Investigation the Effects of Curcumin on Serum Hepatic Enzymes Activity in a Rheumatoid Arthritis Model

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1 Investigtion the Effets of Curumin on Serum Hepti Enzymes Ativity in Rheumtoid Arthritis Model Ftemeh Aghei Borshn 1,, Mino Ilkhnipoor 1, Mohmmd Hshemi 1, Frh Frrokhi 2 1 Deprtment of Biology, Fulty of Siene, Urmi University, Urmi, Irn; 2 Deprtment of Clinil, Fulty of Veterinry, Urmi University, Urmi, Irn. Corresponding uthor. Tel: ; E-mil: Aghei.ftemeh@yhoo.om Astrt Curumin (diferuloylmethne) is the most tive omponent of Curum long L. Severl linil trils hve found urumin to e notle ntiinflmmtory nd ntioxidnt ompound. The present study evlutes the effets of urumin nd indomethin tretment on the inflmmtory responses nd on plsm phosphtse nd trnsminse tivities, employing n djuvnt indued rthritis model in rt. Arthritis ws indued y the suutneous injetion of Freund s omplete djuvnt into the plntr surfe of right hind pw of rts. Development of the inflmmtory response ws ssessed over 21-dys period. In this study smple of 32 mle Wistr lino rts (18-22 gr) were used whih rndomly divided into four groups with eight rts per group. Group I served s norml ontrol rts (untreted), group II rthriti rts whih reeived vehile, group III rthriti rts treted with urumin (3 mg/kg orlly) nd nother group, rthriti rts treted with indomethin (3 mg/kg referene drug orlly). Tretment ws strted from 7 th dy of djuvnt injetion nd ws terminted on dy 21. At the end of the experimentl period, the nimls were nesthetized nd killed; lood smples were olleted nd prepred for mesurement of lkline phosphtse (ALP), sprtte (AST) nd lnine minotrnsferse (ALT) tivities. The results indited tht inflmmtion nd plsm ALP, AST nd ALT tivities were very elevted in the rthriti rts nd urumin tretment to similr indomethin were signifintly redued the swelling of pws nd plsm ALP, AST nd ALT tivities in rthriti rts. In onlusion, the present dt suggest tht minotrnsferses nd lkline phosphtse were signifintly redued in rthriti rts fter the dministrtion of urumin. This reduing effet my e relted to their nti-inflmmtory tivity. Keywords: Curumin; Aminotrnsfernses; Inflmmtory; Arthritis. 1. Introdution Rheumtoid rthritis (RA) is hroni utoimmune disese hrterized y joint swelling, synovil inflmmtion nd rtilge destrution nd ommonly leds to signifint disility nd onsequent redution in qulity of life [1-4]. Epidemiology of the rthritis in femle: mle is 3:1 nd fflits generlly.5-1.% of the popultion round the world [5]. RA used y numer of proinflmmtory moleules relesed y mrophges [6]. These inlude retive oxygen speies nd eiosnoids suh s, prostglndins, leukotrienes nd ytokines [7, 8]. The regultion of these meditors sereted y mrophges nd other immune ells therefore my ontrol the hroni inflmmtory onditions [9, 1]. Mny studies hve demonstrted the involvement of errnt Arhidoni id metolism in the inflmmtory response y generting potent hemil messengers known s eiosnoids. Memrne phospholipids, the mjor soure of rhidoni id re hydrolyzed y phospholipse A2 (pla2); the relesed rhidoni id is further metolized y three different types of oxygenses: ylooxygense (COX), liopxygense (LOX) nd ytohrome P45. Modultion of rhidoni id metolism y inhiiting these enzymes hs een onsidered s n effetive mehnism for hemoprevention [11]. For the tretment of RA, lthough non-steroidl ntiinflmmtory drugs (NSAIDs), steroidl gents nd immunosuppressnts usully used, their side effets nd toxiity ll for new nd more effetive nturl drugs. Curumin (diferuloylmethne) is mjor onstituent found in the spie turmeri, whih is dried powder from the rhizomes of Curum long L. This spie hs een used s trditionl mediine for tretment of inflmmtion, gstrointestinl disorders, hepti disorders, dieti wounds, skin wounds, rheumtism, sinusitis nd other disorders [12] Curumin is lso food oloring nd flvoring gent pproved y the Food nd Drug Administrtion [11]. It is elieved tht urumin is potent ntioxidnt nd nti-inflmmtory gent [13] (Figure 1). ISSN

2 Figure 1. Chemil struture of urumin Curumin ffets rhidoni id metolism y loking the phosphoryltion of PLA2, deresing the expression of COX-2 nd inhiiting the tlyti tivities of 5-LOX. These tivities my ontriute to the nti-inflmmtory tions of urumin nd its nlogs [11]. Evidene suggests tht in some experimentl onditions, urumin n hve similr nti-inflmmtory tivity s some of the ommon NSAIDs like indomethin, Vioxx, Celerex, nd iuprofen, ut without mny of the side effets, suh s gstrointestinl distress nd rdiovsulr omplitions [14, 15]. Adjuvnt indued rthritis (AIA) is experimentl rts, hroni inflmmtory disese hrterized y infiltrtion of the synovil memrne nd ssoited with destrution of the joints resemles losely to the humn rheumtoid rthritis [16-18]. Tissue dmge ws ssessed y mesuring the tivities of enzyme in the serum nd in the respetive orgn, sine liver impirment is lso feture of djuvnt rthritis [19]. The inrese in minotrnsferses is due to their relese from the ells of the dmged orgn [2]. Hyperphosphtsmi hs een oserved osionlly in the ptients with rheumtoid rthritis, nd it hs een suggested tht the serum lkline phosphtse level is relted to the tivity of the disese [21]. The present study is designed to investigte the effets of urumin nd indomethin on inflmmtion nd the reltionship etween serum lkline phosphtse (ALP), sprtte minotrnsferse (AST) nd lnine minotrnsferse (ALT) tivities nd rheumtoid rthritis in AIA rts. 2. Mterils nd Methods 2.1 Mterils Curumin, Freund s omplete djuvnt (FCA), Indomethin were purhsed from Sigm Aldrih, Ether ws purhsed from Merk. Ketmin (Germny Rotex Medi), Xylsin (Hollnd Alfsn woerden). All other hemils used were of the nlytil regent grde. Animls: As smple of 32 mle Wistr lino rts (18-22g; deprtmentl niml house; Fulty of Siene; Urmi University) were used. They were mintined under ontrolled temperture (22±2ºC) nd reltive humidity (4-6%) with stndrd environmentl onditions of 12h light/drk yle in the deprtmentl niml house. Food nd wter were provided d liitum throughout the experiments. Rts were limted to their surroundings over 1 week to eliminte the effet of stress prior to initition of the experiments. 2.2 The indution of rthritis The method desried y Newould in 1963 ws employed with some modifitions [22]. Arthritis ws indued y single suutneous injetion of.1 ml Freund s omplete djuvnt (FCA), suspension of het-killed Myoterium tuerulosis in minerl oil, into footpd of the right hind of mle rts. A glss syringe (1 ml) with 21 GA needle ws used for injetion, while, the vehile ontrol rts were injeted with.1 ml norml sline. The rts were nesthetized with Ketmin (7mg/kg intrperitonelly [I.P]) nd Xylzin (5 mg/kg I.P) prior to nd during djuvnt injetion. 2.3 Experimentl setup Animls were divided into four groups of eight nimls in eh group s follows: Group I: Control rts (untreted) Group II: Adjuvnt indued rthriti rts (.2 ml norml sline) Group III: Arthritis treted y urumin (3 mg/kg/dy, orlly) Group IV: Arthritis treted y indomethin (3 mg/kg/dy, orlly, referene drug) for 15 dys y gvge strting 7 dys fter djuvnt injetion. The hind pw thiknesses (mm) were mesured using vernier liper on dys, 7, 14, 21 fter FCA injetion. At the end of the experimentl period (on dy 22 nd ), rts were fsted overnight nd killed fter generl nesthesi y inhltion of ether. Blood smples were olleted from the dominl ort nd serum immeditely seprted. 2.4 Serum iohemistry mesurement Assy of AST nd ALT tivities ws modified y the Interntionl Federtion of Clinil Chemistry (IFCC) nd eome the reommended methods for mesurement of AST nd ALT tivities in serums [23]. The tivities of the two enzymes re refleted y the derese of NADH in enzyme-oupled retions s shown in the following equtions: L Asprtte (L Alnine) + ketoglutrte AST (ALT) Oxlette (pyruvte) + L Glutmte (1) Oxlette (pyruvte) + NADH + H + MDH (LDH) L mlte (ltte) + NAD + (2) Alkline phosphtse (ALP) ws estimted y the p-nitropheyl phosphte (P-NPP) method using the reommendtions of Germn soiety for Clinil Chemistry (optimized IFCC, P NPP-AMP method) [24]. ALP P-Nitrophenyl phosphte p-nitrophenol +pi 2.5 Sttistil nlysis ISSN

3 All the results were expressed s men ± stndrd error (S.E.M). Dt were nlyzed using one-wy ANOVA followed y Tukey MRT test. p<.5 were onsidered s sttistilly signifint. 3. Results nd Disussion 3.1 Effet on djuvnt-indued rt pws edem Figure 2 depits the nti-inflmmtory effet of urumin nd indomethin on the hnges in pw edem of ontrol nd experimentl nimls. Swelling nd redness developed over 24 h period in the foot injeted with djuvnt. This inflmmtion retion inresed over time when disseminted rthritis ppered nd remins onstnt y the end of 1 week. After urumin nd indomethin tretment the inrese in swelling of the injeted hind pw tht ourred with the pperne of poly rthritis ws suppressed.upon urumin dministrtion similr to indomethin fter seven dys of djuvnt injetion, the inflmmtion were strted to derese signifintly when ompred to AIA rts. pw thikness(mm) time (dy) Figure 2. Pws thikness in rts with djuvnt rthritis. A: ontrol group (- -), B: rthriti rts treted y vehile (- -), C: rthriti rts treted with 3 mg/kg urumin (- -) nd D: rthriti rts treted with 3 mg/kg indomethin (- - ).vlues represent mens ± stndrd error of the men (S.E.M). p<.5 ompred with rthriti rt treted with vehile p<.5 ompred with ontrol rts (n=8). 3.2 Effet on Serum Hepti Aminotrnsfernses Figures 3, 4 nd 5 show the hnges in the tivities of serum minotrnsferses nd ALP in ontrol nd experimentl nimls. As result of inflmmtion indued y djuvnt, the levels of AST, ALT nd ALP were inresed (p<.5) in ll rthritis rts when ompred to ontrol rts. After urumin tretment, the levels of these enzymes were signifintly deresed (p<.5) in group III rts when ompred to AIA rts. ALT (u/l) Tretment groups Figure 3. Effet of urumin nd indomethin on serum lnin minoternsferse in rts with djuvnt rthritis. A: ontrol group, B: rthriti rts treted y vehile, C: rthriti rts treted with 3 mg/kg urumin nd D: rthriti rts treted with 3 mg/kg indomethin.vlues represent mens ± the S.E.M. sme words ren't signifint in p<.5 in TUKEY MRT (n=8). AST (u/l) Tretment groups Figure 4. Effet of urumin nd indomethin on serum sprtt minoternsferse in rts with djuvnt rthritis. A: ontrol group, B: rthriti rts treted y vehile, C: rthriti rts treted with 3 mg/kg urumin nd D: rthriti rts treted with 3 mg/kg indomethin. Vlues represent mens ± the S.E.M. sme words ren't signifint in p<.5 in TUKEY MRT (n=8). ALP (u/l) Tretment groups Figure 5. Effet of urumin nd indomethin on serum lkline phosphtse in rts with djuvnt rthritis. A: ontrol group, B: rthriti rts treted y vehile, C: rthriti rts treted with 3 mg/kg urumin nd D: ISSN

4 rthriti rts treted with 3 mg/kg indomethin.vlues represent mens ± the S.E.M. sme words ren't signifint in p<.5 in TUKEY MRT (n=8). 4 Conlusions In this study, ws investigted effets of urumin nd indomethin of pw swelling nd tivity of hepti enzymes in djuvnt rthritis rts. Freund s omplete djuvnt is generlly used indue rthritis in niml models [25]. Adjuvnt rthritis in rt shows severl linil nd histologil similrities to humn RA [26]. In this study, interplnetry injetion of djuvnt into right hind pw rpidly indued pw swelling nd redness. Spie priniple like urumin ws shown to inhiit the synthesis of proinflmmtory prostglndins nd leukotrienes [27]. Curumin hs een shown to possess enefiil effets in lleviting rthriti symptoms in oth niml nd humn models [12, 28]. Bin Joe et l, hve show tht urumin inhiits the uptke of rhidoni id y mrophges, therey limiting the vilility of the sustrte for eiosnoid prodution [29]. Shimizu et l, hve demonstrted tht urumin lso redues the onversion of linolei id to rhidoni id [16]. Like urumin, other NSAIDs suh s indomethin lower the prodution of potent proinflmmtory gents suh s prostglndins [29]. The results of the present study indited tht the urumin exhiits nti-inflmmtory properties in rts with Freund s djuvnt-indued rthritis. Assessment of the levels of AST, ALT nd ALP provides n exellent nd simple tool to mesure the nti-rthriti tivity of the trget drug [3]. The tivities of minotrnsferses nd ALP were signifintly inresed in rthriti rts, sine these re good indies of liver nd kidney impirment whih is lso onsidered feture of djuvnt rthritis [31]. Serum AST nd ALT hs een reported to ply vitl role in the formtion of iologilly tive hemil meditors suh s rdykinins in inflmmtory proess [32]. Also, Niino-Nnk et l onfirmed positive orreltion etween the inresed tivity of serum lkline phosphtse nd the disese tivity in RA [33]. Elevted levels of serum ALP in AIA rts n e due to inrese in the liver nd one frtion or to n inrese of oth isoenzymes. This in turn implites lolized one loss in the form of one erosion nd peri rtiulr osteopeni, s the enzyme is relesed into irultion in the ourse of one formtion nd resorption [21]. This study hs shown tht n immunologil djuvnt dministered to rts lters hepti iohemistry. Most reports suggested n inrese of the seline serum ALP level in RA, lthough of underlying mehnism remins unertin [34]. Reently, oservtion of serum smple from ptients with RA suggested tht ALP tivity ws detetle t high prevlene of out 3 %[35] Also, Hung et l reported tht djuvnt treted rt showed signifintly higher serum iohemistry mrkers (AST, ALT nd ALP) thn norml rts [26]. Reddy nd Lokesh found tht orl dministrtion of urumin (3 mg/kg ody weight) for 1 dys lowered the liver nd serum lipid peroxide levels, serum ALT, AST enhned y I.P. injetion of iron in rts [36]. Also Arf reported Curumin lso deresed the enzyme tivities of serum AST nd ALT [37]. The results this study indited tht inflmmtory nd plsm ALP, AST nd ALT tivities were very elevted in the rthriti rts whih urumin tretment to similr tht indomethin were signifintly redued the swelling of pws nd plsm ALP, AST nd ALT tivities in rthriti rts. In onlusion, in this study, the development of inflmmtion during indued rthritis my e relted to the iohemil hnges of hepti enzymes nd the present dt suggest tht minotrnsferses nd lkline phosphtse were signifintly redued in rthriti rts fter the dministrtion of urumin. This reduing effet my e relted to their ntiinflmmtory tivity. Aknowledgements This study ws supported y grnt from the reserh ounil of Urmi University, Urmi, Irn. Referenes [1] Kingsley G., Lnhury J. (1991) Immunotherpy of rheumti diseses-prtie nd prospets. Immunology Tody, 12: [2] Lindqvist E., Jonnson K., Sxne T., et l. (23) Course of rdiogrphi dmge over 1 yers in ohort with erly rheumtoid rthritis. Annul of the Rhemti Diseses, 62: [3] Feldmnn M., Brennn F.M., Mini R.N. (1996) Role of ytokines in rheumtoid rthritis. Annul Review of Immunology, 14: [4] Grvllese E.M., Goldring S.R. (2) Cellulr mehnism nd the role of ytokines in one erosions in rheumtoid rthritis. Arthritis nd Rheumtism, 43: [5] Ktz L., Piliero S.J. (1969) A study of djuvnt indued polyrthritis in the rt with speil referene to ssoited immunologil phenomen. Annls of the New York Ademy of Sienes, 147: [6] Henderson B., Pettipher ER., Higgs GA. (1987) Meditors of rheumtoid rthritis. Br Med ull, 42: [7] Ds UN. (1993) Oxyrdils nd their linil implitions. Curr Si, 65: [8] Snedegrd G. (1985) Meditors of vsulr permeility in inflmmtion. Prog Apl Mirori, 7: [9] Knisell JE., Lokesh B. (199) Dietry lipids, eiosnoids nd immune system. Crit Cre Med, 18: S94-S11 3. ISSN

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