Research Article Amelioration of Collagen-Induced Arthritis in Female Dark Agouti Rats by Glucosamine Treatment

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1 Hindwi Publishing Corportion ISRN Phrmcology Volume 2013, Article ID , 7 pges Reserch Article Ameliortion of Collgen-Induced Arthritis in Femle Drk Agouti Rts by Glucosmine Tretment Ngrj Hlegrhr, 1 Dulnthi Tudwe, 2 Srikumr Chkrvrthi, 2 nd Ammu Kutty Rdhkrishnn 2 1 School of Veterinry nd Biomedicl Sciences, Jmes Cook University, Townsville, QLD 4811, Austrli 2 Deprtment of Pthology, Interntionl Medicl University, Kul Lumpur, Mlysi Correspondence should be ddressed to Ngrj Hlegrhr; hsngrj@gmil.com Received 7 December 2012; Accepted 8 Jnury 2013 Acdemic Editors: M. Alkondon, G. Bil, G. Gervsini, nd D. K. Miller Copyright 2013 Ngrj Hlegrhr 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. The present study ssessed the therpeutic efficcy of glucosmine hydrochloride ginst collgen-induced rthritis in femle Drk Agouti rts (DA). Arthritis ws induced by intrdermly injecting collgen nd complete Freund s djuvnt suspension t multiple sites in the rt t dose of 4 mg/kg of body weight nd therefter followed by two more boosters of the sme dose, fter the 1st week nd 2nd week of primry immuniztion. After 21 dys from the dy of primry immuniztion, the rthritic group rts were given orl supplementtion of glucosmine hydrochloride t dose of 300 mg/kg of body weight until dy 45. The rthritic group treted with glucosmine hydrochloride from dy 21 to dy 45 showed significnt reduction in rthritic histopthologicl chnges of the joints, reduction in pw thickness nd lso significnt decrese in C-rective protein nd TNF-lph in the serum. Tretment with 300 mg/kg of glucosmine hydrochloride ws ble to reverse the rthritic chnges, hence suggesting tht glucosmine hs therpeutic effect ginst collgen-induced rthritis. 1. Introduction Rheumtoidrthritis(RA)ischronicinflmmtoryutoimmune disese. It is destructive polyrthropthy tht leds to disbility nd immobility [1]. The min chrcteristic feture of RA is swelling of the joints, nd it is lso sometimes mnifested in other orgn systems in severe disese sttes. Some of the common physicl symptoms of RA re swelling of joint res, pinful nd stiff joints, ftigue, mlise, weight, loss nd sometimes even depression [2]. The exct etiopthogenesis of RA is not known. Risk fctors such s genetic predisposition nd environmentl fctors like cigrette smoking [3], microbil infections [4], nd blood trnsfusions [5] will led to the ctivtion of CD 4 + T helper cells, which will led to the stimultion of vrious cells in the joints tht produce cytokines nd inflmmtory meditors tht ply n importnt role in the development of RA. Inflmmtory meditors like mtrix metlloproteinses (MMP) nd cell dhesion molecules (CAMs), together with cytokines like tumor necrosis fctor-lph (TNF-α) nd interleukin-1 (IL-1), led to joint destruction in RA ptients [2, 6]. At present, there re few tretment options vilble for RA. Nonsteroidl nti-inflmmtory drugs (NSAIDs), cyclooxygense-2-selective NSAIDs [7], glucocorticoids, biologicl response modifying gents, nd disese modifying ntirheumtic drugs (DMARDs) re prescribed by physicins currently s single therpy or combined multiple drug therpy [8]. It hs beenshown to slow the disese progression in some cses, but itis not shown to completely reverse symptomsofrandpreventthediseseonset. Glucosmine is n mino monoscchride, which is derivtive of glucose. It is nturlly found in mucous secretions, connective tissue, skin, tendons, nd ligments, nd it is found in highest concentrtion in crtilge [9]. With ge, the production of glucosmine by the body will decrese; hence, it needs to be replenished by the use of orl glucosmine supplements. Currently glucosmine is used in the therpy for osteorthritis (OA) to synthesize glycosminoglycns (GAGs) [10]. Studies hve lso shown tht glucosmine

2 2 ISRN Phrmcology supplementtion cn repir the crtilge to some extent in OA [11]. Recent studies hve lso shown tht glucosmine hs immunosuppressive properties, which cn successfully suppress the ctivity of T-lymphocytes nd dendritic cells [12] whichreinvolvedindptiveimmuneresponses.the present study investigted the efficcy of glucosmine ginst collgen-induced rthritis in femle Drk Agouti (DA) rts. 2. Methods 2.1. Regents. Glucosmine hydrochloride (D-(+)- glucosmine hydrochloride; G4875; C 6 H 13 NO 5 HCl; mol wt: g/mol; Sigm), complete Freund s djuvnt (CFA), type II collgen, nd ll other nlyticl chemicls used for the experiment were obtined from Sigm Aldrich (St. Louis, MO, USA). The dose received by the tretment group in this study ws 300 mg/kg/rt [13] Animls. Femle Drk Agouti (DA) rts [14] of 6 10 weeks old were obtined from the Institute of Medicl Reserch, Kul Lumpur, Mlysi. The rts were kept in individul ventiltion cges throughout the study. Rts were housed in stndrd lbortory conditions with 12:12 lightdrkcyclewithfreeccesstofoodndwterthroughout thestudy.therewerethreegroupsofnimlsinthisstudy, ech group contining 10 rts. Group 1 control without ny tretment, Group 2 rthritis only group, nd Group 3 rthritis with glucosmine hydrochloride (300 mg/kg/rt). All the experimentl procedures were in ccordnce with theguidelinesforthecrenduseoflbortorynimls, nd pprovl from the Institutionl Reserch nd Ethics Committee ws tken prior to the experiments Collgen-Induced Arthritis. Type II collgen from chicken trchel crtilge obtined from Sigm Aldrich (USA) ws dissolved in 0.1 M of cold cetic cid. The dissolved collgen ws then emulsified with n equl volume of complete Freund s djuvnt (CFA) (Sigm Aldrich, USA). Ech rt ws injected intrdermlly with 4 mg/kg of collgen suspension t multiple joint res [15]ndlsotthebseof the til [16]. A booster dose injection ws given fter week following primry immuniztion, nd second booster ws dministered 15 dys fter first booster injection Tretment of Animls with Glucosmine Hydrochloride. Glucosmine hydrochloride ws dministered orlly using rodent feeding tube with dose of 300 mg/kg/rt. The tretment ws strted 21 dys fter the primry immuniztion nd continued until dy Mesurement of Body Weight nd Pw Thickness Chnges. The severity of rthritis in the nimls ws evluted by quntifying the chnges in pw thickness in the front nd hind pws. Thickness ws mesured using digitl cliper once every five dys (there ws not ny significnt chnge in the pw thickness during the 5-dy period in the erlier studies done in our lbortory) until dy 45. The body weight chnges of the rts were lso recorded once every five dys during the study period Collection of Plsm. At the end of the experimentl period, nimls were nesthetized with sodium pentobrbitl (40 mg/kg body weight) nd blood smples were collected by crdic puncture. A volume of 3-4 ml of blood ws collected in vcutiner tube which contined EDTA. These tubes were then centrifuged t 5000 rpm for 10 minutes t 4 C. The plsm ws seprted nd stored t 30 Cfor further investigtions. Rts were scrificed with n overdose of nesthesi, nd limbs were hrvested for joint histopthology C-Rective Protein nd Tumour Necrosis Fctor-Alph Assy. The C-rective protein (CRP) nd tumour necrosis fctor-lph (TNF-α) concentrtion level in the plsm were quntified using commercil kit ccording to the stndrd protocol recommended by the mnufcturer (Assy Pro, USA, nd ebioscience, USA). In the C-rective protein ssy, 25 μl of the stndrds ws dded to the relevnt wells, nd 25 μl of the diluted smples were dded in duplictes. Biotinylted rt CRP (25 μl) ws dded to ll wells, gently mixed, nd kept for incubtion for 2 h. After incubtion, the plte ws wshed using wsh buffer nd 50 μl ofdiluted streptvidin peroxidse conjugte ws dded nd incubted for30min.thepltewswshedndfilledwith50μlchromogen substrte nd left for 10 min for color development. A stop solution ws dded, nd the bsorbnce produced ws immeditelyredt450nm.thecrpconcentrtionofech ofthesmpleswsclcultedinμg/mlbsedonthestndrd curve obtined. The concentrtion of TNF-α in the plsm ws quntified using commercil rt TNF-α ELISA kit. The previous night, the plte ws coted with diluted cpture ntibody s described in the study protocol provided with this kit. Then, 100 μl of the stndrds were dded to the stndrd wells nd 100 μl of smples ws dded to the smple s wells in duplictes. The plte ws incubted t room temperture for 2h.After wshingwith buffer,100μl of the diluted detection ntibody solution ws dded to ll wells, nd the plte ws incubted for 1 h t room temperture. Afterwrds, 100 μl of the diluted vidin HRP ws dded to ll the wells nd incubted t room temperture for 30 min. Following this, 100 μl ofthesubstrtesolutionwsddedtollthewells. Finlly, 50 μlof2nh 2 SO 4 wsddedndthebsorbncews red t 450 nm Histopthologicl Anlysis. The flesh from the joints ws clered wy, nd the joint smple ws stored in 10% formlin solution for three weeks in specimen bottles. The joint smples were stored in declcifiction gent for 48 hours before processing. The declcified smples were processed using Leic TP 1020 tissue processor mchine nd embedded in prffin blocks using Leic EG 1160 tissue embedding center. The hemtoxylin nd eosin stined slides were visulized ndnlyzedusingnikoneclipse80imicroscope.the joints were evluted nd nlyzed ccording to grding system dopted from previous study [17](Tble 1).

3 ISRN Phrmcology 3 Tble 1: The histopthologicl chrcteristics observed in joint ffected by rheumtoid rthritis nd the grding system for the nlysis of joints Oedem Nil Mild Moderte Severe Cellulr Sheets of inflmmtory Nil Mild scttered infiltrtion Lymphocytes nd mcrophges Infiltrtion cells/grnuloms/mng cells Joint spce No nrrowing Mild nrrowing Moderte nrrowing with very little intermeningel spce Totl oblitertion of joint spce Synovil hyperplsi Nil Mild Moderte Extensive Fibrosis Nil Mild Moderte Extensive/severe Erosion Absent Present 2.9. Sttisticl Anlysis. Sttisticl nlysis ws crried out by one-wy nlysis of vrince (ANOVA) using the SPSS. The histology of the joints nd the plsm levels of C- rective protein nd TNF-μ were lso sttisticlly nlyzed for nonprmetric mesures using the Mnn-Whitney U test. P < 0.05 ws considered sttisticlly significnt. 3. Results 3.1. Effects on the Body Weight. In the rthritis only group, there ws significnt decrese in the body weight (P < 0.05). Arthritis rts tht hd orl supplementtion of glucosmine showed stedy increse in the body weight initilly, but there ws significnt decrese in the weight gin fter 15 dys until dy 25, but weight incresed significntly fter dy 25 in these rts. Even though there ws n increse in the body weight with glucosmine tretment, the weight chnge did not increse significntly compred to control rts (Figure 1) Effects on Gross Arthritic Symptoms. A significnt increse (P < 0.05) in the hind pw thickness ws observed in the rthritic rts from dy 1 to dy 45 when compred to control rts. Glucosmine tretment depicted significnt reduction (P < 0.05) in the pw thickness compred to rthritis lone t the 45th dy. There ws severe swelling of the joints in the hind pw in the rthritic rts. The joints showed redness nd nkylosis. The group of rts tht were strted on orl supplementtion of glucosmine showed only mild to modertesignsndsymptomsofrthritis,whencompred totherthritislonegroup.theswellingofthenklejointsin the hind pw ws significntly less in the treted group thn in the rthritis only group (Figures 2 nd 3) Effects of Glucosmine on C-Rective Protein. Asignificnt increse (P < 0.05) in C-rective protein level ws seen in rthritis only rts until 45 dys compred to control rts. The level of C-rective protein concentrtion ws significntly reduced (P < 0.05) fter orl supplementtion of glucosmine (Figure 4) Effects of Glucosmine on TNF-α. Asignificntincrese (P < 0.05) inthetnf-α level ws seen in the rthritis lone rts compred to controls. There ws significnt Body weight (g) Control Arthritis only Arthritis + glucosmine Time (dys) Figure 1: Chnges in body weight in the two experimentl groups. () P < 0.05 control with other groups; (b) P < 0.05 rthritis with rthritis + glucosmine. Pw thickness (mm) Control Arthritis Arthritis + glucosmine Dys Figure 2: Chnges in pw thickness. () P < 0.05 control with other groups; (b) P < 0.05 rthritis with rthritis + glucosmine.

4 4 ISRN Phrmcology () (b) (c) Figure3:Pwedem ()hindpwndnkleofcontrolrts;(b)rthritiscontrolshowingsevereswellingtthejointsinbothnkles;(c) hind pw nd nkle joint of rthritic rt with glucosmine CRP (μg/ml) 10 TNF-α (pg/ml) Control Arthritis only Arthritis + glucosmine Figure 4: Plsm concentrtion of C-rective protein. () P < 0.05 control with other groups; (b) P < 0.05 rthritis with rthritis + glucosmine. decrese in TNF-α levels in the plsm of the glucosmine treted group (P < 0.05) thntheuntretedrthritisonly control group. Glucosmine hs reduced the concentrtion of this inflmmtory mrker significntly in the treted group (Figure 5) Histopthologicl Chnges. To further evlute the tretment spect of glucosmine hydrochloride ginst rheumtoid rthritis, histopthologicl nlysis ws done s described in Tble 1,which is method dpted from the 0 Control Arthritis only Arthritis + glucosmine Figure 5: Plsm levels of TNF-α.()P < 0.05 control with other groups; (b) P < 0.05 rthritis with rthritis + glucosmine. previous study [17]. Arthritis only group showed severity of grde three, while the glucosmine treted rthritic group hd pthologicl chrcteristics of grde 1 0 severities (Figure 5). Synovil hyperplsi is observed in the untreted rthritis only group, which ppered to hve developed extensive oedem cusing the joint spce to nrrow. The group treted with glucosmine showed the best results, tht is, the reversl of synovil hyperplsi s in these rts nd the joint ppered to be similr to norml joint (Figure 6). There were helthy regenertive synovium nd res of fibrosis, ngiogenesis, nd fibroblsts prolifertion with ccumultion of collgen,

5 ISRN Phrmcology 5 () (A) (b) () (B) (b) () (C) (b) Figure 6: Histopthologicl nlysis of joint morphology (200x). Control synovium (A) nd joint (Ab); rthritis joint nrrowing (B) nd synovil congestion (Bb); rthritis with glucosmine joint (C) nd reduced synovil congestion (Cb). suggesting good wound heling. In the untreted rthritis rts, the joints hd extensive oedem with nrrowing of the spce. The surfce of the joint mrgins showed degenertive chnges. In the glucosmine treted rthritic groups, the synovil spce ws dequte with focl res of degenertive chnges (Figure 6). In the untreted rthritic group, the synovium showed hyperplsi nd incresed ngiogenesis nd fibrosis. The joint vsculrity showed congestion with dilted blood vessels. The group treted with glucosmine showed no synovil congestion compred to the other two groups (Figure 6). There ws n extensive inflmmtion with pnnus formtion in severl plces mong untreted rthritic rts. The pnnus ws composed of grnulomtous ccumultion of chronic inflmmtory cells like lymphocytes, plsm cells, nd mcrophges nd multinuclete gint cells seen in the untreted rthritic group. The glucosmine treted group hd mild to moderte inflmmtion (Figure 6). 4. Discussion Collge-inducedrthritis(CIA)wsinducedwhentypeII collgen ws emulsified with complete Freund s djuvnt (CFA) nd injected into the bse of the rt s til. In this niml model of rthritis study, femle Drk Agouti or DA rts, ged 6 10 weeks, re used. Complete Freund s djuvnt injection will induce n immunologicl hypersensitivity rection to collgen in the rts, leding to the development of systemic

6 6 ISRN Phrmcology effects of chronic rheumtoid rthritis [18]; therefore, the collgen-induced rthritis DA model shows most similrities to the humn rheumtoid rthritis. Drk Agouti rts re especilly susceptible to rheumtoid rthritis. These rts develop chronic nd severe form of utoimmune rthritis, which leds to permnent bone destruction nd nkylosis of ffected joints. Arthritis-induced joint destruction in DA rts is believed to be the consequence of cell medited immune responses, which mimic the pthogenesis of humn rheumtoid rthritis [19 21]. This study evluted the orl dministrtion of glucosmine on histopthologicl nd inflmmtory mrkers. The dose of glucosmine used in this study is 300 mg/kg body weight, which would give n pproximte serum level of glucosmine (0.05 mm) similr to tht in humns [13, 22]. Becuseofthesubstntilmountofgstrointestinllossin rts, higher dily dose of glucosmine is usully selected in niml models of rthritis. Glucosmine is nturlly occurring chemicl component tht is found in its highest concentrtion in joint res [23, 24]. Our study showed significnt decrese in body weight round the 25th dy in rthritis rts s compred to the norml control. Glucosmine tretment (300 mg/kg) showed significnt recovery in the body weight during the lst 10 dys of the tretment when compred to their rthritic controls. Glucosmine tretment lso suppressed the pw thickness from dy 20 to dy 45. Glucosmine cts t the joints by being incorported by the chondrocytes into the crtilge. Here it will stimulte the synthesis of physiologicl proteoglycns nd lso will decrese the ctivity of mtrix metlloproteinses, which re ctbolic enzymes tht ttck the crtilge. Glucosmine cn lso inhibit the degrdtion of crtilge, which is induced through IL-1 [21, 25]. Thus, glucosmine cts s substrte for the biosynthesis of mucopolyscchrides in bones nd joints, which will id in restoring dmged crtilge. Severl studies hve implicted TNF-α s contributor to cellulr dmge in collgen-induced rthritis. The incresed concentrtion of this cytokine in circultion cn be interpreted s progression of crtilge cell injury [26, 27]. The nti-inflmmtory ction of glucosmine might hve lowered circulting TNF-α concentrtion nd consequently reduced the joint dmge. C-rective protein is produced under conditions of inflmmtion in crtilge nd bone nd is useful biomrker in the evlution of disese progression nd response to therpeutic intervention in number of inflmmtory disorders, including RA. Higher concentrtion of C- rective protein will point towrd incresed joint chnges in rthritis [28].Inourstudy,itisshownthtglucosmine reversed inflmmtory dmge in joints when compred with untreted rthritic rts. Histopthologicl chnges in our study correlted with mcroscopic observtions, including chnges in the pw thickness. Arthritis only rts showed mximum degenertive chnges. A significnt reduction in the joint chnges ws observed in glucosmine treted groups. In the treted rts, reversl of mny of the rthritis-induced histologicl chnges ws observed. In terms of the joint nlysis grding system, the untreted rthritic rts mostly showed the grde 3 chrcteristics, which re of the most severity level, nd the glucosmine treted group showed the lest severity (grde 1-2). Observed protective effects my be due to the inhibition of inflmmtory meditors by glucosmine [12]. Glucosmine cts t the joints nd is known to efficiently suppress the ctivity of T-lymphocytes nd dendritic cells, two crucil cells involved in immune responses [12]. Tumour necrosis fctor-α (TNF-α) nd interleukin-1 (IL-1) re produced by mcrophges nd synovil lining cells nd cn be found in high concentrtions in rthritis. Both of these cytokines ct synergisticlly in the production of mtrix metlloproteinse (MMP), expression of vrious cell dhesion molecules (CAMs), nd secretion of prostglndins [6, 29]. The observed joint destruction could be becuse of the ccumultion of MMPs, nd prostglndins nd glucosmine tretment hs suppressed the productions of these inflmmtory meditors. By suppressing joint inflmmtory responses, glucosmine prevents pnnus formtion. During rthritis, the pnnus becomes fibrosed nd will hve miniml vsculriztion, nd this will result in the destruction of the crtilge. Histopthology reveled reduced pnnus nd bony nkylosis in DA rts with glucosmine tretment confirming the protective effect of glucosmine ginst joint dmge. This supports some of the other studies tht hve shown tht glucosmine supplementtion cn repir crtilge dmge to some extent in osteorthritis ptients [10, 11]. Thus, our study evluted the effect of orl dministrtion of glucosmine to Drk Agouti rts on gross, biochemicl, nd histopthologicl chnges. Glucosmine tretment not only suppressed joint swelling, it lso reduced the inflmmtory meditors like TNF-α nd C-rective proteins. There ws reduced histopthologicl chnge in the joints. These observtions support the view tht glucosmine suppresses the chronic inflmmtory phse in collgen-induced rthritis. Glucosmine exerts its nti-inflmmtory effects by suppressing the ctivtion of inflmmtory cytokines. A rpid nd significnt beneficil effect of glucosmine ws observed ginst rthritis in DA rts in this study. Thus, the findings confirmed the joint modifying nd nti-inflmmtory properties of glucosmine in collgen-induced rthritis. 5. Conclusion In conclusion, the findings from this study demonstrte tht glucosmine effectively reduces the degree of edem nd inflmmtion cused by collgen-induced rthritis in femle Drk Agouti rts. Results lso confirm glucosmine s therpeutic gent for the tretment of rheumtoid rthritis. A dose-response study of glucosmine-induced nti-inflmmtory effects nd its moleculr mechnisms re being investigted further. Conflict of Interests The uthors hve declred tht no conflict of interests exists.

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