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Atorvastatin ameliorates inflammatory hyperalgesia in rat model of monoarticular arthritis
Pharmacological Research 61 (2010) 329–333
Contents lists available at ScienceDirect
Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs
Atorvastatin ameliorates inflammatory hyperalgesia in rat model of monoarticular arthritis V.D. Wahane, V.L. Kumar ∗ Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India
a r t i c l e
i n f o
Article history: Received 26 August 2009 Received in revised form 9 November 2009 Accepted 12 November 2009 Keywords: Atorvastatin Arthritis Inflammation Hyperalgesia Anti-arthritic drugs
a b s t r a c t Statins, the cholesterol lowering drugs, have been shown to exhibit anti-inflammatory properties. The aim of the present study was to evaluate the efficacy of atorvastatin in ameliorating joint dysfunction associated with arthritis. Monoarticular arthritis was induced by the intra-articular injection of FCA (0.1 mL of 0.1%). The effect of atorvastatin (10 and 50 mg/kg, A10 and A50) following oral administration was evaluated on joint inflammation, locomotor function and hyperalgesia daily for first 4 days and every 4th day till 28 days. The effect of atorvastatin was compared with that of diclofenac (5 mg/kg, D5). Daily oral administration of atorvastatin produced a significant reduction in joint inflammation (21% in A10 and 33% in A50) and associated hyperalgesia. Atorvastatin also produced a marked improvement in the stair climbing ability and motility of the arthritic rats. The beneficial effect of atorvastatin was also evident from the histological analysis of joint carried out on day 28. Our results show that atorvastatin is more effective in decreasing the joint inflammation and hyperalgesia as compared to diclofenac while the efficacy of both the drugs in ameliorating functional disability was comparable. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Statins are well known for their cholesterol lowering effect and are clinically used not only to ameliorate conditions associated with dyslipidemia but to reduce the cardiovascular morbidity and mortality [1–3]. They are also known for their pleiotropic effects that have implications in a wide variety of clinical conditions [4,5]. They have been reported to suppress acute and chronic inflammation by inhibiting edema formation, leukocyte-endothelial adhesion, production of inflammatory cytokines and transcription factors [6–9]. Their beneficial effect in joint disorders has been attributed to their ability to suppress articular inflammation, preventing bone loss and preserving bone mineral density [10–13]. Arthritis is an inflammatory condition of the joint characterized by pain, stiffness and difficulty in joint movements. It could be induced in animals by intra-articular injection of various inflammagens including Freund’s Complete Adjuvant (FCA) [14]. The FCA induced arthritis is a well established model that has been used to evaluate the efficacy of anti-inflammatory and anti-hyperalgesic drugs. In view of the anti-inflammatory properties of statins, it is interesting to evaluate their efficacy in ameliorating inflammatory hyperalgesia. In present study the effect of atorvastatin, one of the longer acting and clinically used statins, was compared with
that of diclofenac, a standard anti-inflammatory drug. The effect of these drugs was evaluated on parameters like joint inflammation, hyperalgesia and locomotor functions. 2. Materials and methods 2.1. Animals Wistar rats of either sex weighing between 150 and 180 g used for this study were maintained at ambient temperature and had free access to food and water. The study was carried out after a period of 7–10 days of acclimatization during which rats were trained to climb a staircase with steps at 5, 10 and 15 cm following overnight fasting. The experiments were carried out as per the guidelines of Institutional Animal Ethics Committee. 2.2. Reagents and drugs FCA was purchased from Sigma–Aldrich Co., St. Louis, MO, USA. Diclofenac and atorvastatin were obtained from Biochem Pharmaceutical Industries Ltd., New Delhi and Arbro Pharmaceuticals Ltd., New Delhi, respectively. 2.3. Experimental procedure
∗ Corresponding author. Tel.: +91 11 26593681; fax: +91 11 26588663. E-mail addresses: [email protected], [email protected] (V.L. Kumar). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.11.006
Monoarticular arthritis was induced in rats by injecting 0.1 mL of 0.1% FCA into the intra-articular space of left ankle joint (day 0) [14]. Rats were divided into five groups comprising of six animals each.
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Group I and Group II served as normal control (NC) and FCA control (FC) and were given normal saline. Arthritic rats in Groups III–V were treated with drugs; Group III: diclofenac (5 mg/kg, D5); Group IV: atorvastatin (10 mg/kg, A10); Group V: atorvastatin (50 mg/kg, A50). The drugs were administered orally daily for 28 days starting two-hour before injecting FCA on day 0. The effect of drugs was evaluated on joint diameter, stair climbing ability, motility and pain produced by dorsal flexion, daily for first 4 days and thereafter every 4th day until 28 days. At the end of experiment the animals were sacrificed to study the joint histology. 2.4. Parameters measured 2.4.1. Increase in joint diameter The joint diameter was measured by means of micrometer screw gauge on day 0 before injecting FCA and then at time intervals mentioned above. The increase in joint diameter was calculated and expressed in millimeter (mm). 2.4.2. Stair climbing ability (SCA) The trained rats were allowed to climb a staircase and their climbing ability was scored 0, 1, 2, and 3 if the rats did not climb any step, climbed onto step 1, climbed onto step 2 and climbed onto step 3, respectively [15]. 2.4.3. Motility The individual rats were observed for a period of 5 min and their motility was scored 0, 1 and 2 if the rats avoided touching the feet to ground, walked with difficulty with toes touching the ground and walked easily [15]. 2.4.4. Dorsal flexion pain (DFP) The ankle joint of the rat was flexed five times until the toes touched the front of the leg with an inter test interval of 5 s. The test was performed five times and the pain was scored as 0, if there was no squeaking or leg withdrawal; 1, if there was either squeaking or leg withdrawal; 2, if both squeaking and leg withdrawal were present [16]. 2.4.5. Tissue histology Rats were sacrificed on day 28 and the limbs were removed above the stifle joints, degloved and fixed in 1% formaldehyde in saline. The limbs were decalcified in EDTA, processed for paraffin embedding, sectioned, and stained with hematoxylin–eosin. The sections were examined for arthritic changes in the control as well as in the drug-treated rats.
Fig. 1. Effect of atorvastatin on the increase in joint diameter in arthritic rats. Values given are mean ± SEM (n = 6), a p < 0.05; b p < 0.01 vs FC and c p < 0.01 vs D5.
diameter of 0.59 ± 0.07 mm on day 1 that returned to normal on day 4 (Fig. 1). Atorvastatin produced a dose-dependent decrease in joint inflammation on day 3 and the increase in joint diameter in A10 and A50 groups was 3.40 ± 0.09 and 2.90 ± 0.06 mm compared to 4.33 ± 0.08 mm in FCA control group (21% and 33% inhibition). The increase in joint diameter in D5 group on day 3 was 3.05 ± 0.08 mm (30% inhibition). From day 8 onwards the inhibitory effect of atorvastatin was more pronounced than that of diclofenac (Fig. 1). 3.2. Effect of drugs on stair climbing ability Injection of FCA into the ankle joint affected the stair climbing ability of the rats and a median score of 1 was obtained in FC group on day 3 against a median score of 3 in the NC group. A median score of 3 was obtained on day 16 in the FC group. Treatment with atorvastatin produced a dose-dependent improvement in SCA of arthritic rats and a median score of 2 was obtained in A10 group on day 3. Like diclofenac, a score of 3 was obtained in these rats on day 8. The median score of SCA of arthritic rats in A50 group was equivalent to that of rats in the NC group (Fig. 2).
2.5. Statistical analysis The values of increase in joint diameter are expressed as mean ± SEM and one-way ANOVA followed by post hoc analysis was used to compare the groups. The stair climbing ability, motility and dorsal flexion pain are expressed as median score and Kruscal–Wallis test was used to compare the groups. The statistical analysis was carried out by SPSS programme, version 11.5. A value of p < 0.05 was considered statistically significant (a p < 0.05; b p < 0.01 vs FC and c p < 0.01 vs D5). 3. Results 3.1. Effect of drugs on increase in joint diameter Intra-articular injection of FCA produced a peak increase in the diameter of ankle joint on day 3 and thereafter it gradually declined till day 28. Injection of normal saline (0.1 mL) in the intra-articular space on the other hand, produced a marginal increase in joint
Fig. 2. Effect of atorvastatin on stair climbing ability of arthritic rats. The SCA score is illustrated as box plots where bold line represents median values (n = 6) boxes represent interquartile ranges (25th and 75th percentiles), a p < 0.05; b p < 0.01 vs FC.
V.D. Wahane, V.L. Kumar / Pharmacological Research 61 (2010) 329–333
Fig. 3. Effect of atorvastatin on motility of arthritic rats. The motility score is illustrated as box plots where bold line represents median values (n = 6), boxes represent interquartile ranges (25th and 75th percentiles), a p < 0.05; b p < 0.01 vs FC.
3.3. Effect of drugs on motility Injection of FCA produced impairment in the motility of rats and on day 3 a median score of 0 was obtained. Treatment of arthritic rats with atorvastatin (A10 and A50) improved their motility (median score 1) and the effect was comparable to that of diclofenac. In A50 group a median score of 2 was obtained on day 8 while in groups A10 and D5 a motility score of 2 was obtained on day 12 against day 16 in FC group (Fig. 3).
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Fig. 4. Effect of atorvastatin on inflammatory hyperalgesia in arthritic rats. The DFP score is illustrated as box plots where bold line represents median values (n = 6), boxes represent interquartile ranges (25th and 75th percentiles) and whiskers represent extreme values, a p < 0.05; b p < 0.01 vs FC.
the score to 7.5 and 5 at doses of 10 and 50 mg/kg, respectively on day 3 and the effect of atorvastatin at 50 mg/kg dose was comparable to diclofenac. The beneficial effect of atorvastatin was more pronounced than that of diclofenac and a median score of 0 was obtained in A10 and A50 group on day 12 as compared to day 20 in diclofenac group (Fig. 4). 3.5. Histological findings
3.4. Effect of drugs on dorsal flexion pain Rats in the FCA control group exhibited a low threshold for pain and a median score of 9.5 was obtained on day 3. Treatment with atorvastatin significantly ameliorated the pain and brought down
Microscopic examination of joint tissue in FC group on day 28 revealed chronic inflammatory changes with influx of lymphocytes, monocytes and macrophages along with vascular proliferation extending from periosteum to dermis. These changes were atten-
Fig. 5. Effect of atorvastatin on joint histology in arthritic rats. A: NC; B: FC; C: D5; D: A10; E: A50; scale bar = 100 m.
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uated in A10 and D5 groups while the integrity of joint tissue was well preserved in A50 group (Fig. 5).
4. Discussion Arthritis is an inflammatory condition of the joint that is associated with joint pain, deformity and restricted mobility. In view of the anti-inflammatory properties of the statins, the present study was carried out to evaluate the efficacy of atorvastatin in ameliorating inflammatory hyperalgesia induced by FCA and to compare it with that of standard anti-inflammatory drug diclofenac. Intraarticular injection of FCA induced an inflammatory response characterized by fluid exudation, neutrophil infiltration, release of cytokines and mediators like histamine and prostaglandins (PGs) [17–20]. The joint inflammation in the arthritic rats is also associated with granuloma formation that regresses slowly possibly due to oil based adjuvant and the antigenicity of the mycobacterium [21]. In present study, atorvastatin produced a dose-dependent reduction in joint inflammation. Atorvastatin has earlier been shown to inhibit edema formation, neutrophil influx, release of histamine and inflammatory cytokines and to ameliorate pathological changes in arthritic joints [11,22–24]. Statins, other than atorvastatin, have also been shown to be effective in reducing the disease severity, levels of cytokines and chemokines and inhibiting bone destruction [10,12–13]. The beneficial effects of statins in arthritis could be attributed to their ability to inhibit HMG-CoA reductase enzyme. By inhibiting this enzyme, statins not only inhibit cholesterol synthesis but also inhibit the synthesis of isoprenoid intermediates that control various inflammatory pathways [25,26]. Statins have also been reported to decrease the levels of inflammatory markers like CRP, IL-6 and improve the endothelial dysfunction in rheumatoid arthritis patients [27,28]. The anti-inflammatory efficacy of atorvastatin was found to be comparable to that of diclofenac that acts by inhibiting both COX-1 and COX-2 thereby resulting in marked reduction in generation of PGs. The inhibition of PG synthesis has been suggested to contribute to the bone protecting effect of dicofenac in monoarticular arthritis [29]. Intra-articular injection of FCA produces inflammatory hyperalgesia that has been used as a model to evaluate the analgesic activity of the drugs [30]. As a result of hyperalgesia a state of functional disability is attained and the animal avoids weight bearing on the inflamed joint and is unable to freely move and climb [15]. Such impairment was maximum at the time of peak inflammation on day 3. Both atorvastatin and diclofenac produced improvement in motility, stair climbing ability and decreased DFP score in arthritic rats. The anti-hyperalgesic effect of atorvastatin could be attributed to its anti-bradykinin and anti-cytokine properties besides its ability to inhibit PG release [31,32]. Atorvastatin has also been reported to inhibit PG induced hypernociception in rodent model [31]. In a randomized double blind placebo controlled clinical trial where atorvastatin has been given as add on therapy to rheumatoid arthritis patients, a significant improvement in disease activity score, swollen joint count and parameters like CRP and ESR has been reported [27]. It however, did not show any significant improvement in early morning stiffness, tender joint count and visual analogue pain score. In present study the anti-hypernociceptive effect of atorvastatin was found to be more pronounced than that of diclofenac as revealed by DFP score. Diclofenac has been shown to ameriolate hyperalgesia not only through inhibition of PGs but also through central mechanism [33,34]. The present study shows that although the beneficial effects of atorvastatin and diclofenac are comparable in ameliorating functional disability in arthritis, atorvastatin may be more effective in decreasing joint inflammation and hyperalgesia. This study also suggests that atorvastatin could be used for the treatment of
arthritic patients in addition to its use in treating cardiovascular diseases and it may provide dual benefit in patients suffering from both the disease conditions. References [1] Downs JR, Clearfield M, Weis S, Whitney E, Shapiro DR, Beere PA, et al. Primary prevention of acute coronary events with lovastatin in men and women with average cholesterol levels: results of AFCAPS/TexCAPS. Air Force/Texas Coronary Atherosclerosis Prevention Study. JAMA 1998;279:1615–22. [2] Nasu K, Tsuchikane E, Katoh O, Tanaka N, Kimura M, Ehara M, et al. Effect of fluvastatin on progression of coronary atherosclerotic plaque evaluated by virtual histology intravascular ultrasound. JACC Cardiovasc Interv 2009;2:689–96. [3] Sawara Y, Takei T, Uchida K, Ogawa T, Yoshida T, Tsuchiya K, et al. Effects of lipid-lowering therapy with rosuvastatin on atherosclerotic burden in patients with chronic kidney disease. Intern Med 2008;47:1505–10. [4] Amarenco P, Benavente O, Goldstein LB, Callahan III A, Sillesen H, Hennerici MG, et al. Results of the stroke prevention by aggressive reduction in cholesterol levels (SPARCL) trial by stroke subtypes. Stroke 2009;40:1405–9. [5] Xie RQ, Cui W, Liu F, Yang C, Pei WN, Lu JC. Statin therapy shortens QTc, QTcd, and improves cardiac function in patients with chronic heart failure. Int J Cardiol 2008; in press. [6] Garjani A, Andalib S, Ziaee M, Maleki-Dizaji N. Biphasic effects of atorvastatin on inflammation. Pak J Pharm Sci 2008;21:125–30. [7] Niwa S, Totsuka T, Hayashi S. Inhibitory effect of fluvastatin, an HMG-CoA reductase inhibitor, on the expression of adhesion molecules on human monocyte cell line. Int J Immunopharmacol 1996;18:669–75. [8] Ikeda U, Shimada K. Statins and monocytes. Lancet 1999;353:2070. [9] Dichtl W, Dulak J, Frick M, Alber HF, Schwarzacher SP, Ares MP, et al. HMGCoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2003;23:58–63. [10] Leung BP, Sattar N, Crilly A, Prach M, McCarey DW, Payne H, et al. A novel anti-inflammatory role for simvastatin in inflammatory arthritis. J Immunol 2003;170:1524–30. [11] Barsante MM, Roffê E, Yokoro CM, Tafuri WL, Souza DG, Pinho V, et al. Antiinflammatory and analgesic effects of atorvastatin in a rat model of adjuvantinduced arthritis. Eur J Pharmacol 2005;516:282–9. [12] Funk JL, Chen J, Downey KJ, Clark RA. Bone protective effect of simvastatin in experimental arthritis. J Rheumatol 2008;35:1083–91. [13] Yamagata T, Kinoshita K, Nozaki Y, Sugiyama M, Ikoma S, Funauchi M. Effects of pravastatin in murine collagen-induced arthritis. Rheumatol Int 2007;27:631–9. [14] Butler SH, Godefroy F, Besson JM, Weil-Fugazza J. A limited arthritic model for chronic pain studies in the rat. Pain 1992;48:73–81. [15] De Castro Costa M, De Sutter P, Gybels J, Van Hees J. Adjuvant induced arthritis in rats: a possible animal model of chronic pain. Pain 1981;10:173–85. [16] Wang Y, Huang C, Cao Y, Han JS. Repeated administration of low dose ketamine for the treatment of monoarthritic pain in the rat. Life Sci 2000;67:261–7. [17] Keeble J, Blades M, Pitzalis C, Castro da Rocha FA, Brain SD. The role of substance P in microvascular responses in murine joint inflammation. Br J Pharmacol 2005;144:1059–66. [18] Johnston B, Burns AR, Kubes P. A role for mast cells in the development of adjuvant-induced vasculitis and arthritis. Am J Pathol 1998;152:555–63. [19] Burgess GM, Perkins MN, Rang HP, Campbell EA, Brown MC, McIntyre P, et al. Bradyzide, a potent non-peptide B(2) bradykinin receptor antagonist with long-lasting oral activity in animal models of inflammatory hyperalgesia. Br J Pharmacol 2000;129:77–86. [20] Day SM, Lockhart JC, Ferrell WR, McLean JS. Divergent roles of nitrergic and prostanoid pathways in chronic joint inflammation. Ann Rheum Dis 2004;63:1564–70. [21] Kleinau S, Erlandsson H, Klareskog L. Percutaneous exposure of adjuvant oil causes arthritis in DA rats. Clin Exp Immunol 1994;96:281–4. [22] Majlesi Y, Samorapoompichit P, Hauswirth AW, Schernthaner GH, Ghannadan M, Baghestanian M, et al. Cerivastatin and atorvastatin inhibit IL-3-dependent differentiation and IgE-mediated histamine release in human basophils and downmodulate expression of the basophil-activation antigen CD203c/E-NPP3. J Leukoc Biol 2003;73:107–17. [23] Hernández-Presa MA, Martín-Ventura JL, Ortego M, Gómez-Hernández A, ˜ Tunón J, Hernández-Vargas P, et al. Atorvastatin reduces the expression of cyclooxygenase-2 in a rabbit model of atherosclerosis and in cultured vascular smooth muscle cells. Atherosclerosis 2002;160:49–58. [24] Yokota K, Miyazaki T, Hirano M, Akiyama Y, Mimura T. Simvastatin inhibits production of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha in fibroblast-like synoviocytes from patients with rheumatoid arthritis. J Rheumatol 2006;33:463–71. [25] Maher BM, Dhonnchu TN, Burke JP, Soo A, Wood AE, Watson RW. Statins alter neutrophil migration by modulating cellular Rho activity—a potential mechanism for statins-mediated pleotropic effects? J Leukoc Biol 2009;85:186–93. [26] Zhao D, Pothoulakis C. Rho GTPases as therapeutic targets for the treatment of inflammatory diseases. Expert Opin Ther Targets 2003;7:583–92. [27] McCarey DW, McInnes IB, Madhok R, Hampson R, Scherbakov O, Ford I, et al. Trial of atorvastatin in rheumatoid arthritis (TARA): double-blind, randomised placebo-controlled trial. Lancet 2004;363:2015–21.
V.D. Wahane, V.L. Kumar / Pharmacological Research 61 (2010) 329–333 [28] Van Doornum S, McColl G, Wicks IP. Atorvastatin reduces arterial stiffness in patients with rheumatoid arthritis. Ann Rheum Dis 2004;63:1571–5. [29] Seed MP, Gardner CR. The modulation of intra-articular inflammation cartilage matrix and bone loss in mono-articular arthritis induced by heat-killed Mycobacterium tuberculosis. Inflammopharmacology 2005;12:551–67. [30] Pircio AW, Fedele CT, Bierwagen ME. A new method for the evaluation of analgesic activity using adjuvant-induced arthritis in the rat. Eur J Pharmacol 1975;31:207–15. [31] Santodomingo-Garzón T, Cunha TM, Verri Jr WA, Valério DAR, Parada CA, Poole S, et al. Atorvastatin inhibits inflammatory hypernociception. Br J Pharmacol 2006;149:14–22.
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Contents lists available at ScienceDirect
Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs
Atorvastatin ameliorates inflammatory hyperalgesia in rat model of monoarticular arthritis V.D. Wahane, V.L. Kumar ∗ Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India
a r t i c l e
i n f o
Article history: Received 26 August 2009 Received in revised form 9 November 2009 Accepted 12 November 2009 Keywords: Atorvastatin Arthritis Inflammation Hyperalgesia Anti-arthritic drugs
a b s t r a c t Statins, the cholesterol lowering drugs, have been shown to exhibit anti-inflammatory properties. The aim of the present study was to evaluate the efficacy of atorvastatin in ameliorating joint dysfunction associated with arthritis. Monoarticular arthritis was induced by the intra-articular injection of FCA (0.1 mL of 0.1%). The effect of atorvastatin (10 and 50 mg/kg, A10 and A50) following oral administration was evaluated on joint inflammation, locomotor function and hyperalgesia daily for first 4 days and every 4th day till 28 days. The effect of atorvastatin was compared with that of diclofenac (5 mg/kg, D5). Daily oral administration of atorvastatin produced a significant reduction in joint inflammation (21% in A10 and 33% in A50) and associated hyperalgesia. Atorvastatin also produced a marked improvement in the stair climbing ability and motility of the arthritic rats. The beneficial effect of atorvastatin was also evident from the histological analysis of joint carried out on day 28. Our results show that atorvastatin is more effective in decreasing the joint inflammation and hyperalgesia as compared to diclofenac while the efficacy of both the drugs in ameliorating functional disability was comparable. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Statins are well known for their cholesterol lowering effect and are clinically used not only to ameliorate conditions associated with dyslipidemia but to reduce the cardiovascular morbidity and mortality [1–3]. They are also known for their pleiotropic effects that have implications in a wide variety of clinical conditions [4,5]. They have been reported to suppress acute and chronic inflammation by inhibiting edema formation, leukocyte-endothelial adhesion, production of inflammatory cytokines and transcription factors [6–9]. Their beneficial effect in joint disorders has been attributed to their ability to suppress articular inflammation, preventing bone loss and preserving bone mineral density [10–13]. Arthritis is an inflammatory condition of the joint characterized by pain, stiffness and difficulty in joint movements. It could be induced in animals by intra-articular injection of various inflammagens including Freund’s Complete Adjuvant (FCA) [14]. The FCA induced arthritis is a well established model that has been used to evaluate the efficacy of anti-inflammatory and anti-hyperalgesic drugs. In view of the anti-inflammatory properties of statins, it is interesting to evaluate their efficacy in ameliorating inflammatory hyperalgesia. In present study the effect of atorvastatin, one of the longer acting and clinically used statins, was compared with
that of diclofenac, a standard anti-inflammatory drug. The effect of these drugs was evaluated on parameters like joint inflammation, hyperalgesia and locomotor functions. 2. Materials and methods 2.1. Animals Wistar rats of either sex weighing between 150 and 180 g used for this study were maintained at ambient temperature and had free access to food and water. The study was carried out after a period of 7–10 days of acclimatization during which rats were trained to climb a staircase with steps at 5, 10 and 15 cm following overnight fasting. The experiments were carried out as per the guidelines of Institutional Animal Ethics Committee. 2.2. Reagents and drugs FCA was purchased from Sigma–Aldrich Co., St. Louis, MO, USA. Diclofenac and atorvastatin were obtained from Biochem Pharmaceutical Industries Ltd., New Delhi and Arbro Pharmaceuticals Ltd., New Delhi, respectively. 2.3. Experimental procedure
∗ Corresponding author. Tel.: +91 11 26593681; fax: +91 11 26588663. E-mail addresses: [email protected], [email protected] (V.L. Kumar). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.11.006
Monoarticular arthritis was induced in rats by injecting 0.1 mL of 0.1% FCA into the intra-articular space of left ankle joint (day 0) [14]. Rats were divided into five groups comprising of six animals each.
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Group I and Group II served as normal control (NC) and FCA control (FC) and were given normal saline. Arthritic rats in Groups III–V were treated with drugs; Group III: diclofenac (5 mg/kg, D5); Group IV: atorvastatin (10 mg/kg, A10); Group V: atorvastatin (50 mg/kg, A50). The drugs were administered orally daily for 28 days starting two-hour before injecting FCA on day 0. The effect of drugs was evaluated on joint diameter, stair climbing ability, motility and pain produced by dorsal flexion, daily for first 4 days and thereafter every 4th day until 28 days. At the end of experiment the animals were sacrificed to study the joint histology. 2.4. Parameters measured 2.4.1. Increase in joint diameter The joint diameter was measured by means of micrometer screw gauge on day 0 before injecting FCA and then at time intervals mentioned above. The increase in joint diameter was calculated and expressed in millimeter (mm). 2.4.2. Stair climbing ability (SCA) The trained rats were allowed to climb a staircase and their climbing ability was scored 0, 1, 2, and 3 if the rats did not climb any step, climbed onto step 1, climbed onto step 2 and climbed onto step 3, respectively [15]. 2.4.3. Motility The individual rats were observed for a period of 5 min and their motility was scored 0, 1 and 2 if the rats avoided touching the feet to ground, walked with difficulty with toes touching the ground and walked easily [15]. 2.4.4. Dorsal flexion pain (DFP) The ankle joint of the rat was flexed five times until the toes touched the front of the leg with an inter test interval of 5 s. The test was performed five times and the pain was scored as 0, if there was no squeaking or leg withdrawal; 1, if there was either squeaking or leg withdrawal; 2, if both squeaking and leg withdrawal were present [16]. 2.4.5. Tissue histology Rats were sacrificed on day 28 and the limbs were removed above the stifle joints, degloved and fixed in 1% formaldehyde in saline. The limbs were decalcified in EDTA, processed for paraffin embedding, sectioned, and stained with hematoxylin–eosin. The sections were examined for arthritic changes in the control as well as in the drug-treated rats.
Fig. 1. Effect of atorvastatin on the increase in joint diameter in arthritic rats. Values given are mean ± SEM (n = 6), a p < 0.05; b p < 0.01 vs FC and c p < 0.01 vs D5.
diameter of 0.59 ± 0.07 mm on day 1 that returned to normal on day 4 (Fig. 1). Atorvastatin produced a dose-dependent decrease in joint inflammation on day 3 and the increase in joint diameter in A10 and A50 groups was 3.40 ± 0.09 and 2.90 ± 0.06 mm compared to 4.33 ± 0.08 mm in FCA control group (21% and 33% inhibition). The increase in joint diameter in D5 group on day 3 was 3.05 ± 0.08 mm (30% inhibition). From day 8 onwards the inhibitory effect of atorvastatin was more pronounced than that of diclofenac (Fig. 1). 3.2. Effect of drugs on stair climbing ability Injection of FCA into the ankle joint affected the stair climbing ability of the rats and a median score of 1 was obtained in FC group on day 3 against a median score of 3 in the NC group. A median score of 3 was obtained on day 16 in the FC group. Treatment with atorvastatin produced a dose-dependent improvement in SCA of arthritic rats and a median score of 2 was obtained in A10 group on day 3. Like diclofenac, a score of 3 was obtained in these rats on day 8. The median score of SCA of arthritic rats in A50 group was equivalent to that of rats in the NC group (Fig. 2).
2.5. Statistical analysis The values of increase in joint diameter are expressed as mean ± SEM and one-way ANOVA followed by post hoc analysis was used to compare the groups. The stair climbing ability, motility and dorsal flexion pain are expressed as median score and Kruscal–Wallis test was used to compare the groups. The statistical analysis was carried out by SPSS programme, version 11.5. A value of p < 0.05 was considered statistically significant (a p < 0.05; b p < 0.01 vs FC and c p < 0.01 vs D5). 3. Results 3.1. Effect of drugs on increase in joint diameter Intra-articular injection of FCA produced a peak increase in the diameter of ankle joint on day 3 and thereafter it gradually declined till day 28. Injection of normal saline (0.1 mL) in the intra-articular space on the other hand, produced a marginal increase in joint
Fig. 2. Effect of atorvastatin on stair climbing ability of arthritic rats. The SCA score is illustrated as box plots where bold line represents median values (n = 6) boxes represent interquartile ranges (25th and 75th percentiles), a p < 0.05; b p < 0.01 vs FC.
V.D. Wahane, V.L. Kumar / Pharmacological Research 61 (2010) 329–333
Fig. 3. Effect of atorvastatin on motility of arthritic rats. The motility score is illustrated as box plots where bold line represents median values (n = 6), boxes represent interquartile ranges (25th and 75th percentiles), a p < 0.05; b p < 0.01 vs FC.
3.3. Effect of drugs on motility Injection of FCA produced impairment in the motility of rats and on day 3 a median score of 0 was obtained. Treatment of arthritic rats with atorvastatin (A10 and A50) improved their motility (median score 1) and the effect was comparable to that of diclofenac. In A50 group a median score of 2 was obtained on day 8 while in groups A10 and D5 a motility score of 2 was obtained on day 12 against day 16 in FC group (Fig. 3).
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Fig. 4. Effect of atorvastatin on inflammatory hyperalgesia in arthritic rats. The DFP score is illustrated as box plots where bold line represents median values (n = 6), boxes represent interquartile ranges (25th and 75th percentiles) and whiskers represent extreme values, a p < 0.05; b p < 0.01 vs FC.
the score to 7.5 and 5 at doses of 10 and 50 mg/kg, respectively on day 3 and the effect of atorvastatin at 50 mg/kg dose was comparable to diclofenac. The beneficial effect of atorvastatin was more pronounced than that of diclofenac and a median score of 0 was obtained in A10 and A50 group on day 12 as compared to day 20 in diclofenac group (Fig. 4). 3.5. Histological findings
3.4. Effect of drugs on dorsal flexion pain Rats in the FCA control group exhibited a low threshold for pain and a median score of 9.5 was obtained on day 3. Treatment with atorvastatin significantly ameliorated the pain and brought down
Microscopic examination of joint tissue in FC group on day 28 revealed chronic inflammatory changes with influx of lymphocytes, monocytes and macrophages along with vascular proliferation extending from periosteum to dermis. These changes were atten-
Fig. 5. Effect of atorvastatin on joint histology in arthritic rats. A: NC; B: FC; C: D5; D: A10; E: A50; scale bar = 100 m.
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uated in A10 and D5 groups while the integrity of joint tissue was well preserved in A50 group (Fig. 5).
4. Discussion Arthritis is an inflammatory condition of the joint that is associated with joint pain, deformity and restricted mobility. In view of the anti-inflammatory properties of the statins, the present study was carried out to evaluate the efficacy of atorvastatin in ameliorating inflammatory hyperalgesia induced by FCA and to compare it with that of standard anti-inflammatory drug diclofenac. Intraarticular injection of FCA induced an inflammatory response characterized by fluid exudation, neutrophil infiltration, release of cytokines and mediators like histamine and prostaglandins (PGs) [17–20]. The joint inflammation in the arthritic rats is also associated with granuloma formation that regresses slowly possibly due to oil based adjuvant and the antigenicity of the mycobacterium [21]. In present study, atorvastatin produced a dose-dependent reduction in joint inflammation. Atorvastatin has earlier been shown to inhibit edema formation, neutrophil influx, release of histamine and inflammatory cytokines and to ameliorate pathological changes in arthritic joints [11,22–24]. Statins, other than atorvastatin, have also been shown to be effective in reducing the disease severity, levels of cytokines and chemokines and inhibiting bone destruction [10,12–13]. The beneficial effects of statins in arthritis could be attributed to their ability to inhibit HMG-CoA reductase enzyme. By inhibiting this enzyme, statins not only inhibit cholesterol synthesis but also inhibit the synthesis of isoprenoid intermediates that control various inflammatory pathways [25,26]. Statins have also been reported to decrease the levels of inflammatory markers like CRP, IL-6 and improve the endothelial dysfunction in rheumatoid arthritis patients [27,28]. The anti-inflammatory efficacy of atorvastatin was found to be comparable to that of diclofenac that acts by inhibiting both COX-1 and COX-2 thereby resulting in marked reduction in generation of PGs. The inhibition of PG synthesis has been suggested to contribute to the bone protecting effect of dicofenac in monoarticular arthritis [29]. Intra-articular injection of FCA produces inflammatory hyperalgesia that has been used as a model to evaluate the analgesic activity of the drugs [30]. As a result of hyperalgesia a state of functional disability is attained and the animal avoids weight bearing on the inflamed joint and is unable to freely move and climb [15]. Such impairment was maximum at the time of peak inflammation on day 3. Both atorvastatin and diclofenac produced improvement in motility, stair climbing ability and decreased DFP score in arthritic rats. The anti-hyperalgesic effect of atorvastatin could be attributed to its anti-bradykinin and anti-cytokine properties besides its ability to inhibit PG release [31,32]. Atorvastatin has also been reported to inhibit PG induced hypernociception in rodent model [31]. In a randomized double blind placebo controlled clinical trial where atorvastatin has been given as add on therapy to rheumatoid arthritis patients, a significant improvement in disease activity score, swollen joint count and parameters like CRP and ESR has been reported [27]. It however, did not show any significant improvement in early morning stiffness, tender joint count and visual analogue pain score. In present study the anti-hypernociceptive effect of atorvastatin was found to be more pronounced than that of diclofenac as revealed by DFP score. Diclofenac has been shown to ameriolate hyperalgesia not only through inhibition of PGs but also through central mechanism [33,34]. The present study shows that although the beneficial effects of atorvastatin and diclofenac are comparable in ameliorating functional disability in arthritis, atorvastatin may be more effective in decreasing joint inflammation and hyperalgesia. This study also suggests that atorvastatin could be used for the treatment of
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