Dual effect of nitric oxide donor on adjuvant arthritis

International Immunopharmacology 9 (2009) 439–447

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Dual effect of nitric oxide donor on adjuvant arthritis Adel A. Gomaa a,⁎, Mohsen M. Elshenawy a, Noha A. Afifi b, Eman A. Mohammed c, Romany H. Thabit a a b c

Department of Pharmacology, Faculty of Medicine, Assiut University, Assiut, Egypt Department of Microbiology and Immunity, Faculty of Medicine, Assiut University, Assiut, Egypt Department of Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt

a r t i c l e

i n f o

Article history: Received 28 October 2008 Received in revised form 4 January 2009 Accepted 15 January 2009 Keywords: Dual NO Dose Adjuvant arthritis

a b s t r a c t The effect of medical use of NO donors on the pathogenesis of arthritis is still yet unclear. We investigated the effects of the NO donor, sodium nitroprusside (SNP), on the pathogenesis of adjuvant-induced arthritis in rats. Rats were given SNP intraperitoneally either from day 5 to day 14 (as a prophylactic protocol) or from day 16 to day 25 (as a therapeutic protocol) after inoculation of adjuvant. SNP administration, whether prophylactic or therapeutic, in doses of 0.1 and 1 mg/kg/d significantly aggravated pathogenesis of adjuvant arthritis in rats. SNP-treated rats showed significant (P b 0.05) increase in arthritis index, hind paw volume, ankle joint diameter and hyperalgesia compared with control adjuvant arthritic rats. However, in adjuvant rats given the smallest dose of SNP (0.01 mg/kg/d), arthritis index, volume of hind paws, ankle joint diameter, body weight loss, and hyperalgesia were significantly lower than that of control adjuvant rats. After 30 d of the induction of adjuvant arthritis, TNF alpha levels exhibited insignificant changes either in control adjuvant rats or in rats given SNP compared with control non adjuvant rats. IL-10 levels in adjuvant control rats and adjuvant rats given 1 mg or 0.1 mg/kg/d from day 15 to day 25 were significantly lower than that of control non adjuvant rats. Histopathology examination of ankle joint showed that large doses of SNP (1 mg or 0.1 mg/kg/d) increased the mononuclear cells infiltration and erosion of cartilage induced by adjuvant while the infiltration of the inflammatory cells in the synovium of adjuvant rats treated with 0.01 mg/kg/d was minimal and the pannus was inhibited with alleviation of erosion of articular cartilage. Prophylactic small dose of SNP improved the histological status more than the therapeutic small dose. The present work reveals that SNP administration, either prophylactic or therapeutic, was deleterious in higher doses. However, the smallest dose used 0.01 mg/kg/d attenuates joint inflammation, hyperalgesia and body weight loss in adjuvant arthritic rats. These results suggest that small dose of NO donor may exert partial protective effects while the safety of the clinical use of NO donors, in higher doses, in patients with rheumatoid arthritis is questioned. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Cardiovascular diseases have become the main cause of excessive mortality of patients with rheumatoid arthritis [1]. Nitric oxide donors have been used for many years as a vasodilator and symptomatic treatment for cardiovascular diseases as angina pectoris, hypertension and congestive heart failure. Therefore, the nitrate medication may be needed for treatment of cardiovascular diseases in patients with rheumatoid arthritis. On basis of epidemiological studies, NO donor medication may accentuate bone sclerosis and contribute to disease progression if used in the presence of osteoarthritis [2]. In rheumatoid arthritis (RA), it has been demonstrated that serum and synovial fluid nitrite concentrations were significantly higher than control [3]. It has been suggested that endogenous production of NO is enhanced in proportion to the degree of inflammation in patients with RA owing to enhanced iNOS activity [4]. Also, in osteoarthritis, NO production was found to be higher [5]. It mediates many of the destructive effects of ⁎ Corresponding author. E-mail address: [email protected] (A.A. Gomaa). 1567-5769/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.01.009

interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in the cartilage and inhibitors of NO synthesis have demonstrated retardation of clinical and histological signs and symptoms in experimentally induced osteoarthritis and other forms of arthritis [6]. It appears likely that the increase in NO associated with arthritis can be caused by pro-inflammatory cytokines and mechanical stress and molecular oxygen is required for production of NO that is associated with osteoarthritis and RA [7]. Further evidence of the deleterious effects of NO comes from the study of Nagy et al. who supported the NO inhibiting therapeutic strategies for the treatment of chronic inflammatory diseases such as RA and concluded that local inhibition of NO synthesis at the site of synovial inflammation may provide better therapeutic tool than systemic inhibition [8]. Their prior study revealed that overproduction of NO may perturb T cell activation, differentiation and effector response which may contribute in different ways to the pathogenesis of autoimmune diseases [9]. Contrary to the above studies, there also exists the conflicting notion that NO may be protective during an inflammatory process. It has been shown that NO prevents apoptosis in rheumatoid synovial cells by directly inhibiting caspase-3-activation [10] and the local

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production of NO may be protective by the virtue of its ability to regulate the release of pro-inflammatory mediators [11]. In addition, nitric oxide donors were found to increase the production of hyaluronic acid by synovial cells from patients with RA [12]. Other studies found that NO does not mediate the chronic inflammation and joint destruction which occur during the latter phase and the therapeutic administration of a selective inhibitor of iNOS does not ameliorate the chronic inflammation and tissue damage associated with adjuvant arthritis in rats [13]. Furthermore, it has been shown that NO has limited modulating effects in cartilage metabolism with evidence for both protective and deleterious effects [14] and no fundamental relationship between magnitude of NO production and arthritis susceptibility and severity suggesting that NO has no effector role in arthritis [15]. Similarly, it has been found that the relationships between measures of arthritis disease activity and urinary and serum nitric oxide levels were not significant in rheumatoid patients [16]. Numerous studies have unanimously shown an association between RA and impaired endothelium function [17]. Endothelial dysfunction is defined as loss of NO bioavailability in patients with chronic inflammatory conditions [18]. Some investigators have reported that activation of iNOS may lead to endothelial dysfunction by depleting the bioavailability of tetrahydrobiopterin from endothelial nitric oxide synthase (e NOS) and subsequently uncouple eNOS resulting in production of superoxide anion rather than NO [19,20]. More recently, Maki-Petaja et al. demonstrated an endothelial dysfunction and increased iNOS activity in rheumatoid patients [21]. They suggested that inflammation is a key mediator in the process of endothelial dysfunction possibly via activation of iNOS and increased production of myeloperoxidase enzyme. Interesting studies have implicated that NO has dual effects. It has been reported that relatively low concentration of NO plays a defensive role in the immune system [22] and exerts anti-apoptotic effects via cGMP [23] while higher concentration causes numerous pathological processes including inflammation [24], vascular damage [25] and apoptosis in various cell types [26]. Additionally, Kwak et al. showed that low concentration of SNP suppresses subsequent high concentration SNP-induced apoptosis by inhibiting p38 kinase [27]. It is clear from the aforementioned reports that there is conflicting data about the effect of NO donors and the effect of systemic use of NO donors on pathogenesis of adjuvant-induced arthritis needs to be identified. Therefore, we examine the effect of SNP as a representative of nitric oxide donors on signs, symptoms, histopathology and cytokines in adjuvant-induced arthritis in rats. 2. Materials and methods 2.1. Animals The experimental study was carried out using adult female albino rats of the Sprague–Dawley strain weighing between 160 and 200 g. The animals were acclimatized in a light- and temperature-controlled room with a 12–12 h dark–light cycle. The rats were fed with commercial pelleted rat feed and water was given ad libitum. Food was placed on the floor of the cage to facilitate access, as the pain which accompanies adjuvant-induced arthritis renders the rats immobile and unable to use their hind limbs to obtain food from the cover mesh of the cage. The experimental protocol was approved by the local ethical committee.

2.3. Experimental induction of arthritis In this study, adjuvant arthritis was induced in rats according to previously described methods for the evaluation of rheumatoid arthritis. Based on preliminary experiments, the method of Trentham et al. was modified by intradermal injection of 0.1 ml squalene before inoculation of CFA into a different site in the subplanter surface of right hind paw to increase the sensitivity of rats used to CFA [28]. Rats were divided into 8 groups (6 animals each). The first group (group I) served as normal control which received only 0.1 ml kg− 1 saline. Each rat in the other 7 groups received 0.1 ml of CFA and 0.1 ml of squalene. Rats in group II received intraperitoneally 0.1 ml of distilled water, the vehicle in which SNP was dissolved (Adjuvant arthritic control group). Treatment was initiated on day 5 to day 14 in three groups III, IV and V with SNP, given intraperitoneally, in doses of 1, 0.1 and 0.01 mg/kg/d respectively (prophylactic protocol). In groups VI, VII and VIII, SNP was given i.p. as therapeutic protocol in doses of 1, 0.1 and 0.01 mg/kg/d respectively from day 16 to day 25. The day of inoculation was regarded as day 0 while day 16 was the day in which oedema in the contralateral, non-injected, hind paw was observed. Arthritis index, hind paw height, volume of paw oedema, body weight, rectal temperature and pain threshold to pressure on hind paws, were measured daily from day 0 until day 30 after adjuvant inoculation. At the end of the study, the animals were sacrificed and blood was collected. Blood samples were immediately centrifuged at 3000 rpm for 10 min and serum samples were stored at −80 °C until assayed for TNF-alpha and IL-10. Specimens of ankle joints' tissues were also examined for histopathology. 2.4. Arthritis index Rats were evaluated daily for arthritis. The physical symptoms of arthritis were judged by the following grading system [29]: 0 = normal paws; 1 = erythema of toes; 2 = erythema and swelling of paws; 3 = swelling of ankles; 4 = complete swelling of the whole leg and inability to bend it. The maximum achievable score is thus 16. Arthritis index for each rat was calculated by adding the four scores of individual paws. A sensitized animal was considered to have arthritis when at least one non-injected paw was inflamed [30]. 2.5. Measurement of body weight and temperature in arthritic rats Body weight for each rat was recorded before and daily after adjuvant inoculation to assess food intake and weight gain throughout the period of arthritis. The difference between body weight in each day and that of day 0 was calculated to determine the change in body weight in arthritic rats. Body temperature, as an index of inflammation, was monitored for rats, before and daily after disease induction between 9:00 AM and 11:00 AM, using a rectal thermometer. 2.6. Measurement of ankle diameter and paw volume changes Changes in the ankle diameter of both ipsilateral (injected) and contralateral (non-injected) hind paws, from the height on day 0, were daily assessed using a Vernier scale [31]. Volumes of hind paws were measured before and daily after adjuvant inoculation by using water displacement plethysmometry [32]. The changes of volumes of hind paws, from those of day 0, were calculated.

2.2. Reagents and drugs 2.7. Analgesimetry Complete Freund's Adjuvant (CFA) was purchased from Difco Laboratories, Detroit, Michigan, USA. Squalene was purchased from MP Biomedicals, Inc. Sodium nitroprusside (SNP) was purchased from Sigma chemical, St. Louis, USA. SNP was freely dissolved in water.

Using a Ugo basile analgesimeter (Ugo Basile Biological Research Apparatus, Italy), a crescent pressure (in grams) was applied separately to the posterior paws until the animal displayed a reaction

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that consisted of withdrawing the paw and/or vocalizing [33]. The slide of the device moved at the speed of 16 mm/s. The force on the paw was at the rate of 16 g/s, so a distance of 11.5 mm means 115 g. The pain threshold to pressure on hind paws of rats was measured. 2.8. TNF-alpha and IL-10 assays Animals were sacrificed on day 30 after disease induction and samples of blood were taken to separate sera from control, adjuvant non-treated and SNP-treated arthritic rats. Serum levels of TNF-alpha and IL-10 were determined using enzyme-linked immunosorbent assay (ELISA) kits from (Bender Medsystems). Antibodies specific for rat TNF-alpha and IL-10 were coated onto the wells of the microtiter strips and the samples including standards of known rat TNF-alpha and IL-10 were pipetted into the wells, incubated and washed. Intensity of the colour was determined at (450) nm with a correction wave length of (630) nm. 2.9. Histopathological examination Ankle joint tissues from control, arthritic and treated rats were excised and fixed in 10% buffered formalin, decalcified in 10% EDTA, embedded in paraffin, sectioned and stained with hematoxylin and eosin and then evaluated under light microscope. The evaluation parameters were mononuclear inflammation, vascular proliferation, oedema, synovial hyperplasia and vasculitis causing fibrinoid necrosis on the vessel wall in periarticular and subcutaneous adipose tissue [34]. The pathological evaluation was performed randomly by a pathologist, blind to the specimens.

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Therapeutic administration of SNP in doses of 1 and 0.1 mg/kg/d (groups VI and VII) from day 16 to day 25 after adjuvant inoculation significantly increased the arthritis index. However, SNP in dose of 0.01 mg/kg/d (group VIII) significantly reduced the arthritic scores (4.17 ± 0.17, P b 0.05) than that of adjuvant non-treated arthritic control rats (group II) on day 30 (Fig. 1). Scores of arthritis index in animals treated by 0.01 mg/kg/d SNP either prophylactically or therapeutically were significantly lower (P b 0.05) than that of animals treated by 1 or 0.1 mg/kg/d SNP on days 14, 25 and 30 (Fig. 1). 3.2. Body weight changes In the present work, body weight of non-adjuvant control rats (group I) was increased significantly (P b 0.05) over the observation period. However, body weight of animals inoculated with adjuvant control rats (group II) was markedly decreased starting from day 5 and the maximum reduction was shown on day 30. SNP (0.01, 0.1 and 1 mg/kg/d) in prophylactic protocol significantly reduced (P b 0.05) loss in body weight observed in the control adjuvant arthritic rats (group II). The losses in body weight were 8.5 ± 0.05, 7.8 ± 0.1 and 5.3 ± 0.01 g in groups III, IV and V respectively. The smallest dose of SNP (0.01 mg/kg/d) was the most effective dose in reducing the loss in body weight especially when given prophylactically. The losses of body weight in groups treated by therapeutic SNP protocol

2.10. Statistical analysis The results are presented as the mean ± standard error. Changes of arthritis index, body weight, temperature, hind paw height, clinical oedema volumes, pain threshold to paw pressure and serum levels of cytokines measured in different treatment groups were compared with adjuvant non-treated control group (group II) and non-adjuvant control group (group I) by one way ANOVA and Student-t tests for significance. Also, significance tests were calculated to determine the differences between the effects of different doses. 3. Results 3.1. Arthritis index Arthritis was successfully created in rats by the administration of CFA and squalene. After induction of arthritis in the control non-treated group (group II), the injected hind paw (right one) showed, on day 1, obvious swelling of the ankle and small joints of the foot with marked redness of the inflamed joints while the left non-injected hind paw showed swelling and redness on day 16 after adjuvant inoculation. On day 1 after adjuvant inoculation, arthritis index was 2.5 ± 0.22. Arthritis index peaked on day 18 (6.17 ± 0.17) and slightly decreased on the subsequent days until the end of experiments on day 30 (5.67 ± 0.21). Treatment with SNP in doses of 1 and 0.1 mg/kg/d (groups III and IV respectively) from day 5 to day 14 (prophylactic protocol), leads to significant increase in arthritis index. Arthritis scores were 6.17 ± 0.17 in group III and 5.8 ± 0.17 in group IV on day 30 after adjuvant inoculation. Arthritis scores in group treated with 1 mg/kg/d SNP (group III) was significantly greater (P b 0.05) than that of adjuvant non-treated control group while it was insignificantly greater (P N 0.05) in group treated with 0.1 mg/kg/d SNP (group IV) on day 30. On the contrary, arthritis index in group treated with 0.01 mg/kg/d SNP (group V), was significantly lower (3.83 ± 0.41, P b 0.05) than that of adjuvant non-treated arthritic control rats (group II) on day 30 (Fig. 1).

Fig. 1. Effect of prophylactic (A) and therapeutic (B) administration of SNP on the arthritis index in adjuvant arthritic rats. Saline-treated non-adjuvant rats; adjuvant non-treated rats; SNP (1 mg/kg/d)-treated adjuvant rats; NP (0.1 mg/ kg/d)-treated adjuvant rats; S NP (0.01 mg/kg/d)-treated adjuvant rats. *P b 0.05 for SNP-treated AIA rats vs untreated AIA rats; †P b 0.05 for group V vs group III; ††P b 0.05 for group VIII vs group VI.

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(groups VI, VII and VIIII) were significantly lower (P b 0.05) than that in adjuvant non-treated arthritic rats (group II) on days 25 and 30 after adjuvant inoculation (Fig. 2). The losses of animal body weight in group V (5.3 ± 0.01 g) and group VIII (6.7 ± 0.1 g) treated with the smallest dose of SNP (0.01 mg/ kg/d) were significantly lower (P b 0.05) than that of animals treated with 0.1 or 1 mg/kg/d of SNP either as therapeutic or prophylactic protocol (Fig. 2). 3.3. Temperature changes Inoculation of CFA in control rats showed large, transient febrile response during local, acute inflammation, with a peak on day 1 (38.7 ± 0.1 °C) followed by a return to the control level on day 3. A second bout of fever, peaked on day 16 (38.1 ± 0.1 °C), was observed. Then the body temperature fell toward normal levels until the end of the experiments on day 30 (37.5 ± 0.1 °C). Treatment of adjuvant arthritic rats with SNP from day 5 to day 15 did not abolish the second peak of fever on day 16 after disease induction. The differences in body temperature in different treatment groups compared with group II were nonsignificant (Fig. 3). 3.4. Changes of ankle diameter Swelling and erythema with increase in diameter of ankle joint of the injected right hind paw was evident on day 3 in control non-

Fig. 3. Effect of prophylactic (A) and therapeutic (B) administration of SNP on the body temperature in adjuvant arthritic rats. Saline-treated non adjuvant rats; adjuvant non-treated rats; SNP (1 mg/kg/d)-treated adjuvant rats; SNP (0.1 mg/ kg/d)-treated adjuvant rats; SNP (0.01 mg/kg/d)-treated adjuvant rats. *P b 0.05 for SNP-treated AIA rats vs untreated AIA rats; †P b 0.05 for group V vs group III; ††P b 0.05 for group VIII vs group VI.

Fig. 2. Effect of prophylactic (A) and therapeutic (B) administration of SNP on the change of body weight (g) in adjuvant arthritic rats. Saline-treated non-adjuvant rats; adjuvant non-treated rats; SNP (1 mg/kg/d)-treated adjuvant rats; SNP (0.1 mg/kg/d)-treated adjuvant rats; SNP (0.01 mg/kg/d)-treated adjuvant rats. *P b 0.05 for SNP-treated AIA rats vs untreated AIA rats; †P b 0.05 for group V vs group III; ††P b 0.05 for group VIII vs group VI.

treated adjuvant rats (group II). The change in diameter of the ankle joint significantly increased on day 5 then slightly decreased until day 16. The change of ankle diameter was peaked again on day 20 (0.33 ± 0.01 mm) with no change on the subsequent days until the end of experiments on day 30 (0.32 ± 0.01 mm). Swelling of the ankle joint of the contralateral non-injected left hind paw was observed on day 16. The change in left ankle diameter was significantly increased on day 20 (0.44 ± 0.01 mm) then decreased on the subsequent days until day 30, it was 0.34 ± 0.01 mm (Fig. 4). Treatment with SNP in doses of 1 and 0.1 mg/kg/d either in prophylactic (groups III and IV) or therapeutic protocol (groups VI and VII) significantly increased (P b 0.05) the diameter of the right ankle joint after day 5 compared with that of adjuvant non-treated control animals while the changes of diameter of right ankle of adjuvant rats treated with 0.01 mg/kg/d were insignificantly lower (P N 0.05) in prophylactic protocol (group V) and significantly lower (P b 0.05) in therapeutic protocol (group VIII) compared with that of adjuvant nontreated control animals (Fig. 4). Diameters of left ankle joints of adjuvant rats treated with 1 or 0.1 mg/kg/d either in prophylactic or therapeutic protocol showed significant increase only after day 16 compared with control adjuvant rats. However, in groups treated with the smallest dose (0.01 mg/kg/d). The changes of diameter of left ankle were insignificantly lower (P N 0.05) in prophylactic protocol (group V) and significantly lower (P b 0.05) in therapeutic protocol (group VIII) compared with that of adjuvant non-treated control animals (Fig. 4).

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control non-treated adjuvant rats. No significant differences (P N 0.05) were observed in left hind paw volumes between the groups injected with 0.01 mg/kg/d SNP in prophylactic or therapeutic protocol and control adjuvant group. 3.6. Analgesimetry The results are presented in Fig. 6. Adjuvant inoculation into control rats (group II) was accompanied by hyperalgesia as evidenced by lowering of the pain threshold to paw pressure. The animals presented a reduction of pain threshold until the end of experiments on day 30. The threshold to withdraw the right hind paw, in grams, in the analgesimeter was 71 ± 0.8 g on day 0 and it significantly decreased (P b 0.05) throughout the period of the study. On day 30 after adjuvant inoculation, it was 32 ± 0.1 g. The threshold to withdraw the left hind paw was 73 ± 1.03 g on day 0 and also significantly decreased (P b 0.05) throughout the period of the study. On day 30 after adjuvant inoculation, it was 28 ± 0.1 g. There was a marked mechanical hyperalgesia in the right hind paws following SNP treatment, either prophylactic or therapeutic, with higher doses 1 and 0.1 mg/kg/d (groups III, IV, VI and VII), as shown by a significant reduction (P b 0.05) in paw pressure withdrawal thresholds in groups III, VI and VII and insignificant reduction (P N 0.05) in group IV compared to control adjuvant non-treated animals. On day 30, the thresholds to withdraw right hind paws were 25 ± 0.1, 29 ± 0.1, 15 ± 0.1 and 16 ± 0.1 in groups III, IV, VI and VII

Fig. 4. Effect of prophylactic (A) and therapeutic (B) administration of SNP on the change of right and left ankle diameters (mm) in adjuvant arthritic rats; , change of ankle diameters in saline-treated non-adjuvant rats; , change of ankle diameters in adjuvant non-treated rats; , change of ankle diameters in SNP (1 mg/kg/d)-treated adjuvant rats; , change of ankle diameters in SNP (0.1 mg/ kg/d)-treated adjuvant rats; , change of ankle diameters in SNP (0.01 mg/kg/d)treated adjuvant rats. *P b 0.05 for SNP-treated AIA rats vs untreated AIA rats; †P b 0.05 for group V vs group III; ††P b 0.05 for group VIII vs group VI.

3.5. Changes of paw volume Starting from day 1 after inoculation of CFA, volumes of the right hind paw of non-treated control rats (group II) exhibited significant increases (P b 0.05) compared with that of non-adjuvant control animals (group I). The change in volume of the right paw was 1.38 ± 0.15 ml on day 5 and increased on day 30 to 1.57 ± 0.1 ml. On the other hand, little change was noticed in the volume of left non-injected hind paw before day 16. The increase in volume of the left hind paw was peaked on day 20; it was 0.3 ± 0.03 ml then slightly decreased on the subsequent days to become 0.26 ± 0.04 ml on day 30 after adjuvant inoculation (Fig. 5). SNP administered prophylactically (groups III and IV) or therapeutically (groups VI and VIII) in dose of 0.1 and 1 mg/kg/d markedly increase the swelling of right hind paw after day 5 compared with control non-treated adjuvant rats. The increase in right hind paw volume was 1.8 ± 0.04, 1.76 ± 0.07, 1.9 ± 0.01 and 1.74 ± 0.01 ml in groups III, IV, VI and VII on day 30 respectively. Controversially, SNP in dose of 0.01 mg/kg/d administered either prophylactically (group V) or therapeutically (group VIII) significantly reduced the right hind paw volumes (1.07 ± 0.01 ml and 1.02 ± 0.03 ml respectively) compared with control non-treated adjuvant rats (group II). Fig. 5 showed that the increases in volumes of the contralateral non-injected hind paws were significantly higher (P b 0.05) in groups administered 1 or 0.1 mg/ kg/d SNP either prophylactically or therapeutically compared with

Fig. 5. Effect of prophylactic (A) and therapeutic (B) administration of SNP on the change of hind paw volumes (ml) in adjuvant arthritic rats. , change of hind paw volumes in saline-treated non-adjuvant rats; , change of hind paws volumes in adjuvant non-treated rats; , change of hind paw volumes in SNP (1 mg/kg/d)treated adjuvant rats; , change of hind paw volumes in SNP (0.1 mg/kg/d)treated adjuvant rats; , change of hind paw volumes in SNP (0.01 mg/kg/d)treated adjuvant rats. *P b 0.05 for SNP-treated AIA rats vs untreated AIA rats; †P b 0.05 for group V vs group III; ††P b 0.05 for group VIII vs group VI.

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and 28.6 ± 1.5 pg in groups V and VIII respectively. Prophylactic treatment with 0.01 mg/kg/d SNP (group V) insignificantly increased IL-10 levels (249.8 ± 52.5, P N 0.05) compared with those in salinetreated adjuvant rats (group II). However, there was significant differences between the IL-10 levels in adjuvant rats treated therapeutically with 1 mg/kg/d (147.5 ± 24.1 pg) and 0.01 mg/kg/d (192.4 ± 43.2 pg). 3.8. Histopathological examination

Fig. 6. Effect of prophylactic (A) and therapeutic (B) administration of SNP on pain threshold to pressure on hind paws in adjuvant arthritic rats. , pressure in saline-treated non-adjuvant rats; , pressure in adjuvant non-treated rats; , pressure in SNP (1 mg/kg/d)-treated adjuvant rats; , pressure in SNP (0.1 mg/kg/d)-treated adjuvant rats; , pressure in SNP (0.01 mg/kg/d)-treated adjuvant rats. *P b 0.05 for SNP-treated AIA rats vs untreated AIA rats; †P b 0.05 for group V vs group III; ††P b 0.05 for group VIII vs group VI.

respectively. Hyperalgesia was significantly noticed (P b 0.05) in the left non-injected hind paws in group VI but was insignificant (P N 0.05) in groups III, IV and VII compared with control adjuvant rats (group II). The thresholds to withdraw left hind paws were 24 ± 0.1, 26 ± 0.1, 19 ± 0.1 and 28 ± 0.1 in groups III, IV, VI and VII respectively. Contrarily, hyperalgesia in both right and left hind paws of rats treated with SNP in a dose of 0.01 mg/kg/d, either prophylactic or therapeutic (groups V and VIII), was significantly lower (P b 0.05) compared to adjuvant non-treated arthritic rats (group II). On day 30, the thresholds to withdraw right and left hind paws were 42 ± 0.3and 45 ± 0.2 in group V; 45 ± 0.1 and 41 ± 0.1 in group VIII respectively. 3.7. Effect of SNP on serum TNF-alpha and IL-10 in adjuvant arthritic rats As shown in Table 1, serum TNF-alpha level was insignificantly and slightly greater in adjuvant arthritic rats treated with 1 mg/kg/d SNP than that of control adjuvant animals on day 30 after adjuvant inoculation. However there were no significant differences between the serum levels of TNF-alpha in the different groups of the experiment. Serum IL-10 level was significantly low (P b 0.05) in adjuvant non-treated control rats, (171. ± 34 pg) and therapeutic SNPtreated groups (group VI, VII, VIII) compared with non-adjuvant control rats (345.6 ± 64.4 pg). There was no significant change (P N 0.05) in serum levels of TNFalpha in rats given SNP in a dose of 0.01 mg/kg/d either prophylactically or therapeutically (groups V and VIII) compared with that in saline-treated adjuvant rats (group II). TNF-alpha levels were 31.2 ± 3.7

The histopathological examination of the left ankle joint revealed normal joint space, synovial lining, articular cartilage and subchondral bone in saline-treated non-adjuvant arthritic rats (Fig. 7A). On the contrary, synovium of adjuvant-arthritic control rats was oedematous and thickened with a dense perivascular inflammatory infiltrate composed of lymphocytes, plasma cells and macrophages filled the synovial stroma. The vascularity was increased and the inflamed and hyperemic synovium crept over the articular cartilage forming a pannus and causes erosion of the underlying cartilage (Fig. 7B). In adjuvant arthritic rats treated with higher dose of SNP (1 mg/kg/d) as prophylactic treatment, the examination showed severe destruction of the cartilage and subchondral bone with complete filling of the joint space with pannus producing fibrous ankylosis of the joint (Fig. 7C). Therapeutic treatment of adjuvant rats with 1 mg/kg/d induced much fibrovascular thickening of the synovium and excessive increase of the inflammatory cellular infiltrate, with scattered giant cells. The small blood vessels are obstructed by endarteritis obliterans. Fibrocellular pannus was observed eroding the articular cartilage causing its destruction with penetration into the subchondral bone forming juxta-articular erosions and subchondral cysts (Fig. 7D). The infiltration of the inflammatory cells in the synovium of adjuvant arthritic rats treated with the small dose of (0.01 mg/kg/d) SNP from day 5 to day 14 was minimal. The pannus was partly inhibited with alleviation of the destruction of articular cartilage compared with the adjuvant non-treated arthritic rats (Fig. 7E). Similarly, proliferation and infiltration of inflammatory cells in the synovium of adjuvant arthritic rats treated with 0.01 mg/kg/d SNP from day 16 to day 25 were inhibited and the erosion of the articular cartilage was alleviated (Fig. 7F). 4. Discussion Adjuvant arthritis in rat is an experimental model that shares many features with human RA, such as swelling, cartilage degradation, and loss of joint function. It has been used for many years for evaluation of anti-arthritic/anti-inflammatory agents [35]. In this model, rats develop a chronic swelling in multiple joints, with influx of inflammatory cells, erosion of joint cartilage and bone destruction after inoculation of Complete Freund's Adjuvant (CFA). In this study, Table 1 Effect of i.p. administration of SNP on serum levels of TNF-α and IL-10 in adjuvant arthritic rats Group Drug treatment

Serum levels (picogram) TNF-α

I II III IV V VI VII VIII

IL-10

Saline-treated (non-adjuvant) 30.7 ± 2.3 345.6 ± 64.4 Adjuvant arthritic (non-treated) 31.04 ± 1.4 171.6 ± 34* SNP-treated [1 mg/kg/d] (prophylactic protocol) 33.4 ± 6.8 248.3 ± 71.1 SNP-treated [0.1 mg/kg/d](prophylactic protocol) 32.6 ± 2.4 225 ± 58.6 SNP-treated [0.01 mg/kg/d] (prophylactic protocol) 31.2 ± 3.7 249.4 ± 52.5 SNP-treated [1 mg/kg/d] (therapeutic protocol) 33.7 ± 3.5 147.5 ± 24.1*† SNP-treated [0.1 mg/kg/d] (therapeutic protocol) 31.8 ± 5.6 159.6 ± 38.8* SNP-treated[0.01 mg/kg/d] (therapeutic protocol) 28.6 ± 1.5 192.4 ± 43.2*

Samples were taken from rats on day 30 after adjuvant inoculation. (*) P b 0.05 for SNP-treated rats vs saline-treated non adjuvant rats. (†) P b 0.05 for SNP-treated rats vs 0.01 mg/kg/d SNP-treated adjuvant arthritic rats (group II).

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Fig. 7. Representative histopathology of left ankle joints of (A) normal control rats (normal synovial lining, articular cartilage and subchondral bone), (B) adjuvant arthritic rats nontreated rats, (C) adjuvant arthritic rats treated with SNP (1 mg/kg/d) from day 5 to day 14, (D) adjuvant arthritic rats treated with SNP (1 mg/kg/d) from day 16 to day 25, (E) adjuvant arthritic rats treated with SNP (0.01 mg/kg/d) from day 5 to day 14, and (F) adjuvant arthritic rats treated with SNP (0.01 mg/kg/d) from day 16 to day 25. Note the increase in degree of inflammation with cartilage and bone erosion by high dose of SNP and the marked inhibition of inflammatory cellular infiltration in the synovium with alleviation of articular cartilage erosion by the low dose of SNP.

signs and symptoms of rheumatoid arthritis did not appear in contralateral non-injected hind paw after CFA inoculation so, the method of induction of adjuvant arthritis was modified by intradermal inoculation of squalene in addition to CFA into the subplanter surface of right hind paw, to increase sensitivity of arthritic rats to CFA. Squalene was also used by others to potentiate the effect of CFA [36]. The present study demonstrated protective and deleterious effects for SNP administration on the local inflammatory infiltrate in the adjuvant arthritic rats. It was observed that the higher doses of SNP (1 and 0.1 mg/kg/d) significantly exacerbated inflammatory processes in the injected and non-injected hind paws of arthritic rats whether these doses were administered early (prophylactic) i.e. from day 5 to day 14 or late (therapeutic) i.e. from day 16 to day 25. These doses provoked a significant increase in paw volume, ankle diameter, arthritis score and degree of cartilage erosion. On the contrary, this

study revealed that small dose of SNP (0.01 mg/kg/d) was successful in reducing the inflammatory responses associated with induction of arthritis by adjuvant and squalene. Prophylactic and therapeutic use of 0.01 mg/kg/d SNP decreased significantly the arthritis index, paw volume and ankle diameter of right hind paw of arthritic rats compared with control adjuvant arthritic rats. Furthermore, small dose of SNP reduced markedly the inflammatory cellular infiltration in synovium with alleviation of articular cartilage erosion. High dose effects in the present study are in agreement with many previous studies. Topical application of nitroglycerin has been reported to accelerate both cartilage degeneration and subchondral bone sclerosis in osteoarthritis [37,38]. Lane et al. demonstrated that NO donor medication may accentuate bone sclerosis and contribute to disease progression if used in the presence of osteoarthritis [2]. Other studies reported that SNP mediate chondrocyte death and it has been

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viewed as NO-mediated form of chondrocyte apoptosis. Kim et al. in line with Del Carlo and Loeser, reported that high doses of SNP induce chondrocyte death [39,40]. It was believed that NO was the sole mediator of SNP-induced chondrocyte death and peroxynitrite, a reaction product of NO and superoxide anion, or the primary byproducts of the decomposition of SNP might contribute to its cytotoxicity [41]. Patients with rheumatoid arthritis have documented evidence for increased endogenous NO synthesis [8] and increased iNOS activity in comparison with control [21] supporting the hypothesis that overproduction of NO may be important in pathogenesis of RA. Direct involvement of NOergic mechanisms in the development of adjuvant arthritis was also demonstrated by many investigators [42,43]. Moreover, Vuolteenaho et al. and Nagy et al. suggested that NO mediates many of destructive effects of IL-1 and TNF-alpha in the cartilage and that inhibitors of NO synthesis have demonstrated retardation of clinical and histopathological signs and symptoms in experimentally induced osteoarthritis and other forms of arthritis [6,9]. However, controversies regarding NO role and NO donors' effect have arisen. Laitao et al. demonstrated that locally applied isosorbide dinitrate decreased bone resorption in experimental periodontitis in rats suggesting a local anti-inflammatory effect of isosorbide [44]. In recent observation, nitroglycerin has been shown to stimulate cell proliferation and osteoblastic differentiation of human bone marrowderived mesenchymal stem cells through a direct release of NO [45]. SNP was effective in stimulation of synovial cells from patients with RA and increasing hyaluronic acid production [12]. In addition, nitrate was found to exert a protective effect upon hypochlorus acid-induced chondrocyte toxicity suggesting that NO has a novel cytoprotective role in inflamed joints [46]. Agreeing with such reports, our results showed that the low dose of SNP (0.01 mg/kg/d) used in this study has possible anti-inflammatory effects. Therefore, our findings suggest that SNP produces dual effects (which is dose dependent). Many studies support the present findings. It has been reported that low concentration of SNP suppresses subsequent high concentration SNP-induced apoptosis [27]. Furthermore, Kim et al. reported that SNP in low concentrations protects chondrocytes from toxic effect of the higher concentration [39]. Consistent with the notion of a dual effects of NO donors, several experiments have shown that local application of drugs generating a low NO concentration reduces incision pain through activation of guanylate cyclase while drugs generating high NO concentration intensify pain via guanylate cyclase-independent mechanism [47,48]. With respect to joint pain there is only one clinical study showing that transdermal nitroglycerin provided partial symptomatic pain relief in patient with painful shoulders [49]. Consistent with these data our work demonstrated that high doses of the NO donor, SNP intensified the hyperalgesia induced by CFA in arthritic rats while the small dose of SNP reduced the hyperalgesia. The precise mechanism of how NO is affecting pain transduction pathways remains unclear. However, Duarte et al. reported that NO-promoted analgesia was blocked by cGMP inhibitor [50]. The analgesic effect of sildenafil, a phosphodiesterase-5-inhibitor working via increasing cGMP production, was potentiated by NO donors. Therefore, NO donor low dose could be protective and analgesic through activation of cGMP. In the present experiments, it was observed that, in adjuvant nontreated arthritic rats (group II), there was significant loss of body weights in arthritic animals while the body weight in saline non-adjuvant group (group I) increased gradually over the study duration. This is in agreement with others who reported that body weight loss is an indication of abnormal conditions [51,52]. Koufany et al. also demonstrated that in all arthritic rats, body weight decreased progressively as arthritis settled [53]. This study demonstrated that SNP treatment significantly reduced the loss in body weight observed in the adjuvant non-treated arthritic animals and the smallest dose 0.01 mg/kg/d was the most effective in reducing the decrease in body weight especially when given prophylactically. Weight loss can be explained by the decrease of food intake observed throughout the period of the study due

to immobility accompanying hyperalgesia which was less in animals treated with the low dose of SNP. To our knowledge, no study reported the effect of NO donors on the body weight loss of arthritic animals. Considering the contribution of TNF-alpha and IL-10 to adjuvant arthritis severity, the present study showed that serum TNF-α level in adjuvant arthritic non-treated rats was slightly higher than that of normal rats with no arthritis, on day 30 after induction of arthritis. The differences in TNF-α levels between the groups were insignificant. This observation is consistent with Philippe et al. who reported that the systemic TNF-α concentration had significantly increased 6 h after adjuvant injection, peaked at 12 h, returned to near control concentrations on day 2, and increased slightly until day 20 [30]. Serum IL-10 levels in control adjuvant rats were significantly lower compared with those in saline-treated non-adjuvant rats. This is consistent with the fact that IL-10 has potent anti-inflammatory activity [54]. This study demonstrated that serum IL-10 levels on day 30 after adjuvant inoculation, in rats given SNP therapeutically, were significantly reduced compared with those in saline control rats. However, IL-10 levels in animals given the lower dose, 0.01 mg/kg/d, were higher than those in animals given the higher dose, 1 mg/kg/d. In this study, our histological observations revealed that the administration of large doses of SNP in arthritic rats was associated with the increase in the destruction of the inflamed joint. We speculate that this observed effect may be due to the increase of the formation of peroxynitrite in the synovium where peroxynitrite produces cellular injury and necrosis. However, the small dose of SNP reduced the inflammatory cellular infiltration in the synovium with alleviation of the articular cartilage erosion. We proposed that the protective effect of the small dose is secondary to a reduced formation of peroxynitrite in the synovium, where small dose of SNP may exert negative feed back regulation on iNOS gene expression.[55,56]. In conclusion, the results presented in this study reveal that systemic use of NO donor, SNP, may produce either exacerbation or attenuation of adjuvant arthritis in rats depending on administered dose. The smallest dose of SNP has protective effect against inflammatory processes, hyperalgesia and weight loss in adjuvant arthritic rats. Also, small dose reduces the serum TNF-alpha and insignificantly increases serum IL-10 while higher doses have the reverse actions. These findings suggest that nitrite medication in large dose contributes to disease progression of rheumatoid arthritis but low dose may exert protective effects against the pathogenesis of the disease. References [1] Wallberg-Jonsson S, Ohman ML, Dahlqvist SR. Cardiovascular morbidity and mortality in patients with seropositive rheumatoid arthritis in northern Sweden. J Rheumatol 1997;24:445–51. [2] Lane NE, Williams EN, Hung YY, H ochberg MC, Cummings SR, Nevitt MC. Association of nitrate use with risk of new radiographic features of hip osteoarthritis in elderly white women: the study of osteoporotic fractures. Arthritis Rheum 2003;49:752–8. [3] Jacob T, Morrell M, Manzi S, Verdile V, Simmons RL, Peitzman A. Elevated nitrites in inflammatory joint disease: nitric oxide in the arthritides. Arthritis Rheum 1992;35:R9–15. [4] Yki-Jarvinen H, Bergholm R, Leirisalo-Repo M. Increased inflammatory activity parallels increased basal nitric oxide production and blunted response to nitric oxide in vivo in rheumatoid arthritis. Ann Rheum Dis 2003;62:630–4. [5] Scher JU, Pillinger MH, Abramson SB. Nitric oxide synthases and osteoarthritis. Curr Rheum Reports 2007;9:9–15. [6] Vuolteenaho K, Moilanen T, Knowles RG, Moilanen E. The role of nitric oxide in osteoarthritis. Scand J Rheumatol 2007;36:247–58. [7] Fermor B, Christensen SE, Youn I, Cernancec JM, Davies CM, Weinberg JB. Oxygen, nitric oxide and articular cartilage. Eur cells Mterials 2007;13:56–65. [8] Nagy G, Clark JM, Buzas E, Gorman C, Pasztoi M, Koncz A, et al. Nitric oxide production of T lymphocytes is increased in rheumatoid arthritis. Immunol Lett 2008;118:55–8. [9] Nagy G, Clark JM, Buzas EI, Gorman CL, Cope AP. Nitric oxide, chronic inflammation and autoimmunity. Immunol Lett 2007;111:1–5. [10] Migita K, Yamasaki S, Kita M, Ida H, Shibatomi K. Nitric oxide protects cultured rheumatoid synovial cells from Fas-induced apoptosis by inhibiting caspase-3. Immunology 2001;103:362–7. [11] Paul-Clark MJ, Gilory DW, Willis D, Willoughby DA, Tomlinson A. Nitric oxide synthase inhibitors have opposite effects on acute inflammation depending on their route of administration. J Immunol 2001;166:1169–77.

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