Protective effect of rutin in attenuation of collagen-induced arthritis in Wistar rat by inhibiting...

Indian Journal of Rheumatology 2012 -

Volume -, Number -; pp. 1e8 Original Article

Protective effect of rutin in attenuation of collagen-induced arthritisin Wistar rat by inhibiting inflammation and oxidative stress

Sadiq Umara, Navin Kumar Mishrab, Kaushal Palb, Mir Sajada, Nehaa, Md. Meraj Ansaria,Sayeed Ahmadc, Chandra K. Katiyare, Haider A. Khand,*

aPh. DcAssisResea*CorreReceivCopyrihttp://d

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ABSTRACT

Background/aim: In the present study, the protective effect of rutin in ameliorating the disease process via markedlyreducing the reactive oxygen species in joint destruction was demonstrated in collagen induced arthritis (CIA) model.

Methods: Arthritis was induced in male Wistar rats by CIA method. Rutin was administered at a dose of 25 mg kg�1

body weight once daily for 21 days. The effects of treatment in the rats were assessed by biochemical and histologicalevaluation in joints.

Results: There was a steep elevation in the activity of neutrophils elastase following by the induction of disease. Thedepletion of the cellular defence in the CIA rats was significant decrease in the enzymatic as well as non-enzymaticantioxidants. The study revealed that the treatment with rutin was due to the significant changes on all the parametersstudied in CIA rats as compared to control.

Conclusions: These data confirm that erosive destruction of the joint cartilage in CIA is due to free radicals releasedby activated neutrophils. The beneficial effect of rutin may be due to its antioxidant and anti-inflammatory activity. Thisstudy indicates that the administration of rutin might have potential value in the treatment of rheumatoid arthritis.

Copyright © 2012, Indian Rheumatology Association. All rights reserved.

Keywords: Collagen induced arthritis, Rutin, Inflammation, Oxidative stress, Articular elastase

INTRODUCTION

Inflammation is a physiological response of the organism toinjuries such as trauma, infection, or immune reactions.1 Itoccurs in various diseases ranging from allergies to kidneyfailure, stroke, and rheumatoid arthritis, and many are agerelated.2 Rheumatoid arthritis (RA) is a chronic inflamma-tory and systemic disorder characterized by cellular infiltra-tion and proliferation of synovium, leading to progressivedestruction of the joints through the interaction betweeninfiltrating cells and mediators they produce.3

. Student, bM.sc student, dAssistant Professor, Clinical Toxicologytant Professor, Faculty of Pharmacy, Jamia Hamdard (Hamdard Unrch Lab., Dabur Research Centre, Ghaziabad 201010, U.P., India.sponding author. Tel.: þ91 9910940516; fax: þ91 1126059663, ema

ed: 7.7.2012; Accepted: 3.9.2012; Available online: xxxght � 2012, Indian Rheumatology Association. All rights reserved.x.doi.org/10.1016/j.injr.2012.09.001

e cite this article in press as: Umar S, et al., Protective effect of ruiting inflammation and oxidative stress, Indian Journal of Rheum

Reactive oxygen species (ROS) and reactive nitrogenspecies (RNS) may perpetuate inflammation by facilitatingthe generation of chemotactic factors at the local site. Themajor ROS and RNS generated are the superoxide anionradical ðO2

$�Þ, the hydroxyl radical (OH) and nitric oxideradical (NO).4 Oxygen radical production is comparativelyhigh in the joints of patients affected with RA.5,6 Polymor-phonuclear cells (PMNs) alter IgG by generating free radi-cals, which could in turn activate PMNs to produceadditional superoxides.7,8 NO is a free radical that servesas an essential messenger molecule in inflammatory

Laboratory, Department of Medical Elementology & Toxicology,iversity), New Delhi 110062, eHead and Vice president, Ayurvedic

il: [email protected]

tin in attenuation of collagen-induced arthritis in Wistar rat byatology (2012), http://dx.doi.org/10.1016/j.injr.2012.09.001

mailto:[email protected]

http://dx.doi.org/10.1016/j.injr.2012.09.001

2 Indian Journal of Rheumatology 2012 -; Vol. -, No. - Umar et al.

conditions.9 Pro-inflammatory cytokines induce the expres-sion of iNOS in number of cells, including fibroblasts,10

may have either a toxic or a protective effect.11e15 NOalso induces chemokines that contribute to the diseasedevelopment in arthritis. Decreased production of NO viasuppressing or inhibiting iNOS reduces arthritic symptomsand affords protection.16

Current treatment modalities for RA either producesymptomatic relief (non-steroidal anti-inflammatory drugs;NSAIDs) or modify the disease process (disease-modifyinganti-rheumatic drugs; DMARDs). Though effective, theiruse is also limited by their side effects including gastroin-testinal ulcers and perforation, cardiovascular complica-tions and emergence of opportunistic infections due toimmunosuppressant.17 Due to the long-term use, sideeffects are associated with these agents, patients witharthritis rely on other substitutes like use of complementaryand alternative medicine (CAM) and according to reportsCAM therapy is on rise as 60e90% dissatisfied patientsare likely to seek option of CAM therapy.18

Rutin is a member of bioflavonoid also called vitamin Pwith the excellent therapeutic properties, have been demon-strated in various animal models with antioxidant, anti-inflammatory, neuroprotective, antiallergenic, antiviral, andanti-carcinogenic properties.19e22 In humans, it attaches tothe iron ion (Fe2þ), preventing it from binding to hydrogenperoxide, which would otherwise create a highly-reactivefree radical that may damage cells.21 Inside the intestines ofanimals, rutin is converted to quercetin by microflora andthen it is the quercetin that is absorbed by intestinal cells.23

Recent animal and human studies support the potentialof rutin for the treatment of a variety of inflammatory disor-ders like inflammatory bowel disease, rheumatoid arthritis,and osteoarthritis. Ostrakhovitch and Afanas’ev24 reportedthat rutin inhibited oxygen radical overproduction in rheu-matoid arthritis patients in an efficient manner. Kausset al25 reported that administration of rutin inhibit thetranscription of more than 20 genes encoding criticalpro-inflammatory factors including TNF-a, IL-1, IL-8,TNF-a, MIF, and chemo attracting factors in macrophagesand reversed growth delay and severity of adjuvant inducedarthritis (AIA) development in rats. In this study we usedcollagen induced arthritis (CIA) rat model to confirm theantioxidant activity of rutin.

MATERIALS AND METHODS

Chemicals

Rutin, Freund’s adjuvant complete (CFA), N-methoxysuc-cinyl-Ala-Ala-Pro-Val p-nitroanilide and Griess Reagent

Please cite this article in press as: Umar S, et al., Protective effect of ruinhibiting inflammation and oxidative stress, Indian Journal of Rheum

system were purchased from Sigma Chemical Co. (StLouis, MO, USA). Collagen type II from bovine nasalseptum was purchased from Elastin Products Co, Inc,Owensville, MO, USA. Thiobarbituric acid (TBA), tri-chloroacetic acid (TCA), 5-50dithio-bis-2-nitrobenzoicacid(DTNB), nitrobluetetrazolium (NBT), ethylene diaminetetra-acetic acid (EDTA), xanthine, xanthine oxidase, Trishydrochloride were purchased from SD Fine chemicalsIndia. All other routine chemicals used in this investigationwere of research grade.

Animals

Male Wistar rats weighing 150e170 g were used. Theywere kept in the Central Animal House of Hamdard Univer-sity in colony cages at an ambient temperature of 25 � 2 �Cand relative humidity 45e55% with 12 h light/dark cyclesafter initial acclimatization for about 1 week. They had freeaccess to standard rodent pellet diet and water ad libitum.The experimental study was conducted in accordancewith the Institutional Animal Ethics Committee of theUniversity.

Induction of collagen-induced arthritis (CIA)

Arthritis was induced in rats as described previously.26

Collagen type II from bovine nasal septum was dissolvedin 0.05 M acetic acid at a concentration of 2 mg ml�1

was emulsified with an equal volume of Freund’s adjuvantcomplete (CFA) containing 1 mg ml�1 Mycobacteriumtuberculosis H37 RA and stored on ice before use. Ratswere immunized intradermally at about 1.5 cm distalfrom the base of the tail. All rats were randomly assignedto three groups of six animals each. The first group servedas control and saline was given orally, the second wascollagen induced arthritis (CIA), the third was administeredrutin (25 mg kg�1 body weight) daily for 21 days startingfrom day 0 followed by CIA. The dose of rutin was selectedfrom literature and in vivo studies demonstrating the antiox-idant and anti-inflammatory efficacy in other inflammatorydiseases without any resultant toxicity.21

Measurement of clinical severity of arthritis

For macroscopic assessment of arthritis, the thickness ofeach affected hind paw was measured every day withdigital calliper (YAMAYO, Japan) and the diameter wasexpressed as an average for inflamed hind paws per rats.Evaluation of joint inflammation was performed bya blinded independent observer with no knowledge of thetreatment protocol. The severity of the arthritis was


Rutin attenuates collagen-induced arthritis Original Article 3

quantified every third day by a clinical score measure-ment26 from 0 to 4.

Preparation of cell-free extract of the knee joints

At the end of experiment animals were sacrificed bycervical dislocation. Arthritic and nonarthritic joints wereremoved and cut into small pieces and homogenized in 5vol of 50 mM Tris HCl buffer, pH 7.4 containing 0.1 MNaCl and 0.1% Triton X-100 and 1 vol. of fine glasspowder by using a mortar and pestle. The crude extractthen was sonicated for 20 s. The homogenate was centri-fuged at 3000�g for 5 min, and the resulting supernatantwas stored at �80 �C until further analysis.

Articular elastase (ELA)

ELA levels in the articular joints were evaluated as an index ofpolymorphonuclear leukocyte (PMNs) accumulation and acti-vation in the inflamed tissue as described earlier.26 Briefly,tissue samples were homogenized in a solution containing20 mM potassium phosphate buffer pH 7.0 in a ratio of 1:10(w/v) and centrifuged for 20 min at 10,000�g at 4 �C. Analiquot of each sample was incubated for 24 h at 37 �C with0.1 M Tris-HCl buffer (pH 8.0), containing 0.5 M NaCl and1 mM N-methoxysuccinyl-Ala-Ala-Pro-Val p nitroanilide,a high specific synthetic substrate for neutrophil elastase(ELA). The amount of p-nitroanilide liberated was measuredspectrophotometrically at 405 nm and was considered asneutrophil ELA activity. The ELA activity was convertedand expressed as ng/g protein using molar extinction coeffi-cient (9500) of substrate as permanufacturer recommendation.

Estimation of thiobarbituric acid reactivesubstances (TBARS)

The assay of TBARS was done according to earliermethod,26 adapted to microtiter plates by bringing the finalvolume to 150 ml. In brief, tissue homogenate was preparedin 0.15 M KCl (5% w/v homogenate) and aliquots of 30 mlwere incubated for 0 �C and 37 �C at 1 h. Subsequently,60 ml of 28% w/v TCA was added and the volume wasmade up to 150 ml by adding 60 ml of distilled water fol-lowed by centrifugation at 3000�g for 10 min. The super-natant (125 ml) was taken and colour was developed byaddition of 25 ml of 1% w/v TBA dissolved in 0.05 NNaOH and kept in boiling water bath for 15 min. The absor-bance was read at 532 nm in a plate reader (Bio-Rad,U.S.A.). The result was expressed in mmoles TBARSformed/h/g tissue using a molar extinction coefficient of1.56 � 105 M�1 cm�1.


Reduced glutathione (GSH)

GSH was measured in the groups following the methoddescribed earlier.27 Homogenized joint tissue (10% w/v inphosphate buffer pH 7.4) was deproteinized by adding anequal volume of 10% TCA and was allowed to stand at4 �C for 2 h. The contents were centrifuged at 2000�gfor 15 min 50 ml supernatant was added to 200 ml of0.4 M Tris buffer (pH 8.9) containing 0.02 M EDTA (pH8.9) followed by the addition 20 ml of 0.01 M DTNB.The absorbance was read in a microplate reader at412 nm and results are expressed as mg GSH/g tissue usinga molar extinction coefficient of 13.6 � 103 M�1 cm�1.

Total superoxide dismutase (SOD) activity

Total SOD were measured in joints as described earlier27

adapted to microtiter plates by bringing the final volumeto 100 ml. Reaction mixture consisted of 0.05 M phosphatebuffer (pH 7.4), 1 mM xanthine and 57 mM NBT. Afterincubation at room temperature for 15 min, reaction wasinitiated by addition of 50 mU xanthine. The SOD activityis expressed in units/mg protein using a molar extinctioncoefficient of 4.02 � 103 M�1 cm�1.

Catalase activity

Catalase activity in the joint tissues was assayed according tomethod described earlier27 usingH2O2 as substrate. The reac-tion was adjusted to multiwell flat bottom plates by reducingthe final volume to 200 ml. Briefly reaction mixture consistedof phosphate buffer (0.01M, pH7.0), distilledwater and 10%homogenate (prepared in 0.1 M phosphate buffer). Reactionwas started by adding H2O2 (0.2 M), incubated at 37 �C for1 min and reaction was stopped by addition of dichromate:-acetic acid reagent (1:3). The tubes were kept in a boilingwater bath for 15 min and centrifuged for 10 min at1500�g .The colour developedwas read at 570nm in amicro-plate reader. The enzyme activity was expressed as mmolH2O2 consumed/min/mg protein using a molar extinctioncoefficient of 43.6 M�1 cm�1.

Measurement of nitric oxide (NO): Griessreaction

After the experiment, animals were sacrificed and the jointtissues were washed with PBS (pH 7.4) and placed on ice asthe method described earlier.28 Briefly a 50 ml sample wasadded with 100 ml of Griess reagent and reaction mixturewas incubated for about 5e10 minutes at room temperatureand protects it from light, the optical density was measured



at 540 nm in microplate reader according to the reagentmanufacturer’s protocol. Calculations were done aftergenerating a standard curve from sodium nitrite in thesame buffer as used for preparation of homogenate.

Histological examinations

Rats were sacrificed at day 21 by cervical dislocation. Kneejoints were removed and fixed in 4% formaldehyde. Afterdecalcification in 5% formic acid, the samples were pro-cessed for paraffin embedding.29 Tissue sections (5 mmthick) were stained with haematoxylineeosin for lightmicroscope examination.

Protein content

Protein was determined by Bradford method30 using bovineserum albumin (BSA) as a standard.

Statistical analysis

Results are expressed as mean � SEM. Statistical analysisof the data was done by applying the analysis of variance(ANOVA), followed by Tukey’s test for all parameters.The p-value <0.05 was considered statistically significant.

RESULTS

Effect of rutin on clinical severity and pawswelling

Arthritis developed rapidly in rats immunized with collagentype II (CII). Clinical signs of the disease were erythema of

Fig. 1 Effect of rutin (R) on time course of change in hind paw diimmunized with collagen type II. Values are means � SD for six anim


one ormore ankle joints, followedby involvement of themeta-tarsal and interphalangeal joints, first appeared in the hindpaws between 8 and 9 days after CII immunization, witha 100% incidence by day 13 � 1 (Fig. 1A). Rutin treatmentsuppressed the progression of collagen induced arthritis. Therewas no macroscopic evidence of either hind paw erythema oroedema in the control group. Our data suggested that oral rutinadministration to collagen-immunized rats reduced theprogression of arthritis by inhibiting the increase in arthritisscore (Fig. 1B) and paw swelling compared to RA rats.

Effect of rutin on cartilage elastase activity

Elastase activity assayed at the day 21st in the studiedgroups. Very low ELA concentrations were measured inthe joints of control rats. However, a significant increase(p < 0.01) in the activity of this enzyme was seen inCIA þ vehicle group. Administration of the rutin showeda significant decrease (p < 0.01) in ELA activity resultingreduction in neutrophil activation and infiltration (Fig. 2a).

Rutin treatment decreased TBARS level

The effect of rutin on TBARS level was measured todemonstrate the oxidative damage on lipid (Fig. 2b). Asignificant increase (p < 0.05) in TBARS level wasobserved in CIA þ vehicle group when compared to thecontrol group. Treatment with rutin decreased TBARS levelby inhibiting lipid peroxidation in the cartilage tissue.

Rutin restored the GSH level and SOD activity

The concentration of GSH was evaluated to estimateendogenous defences against hydrogen peroxide formation

ameter (mm) (1A) and mean clinical severity score (1B) ratsals for each group. *(p < 0.05), **(p < 0.01) vs. CIA þ vehicle.


Fig. 2 Effect of rutin (25 mg/kg b.wt.) treatment on (a) articular elastase activity (b) lipid peroxidation (c) catalase activity (d) articularnitrite content in joints of rats immunized with collagen type II. Data are expressed as mean � SEM of 6 rats. ### (p < 0.001),**(p < 0.01) vs. control, #(p < 0.05), ##(p < 0.01) vs. CIA þ vehicle.


and SOD activity to estimate endogenous defences againstsuperoxide anions. Table 1 shows the changes in GSHlevels and SOD activity evaluated in the joints (day 21)in the experimental groups. A marked decrease in GSH(p < 0.05) and SOD (p < 0.01) was found in the joint ofRA rats. Rutin significantly restored the GSH level(p < 0.05) and SOD (p < 0.01) activity significantly.

Table 1 Effect of rutin treatment on the GSH and SOD activityrats immunized with CII emulsified in CFA. Data are expressedas mean � SEM of 6 rats.

Treatment groups GSH level(mg GSH/g tissue)

SOD activity(unit/mg protein)

Catalase activity and its restoration by rutin

Fig. 2c shows catalase activity evaluated at the day 21st inthe joints. In the control group, catalase activity was16.83 � 0.013 mmol H2O2 consumed/min/mg protein. Onthe contrary, a substantial reduction in this enzyme wasobserved in the CIA þ vehicle rats (9.21 � 0.007). Alsoin this case the treatment with rutin was significantly(p < 0.01) effective as compared to CIA þ Vehicle group.

Control 0.844 � 0.054 9.16 � 0.44CIA þ vehicle 0.617 � 0.027* 5.12 � 0.076**CIA þ rutin(25 mg/kg)

0.794 � 0.052# 8.00 � 0.17##

*(p < 0.05), **(p < 0.01) vs. control, #(p < 0.05), ##(p < 0.01) vs.CIA þ vehicle.

Rutin reduced nitric oxide

Analysis of nitrite estimation is summarised in Fig. 2d. Inthe control group, the nitrite concentration was3.02 � 0.019 mmol/mg wet tissue while CIA group showed


high nitrite level (10.39 � 0.035). The treatment with rutinwas able to reduce the nitrite level significantly (p < 0.01).

Effect of rutin treatment on histology

Persistent with the biochemical alterations, the histologicalfindings (Fig. 3) revealed massive cell infiltration in theCIA þ vehicle group. Bone suffered resorption and pannusformation, whereas synovial hyperplasia was consistentfinding. The treatment with rutin ameliorated the changes


Fig. 3 Histological findings. Massive and diffuse polymorphonuclear cellular flux (white arrows) in the collagen-induced arthritic rats(B) in comparison to the control rats. (A) Cellular infiltration leads to the cartilage erosion mainly by inflammatory necrosis. (B)Reduction of cellular flux and cartilage erosion which was evidenced by the minimum necrotic lesions (black arrows) in the rats fedwith rutin. (C) Original magnification 40�.


at histological level and able to restore the changes toa greater extent.

DISCUSSION

Rheumatoid arthritis (RA) induced by collagen inducedarthritis (CIA) is a widely studied animal model of inflam-matory polyarthritis as it has similar characteristics tohuman RA in the aspects of histology and immunology.31

The purpose of the present study was to reveal the effectsand the mechanisms of rutin on CIA. It was found that rutinmarkedly inhibited joint swelling and significantlydecreased the free radical load in CIA rats, which suggestedthat rutin was effective in treating inflammatory disorderslike rheumatoid arthritis.

A number of studies have demonstrated beneficial effectsof various antioxidants and their free radical scavengingproperty in rheumatoid arthritis.32,33 To validate the anti-arthritic activity of the rutin, we evaluated articular elastaseactivity, which is a marker for joint inflammation. Its activityis directly proportional to the accumulation and activation ofpolymorphonuclear leukocytes in the inflamed tissue as itreleased from stimulated granulocytes at the site of injury.The inflammation so caused by the infiltrating cells leads tothe release of reactive oxygen and nitrogen species.34,35

This decrease in elastase activity might be due to the inhibi-tion of lipid peroxidation and the consequent decrease in thereduction of chemotactic peroxide27. During lipid peroxida-tion, a vital mechanism of the injury, lipid peroxyl radicalsare produced that can lead to cell membrane damage. Matrixdegradation arising from cytokine-stimulated chondrocyteshas been shown to be primarily due to lipid peroxidation.

Free radicals production that occurs during developmentof arthritis in the articular cartilage leads to decreased GSH


and SOD levels as a result of their consumption duringoxidative stress and cellular lysis.36 The increase in produc-tion of reactive oxygen species can be detoxified by endog-enous antioxidants, causing their cellular stores to bedepleted.37 A reduction in GSH may impair H2O2 clearanceand promote OH formation, thus increasing the free radicalload and affects the state of homeostasis. Rutin replenishedGSH and SOD level significantly probably by scavengingfree radicals and as a result helped to maintain the integrityof cellular membranes in the injured cartilage.

Nitric oxide (NO) is an important signalling molecule,produced as a part of the inflammatory response from acti-vated cells and macrophages.38 An extreme production ofNO induced by inflammatory cytokines in arthritic jointshas been associated to the initiation of apoptosis in chon-drocytes. Thus, compounds that hamper excessive NOproduction may have beneficial therapeutic effects inarthritis by blocking cartilage degradation.39 In the presentstudy, increased NO level have been detected in arthriticgroup as similar with those previously reported in synovialfluids of patients with rheumatoid arthritis.34 Treatmentwith rutin produced a significant decrease in nitric oxidelevel. The biochemical alterations were further supportedby histopathological observations of the joints. The highernumber of infiltrating cells, extensive bone degradation,and synovial hyperplasia which are hallmarks of RA wasfound in CIA plus vehicle group. Treatment with rutinwas able to reverse the histological findings towardsnormal.

From the present study, it can be concluded that rutinwhich is a potent antioxidant, reduced paw inflammationsignificantly by scavenging free radicals, which are impli-cated in initiation of cellular damage in cartilage in experi-mental animals. The major findings of the present studywere that rutin suppressed the accumulation of lipid



peroxidation, nitric oxide, boosted the antioxidant enzymessystem and eliminated the accumulation and activation ofpolymorphonuclear cell. These observations suggest thatrutin may be a clinically viable protective agent againsta variety of conditions where cellular damage is a conse-quence of oxidative stress.

CONFLICTS OF INTEREST

All authors have none to declare.

ACKNOWLEDGEMENTS

Author is grateful to “Indian Council of Medical Research”,Government of India for providing the financial support inthe form of “Senior Research Fellowship” (SRF).

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