehnism of homeostti regultion of suppression of interleukin-17. Arthritis nd mrophges y IL-27. Journl of Immunology, Rheumtism, 56: :

10 Glol J. Phrmol., 9 (1): 87-96, Tnid, S., H. Yoshitomi, M. Ishikw, T. Kshr, 43. Irhim, E.M., A.M. Bdr, H. Siii, A.S.E. El-Wkkd K. Murt, H. Shiuy, H. Ito nd T. Nkmur, nd S. El-Dee, Dul effet of urumin nd IL-27-produing CD14(+) ells infiltrte inflmed methotrexte tretment on vrious orgns in ollgenjoints of rheumtoid rthritis nd regulte indued rthritis in rts. Journl of Amerin Siene, inflmmtion nd hemotti migrtion. Cytokine, 8: : Buhrmnn, C., A. Mosheri, U. Mtis nd 42. Irhim, E.M., A.M. Bdr, H. Siii, A.S.E. El-Wkkd M. Shkiei, Curumin medited suppression nd S. El-Dee, Protetive effet of urumin of nuler ftor- B promotes hondrogeni tretment on some orgns in ollgen-indued differentition of mesenhyml stem ells in rthritis in rts. Journl of Amerin Siene, high-density o-ulture miroenvironment. Arthritis 8: Reserh nd Therpy, 12: R