Int. J. Exp. Path.

(2017), 97, 438–446

ORIGINAL ARTICLE

Mefenamic acid decreases inflammation but not joint lesions in

experimental osteoarthritis
Grazielle C. Aguiar*, Celso M. Queiroz-Junior*, Giovana L. Sitta*, Fl
avio A. Amaral†,
Mauro M. Teixeira†, Marcelo V. Caliari‡ and Anderson J. Ferreira*
*Department of Morphology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil,
†
Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo
Horizonte, Brazil and ‡Department of General Pathology, Institute of Biological Sciences, Universidade Federal de Minas Gerais,
Belo Horizonte, Brazil

INTERNATIONAL
JOURNAL OF
EXPERIMENTAL
PATHOLOGY
doi: 10.1111/iep.12216
Received for publication: 13 July 2016 namic acid prevented joint oedema and hyperalgesia induced by MIA in the acute
Accepted for publication: 4 December
2016
Correspondence:
Anderson J. Ferreira
Departamento de Morfologia
Instituto de Ci^encias Biol

ogicas
Universidade Federal de Minas Gerais
Av. Ant^ onio Carlos, 6627
Pampulha – Belo Horizonte
Minas Gerais 31.270-901
Brazil
Tel.: 55 31 3409 2811
Fax: 55 31 3409 2771
E-mail: anderson@icb.ufmg.br
SUMMARY
Mefenamic acid is a non-steroidal anti-inflammatory drug able to control the symp-
toms of osteoarthritis (OA), but its effects on protection of cartilage and bone are
still unclear. This study aimed to investigate whether the control of inflammation by
mefenamic acid translates into decreased joint lesions in experimental OA in rats.
OA was induced by injecting 1 mg of monosodium iodoacetate (MIA) into the joints
of rats. The animals were treated with mefenamic acid (50 mg/kg, daily, oral gav-
age) either pre-MIA injection (preventive) or post-MIA injection (therapeutic). Joint
swelling and hyperalgesia were evaluated at baseline and 1, 3, 14 and 28 days after
induction of OA. Intra-articular lavage and kinetics of cell migration into the syn-
ovium were measured 3 and 28 days after OA induction. Histopathological analysis,
Osteoarthritis Research Society International (OARSI) score, total synovium cells
count, cartilage area and levels of proteoglycans in joints were also evaluated. Mefe-
phase (3 days) of the disease. In the chronic phase (28 days), preventive and thera-
peutic regimens decreased the number of mononuclear cells in the joint cavity. In
contrast, thickening of the synovium, bone resorption, loss of cartilage and levels of
proteoglycans were unaffected by mefenamic acid when it was administered either
preventively or therapeutically. Thus, mefenamic acid had anti-inflammatory effects
but did not reduce the progression of OA lesions, thereby indicating that it is only
effective for symptomatic control of OA.
Keywords
bone resorption, cartilage degradation, inflammation, joint lesions, mefenamic acid,
osteoarthritis

After some decades of debate, osteoarthritis (OA) is now
considered an inflammatory disease (Berenbaum 2013).
The imbalance between pro- and anti-inflammatory media-
tors results in degeneration of cartilages and changes in
morphology and function of subchondral bones, meniscus
and synovium (Ruiz-Romero et al. 2008; Katz et al. 2010;
Berenbaum 2013). Pro-inflammatory cytokines stimulate
their own expression which activates chondrocytes. This
leads to amplification of the inflammatory milieu by syn-
thesizing other mediators such as prostaglandins (PG)
(Berenbaum 2013; Lee et al. 2013).
438 International Journal of Experimental Pathology © 2017 International Journal of Experimental Pathology
Prostaglandins are products of the arachidonic acid
metabolization by cyclooxygenases (COX). They have a
pivotal role in homoeostasis of human cartilage and also in
the pathophysiology of OA. In this regard, PGE2 is the
major contributor of inflammatory pain in arthritic condi-
tions. PGE2 may mediate pain by sensitizing nociceptors and
inducing downstream catabolic effects, including stimulation
of IL-6 and nitric oxide synthase (NOS) (Lee et al. 2013).
Accordingly, increased levels of PGE2 are observed in the
synovial fluid and synovial tissue of patients with active OA
(Peng et al. 2006). PG also enhance the production of
© 2017 The Authors.

matrix metalloproteinase-3, inhibit proteoglycans and colla-
gen synthesis and stimulate chondrocyte apoptosis (Lee
et al. 2013; Korotkova & Jakobsson 2014).
In view of these data, especially the direct relationship
between PGE2 and pain (Lee et al. 2013), the prescription
of non-steroidal anti-inflammatory drugs (NSAIDs) is one
of the most relevant therapies for symptomatic OA.
Because inhibition of COX activity by COX-2-selective or
non-selective NSAIDs has analgesic and anti-inflammatory
properties, it represents a pathophysiologically sound
approach in OA (Martel-Pelletier et al. 2003; Korotkova
& Jakobsson 2014). In this regard, mefenamic acid (a
non-selective COX inhibitor), which was ranked as one of
the three most prescribed NSAIDs in the 1990s (Cimolai
2013), has been reported to be equivalent to or better
than other NSAIDs for relieving pain and morbidity in
chronic OA (Myles et al. 1967; Aylward et al. 1985; Ste-
wart & Thomas 1988). Nevertheless, although this is true
of pain relief, as well as to decreases in morning stiffness
and free radical-induced tissue damage (Bhat et al. 2007),
there are no data regarding the effects of mefenamic acid
on cartilage and bone protection in OA. Therefore, the
aim of this study was to investigate whether the control
of inflammation and clinical parameters of OA by mefe-
namic acid influences the progression of joint lesions in
an experimental model of monosodium iodoacetate (MIA)-
induced OA in rats.
Methods
Animals
Thirty male Wistar rats, weighing 200–280 g, were obtained
from the animal facility of the Institute of Biological
Sciences (CEBIO, Universidade Federal de Minas Gerais,
Brazil). The rats were kept in controlled environment with
12-h/12-h light/dark cycle with free access to water and
food (Nuvilab CR1; Nuvital Nutrientes S/A, Brazil).
Ethical approval statement
All experimental procedures described in this study were
performed in accordance with the local ethics committee
(Ethics Committee for Animal Experimentation of the Uni-
versidade Federal de Minas Gerais – CETEA-UFMG).
Monosodium iodoacetate-induced osteoarthritis
This study used the experimental model of MIA-induced
OA, as described previously (Guzman et al. 2003). Ini-
tially, the rats were anaesthetized with 100 mg/kg ketamine
(Syntec, Cotia, SP, Brazil) and 15 mg/kg xylazine (Syntec).
The knees were shaved and swabbed with H2O/EtOH solu-
tion. Then, OA was induced by injecting 1 mg of MIA
(Sigma, S~ao Paulo, SP, Brazil) dissolved in 50 ll of sterile
saline into the intra-articular space of the right knee
(Clarke et al. 1997; Bar-Yehuda et al. 2009). The left knee
International Journal of Experimental Pathology, 2017, 97, 438–446
Mefenamic acid and OA joint lesions 439
joint served as control and received the same volume of
sterile saline.
Mefenamic acid treatment
The rats were treated daily (oral gavage) with mefenamic acid
(Ponstanâ; Pfizer, Guarulhos, SP, Brazil) in the preventive
(2 h before OA induction until the end of the experimental
period) as well as in the therapeutic regimens (3 days after
OA induction until the end of the experimental period).
Oedema and hyperalgesia measurements were performed one
hour after the treatments with mefenamic acid. Importantly,
the half-life of the mefenamic acid is 2–4 h (Cimolai 2013).
Figure 1 contains details of the experimental protocol. The
doses of 50 mg/kg and 200 mg/kg were based on previous
studies (Di Rosa et al. 1971; Bhat et al. 2007).
Joint swelling
Joint swelling measurements were carried out at baseline
and 1, 3, 14 and 28 days after induction of OA (n = 4–8
per group). Knee diameter was measured using a digital cal-
liper (Mitutoyo, Suzano, SP, Brazil). To perform the mea-
surement of the diameter of the knee, fixed and mobile
calliper nozzles were positioned in the medial–lateral direc-
tion of the knee. The values were expressed in millimetres
(mm), and the difference between right and left knees diam-
eters was calculated and used to plot the data.
Hyperalgesia behaviour
The assessment of hyperalgesia was conducted at baseline and
1, 3, 14 and 28 days after induction of OA (n = 4–8 per
group). For this purpose, a digital analgesimeter was used
(Insight, Ribeir~ao Preto, SP, Brazil). The analgesimeter
records the pressure in the paw of the animal in grams (g) in a
0.1–150 g range. Briefly, the rats were placed individually in
acrylic boxes measuring 12 9 20 9 17 cm. Angled mirrors
below the acrylic boxes were positioned for viewing the rats.
All animals underwent a minimum of 15 mins of acclimatiza-
tion. The pressure registered in ipsilateral (injected MIA –
right knee) and contralateral (saline injected – left knee) hin-
dlimbs was assessed. Readings were taken in triplicate and
means calculated. Data were analysed as the D withdrawal
threshold (g), calculated by subtracting zero-time mean mea-
surements from the time-interval mean measurements.
Intra-articular lavage and assessment of the kinetics of
cell migration
The rats were euthanized 3 and 28 days after OA induction
(300 mg/kg ketamine and 45 mg/kg xylazine, i.m.) (n = 4–6
per group). Then, intra-articular lavage was performed with
3 9 20 ll of PBS/0.01% BSA/0.5M EDTA. The lavage was
immediately diluted in 100 ll of the same solution and cen-
trifuged for 5 mins at 1200 rpm at 4 °C. The supernatant
was discarded and the pellet was resuspended for cell count-
ing using a Neubauer chamber.
440 G. C. Aguiar et al.

Histopathological evaluation Results

After euthanasia and lavage, the articular soft tissues and
patella of the joints of each animal were removed. Then, the
tibiofemoral joint was sectioned approximately 2 cm above
and below the midline of the knee. All samples (n = 4–6 per
group) were fixed for 3 days in 10% neutral buffered for-
malin and decalcified in 10% EDTA for 21 days. When the
samples were in advanced decalcification, they were cut into
two similar halves in the sagittal plane. Thereafter, the tis-
sues were washed with deionized water, dehydrated in
graded series of ethanol, cleared in xylene, embedded in
paraffin and cut into 6-lm-thick serial sections. Sections
were stained with toluidine blue or haematoxylin–eosin for
histopathological and morphometric analyses.
The histopathological grading system applied was the
OARSI score, which is a semiquantitative score method
Effects of mefenamic acid on clinical parameters
associated with MIA-induced OA
Intra-articular injection of MIA induced significant joint
swelling in rats especially at 1 day after OA induction (Fig-
ure 2). The treatment with 50 mg/kg of mefenamic acid in
the preventive regimens significantly lowered such swelling.
However, therapeutic regimens, which started after the
acute phase of OA (3 days after MIA injection), did not
influence this parameter (Figure 2). Similar results were
(a)
–2 h 0 1 day 3 days 14 days 28 days

(Pritzker et al. 2006). OARSI score reflects the degree and

extent of lesions in the cartilage (Score = grade 9 stage). ----------------------------------- mefenamic acid -----------------------------------
Image magnification was 2009 and 15–20 fields were
analysed (n = 4–6 per group). Total synovium cells (in- –2 h, 1 and 14 days: hyperalgesia; oedema
3 and 28 days: hyperalgesia; oedema; euthanasia; intra-articular lavage; tissue collection for histopathology
tima synoviocytes, subintima connective tissue cells and
inflammatory cells) were also counted. For this, haema- (b)
toxylin–eosin-stained slides were photographed using the 0 1 day 3 days 14 days 28 days

software (Q-Capture Pro 7, Surrey, BC, Canada), linear

scan of the field, and analysed by the software (Image-Pro -------------------------- mefenamic acid -------------------------
Plus, Rockville, MD, USA). All analysis was performed in
a blinded manner by a previously trained investigator. 1 and 14 days: hyperalgesia; oedema
28 days: hyperalgesia; oedema; euthanasia; intra-articular lavage; tissue collection for histopathology
Haematoxylin–eosin-stained slides were also used to anal-
yse histological changes in the subchondral bone. Figure 1 Experimental protocols. (a) Preventive regimen and (b)
therapeutic regimen. Treatment groups, times and procedures.
Black arrows indicate the time of induction of OA with
Morphometric analysis monosodium iodoacetate. White arrows indicate the time of
euthanasia.
Morphometric analysis (n = 4–6 per group) was conducted
after digitization of the images using a microcamera (Q-
Color3; Olympus Latin America Inc., S~ao Paulo, SP, Brazil)
coupled to a microscope (BX53; Olympus Latin America
Inc.). The cartilage area and level of proteoglycans in the tibia
and femur were evaluated. For this purpose, linear scanning
of 6–30 images per animal was performed using a 209 objec-
tive and the KS300 software (Carl Zeiss, Oberkochen, Ger-
many). The loss of proteoglycans was quantified by
calculating the optical density (OD) expressed in Grey units.
The calculation of OD was based on an algorithmic function
(-LOG I/I0). Thus, the OD values near zero corresponded to
an intense staining by toluidine blue (Costa et al. 2010).

Statistical analysis
Data were evaluated statistically using one-way analysis of
variance (ANOVA) and Kruskal–Wallis test for data with non-
parametric distribution or Newman–Keuls post-test for data
with parametric distribution. For analyses involving two vari-
ables, the two-way ANOVA and the Bonferroni post-test were
used. P < 0.05 was considered significant. Ninety-five per cent
confidence intervals were used. All analyses were performed
using the software (GraphPad Prism 5, San Diego, CA, USA).
Figure 2 Knee diameter/swelling over a 28-day time course
(difference between the diameter of the right and left knees).
Double arrows indicate the starting point of the preventive
regimens, and single arrow indicates the beginning of the
therapeutic regimens. Data are presented as mean
SEM
*P < 0.001 preventive regimens vs. vehicle at day 1; n = 4–8
per group (two-way ANOVA followed by the Bonferroni post-
test).

International Journal of Experimental Pathology, 2017, 97, 438–446


Figure 3 Hyperalgesia over a 28-day time course. Hyperalgesia
was analysed as the D withdrawal threshold (g) calculated by
subtracting zero-time mean measurements from the time-interval
mean measurements. Additionally, the difference between right
and left knee values was calculated and used to plot the data.
Results are presented as mean
SEM *P < 0.001 preventive
regimens vs. vehicle at day 1; n = 4–8 per group (two-way ANOVA
followed by the Bonferroni post-test).
(a) (b)
(c) (d)
(e) (f)
(g) (h)
International Journal of Experimental Pathology, 2017, 97, 438–446
Mefenamic acid and OA joint lesions 441
obtained when the dose of 200 mg/kg of mefenamic acid
was used in the therapeutic regimens (data not shown).
Another clinical parameter evaluated in rats with MIA-
induced OA was hyperalgesia. MIA injection caused a sig-
nificant increase in the joint hypernociception (Figure 3). In
accordance with joint swelling, the treatment with mefe-
namic acid prevented the decrease in the nociceptive thresh-
old observed in the acute phase of OA. In contrast, the
therapeutic regimens, which started after the acute phase,
did not change this parameter. Similar results were obtained
with the dose of 200 mg/kg of mefenamic acid in the thera-
peutic regimens (data not shown).
Effects of mefenamic acid on inflammatory profile of
MIA-induced OA
After investigating the clinical effects of mefenamic acid,
inflammatory parameters were studied. The cell infiltrate in
the synovial tissue, characterized by 99.4% of mononuclear
cells, was prominent in rats injected with MIA at 3 days
(Figure 4a,b). Such cellular infiltrate was more pronounced
in the subintimal layer of the synovial membrane. At this
(i)
(j)
(k)
(l)
442 G. C. Aguiar et al.

Figure 4 Synovial and joint cavity cell counting. (a) and (b): Vehicle – 3 days [control and monosodium iodoacetate (MIA)]; (c) and
(d): preventive regimens – 3 days (control and MIA); (e) and (f): vehicle – 28 days (control and MIA); and (g) and (h): therapeutic
regimens – 28 days (control and MIA). H&E, 2009; bar 40 lm. Insert: H&E, 409; scale bar 400 lm. (i): synovium cell counting
(preventive regimens); (j): total leucocytes counting (preventive regimens); (k): synovium cell counting (therapeutic regimens); and (l):
total leucocytes counting (therapeutic regimens). Data are presented as mean
SEM *P < 0.001 MIA vehicle or MIA preventive
regimens or MIA therapeutic regimens vs. their respective controls; +P < 0.001 MIA vehicle and MIA preventive regimens (28 days)
vs. MIA vehicle and MIA preventive regimens (3 days) respectively; #P < 0.05 MIA preventive or therapeutic regimens vs. MIA
vehicle. n = 4–6 per group (one-way ANOVA followed by the Newman–Keuls post-test).

time point, the preventive regimen with mefenamic acid did
not change the number of cells in the synovia (Figure 4c,d,i)
or the number of inflammatory cells in the joint cavity (Fig-
ure 4j). After 28 days of MIA injection, the cellular infiltrate
was also characterized by mononuclear cells, although their
number decreased when compared to the 3-day time point.
Synovium thickening was also apparent. There was signifi-
cant synovial hyperplasia, connective tissue production in
the subintima and presence of blood vessels (Figure 4e,f).
The preventive or therapeutic regimens with mefenamic acid
did not alter the cellularity in the synovium at this time
point (Figure 4g,h,i,k). In contrast, there was a reduction in
the number of total leucocytes in joint cavity after the pre-
ventive or therapeutic regimens with mefenamic acid (Fig-
ure 4j,l). Accordingly, the number of mononuclear cells,
which comprised 99.9% of the inflammatory infiltrate, pre-
sented the same pattern of distribution as total cell profile.
At 28 days after MIA injection, the percentage of mononu-
clear cells did not reduce significantly in the tissue (MIA
vehicle: 306,5
38,1; preventive regiment: 382
75,72;
therapeutic regimen: 454,7
46,4; P > 0.05), but it
decreased in joint cavity (MIA vehicle: 59,700
10,380;
preventive regiment: 19,830
4063; therapeutic regimen:
37,200
10,280; P < 0.05). The therapeutic regimens of
rats with the dose of 200 mg/kg of mefenamic acid induced
similar results (data not shown).
Mefenamic acid does not affect histopathological changes
induced by MIA
We also investigated whether the anti-inflammatory proper-
ties of mefenamic acid translated into chondroprotection.
Important histological changes were observed in MIA-
induced rats at day 28 after OA induction; for example,
collapsed subchondral bone and cystic structures sur-
rounded by fibrous tissue are seen in Figure 5a,b. Further-
more, resorptive processes were observed (Figure 5c), as
well as new bone formation (Figure 5d). These findings
were viewed in animals treated or not with mefenamic
acid. In the acute phase, no significant histological changes
were observed in MIA-induced OA animals (Figure 6a,b).
In contrast, in the chronic phase MIA-induced OA rats
showed cartilage damage associated with changes in the
subchondral bone. MIA injection induced disorganization
of chondron columns, cationic stain matrix depletion, clus-
ter formation, cyst formation within cartilage matrix, chon-
drocyte death (Figure 6c–f), erosion of cartilage by
stripping (complete erosion of the articular cartilage in

(a) (b)

Figure 5 Histological changes induced

by monosodium iodoacetate in the
subchondral bone of vehicle and
mefenamic acid-treated rats. (a)
Subchondral bone collapse and
fragmentation with replacement by
fibrous tissue (arrows). Necrotic and
fragmented cartilage is viewed
(arrowheads). Cystic structure
surrounded by fibrous tissue (asterisk).
(c) (d) H&E, 409. (b) Subchondral bone
collapse and fragmentation with cystic
structures (arrows) demarcated by
fibrous tissue (asterisks). Regions of
proliferation and loss of chondrocytes
in cartilage are also observed
(arrowheads). H&E, 409. (c) Active
osteoclasts (arrows); necrotic cartilage
(arrowheads). H&E, 2009. (d) Bone
invading cartilage, active osteoblasts
(arrow). Giant clusters (arrowheads).
H&E, 2009. n = 4–6 per group.

International Journal of Experimental Pathology, 2017, 97, 438–446

 Mefenamic acid and OA joint lesions 443

(a) (c) (e)

(b) (d) (f)

(g) (i)

(k)

(h) (j)

Figure 6 Cartilage histological changes induced by monosodium iodoacetate (MIA) in vehicle- and mefenamic acid-treated rats. (a)
Control and (b) MIA – joints treated with vehicle in the acute phase – 3 days. (c) Control; (d) MIA – vehicle; (e) MIA – preventive
regimens; and (f) MIA – therapeutic regimens in the chronic phase – 28 days. Arrows: cationic stain depletion into deep zone and
chondrocyte necrosis. Arrowheads: erosion, loss of articular cartilage tissue with exposure of subchondral bone. Asterisks: eroded cartilage
replacement by reparative tissue. Toluidine blue (40 x); bar 300 lm. (g) OARSI score – preventive regimens; (h) OARSI score – therapeutic
regimens; (i) quantification of proteoglycan loss – preventive regimens; (j) quantification of proteoglycan loss – therapeutic regimens; and
(k) quantification of hyaline cartilage area. *P < 0.001 MIA – vehicle- or MIA-treated preventively or clinically at 28 days vs. their
respective controls. Data are presented as mean
SEM; n = 4–6 per group (one-way ANOVA followed by the Bonferroni post-test).

mineralized cartilage level or bone) (Figure 6e) and cartilage in control rats, as well as in MIA-induced animals
replacement of eroded cartilage by repair tissue (Figure 6d, treated preventively or clinically. These changes were quan-
f). Mefenamic acid treatment did not cause any change in tified by the OARSI score, as shown in Figure 6g,h.

International Journal of Experimental Pathology, 2017, 97, 438–446

444 G. C. Aguiar et al.
Similarly, morphometric quantification of proteoglycans
revealed that MIA-induced OA rats presented significant pro-
teoglycan loss, which was not improved by mefenamic acid
(Figure 6i,j). The cartilage area was also quantified, and no sig-
nificant changes were induced by the treatments (Figure 6k).
The dose of 200 mg/kg of mefenamic acid was also evaluated
in the therapeutic regimens and induced similar results (data
not shown).
Discussion
The management of pain and disability in OA is accomplished
by pharmacologic and non-pharmacologic therapies (Felson
et al. 2000). Regarding the pharmacologic treatment, acetami-
nophen is the first-line analgesic agent, although meta-analyses
and systematic reviews have shown that it is less effective in
pain relief than anti-inflammatory drugs (Lee et al. 2004;
Zhang et al. 2004; Towheed et al. 2006; Verkleij et al. 2011).
NSAIDs are considered the first-line drugs for controlling the
manifestations of OA in clinical practice. These drugs are effec-
tive in reducing inflammation and pain, but their effects on car-
tilage and bone protection are questionable. In the present
study, we investigated the effects of mefenamic acid on inflam-
mation and cartilage/bone joint lesions induced by MIA in rats.
Although rather inflammatory, MIA experimental model
reproduces many joint osteoarthritic aspects of human OA, in
line with mechanical-induced models (van der Kraan et al.
1989; Guzman et al. 2003; Naveen et al. 2013).
Mefenamic acid is a well-known N-phenylanthranilic acid
derivative NSAID, a member of the fenamate family (Cimo-
lai 2013). The effects of mefenamic acid on the inflamma-
tory process have been known for many years. Mefenamic
acid (as expected for a NSAID COX inhibitor) controls
inflammation by inhibiting PG production, leading to cellu-
lar changes, such as for example, decreases in mononuclear
cell migration (Di Rosa et al. 1971; Bray & Gordon 1978).
Mefenamic acid treatment also provides significant protec-
tion against increases in the levels of some cytokines
involved in inflammatory contexts, such as TNF-a and IL-
1b (Armagan et al. 2012; Daniels et al. 2016). Accordingly,
in this study mefenamic acid caused a reduction in the num-
ber of inflammatory cells in the joint cavity of MIA-induced
rats, although it did not decrease the cell number in the syn-
ovia. Thus, taking into account these anti-inflammatory
properties, mefenamic acid has been used for the symp-
tomatic treatment of OA.
Here, mefenamic acid decreased oedema and hypernocicep-
tion in joints of rats in the acute phase of OA. Although
hyperalgesia is more frequently reported by women in human
OA, probably due to hormonal and other factors (Sowers and
Karvonen-Gutierrez, 2012), male rats were used in our study.
This should be considered when translating current findings
to clinical contexts. Overall, it has been reported that mefe-
namic acid is able to control clinical manifestations of OA
(pain, stiffness, oedema and limitation of joint movement)
through its analgesic and anti-inflammatory actions (Aylward
et al. 1985; Stewart & Thomas 1988). One possibility is that
by inhibiting COX mefenamic acid may decrease oedema by
reducing synthesis of PGE2. PGE2 increases vascular perme-
ability through activation of EP3 receptor subtype, which
stimulates TRPV1 and NK1 receptors and PKC and MAPK
pathways (Claudino et al. 2006). These events lead to his-
tamine release and mast cell degranulation, culminating in
oedema (Morimoto et al. 2014). The inhibition of COX and,
consequently, of the production of PGE2 could also account
for the anti-nociceptive action of mefenamic acid. The sensiti-
zation of primary afferent fibres to mechanical stimuli is
reduced when cascading events related to PGE2 are inhibited,
and this improves the nociceptive threshold (Cimolai 2013;
Lee et al. 2013). Accordingly, this effect may be favoured by
the larger sensory deficit observed in the acute phase of MIA-
induced OA (Bryden et al. 2015).
We also evaluated whether these anti-inflammatory effects
of mefenamic acid translated into cartilage and bone protec-
tion in OA. Inflammation contributes to the pathological
condition of cartilage and bone in OA. PG can change the
metabolism of the extracellular matrix of cartilages and
imbalance bone formation and resorption (Korotkova &
Jakobsson 2014). Therefore, inhibiting PG synthesis with
mefenamic acid could be a target to prevent joint damage.
In addition, mefenamic acid has other effects beyond inhibit-
ing COX enzymes. It increases proteoglycan production by
decreasing 30 ,50 -cyclic AMP levels in fibroblasts stimulated
by PGE1 (Peters et al. 1975), reduces ROS production and
free radical nitric oxide accumulation and upregulates anti-
apoptotic proteins in experimental neurodegenerative condi-
tions (Joo et al. 2006). Also, mefenamic acid may reduce
malondialdehyde acid, an oxidative factor of cartilage colla-
gen that alters collagen fibrils (Tiku et al. 2003; Armagan
et al. 2012), and increase the levels of thiol, a protective
mediator for damaging free radicals (Bhat et al. 2007).
However, we failed to demonstrate any beneficial effects of
mefenamic acid on the progression of lesions in cartilage,
subchondral bone and synovium. MIA-induced rats treated
with mefenamic acid showed subchondral bone collapse and
fragmentation with replacement by fibrous tissue and/or cyst
formations. There was also necrotic and fragmented
cartilage with a similar pattern to that observed in vehicle-
treated rats. These observations indicate that control of
inflammation by mefenamic acid, although important for
the clinical management of OA, may not directly translate
into cartilage and bone protection. This feature is in line
with other medications used for the symptomatic control of
OA and joint pain, including the drugs celecoxib (COX-2-
selective NSAIDs), diclofenac (non-selective NSAIDs) and
diacerein (inhibitor of IL-1b). They are effective in suppress-
ing the synthesis of proinflammatory cytokines and improv-
ing physical function in patients, but they do not avoid
cartilage and bone destruction (Gallelli et al. 2013; Gal-
lagher et al. 2015; Scarpignato et al. 2015). Similar results
were also observed with the use of other traditional pharma-
cologic therapies such as acetaminophen and chondroitin
sulphate (Singh et al. 2015; Zhang et al. 2016). In addition,
cell therapy was able to reduce the pain, but the structural
International Journal of Experimental Pathology, 2017, 97, 438–446

damage and synovial inflammation were not affected by the
treatment in the OA model induced by MIA (van Buul et al.
2014).
These findings point to one of the main challenges in OA
therapy. The slowly progressive loss of cartilage, the multi-
factorial nature of the disease and its cyclic course with peri-
ods of active disease followed by remission are important
hurdles to the development of a disease modifying OA drug
(Martel-Pelletier et al. 2012). This complex pathogenesis of
OA hinders effective therapies. For instance, NSAIDs such
as mefenamic acid improve clinical manifestations, but do
not avoid cartilage and bone degeneration (Haseeb & Haqqi
2013).
In summary, mefenamic acid presented anti-inflammatory
effects but did not reduce the progression of OA lesions,
thereby indicating that it is only effective for the symp-
tomatic control of OA. Therefore, search for new agents
capable of modifying OA course should receive attention to
generate new and more effective strategies for the treatment
of OA.
Conflict of interest
The authors declare no conflict of interests.
Funding source
We are grateful to financial support of the Coordenacßa~o de
Aperfeicßoamento de Pessoal de N
ıvel Superior (CAPES-Brazil),
Conselho Nacional de Desenvolvimento Cient
ıfico e Tec-
ogico (CNPq-Brazil) and Fundacß~ao de Amparo a Pesquisa
nol

do Estado de Minas Gerais (FAPEMIG-Brazil).
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