Abstract
Background and purpose : Osteoarthritis (OA) is a chronic and progressive joint disorder characterized by structural damage to one or more joints. However, drugs that could cure or at least stop the progression of this disease are still given no satisfactory outcome. The purpose of this work is to study the potential OA disease-modifying effects of atorvastatin in an experimental model of osteoarthritis and the possible underlining mechanisms if any. Experimental Approach : Seventy-six adult male Sprague-Dawley rats (250-300gms) were used throughout this study. Forty rats were used to assess the effect of atorvastatin in surgically induced OA. While 36 rats were used to assess its anti-inflammatory effect in carrageenan-induced paw edema. In the model of OA; the degree of joint stiffness was assessed by measuring the angle of knee extension besides, the histopathological changes of the OA knee joints and measurement of serum Interleukin-1beta (IL-1β), Matrix metalloproteinase-13 (MMP13), and reduced glutathione (GSH) concentration were biochemically assessed. In the carrageenan-induced paw edema, the paw thickness and pain threshold were assessed in different groups.
Key Results: Atorvastatin was found to produce significant improvement of joint stiffness, the histopathological changes, a significant correction in the increased MMP13 and IL1-β, and the decreased GTH in OA rats. Also, atorvastatin showed a significant improvement in both paw thickness and pain threshold.
Conclusion and Implications: These results present atorvastatin as OA disease-modifying drug worse clinical trials.
Keywords: atorvastatin; carrageenan; IL1-β; MMP13; osteoarthritis; paw edema
Introduction
Osteoarthritis is a progressive joint disease affecting all joint structures. It is estimated to be the fourth leading cause of joint disability worldwide in addition to the high socioeconomic costs in all countries (Johnson and Hunter, 2014). Most available drug therapy can relieve symptoms of OA successfully but drugs that could cure or slow down the progress of the disease called disease-modifying osteoarthritic drugs (DMOAD) still do not produce a satisfactory outcome and considerable conflict and controversy about their effects and tolerability are present (Rovati et al., 2012). Therefore, the development of a new DMOAD is highly recommended.
Statins, inhibitors of 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, have been commonly used in cardiovascular diseases. In addition to the ability of statins to decrease cholesterol synthesis, statins have beneficial effects in various disease conditions, mediated through its cholesterol independent pleiotropic effects such as anti-inflammatory, antioxidant properties, and its attenuating tendency to affect a broad range of MMPs (Ghaisas et al., 2010). These properties make statins a possible candidate to be tried as DMOAD.
The present study aims to investigate the potential OA disease-modifying effect of atorvastatin and compares its effectiveness a previously used drug, glucosamine in an experimental model of OA and to provide a piece of direct experimental evidence for its possible underlying mechanism of‘ action by assessing MMP3, IL1- β, reduced glutathione in addition to the detection of its possible anti-nociceptive and anti-inflammatory effects.
Materials and Methods
Drugs and chemicals
Atorvastatin (tablets 10mg EIPICO), Glucosamine sulfate (capsules 500mg, EMA pharm), Indomethacin (capsules 25mg, Aspen pharma), Carrageenan raw material powder, Sigma Aldrich.
Experimental animals
Sixty-two adult male Sprague-Dawley rats (250-300 gm) obtained from Mansoura experimental Research Center (MERC) and were utilized in this study. They were put in similar optimum housing conditions with free access to food and water. Animals were kept in cages (4 rats/cage) at a room with a controlled temperature of 26°C and on a 12-h light-dark cycle. The institutional research board (IRB), Faculty of Medicine, Mansoura University animal ethics committee has approved all the experimental procedures under approval no (MS/17.08.76). Two experimental models were conducted: surgical induced OA and carrageenan-induced paw edema model.
Surgical induction of OA
Thirty-two rats were used. The surgical induction of OA was conducted on 24 rats according to (Janusz et al., 2002), while the remaining 8 rats served as the SHAM control group.
Experimental designThe rats were divided into 4 groups; the Sham non-OA group received saline; OA non-treated group received saline. Glucosamine treated OA group: received glucosamine sulfate solution at a dose of 250 mg /kg/ day (Wen et al., 2010). Atorvastatin treated OA group: given atorvastatin solution 10 mg/kg daily (Pathak et al., 2015a). All drugs are given by oral gavage daily from 1st day of surgery for six weeks.
Joint stiffness assessment.
After the scarification of the animals, the left knee of the animals was dissected, and the articular cartilage was left intact. The maximum extension angle of each knee was measured after the dissection with a zero degree indicates no stiffness increase value of the angle indicates limited joint movement and points to joint stiffness. The angle measured not the usual angle that is assumed to be, but it’s a complementary angle (figure 1). After joint dissection, the joint was put on a paper and the maximum angle was measured by protractor (Rezende et al., 2006).
Histopathological assessment
The preparation of the knee specimen for the histopathological examination was done according to Schmitz et al. (Schmitz et al., 2010). Sections of five microns of tissue were cut then stained with hematoxylin and eosin (H&E) for the examination of cartilage and subchondral bone. Semi-quantitative histological lesions grading was performed following the scoring system of (Khan et al., 2013).
Biochemical assessment
Blood was withdrawn from the heart of rats and was separated into 2 tubes; a dry tube for collection of sera to measure MMP13 and IL-1β and heparinized one for collection of erythrocyte lysate to measure GSH. Measurement of interleukin1- β using rat Interleukin 1β enzyme-linked immunosorbent assay (ELISA) kit Bioassay technology laboratory, Catalog No E0119Ra. Measurement of matrix metalloproteinase- 13 using rat MMP- 13 kit (Eiaab science com, Catalog No E0099r, China). Measurement of GSH by colorimetric method using a Bio-diagnostic kit (Egypt) (Beutler et al., 1963).
Carrageenan induced paw edema
Thirty rats were used for this model. Induction of paw edema was done by sub-plantar injection of carrageenan in the right hind limb of the 24 rats while 6 non injected rats serve as control normal. The animals were pretreated by oral gavage of the drugs, 1h before carrageenan injection. Then, the rats received 0.1 ml of a 1% (w/v) suspension of carrageenan in the sub-plantar surface of the right hind paw (Winter et al., 1962).
Experimental design
The control normal group received 0.5ml saline orally before sub-plantar injection of 0.1 ml of saline. Control carrageenan-treated group rats pretreated with 0.5ml saline by gavage before sub-plantar injection of 0.1 ml of 1% carrageenan dissolved in saline (Winter et al., 1962). Indomethacin-pretreated group received an indomethacin solution at a dose of 10 mg/kg, served as a standard drug group (Sadeghi et al., 2013). Glucosamine pretreated group received glucosamine sulfate solution by oral gavage at a dose of 250 mg/kg (Singh et al., 2007). The atorvastatin-pretreated group received atorvastatin at a dose of 10 mg/kg (Ghaisas et al., 2010).
Recording of paw thickness:
The paw thickness was recorded from the ventral to the dorsal surfaces using a dial thickness gauge flat contact point 0-10mm tester leather measuring tool (weldingstop,china) at 1, 2, 3, 4 h, and 24h after carrageenan injection (Solanki et al., 2015).
Recording of pain threshold:
Carrageenan-induced hyperalgesia was assessed using an analgesimeter (Ugo Basile, Comerio, Italy) which was composed of a cone-shaped paw-pressor with a rounded tip that was used to apply linear increasing force to test paw at the metacarpal level of right dorsal surface oh hind paw. The nociceptive threshold was taken as the point at which the rat vocalized or struggled vigorously, expressed as the force in grams (g). The nociceptive threshold (g) was recorded at 1, 2, 3, 4 h, and 24h after carrageenan injection (Marius et al., 2018).
Statistical Analysis
The results were statistically analyzed using the Statistical Package for Social Science (SPSS) program. The parametric results were expressed as Mean ± SD. One-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple comparisons was used for statistical analysis between groups. Kruskal Wallis test was used for comparison of means of more than two different groups of non-parametric data followed by post-hoc Dunne’s test, data were presented as median & range (minimum-maximum). For all above mentioned statistical tests done, the threshold of significance is fixed at 5% level (p-value) or less.
  1. Results
  2. Model of OA:
  3. Assessment of degree of joint stiffness by recording the maximum angle of knee extension in different groups :
Surgical induction of OA in rats produced a statistically significant increase in the maximum angle of knee extension compared to the control normal rats. Glucosamine and atorvastatin showed a statistically significant correction of the increased maximum angle of knee extension found in the non treated OA group. Atorvastatin treated OA group is significantly less than the glucosamine treated OA group, (Table 1).
Effect of different drugs on histopathological changes in osteoarthritic rats:
Histopathological examination of the control osteoarthritic rats showed loss of superficial layer, cell death, hypocellularity, the maximum loss of cellular layers and matrix, erosion and cavitation in the subchondral bone (figure 2) and showed a significant increase in the histopathological score as compared to control non-arthritic rats, (Table 1).
The histopathological examination of the glucosamine and atorvastatin treated OA groups showed improvement of osteoarthritic changes as shown in (figure 2), and histological score as compared to the non-treated OA group, but still significantly higher than the control normal
Effects of the tested drugs on serum interleukin 1-β level of osteoarthritic rats:
Surgical induction of OA in rats produced a significant rise in serum IL1-β compared to the control normal rats. Glucosamine and atorvastatin treated OA groups showed a statistically significant reduction in the raised IL1-β noticed in the non-treated OA group( Table1).
Serum matrix metalloproteinase 13 levels in different groups:
Osteoarthritic rats showed a significant increase in serum MMP13 compared to the control non-osteoarthritic rats. Glucosamine and atorvastatin treated OA groups showed a statistically significant correction of the increased MMP-13 found in the non-treated OA group. Atorvastatin reduction is significantly less than other treated groups, (Table1).
Effect of tested drugs on GSH level in osteoarthritic rat groups:
Surgical induction of OA in rats produced a statistically significant decrease in GSH compared to the control normal rats. Glucosamine and atorvastatin OA treated groups showed a statistically significant correction of the reduced GSH found in the non-treated OA group. Atorvastatin correction of GSH is significantly higher than glucosamine treated OA groups, (Table 1).