Research Article Possible Potentiation by Certain Antioxidants of the Anti-Inflammatory Effects of Diclofenac in Rats

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1 e Scientific World Journl, Article ID , 9 pges Reserch Article Possible Potentition by Certin Antioxidnts of the Anti-Inflmmtory Effects of Diclofenc in Rts Smh S. Abbs, 1 Mon F. Schln, 2 Ashrf K. Bhgt, 3 nd Ezzeddin S. El-Denshry 3 1 Deprtment of Phrmcology nd Toxicology, Misr Interntionl University (MIU), Km 28, Ciro-IsmiliRod,P.O.Box1,Heliopolis,Ciro,Egypt 2 Deprtment of Biochemistry, Fculty of Phrmcy, Misr Interntionl University (MIU), Km 28, Ciro-Ismili Rod, P.O. Box 1, Heliopolis, Ciro, Egypt 3 Deprtment of Phrmcology nd Toxicology, Fculty of Phrmcy, Ciro University, Giz, Egypt Correspondence should be ddressed to Mon F. Schln; m.schln@gmil.com Received 30 August 2013; Accepted 22 Jnury 2014; Published 12 Mrch 2014 Acdemic Editors: P. Cmpigli, G. Iccrino, M. Illrio, nd N. Montuori Copyright 2014 Smh S. Abbs et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. In the present study, we investigted the potentil beneficil impct of the ddition of ntioxidnt supplements to diclofenc regimen in model of crrgeenn-induced pw. Rts were treted dily with ntioxidnts, tht is, -lipoic cid (50 mg/kg), selenium (2.5 mg/kg), vitmin C (1 g/kg), vitmin E (300 mg/kg), or zinc (25 mg/kg) on seven successive dys nd then received single tretment with diclofenc or sline before crrgeenn ws injected to induce pw inflmmtion. The results indicted tht these combintions did not significntly ffect the percentge inhibition of pw edem cused by diclofenc lone; however, some combintion tretments meliorted signs of concomitnt oxidtive stress (such s ltertions in plsm mlondildehyde (MDA) levels, hemolyste reduced glutthione levels, nd erythrocytic superoxide dismutse enzyme ctivities) imprted by diclofenc lone. In some cses, few tested ntioxidnts in combintion with diclofenc resulted in incresed plsm levels of interleukin- (IL-) 6 nd C-rective protein (CRP). In conclusion, the results of these studies suggested to us tht the dded presence of nturl ntioxidnts could be beneficil s stndrd nti-inflmmtory therpeutics for ptient under diclofenc tretment, lbeit tht these effects do not pper to significntly build upon those tht could be obtined from this common nti-inflmmtory gent per se. 1. Introduction During the lst decde, gret dvnces hve been mde for understnding the pthophysiology of inflmmtion nd the involvement of rective oxygen species (ROS) in its pthogenesis. Inflmmtion is complex defense mechnism in which leukocytes migrte from the vsculture into dmged tissues to destroy the gents tht cn potentilly cuse tissue injury [1]. Millions of people ll over the world re suffering from inflmmtory disorders, mking them use huge mounts of nti-inflmmtory gents (i.e., diclofenc, nonselective cyclooxygense [COX] inhibitor) for mny yers in their lives. However, the serious side effects nd the induced intolernce of the nti-inflmmtory drugs hve led to the serch for methods in order to decrese their effective doses nd improve their sfety ptterns. A plethor of evidence hs shown tht overproduction of ROS occurs t sites of inflmmtion nd this contributes to overll tissue dmge. A subsequent oxidtive stress (OS) predomintes when production of ROS exceeds the cpcity of cellulr ntioxidnt defenses to remove these toxic species [2]. Due to their high rectivity, ROS re potentilly cusing dmge to biomolecules such s DNA, lipids, nd proteins. Thus, there is incresing interest in exmining the potentil benefits from providing ptients ntioxidnts, such s αlipoic cid (α-la), selenium (Se), Vitmin C (Vit C), Vitmin E (Vit E), nd zinc (Zn)-supplements, s dd-ons to their diclofenc regimen. Accordingly, the im of this study ws to investigte the bility of severl vitmins nd elements (commonly tken s food supplements) to reduce ny deleterious side effects from diclofenc or to potentilly bolster the desired effects from

2 2 The Scientific World Journl thedrug.whileourmininterestwstoscreenthepotentil efficcy of these vitmins s stnd-lone gents in plce of diclofenc or s dd-ons to diclofenc ginst n induced inflmmtion in situ, we lso hoped to begin to define ny potentilmechnismsofctionthtthesegentsmightutilize in bringing bout the observed effects. This included nlyses of effects of ech gent lone/in combintion with diclofenc on some key cytokines\inflmmtory meditors (i.e., interleukin [IL]-6 nd C-rective protein [CRP]) relesed during n inflmmtory event). While we focused on the potentil for these gents to reduce ny undesired toxicities from the chronic use of diclofenc, we were lso mindful tht these studies could provide informtion tht would llow for n unintended benefit potentilly llowing for decrese in the effective dose of diclofenc needed by ptient nd subsequent decrese in the risk of development of dverse effects. 2. Mterils nd Methods 2.1. Animls. Mle Sprgue Dwley rts (Ntionl Reserch Center, Giz, Egypt) weighing g were used in this study. Rts were housed in room with controlled temperture (18 22 C), humidity (60 ± 10%), nd light/drk (12 hr/d) cycles for t lest 1 week before being rndomized into vrious experimentl groups. Throughout the study, rts were provided d libitum ccess to stndrd pellet chow (El- Nsr chemicl Co., Ciro, Egypt) nd filtered wter. All experiments were conducted in ccordnce with the principles nd procedures outlined in the Interntionl Guide for the Cre nd Use of Lbortory Animls nd were pproved by the Ethics Committee of the Fculty of Phrmcy Ciro University Drugs. Diclofenc (Diclofenc K; Novrtis, Ciro, Egypt) ws dissolved in distilled wter to concentrtion tht would ssure tht rt would receive per os dose of 5 mg diclofenc/kg body weight. In these studies, the single diclofenc tretment occurred lone or in combintion with (i.e., 2 hr fter the finl of the 7 dily doses) the test ntioxidnts. α-lipoic cid (α-la; EVA Phrm, Giz, Egypt; in norml sline) ws delivered by intrperitonel injection t dose of 50 mg α-la/kg [3]. Ech dily per os delivery of sodium selenite (Sigm, St. Louis, MO; in wter) t 2.5 mg sodium selenite/kg [4]; Vit C (ADWIC, Al Qlyubiyh, Egypt; in wter) t 1 g/kg [5], Vit E (MP Biomedicls, Bs- Rhin, Frnce; in olive oil) t 300 mg/kg [6], nd zinc sulfte (ADWIC;inwter)t25mgZnSO4/kg[7] wsperformed without nesthesi nd with delivery volume of 1 ml/rt. The diclofenc volume (lone or fter the finl ntioxidnt dosing) ws 1 ml/rt s well Experimentl Design. Rts were rndomly divided into 12 groups (n = 8/group): crrgeenn control, rts given diclofenc nd then crrgeenn, rts given Vit C, Zn, α- LA, Vit E, or Se dily for 7 d nd then crrgeenn, nd rts given Vit C, Zn, α-la, Vit E, or Se for 7 d, followed by diclofenc nd then crrgeenn. In the combintion studies, tretment with diclofenc ws lwys 2 hr fter the finl ntioxidnt/vehicle tretment; rts in the crrgeenn-only nd ntioxidnt-only groups received sline t this timepoint. The crrgeenn injection ws performed 2 hr lter (i.e., 4 hr fter the finl dily dministrtion with the test ntioxidnts (or vehicle)) Induction of Inflmmtion. Rts were injected subcutneously in the plntr side of their left hind pws with 0.05 ml of 1% crrgeenn solution. Footpd swelling ws then ssessed (by monitoring chnges in pd thickness) using vernier clipers (Mitutoyo, Tokyo, Jpn). Pw thickness ws mesuredjustbeforetheinjectionnd1,2,3,nd4hr fter injection. Bsed on preliminry studies tht showed tht mximum pw thickness increse occurred t 3hr fter injection of crrgeenn; the vlues obtined t tht timepoint were used here to clculte vlues of percentge inhibition of pw thickness increse Preprtion of Blood Smples. Four hours fter the crrgeenn injection, 4 ml blood ws collected from the retroorbitl plexus into heprinized tubes. A 0.5 ml liquot ws immeditely trnsferred to nother heprinized tube for erythrocyte seprtion nd for determintion of superoxide dismutse (SOD) ctivity. A 100μL liquot of the originl blood ws hemolyzed for determintion of reduced glutthione (GSH) content. The remining originl blood ws centrifuged (3000 rpm, 4 C, 15 min) to isolte plsm; this ws stored t 20 C for subsequent determintions of levels of mlondildehyde (MDA), C-rective protein (CRP), nd IL Erythrocyte Seprtion. The liquot spred for determintion of SOD ctivity ws centrifuged (3000 rpm, 4 C, 15 min); the precipitted RBC were wshed with 3 ml cold sline nd then centrifuged gin t 3000 rpm for 10 min. After the superntnt ws discrded, lysing of the RBC ws performed by resuspension of the cells in 1.75 ml ice-cold distilledwterndthenvigorousshking;thecellswerethen left to stnd for 15 min t 4 C. The resulting hemolyste ws used for determintion of SOD ctivity Determintion of Plsm Lipid Peroxides (MDA), CRP, nd IL-6, Erythrocyte Superoxide Dismutse (SOD) Activity, nd Blood Reduced Glutthione (GSH) Levels. The determintion of lipid peroxide levels (s reflected in mounts of mesurblemda)wsdonesprescribedbefore[8]. In brief, to 0.2mL plsm, 1.2mL of 1% (w/v) o-phosphoric cid nd 0.4 ml of 0.67% (w/v) thiobrbituric cid were dded nd mixed, nd then the mixture ws heted for 45mininboilingwterbth.Aftercooling,1.6mLnbutnol ws dded nd the smple ws mixed vigorously. The butnol lyer ws seprted by centrifugtion t 3000 rpm for 15 min. The bsorbnce of the pink product in the butnol frctionwsthenmesuredt535nd520nminshimdzu double bem spectrophotometer (UV ); ll smples were red ginst blnk processed in prllel contining 0.2 ml distilled wter insted of smple. The difference in

3 The Scientific World Journl 3 bsorbnce between the two redings (i.e., ΔA )ws tken s reflection of the level of MDA (nmol/ml) in the smple. The pyrogllol utoxidtion method ws dopted for determintion of erythrocyte SOD ctivity [9]. In brief, from the previously seprted hemolyste of erythrocytes, 250 μl ws mixed vigorously with 0.75 ml chloroform-ethnol mixture (3 : 5 v/v) to precipitte hemoglobin in the smple. In microcuvette, 1 ml Tris-HCl buffer (ph 8.2) ws dded to 30 μl of10μm pyrogllol solution nd 100 μl distilled wter; the bsorbnce t 420 nm ws then mesured 30 nd 90 sec therefter. The difference in bsorbnce (ΔA) ws used to reflect the rte of pyrogllol utoxidtion (in 1 min) nd ws considered the experiment blnk. The sme procedure ws then crried out using the prepred blood smples or stndrds contining SOD in the plce of distilled wter. The reduction or inhibition of rte of utoxidtion in 1 min (compredtotheblnk)wsusedsnindexofthesod ctivity.thepercentgechngeinpyrogllolutoxidtion ws clculted s follows: % chnge in pyrogllol utoxidtion = (ΔA T or S / min)/δa B / min, where ΔA T / min = chnge in bsorbnce of the test smple in 1 min. ΔA S /min= chnge in bsorbnce of the stndrd smple in 1 min, nd ΔA B /min=chngeinbsorbnceoftheblnksmplein 1min. Hemoglobin (Hb) ws mesured vi the cynomethemoglobin [10]. Hemoglobin in smple liquot (20 μl) ws converted to cynomethemoglobin by ddition of 5 ml Drbkin s regent (0.6 mm potssium ferricynide\0.77 mm potssium cynide); bsorbnce of the cynomethemoglobin ws then monitored t 540 nm. From these ltter vlues, the concentrtion of Hb ws clculted s follows: Hb concentrtion = A smple (g/dl), where A smple is smple bsorbnce t 540 nm, nd is unitless constnt fctor. Bsed on these vlues, the SOD ctivities were then reexpressed s (U SOD/g Hb) in blood smple) ccording to the following eqution: SOD ctivity in blood smple (U/g Hb) = SOD conc. (U/mL)/g Hb in smple. GSH levels in blood were determined s prescribed before [11]. In brief, 1 ml of hemolyste (0.1 ml originl blood ml distilled wter) ws combined with 1.5 ml of protein precipitting solution (1.67 g m-phosphoric cid, 0.2 g EDTA, nd 30 g NCl in 100 ml distilled wter) nd then plced t room temperture for 5 min. The smple ws then centrifuged (3000 rpm, 15 min) nd the resultnt superntnt ssyed for GSH, tht is, 1mL superntnt (or stndrd GSH solution) ws combined with 4 ml of the phosphte solution nd 0.5 ml Ellmn s regent (40 mg 5,5 -dithiobis- (2-nitrobenzoic cid) [DTNB] in 100 ml 1% sodium citrte solution). The bsorbnce of the resulting yellow solution wsthenmesuredt412nm(within5min)inspectrophotometer. From the bsorbnce vlues of stndrd GSH solutions, stndrd curve ws prepred nd levels of GSH in ech smple were then extrpolted (mg %). Plsm CRP nd IL-6 levels were mesured ccording to the procedures described in IMMULITE CRP nd IL-6 ssy kits, respectively (Dignostic Product Corportion (DPC), Los Angeles, CA). The sensitivity of the CRP nd IL-6 ssy Tble 1: Effects of diclofenc, α-la, selenite, Vit C, Vit E, or zinc on pw thickness increse fter 3 hr in rt crrgeenn-induced pw edem model. Groups Pw edem (thickness increse [cm] fter 3 hr) Inhibition % Crrgeenn (50 μl of 1% solution) 0.26 ± Diclofenc (5 mg/kg, per os) 0.16 ± # 38% α-la (50 mg/kg, IP) 0.20 ± # 23% Se (2.5 mg/kg, po) 0.21 ± # 19% Vit C (1 g/kg, po) 0.21 ± # 19% VitE(300mg/kg,po) 0.18 ± # 31% ZnSO 4 (25 mg/kg, po) 0.20 ± # 23% Vlues re mens of 8 rts (±SD). # Vlue significntly different from crrgeenn control (P < 0.05). Vlue significntly different from diclofenc group (P < 0.05). kits defined s the concentrtion two stndrd devitions bove the response t zero dose ws 1pg/mL Sttisticl Anlysis. Vlues were expressed s men ± SD. Results were nlyzed using one-wy nlysis of vrince (ANOVA) followed by lest significnt difference test (LSD) to compre between the different groups. A P vlue < 0.05 ws ccepted s significnt in these tests. SPSS softwre (Chicgo, IL) ws used to crry out ll nlyses. 3. Results 3.1. Effects of Vrious Agents on the Rt Pw Edems. Norml rts injected subcutneously with crrgeenn hd n verge 0.26 cm increse in pw thickness. Administrtion of diclofenc 2 hr prior to the crrgeenn mrkedly inhibited induced edem by 39%; tht is, increse in pw thickness ws 0.16 cm (Tble 1). Dily dministrtion of α-la, Se, Vit C, Vit E, or Zn for 7 d prior to the injection of crrgeenn significntly inhibited the induced pw edem by 23, 19, 19, 31, nd 23%, respectively (i.e., thickness vlues were 0.20, 0.21, 0.21, 0.18, nd 0.20 cm, resp.). Administrtion of the following tretment combintions, diclofenc + α-la, diclofenc + Se, diclofenc + Vit C, diclofenc + Vit E, or diclofenc + Zn, prior to injection of crrgeenn mrkedly inhibited induced pw edem by 19, 27, 31, 27, nd 46%, respectively, reltive to those in hosts tht hd received no drug tretment (i.e., Group I). Increses in pw thicknesses in these hosts were 0.21, 0.19, 0.18, 0.19, nd 0.14cm, respectively (Tble 2). The inhibitory effects imprted by the combintions were significntly no better thn the diclofenc lone; only in the cse of combintion with Zn the inflmmtion ws reduced better thn the outcome induced by diclofenc lone Effects of Test Agents on Plsm MDA Levels. Injection of 1% crrgeenn solution into the pws of nive rts induced n oxidtive stress reflected s significnt increse

4 4 The Scientific World Journl Tble 2: Effects of combintion tretments of diclofenc nd the test ntioxidnts on pw thickness increse fter 3 hr in rt crrgeenn-induced pw edem model. Groups Crrgeenn (50 μl of 1% solution) Diclofenc (5 mg/kg, per os) Diclofenc + α-la (50 mg/kg, IP) Diclofenc + Se (2.5 mg/kg, po) Diclofenc + Vit C (1 g/kg, per os) Diclofenc + Vit E (300 mg/kg, po) Diclofenc + Zn (25 mg/kg, po) Pw edem (thickness increse [cm] fter 3 hr) 0.26 ± 0.06 Inhibition % 0.16 ± # 38% 0.21 ± # 19% 0.19 ± 0.03 # 27% 0.18 ± 0.01 # 31% 0.19 ± 0.04 # 27% 0.14 ± # 46% Vlues re mens of 8 rts (±SD). # Vlue is significntly different from crrgeenn control (P < 0.05). Vlue is significntly different from diclofenc group (P < 0.05). in plsm MDA; vlues in these rts were 2.77-fold bove bckground (Tbles 3 nd 4). Rts tht received diclofenc 2hrbeforethecrrgeenninjectionhdmrkeddecrese in plsm MDA, tht is, 56% reltive to vlues in slinetreted crrgeenn-injected rts. Dily injection of α-la for 7dpriortothecrrgeennlsocusedsignificntdecrese in plsm MDA levels (47.6%) induced by the crrgeenn itself. In rts tht received diclofenc + α-la combintion, thedecreseinmdalevels(reltivetocrrgeenn-only rt levels) reched 69.4%. Selenite dministrtion for 7 d prior to induction of inflmmtion resulted in MDA levels being significntly lower (51.4%) thn those in nondrugtreted counterprts; in comprison, the diclofenc + selenite combintion significntly decresed MDA levels by just 40.7%. Tretment with Vit C for 7 d before induction of cute inflmmtion cused significnt 55.9% decrese in MDA levels; the diclofenc + Vit C combintion significntly reduced (by 67.8%) MDA levels to vlues pproximting those in norml rts. Vit E given for 7 d prior to inflmmtion inductionlsoresultedinsignificnt67.8%decresein plsmmdalevelscompredtothelevelinthecrrgeennonly rts. Unlike with Vit C, the diclofenc + Vit E tretment ws less effective thn the ntioxidnt lone; tht is, the chnge compred to the inflmed rt vlues ws now only 40.0%. Lstly, dosing with Zn for 7 d before injection of crrgeenn significntly decresed plsm levels of MDA (43.0%) reltive to tht in the nondrug-treted hosts; in this cse, the dditionl presence of diclofenc resulted in 69.5% reduction in MDA vlues Effects of Test Agents on Hemolyste GSH Levels nd Erythrocyte SOD Activity. Significnt decreses in hemolyste GSH levels nd erythrocyte (RBC) SOD ctivity (72 nd 86%, respectively, were noted in rts tht received the crrgeenn injection compred to vlues ssocited with the Plsm level (pg/ml) 9 # # 8 # # # 3 # Norml Crrg Diclo α-la Se Vit C Vit E Zn CRP IL-6 Figure 1: Effect of tretments on plsm CRP nd IL-6 levels.all rts were provided dily orl doses of diclofenc (5 mg/kg), selenite (2.5 mg/kg), Vit C (1 g/kg), Vit E (300 mg/kg), or ZnSO 4 (25 mg/kg); α-la (50 mg/kg) ws dministered IP. # Vlue is significntly different from norml control nd crrgeenn-only rts, respectively (P < 0.05). Vlue is significntly different from tht of diclofenctreted hosts (P < 0.05). blood/cells of nïve [norml] rt) (Tbles 3 nd 4). Rts tht received diclofenc 2 hr before injection of crrgeenn hd GSH levels nd SOD ctivity tht were significntly greter (5.70- nd 2.90-fold, resp.) compred to those in the sline-injected inflmed rts. The dily α-la regimen led to significnt increses in the hemolyste GSH levels (2.15-fold versus control) nd SOD ctivity (7.20-fold versus control) s well; the diclofenc + α-la combintion led to significnt increses in hemolyste GSH nd RBC SOD of nd 2.44-fold, respectively. Selenite tretments prior to induction of inflmmtion resulted in significnt increses in the GSH (2.17-fold) nd SOD ctivity (4.00-fold) levels; the diclofenc + selenite regimen led to significnt increse in hemolyste GSH (3.99 fold) levels, but the chnge in RBC SOD ctivity ws insignificnt s compred to the vlues for the crrgeenn-only rts. Tretments with Vit C or Vit E for 7 d before induction of inflmmtion, ech cused significnt increses in hemolyste GSH (3.00- nd fold, resp.) nd RBC SOD ctivity (4.75- nd 2.60-fold, resp.) compred to vlues in the rts injected with crrgeenn only. The dditionl presence of diclofenc resulted in significnt increses in hemolyste GSH of 4- nd 2.45-fold for Vit C nd Vit E, respectively; RBC SOD ctivity ws incresed by 4- nd 4.28-fold, respectively, s compred to levels in nondrug-treted inflmed rts. Lstly, Zn tretment prior to the crrgeenn injection resulted in significnt increses in hemolyste GSH levels ( 2-fold) nd RBC SOD ctivity (4- fold) versus those in the crrgeenn-only rts (Tble 3). The diclofenc + Zn combintion lso led to significnt increses in hemolyste GSH ( 3-fold) nd RBC SOD ctivities ( 5- fold) Effects of Test Agents on Plsm CRP nd IL-6 Levels. Levels of CRP nd IL-6 in the plsm were lso significntly incresed(by2.30-nd6.50-foldbovenivelevel,resp.)

5 The Scientific World Journl 5 Tble 3: Effects of diclofenc, α-la, selenite, Vit C, Vit E, or zinc on plsm levels of MDA, hemolyste GSH, nd erythrocyte SOD ctivity in rt crrgeenn-induced pw edem model. Groups MDA (nmol/ml) GSH (mg %) SOD (U/g Hb) Norml (50 μlsline) 0.21 ± ± ± 0.91 Crrgeenn (50 μl of 1% solution) 0.59 ± ± ± 0.21 Diclofenc (5 mg/kg, per os) 0.26 ± 0.05 # 3.16 ± 0.34 # 1.61 ± 0.16 # α-la (50 mg/kg, IP) 0.31 ± 0.09 # 1.19 ± 0.26 # 3.93 ± 0.87 # Se (2.5 mg/kg, po) 0.29 ± 0.02 # 1.59 ± 0.12 # 2.10 ± 0.44 # Vit C (1 g/kg, po) 0.26 ± 0.03 # 1.69 ± 0.30 # 2.59 ± 0.32 # Vit E (300 mg/kg, po) 0.19 ± 0.03 # 1.35 ± 0.07 # 1.44 ± 0.25 # ZnSO 4 (25 mg/kg, po) 0.33 ± 0.05 # 1.20 ± 0.16 # 2.23 ± 0.57 # Vlues re mens of 8 rts (±SD). Vlue is significntly different from norml control (P < 0.05). # Vlue is significntly different from crrgeenn control (P < 0.05). Vlue is significntly different from diclofenc group (P < 0.05). Tble 4: Effects of combintion tretments of diclofenc nd the test ntioxidnts on plsm levels of MDA, hemolyste GSH, nd erythrocyte SOD ctivity in rt crrgeenn-induced pw edem model. Groups MDA (nmol/ml) GSH (mg %) SOD (U/g Hb) Norml (0.05 ml, 1% sline) 0.21 ± ± ± 0.91 Crrgeenn (50 μl of 1% solution) 0.59 ± ± ± 0.21 Diclofenc (5 mg/kg, po) 0.26 ± # 3.16 ± 0.34 # 1.61 ± 0.16 # Diclofenc + α-la (50 mg/kg, IP) 0.18 ± 0.03 # 2.19 ± 0.18 # 1.33 ± 0.27 # Diclofenc + Se (2.5 mg/kg, po) 0.35 ± 0.08 # 2.21 ± 0.17 # 0.51 ± 0.28 Diclofenc + Vit C (1 g/kg, po) 0.19 ± 0.02 # 2.20 ± 0.30 # 2.12 ± 0.55 # Diclofenc + Vit E (300 mg/kg, po) 0.36 ± 0.09 # 1.36 ± 0.22 # 2.33 ± 0.39 # Diclofenc + Zn (25 mg/kg, po) 0.18 ± 0.03 # 1.62 ± 0.20 # 2.65 ± 0.21 # Vlues re mens of 8 rts (±SD). Vlue is significntly different from norml control (P < 0.05). # Vlue is significntly different from crrgeenn control (P < 0.05). Vlue is significntly different from diclofenc group (P < 0.05). s result of crrgeenn injection (Figures 1 [single gent tretments] nd 2 [combintion tretments]). Rts tht received diclofenc 2 hr before injection of crrgeenn hd insignificnt decreses in the now-elevted plsm levels of CRP nd IL-6 (25.27 nd 52.35% decrese, resp.) noted in the crrgeenn-only-treted rts; interestingly, these ltter levels were incresed nd 6.50-fold, respectively, bove nïve rt vlues. Dily α-la injections prior to the crrgeenn cused significnt increse in plsm levels of CRP (5.0- nd 2.2-fold) nd IL-6 ( nd 1.98-fold) compred to the levels in nive nd crrgeenn-only-treted rts, respectively (Figure 1). Rts tht received the diclofenc + α-lacombintion lso hd significnt increses in plsm CRP (3.77- nd 1.65 fold) nd IL-6 (8.30- nd 1.28-fold) levels compred, respectively, to levels in nive nd crrgeennonly rts (Figure 2). Selenite tretments lso cused significnt increses in plsm CRP (3.75- nd 1.50-fold) nd IL-6 ( nd 1.86-fold) levels; the diclofenc + selenite combintion resulted in slightly greter significnt increses in CRP (5.00- nd 2.20-fold) nd IL-6 ( nd fold) levels. Vit C dministrtion (lone or in combintion with diclofenc) did not significntly ffect plsm levels of CRP nd IL-6 s compred to the levels in crrgeennonly rts. In contrst, Vit E given for 7 d prior to induction of inflmmtion resulted in significnt increses in plsm CRP (6.00- nd 2.60-fold) nd IL-6 ( nd 1.86-fold) levels reltive to levels in norml nd crrgeenn-only rts, respectively; the diclofenc + Vit E regimen cused slightly lower, lbeit still significnt, increses in CRP (5.5- nd 2.4- fold) nd IL-6 (9.7- nd 1.5-fold) levels. Administrtion of Zn (lone or in combintion with diclofenc) before the crrgeenn did not significntly ffect plsm CRP nd IL-6 levels reltive to those in the inflmed nondrug-treted rts. 4. Discussion Interest in the reltionship between inflmmtion nd oxidtive stress hs risen in recent yers s they shre common role in the etiology of vriety of chronic diseses. Mny of these disorders shre common pthophysiologicl link in terms of chronic low-grde inflmmtion nd overproduction of rective oxygen nd nitrogen species (ROS nd RON).

6 6 The Scientific World Journl Plsm level (pg/ml) Norml CRP IL-6 Crrg Diclo # # Diclo + α-la # # Diclo + Se Diclo + vit C # # Diclo + vit E Diclo + Zn Figure 2: Effect of combintionl tretments on plsm CRP nd IL-6 levels. All rts were provided dily orl doses of diclofenc in combintion with selenite, Vit C, Vit E, or ZnSO 4 ; α-la (50 mg/kg) ws dministered IP. Doses were the sme s outlined in Figure 1 legend. # Vlue is significntly different from norml control nd crrgeenn-only rts, respectively (P < 0.05). Vlue is significntly different from tht of diclofenc-treted hosts (P < 0.05). Bsed on this, studies were initited to investigte the efficcy of severl ntioxidnts to potentilly reduce inflmmtion nd cytokine meditors tht occur in model of cute inflmmtion (i.e., crrgeenn-induced rt pw edem) [2]. In the study here, treting the cutely inflmed rts with diclofenc cused significnt inhibition in pw thickness increses, finding previously noted using n orl dose dissimilr to tht here [12]. Another study lso showed tht diclofenc ws effective in reducing pw edem in both irrdited nd nonirrdited rts using celecoxib [13]. The ssessment here of the nti-inflmmtory efficcy of ntioxidnts α-la, Se, Vit C, Vit E, nd Zn reveled significnt inhibition in pw thickness increses to different extents, tht is, Vit E- > α-la, Zn > Se\Vit C. In ll cses, these gents (lone or in combintion with diclofenc) yielded chnges in thickness increses significntly no better thn the diclofenc lone; only in the cse of combintion with Zn the inflmmtion ws reduced better thn the outcome induced by diclofenc lone. This finding gve us puse s to the potentil significnce of use of these ntioxidnts s djunctive therpeutics. However, on their own, these gentsdidinducesignificntreductionsinpwthickness;s such, our interest in their potentil use s stnd-lone ntiinflmmtory gents remined intct. Our findings with the ntioxidnts lone were in ccordnce with the study tht investigted the nti-inflmmtory effects of α-la in crrgeenn-pw edem model [3] nd nother study, which showed tht dministrtion of Vit C reduced expected increses in hind pw inflmmtory edem in model of djuvnt-induced rthritis in rts [5]. Abou- Mohmed et l. concluded tht ZnSO4 supplementtion reduced crrgeenn-pw edem in rts s well [7]. An ssocition between oxidtive stress nd inflmmtory responses induced by crrgeenn in our study ws reflected in significnt increses in plsm levels of MDA nd decreses in both hemolyste GSH levels nd RBC SOD ctivities. Such outcomes re similr to those reported by other studies [3, 14]. Another evidence indicted tht diclofenc induced meliortion of elevted plsm MDA levelsndincresedthectivityoftotlplsmsodin rt djuvnt rthritis model[15]. However, Khyyl et l. presentedcontrstingresults;thtis,diclofencfiledto lter plsm levels of MDA or ffect GSH levels or SOD ctivity; it hs been suggested tht this contrsting outcome my be ttributed to the smll dose of diclofenc tht these investigtorsused [13]. The ntioxidnt efficcy of the studied gents in terms of reduction of elevted MDA levels nd elevtion in both hemolyste GSH nd erythrocyte SOD ctivity reitertes the findings of severl previous studies [16 18]. Nevertheless, though the djunctive therpy of the test gent with diclofenc filed to present ny significnt incresed efficcy ginst chnges in pw thickness edem mesure, the findings still proved significnt promising in regrd to ntioxidnt ctivity. In generl, plsm CRP levels correlte with severity of inflmmtory disese or tissue injury [19]. In vitro studies hve shown tht control of this response is primrily regulted by IL-6 [20]. However, Jones et l. showed tht the reltionship between IL-6 nd CRP is more complex thn previously thought, since IL-6R shedding in response to CRP likely contributes to the formtion of n gonistic sil-6r/il- 6complex[21]. Thus, CRP not only cts s n cute phse rectntbutitlsomyhveprofoundeffectondistlil-6 medited events tht occur during the inflmmtory process. Indeed, CRP levels in severl diseses hve been found to correlte with those of sil-6r [22]. The consensus tht the increse in plsm CRP nd IL- 6levelscorrelteswithseverityofinflmmtorydisesews confirmed in the current study. Another study demonstrted n increse in IL-6 levels in blood 3 hr fter injection of crrgeenn in model of crrgeenn-induced hyperlgesi. Suchoutcomesresuggestiveofpossibilitythtcirculting IL-6 could ct s messenger of informtion from peripherl inflmmtory sites to the CNS [23]. Furthermore, nother study demonstrted tht IL-6 serum levels were significntly incresedt24hrfollowingedeminduction,butnotfter 3 hr, in model of crrgeenn-induced rt pw edem [24]. However, contrdictory results indicted tht LPS-induced inflmmtory pw edem in rts, but not the type induced by crrgeenn, resulted in mesurble levels of IL-6 in serum within 3 hr of induction [25]. Orl dministrtion of diclofenc here lowered the elevted plsm levels of CRP nd IL-6, but the chnges were not significnt. Similrly, other investigtors noted tht circulting IL-6 levels remined unffected fter intrrteril or peritonel injection of diclofenc [26]. Moreover, investigting the modultory effects of diclofenc on IL-6 nd prostglndin (PG) levels showed tht diclofenc significntly decresed PGE2 production but hd no significnt effect on IL-6 levels [27]. As the nti-inflmmtory effect of diclofenc ws reflected only in the inhibition of the pw thickness increse s compred to tht in the untreted inflmedrtsbutnotinthedecresesinplsmil-6,it could be concluded tht the cytokine inhibition does not

7 The Scientific World Journl 7 completely explin the efficcy of cyclooxygense (COX) inhibitors (like diclofenc) in downregulting cute inflmmtion (i.e., suggesting nother mechnism independent on COX inhibition). This observtion ppers to confirm erlier observtions [28, 29]. To our best understnding, the study here ws the first ttempt to test the dditive nti-inflmmtory impct on the studied test gents on diclofenc through ssessment of CRP nd IL-6. Interestingly, α-la, Vit E, sodium selenite, nd their combintions with diclofenc (contrry to with diclofenc monotherpy) cused significnt increses in plsm levels of IL-6 nd CRP s compred to levels mesured in norml rts s well s in crrgeenn-only-treted rts. Such immunostimultory effects of these ntioxidnts, in prticulr, Vit E, hve been demonstrted previously [30, 31]. A plethor of evidence indictes tht CRP lso performs nti-inflmmtory functions in situ [32 37]. Moreover, it hs been documented tht IL-6 exhibits two contrsting fetures; it cts s proinflmmtory cytokine in models of chronic inflmmtory diseses, tht is, collgeninduced rthritis, murine colitis, or experimentl utoimmune encephlomyelitis [38]. In contrst, in models of cute inflmmtion, IL-6 exhibits n nti-inflmmtory profile [39]. It hs lso been reported tht IL-6 is involved in T- cell ctivtion nd represents n essentil competence fctor tht synergizes with IL-1 to control initil steps of T-cell ctivtion, including induction of IL-2 nd enhncement of responsiveness to IL-2 [40, 41]. As IL-2 production is dependent on the relese of IL-6, nd (of the gents tested here) Vit E supplementtion incresed IL-2 plsm levels [30, 31], this could explin the current interesting finding tht VitEcusedsignificntriseinplsmIL-6levelsinour inflmed hosts. In conclusion, the results of the present study (when tken in the context of the bove-noted studies) showed tht the immunostimultory effects of ntioxidnts, nmely, α-la, VitE,ndselenite,mightbereltedtoninducedreleseof IL-6 nd subsequent induction of CRP relese. The current results showed tht the dministrtion of other ntioxidnts, nmely, Vit C, Zn, nd their combintions with diclofenc, did not significntly ffect plsm IL-6 nd CRP levels. It is worth mentioning tht the dministrtion of diclofenc nd α-la, Se, nd Vit E cused significnt increses in plsm CRP nd IL-6 levels s compred to vlues seen in untreted crrgeenn-only injected rts. However, these levels were lower thn those cused by dministrtion of the ech ntioxidnt individully. In summry, the combintion of diclofenc nd ny of the nti-inflmmtory gents tested here ppers to preserve ny immunomodulting effect of the ntioxidnt lone. Thus, we conclude tht the ddition of ntioxidnts to ny tretment regimen using this prticulr drug could hve potentil beneficil effects for the ptient under tretment, lbeit tht it is not one tht builds upon the effects from the diclofenc per se. The presented findings re complementry to the conclusion drwn from the previously published systemtic review of Cnter et l. on evidenced rndomized clinicl trils (RCTs) for the effectiveness of the ntioxidnt Vitmins A, C, E, or selenium or their combintion in the tretment of rthritis. Clinicl trils testing the efficcy of Vitmin E in the tretment of rthritis hve been methodologiclly wek nd hve produced contrdictory findings. There is presently no convincing evidence tht selenium, Vitmin A, Vitmin C, or the combintion product selenium ACE re effective in the tretment of ny type of rthritis [42]. Conflict of Interests The uthors declre tht there is no conflict of interests regrding the publiction of this pper. References [1] C. 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9 The Scientific World Journl 9 [40] J. vn Snick, Interleukin-6: n overview, Annul Review of Immunology, vol. 8, pp , [41] A. Vink, C. Uyttenhove, P. Wuters, nd J. vn Snick, Accessory fctors involved in murine T cell ctivtion. Distinct roles of interleukin 6, interleukin 1 nd tumor necrosis fctor, Europen Journl of Immunology,vol.20,no.1,pp.1 6,1990. [42] P. H. Cnter, B. Wider, nd E. Ernst, The ntioxidnt vitmins A, C, E nd selenium in the tretment of rthritis: systemtic review of rndomized clinicl trils, Rheumtology,vol.46,no. 8, pp , 2007.

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