Evaluation of the anti-inflammatory/chondroprotective activity of aldose reductase inhibitors in human chondrocyte cultures

Article abstract of DOI:10.1039/c4md00556b

(5-Arylidene-4-oxo-2-thioxothiazolidin-3-yl)acetic acids (6) and 5-arylidene-4-oxo-2-thioxothiazolidines (7), which we recently synthesised and assayed as aldose reductase inhibitors, were evaluated as anti-inflammatory/antidegenerative agents in cultures

Full text of DOI:10.1039/c4md00556b

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CONCISE ARTICLE
Evaluation of the anti-inflammatory/
chondroprotective activity of aldose reductase
Cite this: DOI: 10.1039/c4md00556b
inhibitors in human chondrocyte cultures
a
Annamaria Panico, Rosanna Maccari, b Venera Cardile,c Sergio Avondo,d
*
Lucia Crascìa and Rosaria Ottanàb
IJ5-Arylidene-4-oxo-2-thioxothiazolidin-3-yl)acetic acids (6) and 5-arylidene-4-oxo-2-thioxothiazolidines
(7), which we recently synthesised and assayed as aldose reductase inhibitors, were evaluated as anti-
inflammatory/antidegenerative agents in cultures of human chondrocytes stimulated by IL-1β. In this
screening, most of the tested compounds were able to control key components of the IL-1β-induced
inflammatory signalling, by reducing the levels of NF-kB, ICAM-1, and NO as well as increasing the produc-
tion of glycosaminoglycans by chondrocytes. Moreover, these 4-thiazolidinone derivatives exhibited anti-
oxidant properties and were shown to inhibit MMP-3 and MMP-13 at micromolar concentrations, with a
generally marked preference toward MMP-13 which plays a major role in cartilage degeneration. Thus, on
the whole, compounds 6 and 7 were shown to be capable of both counteracting inflammatory events and
contributing to restore normal levels of cartilage components. This anti-inflammatory/antidegenerative
profile makes them interesting cell-permeable molecules that can be assumed as lead compounds in the
search for novel anti-inflammatory agents.
Received 12th December 2014,
Accepted 1st February 2015
DOI: 10.1039/c4md00556b
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1. Introduction
Inflammation is a complex pathophysiological process criti-
cally implicated in the etiology of numerous degenerative
human diseases, such as osteoarthritis, cancer, and diabetes
mellitus, which are major causes of morbidity and mortality
in worldwide populations.
Osteoarthritis (OA), the leading cause of joint disorder in
elderly patients, is characterized by progressive loss of articu-
lar cartilage as the result of complex inflammatory/oxidative
processes responsible for degradation of various components
of the cartilage matrix.
Articular cartilage is mainly composed of an extracellular
matrix (ECM), which is largely formed by type II collagen and
proteoglycans (PGs), with the latter being composed of
sulphated glycosaminoglycans (GAGs) linked to a protein
core. Chondrocytes, the only type of cells present in the carti-
lage, produce and secrete PGs, collagen and enzymes
Fig. 1 Role of NF-kB and ALR2 in the IL-1β-induced oxidative/inflam-
matory signalling pathways. ALR2: aldose reductase; COX: cyclooxy-
genase; GS-DHN: glutathionyl-1,4-dihydroxynonene; GS-HNE: gluta-
thione-4-hydroxy-trans-2-nonenal; HNE: 4-hydroxy-trans-2-nonenal;
ICAM: intercellular adhesion molecule; IKK: inhibitor-kappa B kinase;
iNOS: inducible nitric oxide synthase; MAPK: mitogen activated protein
kinase; MMPs: matrix metalloproteinases; NF-kB: nuclear transcription factor-
kappa B; PI3K: phosphoinositide 3-kinase; PKC: protein kinase C; RNS:
reactive nitrogen species; ROS: reactive oxygen species; TNF-α: tumor
necrosis factor α.
a Department of Drug Sciences, University of Catania, V.le A. Doria 6, 95125
Catania, Italy
b Department of Scienze del Farmaco e dei Prodotti per la Salute, University of
Messina, Polo Universitario dell'Annunziata, 98168 Messina, Italy.
E-mail: rmaccari@unime.it; Tel: +39 90 6766406
c Department of Biomedical Sciences, University of Catania, V.le A. Doria 6, 95125
Catania, Italy
d Department of Surgery, University of Catania, V.le A. Doria 6, 95125 Catania,
Italy
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involved in cartilage metabolism.1,2 Under OA conditions,
cartilage destruction arises from PG degradation and depriva-
tion of cartilage-specific type II collagen, as well as from
increased production of reactive oxygen species (ROS), reac-
tive nitrogen species (RNS) such as nitric oxide (NO) and its
free radical derivative peroxynitrite, adhesion molecules (e.g.,
ICAM-1) and matrix metalloproteinases (MMPs). ROS, RNS,
cytokines such as interleukin-1β (IL-1β) and tumor necrosis
factor α (TNF-α), and other pro-inflammatory stimuli pro-
mote a persistent activation of the nuclear transcription
MMP family play key roles in the loss of structural and func-
tional integrity of the cartilage, particularly MMP-3 and
MMP-13 which are considered attractive targets in the search
for novel therapeutic agents.10,11 MMP-3, also known as
stromelysin 1, acts directly on the cartilage, by degrading var-
ious components of the matrix, such as PGs, fibronectin, col-
lagen type IV, laminin and collagen type IX. MMP-13 (collage-
nase-3) acts by degrading the collagen fibrils, especially those
of type II that are the most abundant in the cartilage.12
Furthermore, recent studies have demonstrated that
aldose reductase (AKR1B1, ALR2), an enzyme which is impli-
cated in the onset and progression of diabetic complica-
tions,13,14 acts as a key component in the mediation of oxida-
tive stress-induced inflammation as well as in the
pathophysiology of inflammatory diseases, both under
normoglycemic and hyperglycaemic conditions.15,16 This
aldo-keto reductase is overexpressed under various oxidative
conditions and catalyses the reduction of aldehydes derived
from the ROS-mediated peroxidation of lipids. Certain
resulting metabolites, such as GS-DHN, are responsible for
the activation of NF-kB, via the PKC/PI3K/MAPK/IKK cascade,
thus triggering inflammatory response and tissue damage
(Fig. 1). Therefore, based on these findings, it has been pro-
posed that ALR2 inhibitors (ARIs) could be developed as new
anti-inflammatory agents for the management of a number
of inflammatory disorders, including sepsis, asthma, cancer,
diabetes and arthritis.14–16
factor-kappa
B (NF-kB) and, consequently, the NF-kB-
dependent expression of inflammatory cytokines, growth fac-
tors and MMPs.3–6 This creates a cascade of events that sus-
tain inflammatory response and tissue damage (Fig. 1).
Proinflammatory cytokine IL-1β also leads to loss of GAGs,
fundamental components of PGs, thus sustaining cartilage
damage. The degree of GAG decrease is considered indicative
of the severity of articular cartilage damage.7,8 Moreover, the
NF-kB-dependent expression of ICAM-1, a transmembrane
glycoprotein belonging to the immunoglobulin superfamily
of adhesion molecules, was shown to be higher in the
synovium of OA patients than in that of healthy individuals.9
ICAM-1 plays a predominant role in recruitment and traffick-
ing of leucocytes to sites of inflammation and is expressed in
a variety of inflammatory diseases.
Another consequence of NF-kB activation is the produc-
tion of MMPs by chondrocytes. Specific members of the
Fig. 2 General structure of previously reported anti-inflammatory 4-thiazolidinones 1–5 (ref. 17–20) and structures of IJ5-arylidene-4-oxo-2-thio-
xothiazolidin-3-yl)acetic acids (6a–d) and 5-arylidene-4-oxo-2-thioxothiazolidines (7a–d).
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In the past few years, we have reported 5-arylidene-2-
phenylimino-3-propyl-4-thiazolidinones (1) (Fig. 2), which
showed promising anti-inflammatory activity in animal
models of acute inflammation,17 and their 3-hydroxyalkyl
substituted analogues 2 (Fig. 2), which displayed anti-inflam-
matory/chondroprotective effects by blocking degenerative
cellular events induced by IL-1β in human chondrocyte cul-
tures.18 More recently, we have described the anti-inflamma-
tory/antidegenerative activity of 4-ij(5-arylidene-4-oxo-2-phenyl-
and appropriate aldehydes (Scheme 1).21 Similarly to
analogous 5-arylidene-2,4-thiazolidinediones and 5-arylidene-
2-phenylimino-4-thiazolidinones,22,23 only the preferred Z
isomers were obtained. The chemical–physical data of com-
pounds 6 and 7 and the source of starting materials were
previously reported in a recent article by some of us.21
The anti-inflammatory/anti-degenerative effectiveness of
2-thioxo-4-thiazolidinones 6 and 7 was assessed in cultures of
human chondrocytes stimulated by IL-1β. This is a useful
experimental model to reproduce the mechanisms involved
in arthritis and other inflammatory diseases, which we uti-
lized to investigate previously reported analogues.1,18,19 The
effects of the tested compounds on the production of NF-kB,
ICAM-1, NO and GAGs were evaluated.
Considering the predominant role of oxidative stimuli in
sustaining inflammatory signalling in IL-1β-activated
chondrocytes,5,6 we also evaluated the antioxidant activity of
derivatives 6 and 7 by means of the Oxygen Radical Absor-
bance Capacity (ORAC) method. Finally, we assessed their
in vitro inhibitory capability towards MMP-3 and MMP-13.
iminothiazolidin-3-yl)methyl]benzoic
acids
(3),
4-ij(5-
arylidene-2,4-dioxothiazolidin-3-yl)methyl]benzoic acids (4)
and IJ5-arylidene-4-oxothiazolidin-3-yl)acetic acids (5) (Fig. 2),
which also produced interesting effects in chondrocyte cul-
tures stimulated by IL-1β.19,20 Among these, acetic acid deriv-
atives 5a–c (Fig. 2) provided encouraging results, being endo-
wed with greater potency than that of indomethacin in
reducing the NO production induced by IL-1β and with a
chondroprotective effect similar to that of indomethacin.19
Analogues 5d–f (Fig. 2) significantly reduced the NF-kB acti-
vation induced by interferon-γ plus histamine in human
keratinocytes, and moreover, 5e and 5f were able to reduce
iNOS expression in the same cells.20 On the whole, our
results indicated that the anti-inflammatory/antidegenerative
effects of these derivatives could be mainly related to the
4-thiazolidinone core, whereas the 5-arylidene group
appeared to be able to modulate their anti-inflammatory
activity.17–20 The IJ4-oxothiazolidin-3-yl)acetic acid moiety of
compounds 5 was shown to be particularly favourable.19,20
The promising activity of acetic acid derivatives 5 encour-
aged us to evaluate the anti-inflammatory/anti-degenerative
effects of selected analogous IJ5-arylidene-4-oxo-2-thio-
xothiazolidin-3-yl)acetic acids (6) (Fig. 2). In addition, in
order to further investigate the influence of the acetic acid
chain on the activity, we tested the corresponding
N-unsubstituted 5-arylidene-4-oxo-2-thioxothiazolidines (7)
(Fig. 2).
2. Results and discussion
2.1 Cellular assays
Compounds 6 and 7 were assayed at 2.5 μM and 10 μM con-
centrations in cultures of IL-1β-stimulated chondrocytes.
The cytotoxic effect of the tested compounds was evalu-
ated by means of a cell viability test based on the cleavage of
3-IJ4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) by mitochondrial dehydrogenases of metabolically
active cells.24 The results showed that the compounds under
study did not affect the ability of chondrocytes to metabolize
tetrazolium salts, thus demonstrating that they do not inter-
fere with cell viability (data not shown).
2.1.1 Determination of NF-kB levels. The treatment of
human chondrocytes with IL-1β (10 ng ml−1) increased NF-kB
levels (measured as NF-kB p65 subunit levels), compared to
untreated cells. IL-1β-stimulated chondrocytes were then
treated with compounds 6a–d and 7a–d and the levels of NF-
kB were measured after 72 h. All compounds reduced NF-kB
levels at both 2.5 μM and 10 μM concentrations, compared to
cells treated only with IL-1β (Fig. 3). The best results were
obtained with both 5-IJ3-phenoxybenzylidene) substituted
derivatives 6b and 7b, which reduced NF-kB to the levels of
the control, at both concentrations (Fig. 3). The correspond-
ing 5-IJ4-phenoxybenzylidene) analogues (6a and 7a) displayed
lower efficacy. In particular, 5-ij(4-phenoxybenzylidene)-4-oxo-
2-thioxothiazolidin-3-yl]acetic acid (6a) restored the NF-kB
levels of the untreated cells only at 10 μM, whereas at lower
concentration it provided about 50% reduction of NF-kB
It appeared to be of interest that, similarly to compounds
5, most selected derivatives 6 and 7 had been shown to be
potent ARIs, with IC50 values ranging from 0.11 μM to 2.94 μM,
as we reported in a recent article.21 Moreover, their ALR2
inhibitory effectiveness had been shown to be generally higher
than that of the corresponding 2,4-thiazolidinediones.21–23
Both compounds 6 and 7 were synthesised in high yields
by the Knoevenagel condensation between IJ4-oxo-2-thio-
xothiazolidin-3-yl)acetic acid or 4-oxo-2-thioxothiazolidine
levels
compared
to
IL-1β-treated
cells
(Fig.
3).
N-Unsubstituted analogue 7a was not able to restore the NF-
kB levels of the control, although it possessed moderate activ-
ity compared to the IL-1β-treated cells at both concentrations.
Benzyloxybenzylidene analogues (6c, 6d, and 7c) generally
caused a more moderate decrease of NF-kB levels than
Scheme 1
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Fig. 3 Effects of 6a–d and 7a–d on NF-kB levels induced by IL-1β on
human chondrocytes. a) Representative blot. b) Nuclear factor-kappa
Fig. 4 Effects of 6a–d and 7a–d on ICAM-1 expression. a) Representa-
tive blot. b) ICAM-1 expression in IL-1β-stimulated human articular
chondrocytes 72 h after the addition of compounds 6a–d and 7a–d at
2.5 μM and 10 μM concentrations, determined by Western blot analysis.
The data show the relative expression (mean SEM) of ICAM-1, calcu-
lated as arbitrary densitometric units (A.D.U.), from three independent
experiments. *P < 0.05, compared with respective IL-1β values.
B
(NF-kB) levels in cultures of IL-1β-stimulated human articular
chondrocytes 72 h after the addition of compounds 6a–d and 7a–d at
2.5 μM and 10 μM concentrations, determined by Western blot analysis.
The data show the relative expression (mean SEM) of NF-kB, calculated
as arbitrary densitometric units (A.D.U.), from three independent experi-
ments. *P < 0.05, compared with respective IL-1β values.
phenoxy substituted analogues (6a, 6b, 7a, and 7b), with the
exception of compound 7d which provided effects similar to
those of 3-phenoxy homologue 7b.
NOS. In human chondrocytes treated with IL-1β, a noticeably
increased production of NO was observed (Fig. 5).
All compounds 6a–d and 7a–d exhibited a significant
The significant ability of compounds 6b, 7b and 7d to
reduce NF-kB levels in IL-1β-activated human chondrocytes
appears to be of interest because NF-kB activation is an
upstream event in the inflammatory cascade. In fact, the
reduction of NF-kB-dependent gene transcription can lead to
decreased levels of other proinflammatory mediators, such as
cytokines and diverse enzymes (such as MMPs and iNOS),
thus interfering with downstream signaling components cru-
cial for tissue inflammatory response and subsequent degen-
erative processes.
dose-dependent
capability
to
reduce
NO
release,
N-unsubstituted 4-oxo-2-thioxothiazolidines 7 being generally
more effective than the corresponding acetic acid derivatives
6. Once again, compounds 6b, 7b and 7d showed the best
activity, almost restoring the NO levels of the untreated cells
at both tested concentrations. Analogously to the observed
reduction of ICAM-1 expression, this effect could be corre-
lated to the capability of reducing NF-kB levels, being a con-
sequence of lower NF-kB-induced iNOS expression.
2.1.2 Determination of ICAM-1 levels. Resting
chondrocytes did not express ICAM-1, whereas their incuba-
tion with IL-1β induced marked ICAM-1 expression. The
treatment of the IL-1β-stimulated chondrocytes with all com-
pounds 6a–d and 7a–d, at both 2.5 μM and 10 μM concentra-
tions, reduced ICAM-1 expression (Fig. 4). Compounds 6b, 7b
and 7d exhibited the greatest potency, providing at least 50%
reduction of ICAM-1 levels at both tested concentrations, and
this finding may be correlated to their significant capability
of counteracting NF-kB production. Analogues 6a and 6d pro-
duced the same effect only at 10 μM concentration, whereas
6c, 7a and 7c, which were only moderately capable of reduc-
ing NF-kB levels, showed poor activity in this assay (Fig. 4).
2.1.3 Determination of nitrite levels. NO production was
measured as the amount of nitrite released by the cells in the
culture medium, determined by using the spectrophotomet-
ric method based on Griess reaction.25 NO production by
chondrocytes under basal conditions (untreated cells) was
very low and mainly due to the activity of the constitutive
Fig.
5 Determination of NO levels. NO production (measured as
nitrite, μM) (means SEM) in the culture medium of IL-1β-stimulated
articular chondrocytes 72 h after the addition of compounds 6a–d and
7a–d at 2.5 μM and 10 μM concentrations. *Significantly different from
IL-1β-treated samples (P < 0.05).
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Table 1 Antioxidant activity of compounds 6a–d and 7a–d
However, the reduction of NO release provoked by the
other tested analogues was also appreciable, especially at
10 μM (Fig. 5), thus suggesting that different factors other
than NF-kB activation might be involved in determining
NO production.
2.1.4 Determination of GAG levels. The treatment with
IL-1β induced a marked reduction in the GAG amount pro-
duced by chondrocytes. Conversely, the tested com-
pounds prevented the depletion of GAGs induced by IL-1β in
a dose-dependent manner. In particular, at 10 μM concentra-
tion, they restored GAG production to almost the levels of
the control, with the exception of compound 7b which was
not able to provide high GAG levels (Fig. 6). It may be
hypothesised that the behaviour of compound 7b is related
to its poor ability to inhibit MMP-13 and to its irrelevant anti-
oxidant capability (see below). In fact, MMP-13 activity is
strongly related to the baseline turnover of GAGs.
Compounds
ORAC unit (TE/μM)a
6a
6b
6c
6d
7a
7b
7c
7d
0.76 0.06*
0.61 0.10*
1.078 0.30*
0.65 0.09*
0.52 0.07*
0.39 0.03*
0.60 0.10*
0.63 0.09*
a
The ORAC values are reported as ORAC unit, corresponding to
μmol of Trolox equivalents (TE) per μM of sample. Reported values
are the means SEM (n = 3). *Significantly different at P < 0.05
compared to Trolox.
susceptible to the inhibitory effect of all compounds (except
7b) than MMP-3. In particular, 5-IJ4-benzyloxybenzylidene)
substituted analogues 6c and 7c were shown to be the most
selective MMP-13 inhibitors in the respective series of acetic
2.2 Radical scavenging activity
acid derivatives
6
and N-unsubstituted 4-oxo-2-thio-
ORAC assay is an established in vitro method used to quan-
tify the antioxidant effectiveness of compounds. The assay
measures the reduction of fluorescence resulting from the
oxidative degradation of fluorescein by the free radical-
xothiazolidines 7. Compound 7c was also the most effective
MMP-13 inhibitor among all tested analogues 6 and 7, with a
submicromolar IC50 value (Table 2).
The replacement of the 4-benzyloxy group (6c, 7c) with a
phenoxy one (6a, 7a) led to a significant increase in MMP-3
inhibitory activity and, consequently, a considerable reduc-
tion in selectivity (Table 2). A similar effect was observed by
displacing the benzyloxy substituent from the para to the
meta position of the 5-benzylidene ring. In particular,
3-benzyloxy substituted derivative 6d was the most potent
inhibitor of both MMP-3 and MMP-13 among the tested com-
pounds, displaying scarce selectivity (Table 2). In addition,
the removal of the terminal phenyl ring from 5-IJ3/4-
benzyloxybenzylidene) substituted derivatives led to 5-IJ3/4-
methoxybenzylidene) substituted analogues which showed a
marked decrease in the inhibitory potency, especially towards
MMP-13 (data not shown).
generator
2,2′-azobisIJ2-amidino-propane)dihydrochloride
(AAPH), using Trolox as a standard. Both series of com-
pounds 6a–d and 7a–d exhibited from moderate to marked
antioxidant activity, being able to delay the reduction of fluo-
rescence of a fluorescein–AAPH solution (Table 1). In particu-
lar, compound 6c showed excellent antioxidant effectiveness,
producing the same effect of the reference Trolox (Table 1).
2.3 Inhibition of matrix metalloproteinases MMP-3 and
MMP-13
The tested compounds inhibited the activity of both MMP-3
and MMP-13 in vitro, with IC50 values generally in the low
micromolar range. MMP-13 was observed to be more
On the whole, the 5-arylidene moiety was able to apprecia-
bly modulate the MMP inhibitory activity, whereas the influ-
ence exerted by the acetic acid residue on N-3 (compounds
Table 2 In vitro inhibitory activity of compounds 6a–d and 7a–d towards
MMP-3, MMP-13 and ALR2
IC50 (μM)
Compounds
MMP-3
MMP-13
ALR2a
6a
6b
6c
6d
7a
7b
7c
7d
8.96 0.9
14.2 0.3
58.5 0.5
4.19 0.09
48.40 1.6
17.50 1.1
77.43 2.1
n.a.
3.8 0.6
4.7 0.8
0.11 (0.07–0.16)
0.41 (0.14–1.22)
0.19 (0.13–0.29)
0.13 (0.09–0.18)
1.10 (0.70–1.71)
2.94 (1.90–4.56)
7% (11 μM)
6.97 0.07
1.59 0.6
3.99 0.5
55.27 1.2
0.64 0.2
5.89 0.7
Fig. 6 Determination of GAG release. GAG release (μg ml−1) in the
culture medium of IL-1β-stimulated articular chondrocytes 72 h after
the addition of compounds 6a–d and 7a–d at 2.5 μM and 10 μM con-
centrations. *Significantly different from IL-1β-treated samples (P <
0.05).
1.08 (0.73–1.70)
Representative results of at least 3 independent experiments are
reported. IC50 (μM) (95% C.L.) or
%
inhibition at the given
concentration. n.a = not active.a Ref. 21.
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6a–d) did not appear to be univocal. In fact, its removal (com-
pounds 7a–d) led to generally less active MMP-3 inhibitors
with different potencies against MMP-13 (Table 2).
capability of reducing NF-kB levels. This suggested that 6b,
7b and 7d might also inhibit NF-kB activation through differ-
ent mechanisms involved in the complex IL-1β-induced NF-
kB activation pathways, besides via ALR2 inhibition.
Compounds 6 and 7, with the exception of 7b, were also
able to oppose or mitigate catabolic events triggered by IL-1β
stimulation, in particular by improving GAG production by
chondrocytes and contributing to restore a normal PG syn-
thesis. In addition, they were shown to inhibit MMP-3 and
MMP-13, two enzymes critically involved in the loss of carti-
lage integrity in OA.
Finally, comparing the MMP-13 inhibitory activity of
2-thioxo-4-thiazolidinone derivatives 6b and 6c (IC50 = 4.7 μM
and 6.97 μM, respectively) with those of the corresponding
2,4-thiazolidinedione counterparts (IC50 = 11.1 μM and 22.3 μM,
respectively)19 highlighted that the replacement of the car-
bonyl group in position 2 of the thiazolidinedione ring with
the bioisostere thiocarbonyl one provided a 2- to 3-fold gain
in potency.
These findings contribute to extend our knowledge on the
biological profile of the 4-thiazolidinone class of ARIs and to
delineate their potential as anti-inflammatory agents. On the
whole, the multiple anti-inflammatory/antidegenerative pro-
file of 2-thioxo-4-thiazolidinones 6 and 7 along with their
marked ALR2 inhibitory effectiveness make them interesting
cell-permeable molecules that can be assumed as lead com-
pounds in the search for novel agents targeting inflammatory
diseases.
3. Conclusions
In the context of the search for new anti-inflammatory
agents, it is desirable to identify agents which are able to
block or reduce the actions of mediators involved in oxida-
tive/inflammatory cascade and, at the same time, to protect
tissues against degenerative damage.
The necessity to develop new agents able to target both
inflammatory and degenerative events is also emphasized by
considering that the effects of classical non-steroidal anti-
inflammatory drugs (NSAIDs) on cartilage metabolism are
still debated. Beneficial actions on the cartilage are not
established for most NSAIDs, whereas some findings
suggested that a certain number of these drugs might accel-
erate joint damage.26,27
4. Experimental section
4.1 Human articular chondrocyte culture
Normal human articular cartilage was obtained at replace-
ment surgery from some patients with femoral neck acciden-
tal fractures whose informed consent was obtained under an
approved Institutional Review Board protocol.28
In the present work, IJ5-arylidene-4-oxo-2-thioxothiazolidin-
3-yl)acetic acids (6a–d) and 5-arylidene-4-oxo-2-thio-
xothiazolidines (7a–d) were shown to control certain key
inflammatory events induced by IL-1β in human
chondrocytes. Among them, ij5-IJ3-phenoxybenzylidene)]
The isolation procedure was conducted under antiseptic
conditions. The cartilage was cut into small fragments and
carefully washed with Dulbecco's modified Eagle's medium
(DMEM) culture medium containing NaHCO3, 25 mM Hepes,
1 mM sodium pyruvate, 50 mg ml−1 gentamycin, 100 U ml−1
penicillin, 100 mg ml−1 streptomycin and 2.5 mg ml−1
amphotericin B. Chondrocytes were isolated through three
sequential passages of enzymatic digestion of the extracellu-
lar matrix: incubation with 0.1% hyaluronidase type III (1 mg
ml−1 for 100 mg of cartilage), for 30 min at 37 °C; incubation
with 0.5% pronase type XIV (5 mg ml−1 for 100 mg of carti-
lage), for 60 min at 37 °C; finally incubation with 0.2% colla-
genase type IA (2 mg ml−1 for 100 mg of cartilage), for 45 min
at 37 °C. The obtained cellular suspension was filtered (filters
from 100 and 70 mm) to eliminate the residues of the diges-
tion, cellular aggregates. Freshly isolated chondrocytes were
seeded into the monolayer culture at a cell density of 2 × 105
cells and cultured in 1 ml of DMEM supplemented with 10%
foetal bovine serum (FCS) at 37 °C in 5% CO2/95% air. Con-
fluent chondrocytes of primary culture were used for all the
experiments: the tested compounds were assayed and
dissolved in DMSO, appropriately diluted in DMEM and
dispensed to the wells treated as follows: control, IL-1β
(10 ng ml−1), 6, 7a–d (10, 2.5 μM) + IL-1β (10 ng ml−1). After
72 h, the supernatants of the cartilage culture were collected
for different assays.
substituted
derivatives
6b
and
7b
and
ij5-IJ3-
benzyloxybenzylidene)] analogue 7d exhibited the most prom-
ising anti-inflammatory profile, being able to reduce both
NF-kB and NO levels to those of the IL-1β-untreated control
and to lower ICAM-1 expression significantly. The observed
capability of reducing the levels of NF-kB is an attractive
aspect, because this ubiquitously expressed nuclear transcrip-
tion factor is critically involved in inflammation and can play
a crucial role in the development and progression of many
human diseases, such as atherosclerosis, diabetes, cancer,
and arthritis. The comparable activity trend of compounds 6
and 7 in reducing NF-kB levels, ICAM-1 expression and NO
production highlights that these events are closely correlated
in IL-1β-stimulated chondrocytes. In particular, it is likely
that the appreciable ability of derivatives 6b, 7b and 7d to
reduce ICAM-1 and NO amounts is a consequence of the
reduced NF-kB levels. As described above, NF-kB activation
can be promoted by both ROS and ALR2, through lipid perox-
idation, the subsequent production of GS-DHN and the
downstream PKC/PI3K/IKK cascade. Compounds 6b, 7b and
7d are potent ARIs (Table 2); therefore, they may reduce
ALR2-mediated NF-kB activation. However, we found that
compounds with more marked ALR2 inhibitory and antioxi-
dant properties, such as 6a, 6c and 6d, showed lower
4.1.1 Cell viability assay. The cytotoxic effect of the tested
substances was evaluated by a cell viability test based on the
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cleavage of 3-IJ4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium
bromide (MTT) by mitochondrial dehydrogenases of metabol-
ically active cells.24
at 37 °C at pH 7.0 with Trolox (12.5 μM) as control standard
and phosphate buffer as blank. Compounds 6, 7a–d were
appropriately diluted with DMSO/buffer (1 : 10) prior to analy-
sis. The fluorescein fluorescence was recorded every 2 min
after addition of AAPH. All measurements were expressed rel-
ative to the initial reading. One blank, one standard, and a
maximum of 10 samples were analyzed at the same time.
Each measure was repeated at least three times. The ORAC
value refers to the net protection area under the quenching
curve of fluorescein in the presence of an antioxidant. The
final results (ORAC value) were calculated and expressed
using Trolox equivalents (TE) for μM of sample (TE/μM):
4.1.2 Western blot analysis: NF-kB and ICAM-1.
Chondrocytes were washed twice with ice-cold PBS and col-
lected with lysis buffer (10 mM tris-HCl plus 10 mM KCl, 2
mM MgCl2, 0.6 mM PMSF and 1% SDS, pH 7.4). After cooling
for 10 min at 0 °C, cells were sonicated. Equal amounts of
total protein (60 μg per lane) were separated by 4–12% Novex
bis–tris gel electrophoresis (NuPAGE, Invitrogen, Italy) and
transferred into nitrocellulose membranes (Invitrogen, Italy)
in a wet system. Protein transfer was verified by staining the
nitrocellulose membranes with Ponceau S. Membranes were
blocked with Tris buffered saline containing 0.01% Tween 20
(TBST) and 5% nonfat milk, washed briefly, incubated with
primary antibodies at 4 °C overnight, and then incubated
with corresponding HRP-conjugated secondary antibodies for
1 h at room temperature. Mouse monoclonal anti-ICAM-1
(1H4: sc-51632, Santa Cruz Biotechnology, Milan, Italy), anti-
NF-kB p65 (ab95020, Abcam, Prodotti Gianni, Milan, Italy)
and anti-α-tubulin (Sigma, Milan, Italy) antibodies were
diluted in TBST at 1 : 200, 1 : 150 and 1 : 5000, respectively.
Protein bands were visualized by incubating membranes with
the enhanced chemiluminescence detection Supersignal West
Pico Chemiluminescent Substrate (Pierce Chemical Co.,
Rockford, IL) before exposure to an X-ray film. Chemilumi-
nescence signals were densitometrically analyzed using
Image-Pro Plus version 6.0 software (Media Cybernetics Inc.,
Bethesda, MD) and their relative density was calculated based
on the density of the α-tubulin bands in each sample. Values
were expressed as arbitrary densitometric units (A.D.U.) cor-
responding to signal intensity.
ORAC units (TE/μM) = K(Ssample – Sblank)/(STrolox − Sblank
)
where K is a sample dilution factor and S is the area under
the fluorescence decay curve of the sample, Trolox or blank.
This area was calculated with Origin®7 (OriginLab Corpora-
tion, Northampton, USA).
4.3 MMP-13/3 fluorimetric assay
The compounds were evaluated for their ability to inhibit the
hydrolysis of fluorescence-quenched peptide substrate Mca-
Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (Vinci Biochem s.r.l.). The
MMP-13 assays were performed in 50 mM HEPES buffer
containing 5 mM CaCl2, 0.1 mM ZnCl2, and 0.05% Brij-35, at
pH 7, using 10 nM proteolytic enzyme (catalytic domains of
MMP-13) (Vinci Biochem s.r.l.) and 350 nM peptide. To pre-
pare MMP-3 buffer, HEPES was replaced with 50 mM MES, at
pH 6.
The enzyme was incubated at 25 °C with increasing con-
centration of the inhibitor and the fluorescence (excitationmax
328 nm; emissionmax 393 nm) was measured for 3 min after
the addition of the substrate using a VICTOR Wallac 1420
Multilabel Counters fluorimeter (Perkin Elmer, USA). Fitting
of rates as a function of inhibitor concentration provided
IC50 values.31
4.1.3 Determination of nitrite. Nitrite was determined by
adding 100 μl of Griess reagent (1% sulphanylamide and
0.1% naphthylethylenediamine dihydrochloride in 5% of
hydrochloric acid) to 100 μl of samples.25 The optical density
at λ = 540 nm was measured using a microtiter plate reader.
Nitrite concentrations were calculated by comparison with
respective optical densities of standard solutions of sodium
nitrite standard curve (0–120 μM).
4.4 Statistical analysis
All data are presented as mean SEM of at least three sepa-
rate experiments. The statistical analysis was performed by
using one-way ANOVA followed by Dunnett's post-hoc test for
multiple comparisons with control. All statistical analyses
were performed using the statistical software package
SYSTAT, version 9 (Systat Inc., Evanston, IL, USA). The
unpaired Student's t-test was used to compare two different
groups. A P value of <0.05 was considered to be statistically
significant.
4.1.4 Determination of GAGs. The level of GAGs, an index
of cartilage damage, was measured by spectrophotometry
with a solution of 1,9-dimethylmethylene blue at λ = 535 nm
using the method of Farndale et al.29 The amount of GAGs
was calculated from a standard curve (100–500 μg ml−1)
obtained for shark chondroitin sulphate C, derived from
shark cartilage.
4.2 Oxygen radical absorbance capacity (ORAC) assays
The ORAC assay was performed as previously reported by Cao
et al.30 The measurements were carried out using a VICTOR
Wallac 1420 Multilabel Counters fluorimeter (Perkin Elmer,
USA) with fluorescence filter (excitation 540 nm, emission
570 nm). Fluorescein (10 nM) was the fluorescence probe and
target molecule for free radical attack from AAPH (100 mM)
as the peroxyl radical generator. The reaction was conducted
Abbreviation list
ALR2
ARIs
COX
GAG
Aldose reductase
Aldose reductase inhibitors
Cyclooxygenase
Glycosaminoglycan
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MedChemComm
GS-DHN Glutathionyl-1,4-dihydroxynonene
GS-HNE Glutathione-4-hydroxy-trans-2-nonenal
M. Stefek, Transworld Research Network, Kerala, India,
2010, pp. 219–245.
HNE
ICAM
IKK
iNOS
MAPK
MMPs
NF-kB
PI3K
PG
4-Hydroxy-trans-2-nonenal
Intercellular adhesion molecule
Inhibitor-kappa B kinase
Inducible nitric oxide synthase
Mitogen activated protein kinase
Matrix metalloproteinases
Nuclear transcription factor-kappa B
Phosphoinositide 3-kinase
Proteoglycan
15 S. K. Srivastava, U. C. S. Yadav, A. M. Reddy, A. Saxena, R.
Tammali, M. Shoeb, N. H. Ansari, A. Bhatnagar, M. J.
Petrash, S. Srivastava and K. V. Ramana, Chem.-Biol.
Interact., 2011, 191, 330–338.
16 S. Pandey, S. K. Srivastava and K. V. Ramana, Expert Opin.
Invest. Drugs, 2012, 21, 329–339.
17 R. Ottanà, R. Maccari, M. L. Barreca, G. Bruno, A. Rotondo,
A. Rossi, G. Chiricosta, R. Di Paola, L. Sautebin, S. Cuzzocrea
and M. G. Vigorita, Bioorg. Med. Chem., 2005, 13, 4243–4252.
18 R. Ottanà, R. Maccari, R. Ciurleo, M. G. Vigorita, A. M.
Panico, V. Cardile, F. Garufi and S. Ronsisvalle, Bioorg. Med.
Chem., 2007, 15, 7618–7625.
PKC
RNS
ROS
Protein kinase C
Reactive nitrogen species
Reactive oxygen species
TNF-α
Tumor necrosis factor α
19 A. M. Panico, R. Maccari, V. Cardile, L. Crascì, S. Ronsisvalle
and R. Ottanà, Med. Chem., 2013, 9, 84–90.
20 R. Maccari, R. M. Vitale, R. Ottanà, M. Rocchiccioli, A.
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Mura and R. Ottanà, Bioorg. Med. Chem. Lett., 2011, 21,
200–203.
22 G. Bruno, L. Costantino, C. Curinga, R. Maccari, F.
Monforte, F. Nicolò, R. Ottanà and M. G. Vigorita, Bioorg.
Med. Chem., 2002, 10, 1077–1084.
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