The Egyptian Rheumatologist (2016) xxx, xxx–xxx

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HO ST E D BYEgyptian Society of Rheumatic Diseases

The Egyptian Rheumatologist

www.rheumatology.eg.netwww.elsevier.com/locate/ejr

ORIGINAL ARTICLE

Therapeutic effects of spironolactone on a collagen-

induced arthritis model of rheumatoid arthritis

* Corresponding author at: Department of Pharmaceutical Sciences

and Drug Research, Punjabi University, Patiala 147002, Punjab, India.

E-mail address: pawankrishan45@gmail.com (P. Krishan).

Peer review under responsibility of Egyptian Society of Rheumatic

Diseases.

http://dx.doi.org/10.1016/j.ejr.2016.06.0021110-1164 � 2016 Egyptian Society of Rheumatic Diseases. Publishing services provided by Elsevier B.V.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Verma I et al. Therapeutic effects of spironolactone on a collagen-induced arthritis model of rheumatoid arthritis, The ERheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.06.002

Inderjeet Verma a, Ashit Syngle b, Pawan Krishan a,*

aDepartment of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, IndiabCardio Rheuma and Healing Touch City Clinic, Chandigarh and Consultant Rheumatologist Fortis Multi Specialty

Hospital, Mohali, India

Received 22 June 2016; accepted 25 June 2016

KEYWORDS

Spironolactone;

Methotrexate;

Collagen-induced arthritis;

Inflammation;

Inflammatory cytokines;

Oxidative stress

Abstract Background: Rheumatoid arthritis (RA) is an autoimmune disease characterized by

increased inflammation of synovial joint. The collagen-induced arthritis (CIA) is a widely used ani-

mal model for RA. Spironolactone possesses potent anti-inflammatory and immune modifying

properties that might make it an excellent medical intervention for rheumatic diseases.

Aim of the work: The present study was conducted to evaluate the therapeutic effect of spirono-

lactone (SPIR) in collagen-induced arthritis model in mice.

Materials and methods: DBA/1mice were divided into eight groups and CIA mice treated with

SPIR (20, 40 and 80 mg/kg/day), methotrexate (MTX) and vehicle was administered beginning

on day 21 (arthritis onset) until day 42. The effects of treatment in the mice were assessed by clinical,

oxidative markers, inflammatory cytokines; tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-

6) as well as histological changes in ankle joints.

Results: Mice immunized with collagen II with complete and incomplete Freund’s adjuvant

developed inflammatory arthritis. Spironolactone (40 and 80 mg/kg/day) was effective in bringing

significant changes on all the parameters (paw swelling, arthritis score, oxidative markers) studied.

Oral administration of SPIR significantly reduced the level of TNF-a and IL-6. The protective

effect of SPIR against RA was also evident from the ankle joint histopathology and its effect

was found comparable to that of MTX.

Conclusion: Amelioration of paw swelling, antioxidant properties, inflammatory mediators

TNF-a and IL-6 and histopathological changes indicates that SPIR can be considered with

MTX among the treatment armamentarium of arthritis. Spironolactone may be considered for

use as a novel therapeutic treatment against human arthritis.� 2016 Egyptian Society of Rheumatic Diseases. Publishing services provided by Elsevier B.V. This is an

open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Rheumatoid arthritis (RA) is an autoimmune and chronicinflammatory disorder which results in inflammation of multi-ple joints with subsequent destruction of joint cartilage and

gyptian

2 I. Verma et al.

erosive destruction of bone with evolving treatment strategies[1]. RA is a potentially devastating condition which lacks goodtreatment options. Although a number of synthetic disease-

modifying antirheumatic drugs (DMARDs) are used for theclinical treatment of RA, an effective drug with few side effectsand of low cost is still lacking [2–4]. Now a days, much atten-

tion has been focused on biologic agents as anti-tumor necrosisfactor-a (anti-TNF-a), interleukin-6 (IL-6) and IL-1 that inhi-bit the activity of proinflammatory cytokines, which are

believed to play a primary role in joint destruction [5]. BiologicDMARD therapy seems to be even more effective than syn-thetic DMARDs in RA, ankylosing spondylitis (AS) and pso-riatic arthritis (PsA) [5,6]. Despite their efficacy, biologic

DMARDs have major limitations as parenteral administra-tion, risk of infection, neutralizing antibodies and the cost[3,7]. Due to the chronic nature of disease and side effects asso-

ciated with these drugs, patients with RA rely on other optionlike use of complementary and alternative safe, economicaland acceptable oral therapeutic agents for treating arthritis

to achieve remission and to prevent collateral damage.Spironolactone was first known to possess anti-

inflammatory potential as early as 1961 [8]. However, that

observation seemed to have gone largely unnoticed until thelast few years. However, in a Danish study populationspironolactone significantly reduced the proinflammatorycytokines as well as decreased gene transcription for many reg-

ulators of inflammation and also demonstrated reduction ininterferon system in incubated human whole blood in arthritispatients [9]. In 2006, Miura et al. reported the potential anti-

inflammatory effect of spironolactone for reduction ininflammation-related cardiovascular risk factors [10]. Inanother study, the immunomodulatory and anti-

inflammatory properties of spironolactone have been shownin autism [11]. Several researchers have shown that spironolac-tone has the potential to improve endothelial dysfunction in

RA, AS and polycystic ovary syndrome [12–14] and to atten-uate alleviated oxidative stress [15,16]. This is relevant becauseoxidative stress has been well described in RA [17–19]. Despiteits widespread use in the treatment of different disorders, there

is no dearth of scientific evidence regarding its anti-inflammatory, immunomodulatory and antioxidant activityin experimental animal model of arthritis.

In view of above research, the present study was carried outto evaluate the immunomodulatory and anti-inflammatoryeffect of spironolactone on type II collagen-induced arthritis

(CIA) in DBA/1 mice. Collagen-induced arthritis model isthe most extensively studied autoimmune model of RA. It isinduced by immunization with an emulsion of complete Fre-und’s adjuvant (CFA) and type II collagen (CII) [20].

2. Materials and methods

2.1. Animals

DBA/1 mice (7–10 weeks of age) were purchased from Central

Drug Research Institute (CDRI), Lucknow, India. The ani-mals were housed in a controlled environment and providedwith standard rodent chow and distilled water. The experimen-

tal protocol was approved by Institutional Animal Ethics

Please cite this article in press as: Verma I et al. Therapeutic effects of spironolactoRheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.06.002

Committee (IAEC) and animal care was done in compliancewith Committee for the purpose of control and Supervisionof Experimental on Animals (CPCSEA) regulations on the

protection of animals used for experimental and other scien-tific purposes (Reg. No- 107/99/CPCSEA).

2.2. Reagents and drugs

Bovine type II collagen (CII), CFA and Freund’s incompleteadjuvant (FIA) were purchased from Sigma Aldrich Co., St.

Louis, MO, USA. Spironolactone (SPIR) and methotrexate(MTX) were obtained from R.P.G life Sciences, Mumbai,India and Cadila Pharmaceutical Ltd., Mumbai, India, respec-

tively. All the reagents used in the present study were of ana-lytical grade.

2.3. Collagen-induced arthritis (CIA)

Bovine type II collagen was dissolved overnight at �4 �C in0.01M acetic acid at a concentration of 2 mg/ml. For theinduction of CIA, mice were immunized with 100 lg of bovine

CII in CFA containing 4 mg/ml heat-killed mycobacteriumtuberculosis by 50 ll volume intradermal injection at the baseof the tail of DBA/1 mice on day 0. On day 21st, a booster

injection of CII in FIA was administered at the base of the tail,but proximal to the primary injection site [20].

2.4. Experimental protocol

In total, forty-eight DBA/1 mice were randomly divided intoeight groups each comprising six animals. Group I and groupII served as normal control and arthritic control/positive con-

trol. Groups III–V arthritic mice were treated orally with SPIR20, 40 and 80 mg/kg/day, respectively given for 21 days afterthe administration of booster injection of CII in FIA. Group

VI arthritic mice treated with MTX (5 mg/kg/week). GroupsVII and VIII arthritic mice were treated with carboxymethylcellulose (CMC) (SPIR groups) and phosphate buffered saline

(PBS) (MTX group) respectively and served as a vehicle con-trol. The animals of different groups were sacrificed by cervicaldecapitation. The blood samples from all groups were col-lected from the orbital vein at the end of experiment for oxida-

tive markers and inflammatory mediators. The levels ofoxidative stress markers including glutathione (GSH),superoxide-dismutase (SOD), thiobarbituric acid reactivity

(TBARS) and nitric oxide were determined.

2.5. Evaluation of arthritis

To evaluate the incidence and severity of arthritis, mice wereexamined 3 times/week. The severity of arthritis was scoredon a 4-point scale, in which 0 = normal appearance, 1 = ery-

thema and/or mild swelling, 2 = erythema and/or moderateswelling, and 3 = erythema and moderate swelling extendingfrom the ankle to metatarsal joints, and 4 = erythema and sev-ere swelling encompass the ankle, foot and digits, or ankylosis

of the limb. An arthritis score for each mice was calculated bythe sum of the four paws. The joint diameter was measured by

ne on a collagen-induced arthritis model of rheumatoid arthritis, The Egyptian

Therapeutic effects of spironolactone 3

means of micrometer vernier calipers placed on the right andleft hind paws [20].

2.6. Measurement of serum TNF-a and IL-6

Were measured in mouse serum using enzyme-linkedimmunosorbent assay (ELISA) method. TNF-a and IL-6 were

estimated by ELISA kit (Ray biotech Inc. Parkway Lane, Suite100, Norcross GA) according to the manufacturer’s instruc-tions. The lower limit of detection was 10 pg/ml. Their concen-

trations were expressed in pg/ml.

2.7. Histopathology

Mice were sacrificed at the end of experiment; the ankle jointswere removed and fixed in 10% formalin, then decalcified andembedded in paraffin. The knee joints were sliced and stainedwith hematoxylin and eosin (H and E). Histopathological

changes were analyzed under a light microscope. The inflam-matory changes were assessed in the arthritis control as wellas in the drug treated arthritic mice. Analysis of histopathol-

ogy was observed in a blind manner by 2 independentpathologists.

2.8. Statistical analysis

Results were expressed as mean ± standard error of mean(SEM). The significance of the differences of the mean values

with respect to positive control was analyzed using a one-way analysis of variance (ANOVA) followed by post hoc anal-ysis. All statistical analysis was performed using Graph padprism Version 6.0 (Graph pad prism, San Diego, CA, USA).

The p-value <0.05 was considered statistically significant(*p 6 0.05, **p 6 0.01 and ***p 6 0.001 vs. arthritis control).

3. Results

3.1. Changes in arthritis score and paw thickness

Arthritis symptoms were first observed about 3 weeks after ini-tial immunization of mice with CII (Fig. 1A). The arthritis

score was taken as the main parameter of arthritis diseasebetween the different treatment groups. Arthritis mice treatedwith SPIR significantly inhibited diseases severity compared

with the positive control group (p < 0.05). In the arthritic micegroup treated with high dose (80 mg/kg) of SPIR, there wasmarked reduction of arthritis score (p< 0.001) and when trea-ted with lower dose of SPIR (20/40 mg/kg) (Fig. 1). The arthri-

tis score of SPIR 80 group was slightly higher than that of theMTX group, suggesting that the therapeutic effect of SPIR 80on CIA was almost similar as MTX. There was no change in

arthritis score in PBS and CMC groups. At the end of exper-iment, paw thickness was significantly lower in the SPIR (40and 80 mg/kg/day) compared with positive control (Fig. 1B).

Mice treated with SPIR 20 had slightly thinner paws, butnot the significant difference compared with positive controlgroup. The inhibitory effect of SPIR 80 group (66%) was com-parable to that of MTX where 74% inhibition in paw thickness

was observed.

Please cite this article in press as: Verma I et al. Therapeutic effects of spironolactonRheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.06.002

3.2. Effect of drugs on serum cytokines (Fig. 2)

A substantial increase in the production of TNF-a and IL-6was found in mice with CIA compared with normal controlgroup on day 42. The serum level of TNF-a and IL-6 were sig-

nificantly reduced in SPIR 40 (all p < 0.05), SPIR 80 (allp 6 0.01) and MTX (all p 6 0.01) treated mice groups com-pared with positive control group. The effect of SPIR 80(36%) was slightly higher than MTX where 25% reduction

in levels of TNF-a was observed, while the effect of SPIR 80group (27%) on the reduction of IL-6 level was almost similarto MTX (31%) treated mice.

3.3. Effects of drugs on parameters of oxidative markers and

nitric oxide (Fig. 3)

Induction of arthritis in the positive control group produced amarked reduction in the levels of GSH and SOD comparedwith normal control. Treatment of arthritic mice with SPIR

40 and 80 significantly (p < 0.05) increased the levels ofGSH. Like glutathione, the SOD activity significantlyincreased in SPIR 40 (p < 0.05) and 80 (p 6 0.01) treated micegroups. TBARS level was significantly higher in positive con-

trol group compared to the normal control. Spironolactone(SPIR 40 and 80, p 6 0.01) significantly reduced levels ofTBARS. Level of NO was higher (p 6 0.01) in the positive con-

trol group than normal control. The level of NO significantlyreduced in both spironolactone (SPIR 40, p< 0.05 and 80,p 6 0.01) and MTX (p< 0.05) treated mice. In MTX treated

group, there was no statically change in the levels of GSH,SOD and TBARS.

3.4. Histological findings (Fig. 4)

Results of histopathology of the ankle joints of all experimen-tal groups are demonstrated in Fig. 4 as analyzed by patholo-gists. It was clearly indicated that the ankle joints of DBA/1

mice in the control group exhibited no pathological changes(Fig. 4A) However, staining revealed multiple pathologicalchanges in the arthritis control group (Fig. 4B), including car-

tilage degradation, bone destruction and inflammatory cellinfiltration. Treatment with MTX produced a marked reduc-tion in cartilage degradation, bone destruction and inflamma-

tory cell infiltration compared (Fig. 4C) with arthritis control.Similar attenuation of these parameters was also observed withhigh dose of spironolactone (SPIR 80) treated mice (Fig. 4F).In the SPIR 40 group, infiltration of synovial cells was present

and moderate cartilage degradation and bone destructivechange was observed (Fig. 4E). There was no significant differ-ence of synovial cells infiltration, cartilage and bony destruc-

tive changes in the SPIR 20 (Fig. 4D), PBS and CMCcontrol groups (Figure not shown).

4. Discussion

Several anti-inflammatory drugs are known to treat arthritis,joint swelling and to improve joint functions. Spironolactone

is a potassium sparing diuretic that has shown immunomodu-latory, anti-inflammatory and antioxidant properties with a

e on a collagen-induced arthritis model of rheumatoid arthritis, The Egyptian

Figure 1 (A) Arthritis scores of collagen-induced arthritis (CIA) and normal mice in different study groups. (B) Paw thickness of normal

and CIA mice after treatment. NC: normal control, PC: positive/arthritis control, MTX: methotrexate control, SPIR: spironolactone (20/

40/80 mg/kg/day). Values are given as mean ± SEM (n = 6), **p 6 0.01 and ***p 6 0.001 vs. arthritis control.

Figure 2 Reduction of tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) level in collagen-induced arthritis mice by

spironolactone and methotrexate treatment. All the values are the mean and SEM. *p < 0.05, **p 6 0.01 compared with positive control

group.

4 I. Verma et al.

potential to improve endothelial dysfunction [9,11–14]. In viewof the pleiotropy exhibited by SPIR and their anti-

inflammatory property, the present study was carried out toevaluate the effect of SPIR on the clinical, biochemical and his-tological parameters in the experimental mice model of CIA.

CIA is a well established animal model widely used for phar-macological evaluation of anti-arthritic agents, as it possessesmany immunological and pathological similarities to human

RA and therefore is widely utilized as an animal model ofRA. In the present study, DBA/1 mice were used to establishthe model.

In the present study, the arthritis score of the SPIR groupwas slightly higher than that of the MTX group, however, amarked reduction in the arthritis score was observed thanthe positive control. Treatment with arthritic mice with SPIR

40 was also significantly lower than that of the positive control

Please cite this article in press as: Verma I et al. Therapeutic effects of spironolactoRheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.06.002

group. This suggests that, similarly to MTX, SPIR possessesthe potential to treat RA. MTX is the most commonly used

as a synthetic disease modifying anti-rheumatic drug(DMARD) and has immunomodulatory and anti-inflammatory effects [21]. Although, there are several disad-

vantages, including the adverse effects and significant numbersof patients require additional treatments in order to controlthe disease process, either concurrently, or following treatment

with MTX.The serum cytokines measured in the present study were

TNF-a and IL-6. There is plenty of evidence that key cytokines

(TNF-a and IL-6) contribute to the inflammation in the patho-genesis of RA [22–25]. The results showed that, followingtreatment with MTX and SPIR (40 and 80), TNF- a and IL-6 significantly recovered as compared to model group. How-

ever, the reduction of TNF- a in SPIR treated mice is slightly

ne on a collagen-induced arthritis model of rheumatoid arthritis, The Egyptian

Figure 3 Effect of spironolactone and methotrexate on the levels of oxidative markers in collagen-induced arthritis mice treated with

SPIR 20, 40 and 80 mg/kg/day and MTX 5 mg/kg/week for 3 weeks. SPIR 80 produced significant effect on glutathione (GSH),

superoxide dismutase (SOD)), thiobarbituric acid reactivity (TBARS) and nitric oxide (NO) level. MTX does not have shown any

significant change in GSH, SOD and TBARS but significantly reduced NO level (p< 0.05). Values are given as mean ± SEM (n= 6).*p< 0.05, **p 6 0.01.

Therapeutic effects of spironolactone 5

higher than MTX treated mice. This suggests that SPIR maymodulate the progression of RA via regulation of these pro-inflammatory cytokines through inhibition of nuclear factor

jB (NF-jB). NF-jB is one of the most important regulatorsof proinflammatory cytokines i.e. TNF-a, IL-6, IL-1b andIL-8, regulates osteoclastic differentiation and participate in

the chronic inflammatory processes that often aggravate boneloss, such as RA [26,27]. The increased level of NF-jB hasbeen demonstrated in the CIA animal model and it gradually

increases during the evolution of disease [28]. NF-kB has beendemonstrated as a potential therapeutic target to controlinflammation. More recently, it has been shown that spirono-

lactone inhibits production of proinflammatory mediators viainactivation of NF-jB [29–31]. Inhibition of NF-jB is inde-pendent of the mineralocorticoid receptor (MR), not onlyMR, but also androgen receptor or progesterone receptor

[29,31].In the current study, histopathological changes revealed by

bone erosion, cartilage destruction and marked cellular infil-

tration of the CIA mice treated with MTX significantlyimproved. We also found that SPIR (80 mg/kg/day) almostcompletely prevented joint destruction, bone erosion and car-

Please cite this article in press as: Verma I et al. Therapeutic effects of spironolactonRheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.06.002

tilage degradation in CIA mice, almost unanimously corre-sponding to the MTX group suggesting that SPIR may alsoexhibit immunomodulatory and anti-inflammatory properties.

The role of antioxidant in the treatment of chronic inflam-matory arthritis has been investigated by several researchers[17,32–37]. Formation of reactive oxygen species (ROS) and

reactive nitrogen species as a result of disease activity may playan important role. Oxidative stress, decreased antioxidant sta-tus and increased NO are the hallmarks in RA patients [32],

which lead to connective tissue degradation leading to jointand periarticular deformities in RA [17]. Increased level ofROS and NO has a role in the activation of transcription fac-

tors such as NF-jB [35]. Our study, showed that SPIR (40 and80 mg/kg/day) treatment, was associated with significantimprovement in GSH and SOD and a significant reductionin levels of TBARS and NO, but in MTX treated mice group

has not shown any significant change in oxidative markersexcept NO. Our study results suggested that SPIR may haveanti inflammatory and antioxidant potential to treat RA and

are in agreement with earlier studies that reported anti-inflammatory and antioxidant effect of spironolactone in var-ious diseases [15,16,38,39].

e on a collagen-induced arthritis model of rheumatoid arthritis, The Egyptian

Figure 4 Histopathological images of the ankle joint of DBA1 mice in different experimental groups. (A) Normal control group: normal

bone, cartilage and joint tissue. (B) Arthritis control group: infiltration of inflammatory cells, erosive bone and cartilage destruction. (C)

Methotrexate treated group: no infiltration of inflammatory cells with minimal cartilage destruction and inflammation. (D)

Spironolactone 20 treated group: erosive bone and cartilage destruction showing marked infiltration of inflammatory cells in synovial

tissue. (E) Spironolactone 40 treated group: moderate erosive bone and cartilage destruction with moderate infiltration of inflammatory

cells in synovial tissue. (F) Spironolactone 80 treated group: mild infiltration of inflammatory cells in the synovial tissue. There is no bone

and cartilage destruction.

6 I. Verma et al.

5. Conclusion

The current study highlights that anti-inflammatory and

immunomodulatory effect of SPIR has extremely similar ther-apeutic effects to MTX, suggesting that SPIR may be a poten-tial alternative DMARD for the treatment of RA. However,

further pharmacological investigations at a molecular level toestablish the mechanism of the action of the drug are war-ranted. Furthermore, studies on the toxicity of SPIR arerequired to demonstrate the clinical applications of this

therapy.

Conflict of interest

All the authors responsible for this work declare no conflict ofinterest.

Acknowledgements

We thank Dr. B.N. Datta, (Former Professor, Department of

Pathology, Postgraduate Institute of Medical Education andResearch, Chandigarh, India) and Dr. Asif Baliyan (JuniorResident, Department of Pathology, Government Medical

College and Hospital, Chandigarh, India for the technical sup-port and histopathology analysis. We are grateful to Dr. JavedN Agrewala (Chief Scientist, Department of Immunology,

Institute of Microbial Technology, Chandigarh, India) forguidance and Collagen Induced arthritis Model establishment.We are also grateful to University Grant Commission, NewDelhi (Govt. of India) for providing the research fellowship

[No. F.10-15/2007 (SA-I)].

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