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.
- Results
- Model of OA:
- 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).