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Interleukin-1β-Induced Extracellular Matrix Degradation and Glycosaminoglycan Release Is Inhibited by Curcumin in an Explant Model of Cartilage Inflammation



Osteoarthritis (OA) is a degenerative and inflammatory disease of synovial joints that is characterized by the loss of articular cartilage, for which there is increasing interest in natural remedies. Curcumin (diferuloylmethane) is the main polyphenol in the spice turmeric, derived from rhizomes of the plant Curcuma longa. Curcumin has potent chemopreventive properties and has been shown to inhibit nuclear factor kappaB-mediated inflammatory signaling in many cell types, including chondrocytes. In this study, normal articular cartilage was harvested from metacarpophalangeal and metatarsophalangeal joints of eight horses, euthanized for reasons other than research purposes, to establish an explant model mimicking the inflammatory events that occur in OA. Initially, cartilage explants (N= 8) were stimulated with increasing concentrations of the proinflammatory cytokine IL-1beta to select effective doses for inducing cartilage degeneration in the explant model. Separate cartilage explants were then cotreated with IL-1beta at either 10 ng/mL (n= 3) or 25 ng/mL (n= 3) and curcumin (0.1 micromol/L, 0.5 micromol/L, 1 micromol/L, 10 micromol/L, and 100 micromol/L). After 5 days, the percentage of glycosaminoglycan (GAG) release from the explants was assessed using a dimethylmethylene blue colorimetric assay. Curcumin (100 micromol/L) significantly reduced IL-1beta-stimulated GAG release in the explants by an average of 20% at 10 ng/mL and 27% at 25 ng/mL back to unstimulated control levels (P < 0.001). Our results suggest that this explant model effectively simulates the proinflammatory cytokine-mediated release of articular cartilage components seen in OA. Furthermore, the evidence suggests that the inflammatory cartilage explant model is useful for studying the effects of curcumin on inflammatory pathways and gene expression in IL-1beta-stimulated chondrocytes.
Interleukin-1β–Induced Extracellular Matrix
Degradation and Glycosaminoglycan Release
Is Inhibited by Curcumin in an Explant
Model of Cartilage Inflammation
Abigail L. Clutterbuck,aAli Mobasheri,aMehdi Shakibaei,b
David Allaway,cand Pat Harrisc
aDivision of Veterinary Medicine, School of Veterinary Medicine and Science, University
of Nottingham, Leicestershire, United Kingdom
bInstitute of Anatomy, Ludwig-Maximilians-University Munich, Munich, Germany
cWALTHAM Centre for Pet Nutrition, Waltham-on-the-Wolds, Melton Mowbray,
United Kingdom
Osteoarthritis (OA) is a degenerative and inflammatory disease of synovial joints that
is characterized by the loss of articular cartilage, for which there is increasing interest
in natural remedies. Curcumin (diferuloylmethane) is the main polyphenol in the spice
turmeric, derived from rhizomes of the plant Curcuma longa. Curcumin has potent
chemopreventive properties and has been shown to inhibit nuclear factor κB-mediated
inflammatory signaling in many cell types, including chondrocytes. In this study, normal
articular cartilage was harvested from metacarpophalangeal and metatarsophalangeal
joints of eight horses, euthanized for reasons other than research purposes, to estab-
lish an explant model mimicking the inflammatory events that occur in OA. Initially,
cartilage explants (N=8) were stimulated with increasing concentrations of the proin-
flammatory cytokine IL-1βto select effective doses for inducing cartilage degeneration
in the explant model. Separate cartilage explants were then cotreated with IL-1βat ei-
ther 10 ng/mL (n=3) or 25 ng/mL (n=3)andcurcumin(0.1μmol/L, 0.5 μmol/L, 1 μmol/L,
10 μmol/L, and 100 μmol/L). After 5 days, the percentage of glycosaminoglycan (GAG)
release from the explants was assessed using a dimethylmethylene blue colorimetric
assay. Curcumin (100 μmol/L) significantly reduced IL-1β-stimulated GAG release in the
explants by an average of 20% at 10 ng/mL and 27% at 25 ng/mL back to unstimulated
control levels (P<0.001). Our results suggest that this explant model effectively simu-
lates the proinflammatory cytokine-mediated release of articular car tilage components
seen in OA. Furthermore, the evidence suggests that the inflammatory cartilage explant
model is useful for studying the effects of curcumin on inflammatory pathways and gene
expression in IL-1β-stimulated chondrocytes.
Key words: osteoarthritis; cartilage; inflammation; polyphenol; equine; glycosamino-
glycan; cytokine; curcumin
Address for correspondence: Abigail L. Clutterbuck, B.Sc., M.Sc., Di-
vision of Veterinary Medicine, School of Veterinary Medicine and Sci-
ence, University of Nottingham, Sutton Bonington Campus, Sutton Bon-
ington, Leicestershire, LE12 5RD, UK. Voice: +44-01159516469; fax:
Articular cartilage covers the end surfaces of
long bones in synovial joints to allow smooth
frictionless movement and cushioning.1Carti-
lage is composed of a dense extracellular matrix
(ECM), water, and chondrocytes. The ECM
confers strength and compressive resistance to
loads placed upon the joint as well as having a
Natural Compounds and Their Role in Apoptotic Cell Signaling Pathways: Ann. N.Y. Acad. Sci. 1171: 428–435 (2009).
doi: 10.1111/j.1749-6632.2009.04687.x c
2009 New York Academy of Sciences.
et al.:
Curcumin Inhibits IL-1
–Stimulated GAG Release
regulatory role. Chondrocytes are the only cell
type present in the ECM and synthesize the
components of this highly adapted structure.
Water is attracted to the negatively charged
proteoglycans in the ECM, causing them to
swell, thus providing elasticity to the joint while
the collagen network, in which the proteo-
glycans are trapped, confers strength. These
proteoglycans are made up of a core protein
to which sulfated glycosaminoglycans (GAGs),
such as chondroitin sulfate and keratin sulfate,
are covalently attached.
Osteoarthritis (OA) is a degenerative disease
which affects humans and companion animals
with significant economic and welfare conse-
quences. It is characterized by articular car-
tilage destruction where components of the
ECM are degraded by proteolytic enzymes,
causing reduced strength and elasticity in the
joint.2In agreement with this, increased levels
of cartilage constituents, such as GAGs, have
been found in the synovial fluid of OA horse
joints compared to normal joints.3Although
OA is thought to be a consequence of aging,
it can be initiated by trauma to the joint ei-
ther through poor conformation or abnormal
loading of the joint.
Inflammation is a key contributory factor in
the pathogenesis of OA. Catabolic events re-
sponsible for cartilage matrix degradation com-
prise the release of catabolic cytokines, such
as IL-1β, IL-6, and tumor necrosis factor-α
(TNF-α), which in turn induce the release
of matrix-degrading enzymes, such as matrix
metalloproteinases (MMPs) and aggrecanases
(ADAM-TS4, ADAM-TS11), by chondrocytes
and by synoviocytes in early OA.4Cartilage
sections from OA patients positively stain for
inflammatory mediators, such as the cytokines,
IL-1β,andTNF-α, unlike sections from healthy
cartilage.5This is supported by work from the
equine field, which has demonstrated a positive
correlation between grade of OA and percent-
age of chondrocytes positively staining for IL-
1βin cartilage from equine joints.6In addition,
cells cultured from synovial tissue of human
patients with OA produce IL-1βand TNF-α,
most significantly in the early stages of OA.7
These studies all contribute to the evidence for
cytokine involvement in OA.
Inflammatory cytokine gene expression is
upregulated in OA cartilage and synovium.
The resultant cytokines, in turn, may perpetu-
ate inflammatory cytokine gene expression, as
has been demonstrated in equine chondrocytes
stimulated with IL-1β.8Proinflammatory cy-
tokines can reduce normal cartilage synthesis as
well as promote cartilage degradation through
the expression of the proteolytic MMPs and
aggrecanases.9This effect is facilitated through
various transcription factors, such as nuclear
factor-κB (NF-κB).10 In fact, NF-κB has been
described as the link between inflammation and
joint hyperplasia in arthritis.11 Thus,inanin-
flammatory state, cytokine activity can activate
transcription factors, which, in turn, can reduce
cartilage synthesis as well as promote cartilage
Thus far, attempts by the pharmaceutical in-
dustry have tended to target the signs or symp-
toms of OA rather than halting or reversing
the progression of the disease. Synthetic anti-
inflammatory and analgesic treatments are of-
ten costly and seek to reduce the inflammation
as well as the pain associated with the condition
but can have deleterious side effects. Thus, re-
searchers have increasingly turned to or are
increasingly looking at natural plant-derived
remedies as potentially cheaper and safer nat-
ural alternatives that may modify the disease
Curcumin (diferuloylmethane) is the main
polyphenol found in the spice turmeric, de-
rived from rhizomes of the plant Curcuma longa.
Botanicals, such as curcumin, have been shown
to have potent anti-inflammatory and anti-
catabolic effects on a number of cell types
through their effects on the NF-κBpath-
way12,13 and AP-1 transcription factor acti-
vation.10 These effects include reducing the
IL-1β-mediated upregulation of NF-κB targets,
such as MMP-1, MMP-3, MMP-9, and cyclo-
oxygenase-2 (COX-2),1416 as well as reducing
chondrocyte apoptosis.17 In addition to these
Annals of the New York Academy of Sciences
anticatabolic effects, curcumin may concur-
rently support anabolism, for example, by re-
versing the cytokine-induced suppression of
collagen type II and β1-integrin synthesis.15,17
Curcumin also exerts inhibitory effects on the
JNK mitogen-activated protein (MAP) kinase
pathway leading to suppression of MMP-3
and MMP-13 mRNA upregulation in human
OA and bovine articular chondrocytes.10 Cur-
cumin is currently being investigated for its
therapeutic potential in OA as it reverses in-
flammation and apoptotic signaling in chon-
drocytes in vitro. However, further evaluation in
explant models mimicking OA cartilage degra-
dation is recommended before embarking on
the direct treatment of patients. Explant cul-
ture closely resembles the physiological envi-
ronment of cartilage without a change in the
phenotype of the chondrocytes, which can oc-
cur in primary culture.
Here we hypothesized that curcumin would
reduce the inflammatory cytokine-driven re-
lease of sulfated GAGs from cartilage explants.
Therefore, we established an in vitro model
of cartilage inflammation and evaluated the
potential anti-inflammatory influence of cur-
cumin on explants within this system.
Materials and Methods
Cartilage Explant Culture
Normal articular cartilage was obtained
from the weight-bearing regions of the meta-
carpophalangeal and metatarsophalangeal
joints of eight horses of mixed breed and un-
known age, euthanized for purposes other than
for research. Cartilage samples from the joints
of the same horse were combined during col-
lection, but explants from each individual ani-
mal were kept separate from the other animals
throughout the in vitro phase of this study. Car-
tilage shavings from the joints of each horse
were aseptically harvested into Dulbecco’s
Modified Eagle’s medium (DMEM) (Hyclone,
Fisher Scientific UK Ltd., Loughborough, UK)
containing 4% penicillin/streptomycin (Sigma
Aldrich, Gillingham, UK) before washing in
PBS with 10% penicillin/streptomycin on a
roller for 20 min. The cartilage shavings were
then cut into 3-mm disks with a sterile biopsy
punch, and in each case, three disks from the
same animal were placed into the wells of 24-
well plates containing 1 mL of DMEM sup-
plemented with 2% penicillin/streptomycin.
Plates were incubated at 37Cand5%CO
overnight. The following day, culture media
was removed from the wells and replaced with
fresh media before the experiments began.
Determination of Optimal
IL-1βDose Ranges
Cartilage disks were subjected to a range of
human recombinant IL-1β(Roche Diagnostics,
Ltd., West Sussex, UK) concentrations. Each
treatment used three wells of a 24-well plate,
and space constrictions resulted in different n
numbers for the various treatments. Control
wells (N=8) contained 1 mL of DMEM sup-
plemented with 2% penicillin/streptomycin.
IL-1βstocks were made in the following con-
centrations and added to each of the three wells
per horse: 0.5 ng/mL (n=5), 10 ng/mL (n=8),
17 ng/mL (n=4), 25 ng/mL (n=4). Explants
were then placed in an incubator at 37Cand
5% CO2for 5 days. Culture supernatants were
placed into individual Eppendorfs and stored
in a freezer at 20C before assaying. For ma-
trix GAG evaluation, the cartilage disks were
placed in Eppendorfs (one Eppendorf per well)
and subjected to a 16-h overnight digestion in
1 mL papain buffer (0.1 M sodium phosphate
buffer, 5 mmol/L N-acetyl cysteine, 5 mmol/L
EDTA) containing 1.5 mg/mL papain (Sigma
Curcumin Treatment
Cartilage from six of the horses was used
for curcumin treatment studies. Curcumin
(Sigma Aldrich) was made into a stock solu-
tion of 1 mmol/L from which the following
concentrations were prepared: 0.1 μmol/L,
et al.:
Curcumin Inhibits IL-1
–Stimulated GAG Release
0.5 μmol/L, 1 μmol/L, 10 μmol/L, and
100 μmol/L. Each treatment used three wells
of the plates of 2 x 24-well plates per horse.
The first plate contained the cartilage in cul-
ture media with the extracts to assess the ef-
fect of curcumin on “normal” cartilage. The
second plate contained the cartilage in culture
media cotreated with the extracts and either
10 ng/mL (n=3) or 25 ng/mL (n=3) hu-
man recombinant IL-1β(to induce cartilage
inflammation) and matrix degradation serving
as a negative control. These two concentra-
tions were used so that the effect of curcumin
could be assessed at different stages of the IL-1β
dose-response curve. Both plates contained the
culture media, which acted as the control, and
the base media for other treatments. Insulin-
like growth factor-1 (IGF-1) (Roche Diagnos-
tics) was selected as a positive control in both
plates at 10 ng/mL because of its known an-
tagonistic action on cytokine-induced proteo-
glycan release.18 Plates were incubated at 37C
and 5% CO2for 5 days. Supernatants were
placed into individual Eppendorfs and stored in
a freezer at 20C for subsequent assays within
7 days. The cartilage disks were removed from
the wells into Eppendorfs (one Eppendorf per
well) and were digested in 1 mL papain buffer
as described above.
Glycosaminoglycan Release Assay
Papain-digested cartilage explants and their
corresponding defrosted supernatants were as-
sayed in 96-well plates using the dimethyl-
methylene blue (DMMB) method as described
by Farndale et al.19 Briefly, each sample was
diluted in distilled water to a total volume of
40 μL per well, in duplicate wells on the plate.
Shark chondroitin sulfate (Sigma Aldrich) was
used as a standard (0–70 ng). DMMB solu-
tion (200 μL) was added to each well on the
plate before being immediately transferred to
a Multiskan Ascent Scanner (Thermo Labsys-
tems, Basingstroke, UK) using Ascent Software
(version 2.6, Thermo Labsystems). Total GAG
release was obtained from a spectrophotomet-
Figure 1. The percentage release of cartilage
glycosaminoglycans (GAGs) into culture medium in
response to various doses of the cytokine IL-1β. Sig-
nificance compared to IL-1βis indicated by ∗∗(
0.05) and ∗∗∗(
<0.001). Results of the dimethyl-
methylene blue assay showed that this response was
dose dependent and that all concentrations of IL-1β
significantly increased percentage GAG release com-
pared to control (
=8) levels [IL-1β0.5 ng/mL
<0.05), 10 ng/mL (
17 ng/mL (
<0.001) and 25 ng/mL (
<0.001)]. Bars represent SEM.
ric reading of the digested cartilage and its cor-
responding supernatant at 540 nm. The per-
centage GAG release was calculated by divid-
ing the supernatant value from the total GAG
release for each well. The results were statisti-
cally analyzed using a one-way ANOVA with
Tukey’s multiple comparison post hoc test us-
ing GraphPad Instat (version 3.05, GraphPad
Software, San Diego, CA) and graphically plot-
ted using GraphPad Prism (version 4, Graph-
Pad Software). Statistical significance was set
at P<0.05. Values are reported as means of
combined animals +SEM.
Testing the IL-1βDose Range
There was a significant increase in the per-
centage of GAGs released into the culture
media in response to all the IL-1βconcen-
trations tested compared to control values
Annals of the New York Academy of Sciences
(P<0.001). This response was dose depen-
dent (see Fig. 1). IL-1βsignificantly increased
percentage GAG release at IL-1β0.5 ng/mL
(n=5, P<0.05), 10 ng/mL (N=8, P<0.001),
17 ng/mL (n=4, P<0.001), and 25 ng/mL
(n=4, P<0.001) compared to control
(N=8) levels. IL-1βat 0.5 ng/mL failed to
consistently induce significant GAG release in
the individual animals, although it achieved
significance when animals were combined.
Therefore, 10 ng/mL IL-1βwas selected as
the minimum dose and 25 ng/mL IL-1βas the
subsequent assays.
Effects of Curcumin and IGF-I on
IL-1β-Stimulated GAG Release
Both IL-1βat 10 ng/mL and 25 ng/mL sig-
nificantly increased GAG release compared to
control levels (P<0.001) (see Fig. 2). IGF-1
alone had no effect on GAG release, but it
significantly reduced IL-1β-stimulated GAG
release down to near control levels at both
10 ng/mL IL-1β(P<0.01) and 25 ng/mL
IL-1β(P<0.001). Curcumin alone had no ef-
fect on the percentage of GAG release at any of
the doses tested. Curcumin, at concentrations
between 0.1 and 10 μmol/L, had no significant
effect on reducing GAG release from explants
cotreated with IL-1β. However, curcumin at
100 μmol/L significantly (P<0.001) reduced
the percentage of IL-1β-stimulated GAG re-
lease down to unstimulated control levels when
exposed to either 10 ng/mL or 25 ng/mL IL-
1β. This meant a reduction in GAG release
from the explants by an average of 20% at
10 ng/mL (P<0.001) and 27% at 25 ng/mL
(P<0.001). Although control levels of GAG re-
lease in individual animals varied, as would be
expected with cartilage samples from individu-
als of unknown age, breed, and background, ev-
ery animal displayed significant increased GAG
release in the presence of IL-1β.ThisIL-1β-
stimulated GAG release was always reduced to
the control level for each particular animal by
the addition of 100 μmol/L curcumin. This
Figure 2. The percentage release of cartilage
GAGs into culture medium at various curcumin con-
centrations, either in the absence of IL-1β(gray
columns) or in the presence of IL-1β(black columns)
at (A)10ng/mL(
=3) and (B)25ng/mL
=3). Significance compared to IL-1βis indicated
by (
<0.05) and ∗∗∗ (
<0.001). Control
indicates cartilage disks incubated in the culture
medium alone. Both IL-1βconcentrations signifi-
cantly increased GAG release compared to control
<0.001). Insulin-like growth factor-1 (IGF-1) alone
at 10 ng/mL had no effect on GAG release com-
pared to control but significantly reduced the IL-1β-
stimulated GAG release compared to IL-1βalone at
both 10 ng/mL (
<0.01) and 25 ng/mL
<0.001). Curcumin alone had no sig-
nificant effect on GAG release compared to control.
Curcumin at 100 μmol/L with both concentrations of
IL-1βsignificantly reduced GAG release compared to
IL-1βalone at 10 ng/mL (
<0.001) and 25 ng/mL
<0.001). Bars represent SEM.
suggests that results of this model are repro-
ducible in spite of the biological variation from
animal to animal.
et al.:
Curcumin Inhibits IL-1
–Stimulated GAG Release
Previous studies in our laboratories have
shown that 50 μmol/L curcumin exerts
potent anti-apoptotic and anticatabolic ef-
fects on IL-1β-stimulated cultures of artic-
ular chondrocytes. Curcumin has been re-
ported to protect human chondrocytes from
IL-1β-induced activation and nuclear translo-
cation of NF-κB.15 Thus it can also pro-
tect chrondocytes from the negative down-
stream effects of IL-1βsuch as inhibition of
collagen type II and β1-integrin expression
and up regulation of COX-2, MMP-9, and
MMP-3.15,20 Curcumin also inhibits IL-1β-
induced activation of caspase-3 and human
chondrocyte apoptosis.17 Studies by other in-
vestigators have shown that curcumin inhibits
MMP-1, MMP-3, MMP-13, ADAM-TS4, and
tissue inhibitor of metalloproteinase-3 (TIMP-
3) gene expression in chondrocytes stimulated
with IL-1β,TNF-α,andoncostatinM(OSM)
(a member of the IL-6 superfamily of pro-
inflammatory cytokines).10,2123 The aim of the
present study was to establish an in vitro explant
model of equine cartilage inflammation and
extend our knowledge of the biological actions
of curcumin by determining if it can inhibit IL-
1β-stimulated degradation and release of ECM
Using this culture system we have shown that
human recombinant IL-1βeffectively stimu-
lates sulfated GAG release from equine car-
tilage explants at 10 ng/mL and 25 ng/mL.
These observations concur with several stud-
ies using equine cartilage.24,25 Similar studies
using recombinant equine IL-1βhave shown
a more potent effect at lower IL-1βconcen-
trations26 and have reported a reduced effect
of human IL-1βon equine articular chondro-
cytes compared to equine IL-1β.27 However,
the significant effects observed in this study sug-
gest that human IL-1βis a suitable catabolic
agent in this model. Although species-specific
cytokines are more biologically relevant and
hence may be more appropriate for models
studying the pathogenesis of OA in a particular
species, this study supports the use of human
recombinant IL-1βfor high throughput inves-
tigation of anti-inflammatory botanicals, such
as curcumin, that might support joint health in
commercial availability of human IL-1βover
species-specific IL-1βare distinct advantages.
Thus, the significant increase in GAG re-
leased from cartilage explants in response to the
IL-1βconcentrations tested shows that IL-
1βeffectively mimicked the cytokine-induced
GAG loss observed in OA.
IGF-1 is a growth factor involved in carti-
lage anabolism and can counteract the degen-
erative effects of proinflammatory agents.18 In
this study we showed that 10 ng/mL IGF was
an effective positive control in reducing GAG
release stimulated by 10 ng/mL and 25 ng/mL
IL-1βwithout affecting cartilage GAG release
in the absence of IL-1β. This is in agreement
with previous studies that have found IGF-1
to counteract the IL-1α-orIL-1β-stimulated
proteoglycan release in porcine cartilage28 and
equine cartilage.29
The anti-inflammatory30 effects of curcumin
have generated increasing interest in its po-
tential for the treatment of inflammatory dis-
eases. Its suppressive effects on neutrophil and
MMP activation in chondrocyte cultures have
led to it being suggested as a treatment for
rheumatoid arthritis.14 In this study we ex-
amined the effects of curcumin on IL-1β-
stimulated and unstimulated equine cartilage.
We found that curcumin at concentrations be-
tween 0.1 μmol/L and 100 μmol/L had no ef-
fect on unstimulated cartilage, suggesting that
it has no gross effect on altering normal car-
tilage. However, curcumin at 100 μmol/L ef-
fectively reduced IL-1β-stimulated GAG loss
down to control levels in the presence of both
10 ng/mL and 25 ng/mL IL-1β. The reduced
GAG loss observed in our study may be at-
tributed to the inhibitory effect of curcumin
on the NF-κB inflammatory pathway and the
mediators of cartilage degradation, such as the
MMPs and ADAM-TSs. This effect could ini-
tially occur between the doses of 10 μmol/L
Annals of the New York Academy of Sciences
and 100 μmol/L used in this study, and we
are currently investigating whether the bene-
ficial effect in reducing GAG release is seen
within this concentration range. It is impor-
tant to emphasize that our observation of re-
duced GAG release from the explants in the
presence of higher concentrations of curcumin
may not be a specific biological effect of cur-
cumin. High concentrations of curcumin may,
in fact, be toxic to chondrocytes.31 Therefore,
any curcumin-induced cytotoxicity is bound to
interfere with proteoglycan metabolism in the
explant model and this may be reflected in
the results of the GAG assays. Consequently,
the observed reduction in GAG loss from the
explants in the presence of high concentra-
tions of curcumin may be a result of curcumin-
induced cell death, which would, in turn, in-
fluence the expression of matrix degrading
enzymes. Thus, chondrocyte viability testing
will be an essential prerequisite in future stud-
ies in order to determine the precise mode of
action of curcumin in the explant model.
Despite biological variation between ani-
mals, the reproducibility of the results suggests
that this model may be an effective in vitro sys-
tem for evaluating the potential beneficial ef-
fects of botanical extracts. These results suggest
that curcumin antagonizes GAG release in vitro.
However, further work is clearly needed to in-
vestigate the biological effects of curcumin at
higher concentrations in order to determine its
true efficacy and potential safety.
This work was supported by the Biotechno-
logy and Biological Sciences Research Coun-
cil (BBSRC) and the WALTHAM Centre for
Pet Nutrition (MARS) (BBSRC Grant No.
Conflicts of Interest
The authors declare no conflicts of interest.
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... The signs of disease progression include narrowing of the joint space, cartilage lesions and surface fibrillation, loss of PG and collagen contents and increases in the fluid content in cartilage, formation of osteophytes, possible ligamentous and meniscal lesions, cement line advancement, subchondral sclerosis and cysts, chondrocyte clustering, increased subchondral bone plate thickness, fibrosis, and thickening and increased vascularity of synovium [3, 8, [248], and genome editing [249]. Experimental research has discovered that in biomechanical terms, cartilage stiffness decreases after a mechanical injury [250] and that in biochemical terms, the introduction of exogenous pro-inflammatory cytokines leads to PG loss in a dose-dependent manner [251]. However, there is no overall consensus in the OA scientific community about the pathogenesis of PTOA. ...
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Osteoarthritis is a debilitating musculoskeletal whole-joint disease affecting the quality of life of over 340 million people globally. One of the most common disease sites is the knee joint. The disease is characterized by degradation of the articular cartilage covering the ends of bones. Degraded cartilage exhibits detrimental changes in its structure and composition, such as loss of proteoglycans (PG). The changes compromise the functioning of the cartilage. Ultimately, articular cartilage degradation leads to pain, joint space narrowing, joint stiffness, and a restricted range of motion, rendering the disease as a leading cause of disability. The disease phenotype initiated by traumatic injuries, i.e. post-traumatic osteoarthritis (PTOA), has been suggested to be triggered by two intertwined mechanisms: (1) biomechanics-related degradation resulting from impact damage, injuries, joint overloading, as well as mechanical shearing and wearing of cartilage, and (2) inflammation-related changes in cellular behavior and subsequent biochemical degradation. These two fundamental mechanisms of cartilage degradation and their dynamics are not fully understood. As a result, the current clinical treatment options are limited, diagnosis is delayed to the late phases of the disease where extensive and irreparable degradation has already occurred, and there are no known ways to restore cartilage back to its healthy state. The endeavors to treat symptoms, but not the causes, are also expensive. Therefore, the best and most cost-effective cure would be prevention. In order to achieve this goal, there is a need for a novel computational framework capable of predicting the progression of PTOA. Computational models incorporating biomechanical cartilage degradation mechanisms have recently been a focus of interest. However, there is a myriad of evidence underlining the importance of biochemical mechanisms. Thus, biomechanics-only models are likely to be incomplete in providing a comprehensive picture of the disease progression, and should be enriched with biochemical factors. When validated with experiments where both mechanisms are examined at several time points, such comprehensive models would represent promising tools for predicting PTOA progression and designing personalized intervention strategies. The first aim of this thesis was to develop tissue-level finite element models to predict PG matrix damage of injured knee cartilage explants incorporating both biomechanical and biochemical degradation mechanisms based on previous in vitro findings. With respect to biomechanical degradation, several mechanical strain measures and one stress measure were investigated; for biochemical degradation, the net catabolic effect of inflammation was modeled with the diffusion of pro-inflammatory messenger proteins into cartilage (interleukin-1 cytokines) followed by cell-level perturbations in the biosynthesis levels of enzymatic proteins (aggrecanases) and PGs (aggrecan). These two approaches were combined, i.e. excessive levels of maximum shear strain were used in tandem with assessments of cytokine diffusion. The second aim was to study in more detail the biomechanical and biochemical degradation mechanisms by conducting a new set of in vitro experiments in a bovine cartilage PTOA model. We investigated these mechanisms at several early time points up until 12 days of explant culture incorporating a combination of injurious loading, cytokine challenge, and a cyclic loading protocol that was considered healthy and mimicking daily activities. The experiments provided important information about the early-stage disease progression in terms of changes in tissue glycosaminoglycan content (GAG, a building block of PGs), aggrecan biosynthesis, and cell viability. The final aim was to generate, for the first time, a subject-specific knee joint-level computational model with both biomechanical (chondral injury, gait information) and biochemical (synovial fluid cytokine concentrations) aspects in a patient who had undergone an anterior cruciate ligament reconstruction surgery. These results were compared to quantitative magnetic resonance imaging (MRI) findings at the 3-year follow-up. The results of this thesis confirm that inflammation plays a crucial role alongside biomechanical factors in early PTOA progression. Thus, including both degradation mechanisms into adaptive models of cartilage allows a more comprehensive prediction of disease progression than is possible with biomechanics-only models. Specifically, injury-related PG loss and cell death localized near to lesions, cytokine-induced PG loss occurred also near to explant edges, and the combination of injurious loading and the presence of excess cytokine levels caused more PG matrix damage than either of these conditions alone. All of these findings were captured by the tissue-level computational models with elevated maximum shear strain as a biomechanical damage biomarker. Shear strain-driven PG loss was also prominent near a lesion in the joint-level model, corresponding with MRI findings which detected a localized substantial increase of T1rho relaxation time. Relaxation times were also increased in areas away from the lesions, possibly affected by the presence of pro-inflammatory cytokines as suggested in the new model. Interestingly, in the new experiments, the chosen cyclic loading regime (15% strain amplitude, 1 Hz frequency) was beneficial for GAG retention in inflamed cartilage within the first four days of loading, but became deleterious after 12 days despite the increasing aggrecan biosynthesis rate. The early-stage (1--4 days) protecting effect of cyclic loading is suggested to be prominent in the lower transitional and deep cartilage zones. In conclusion, the novel biomechanical and inflammation models of cartilage degradation presented in this thesis could be used to predict PTOA progression. In the future, such deterministic, physics-based models could help clinicians to assess either patient-specific or even population-specific risks of PTOA progression based on non-invasively imaged cartilage geometries, gait patterns if available, and synovial fluid biomarker profiles. The effects of different interventions could also be evaluated with the predictive model. Moreover, the new experimental findings provide a foundation for further in vivo studies where anti-catabolic drug treatment could be combined with early well-timed rehabilitation, but physical rehabilitation would not be continued if there were signs of chronic inflammation. The effectiveness of disease-modifying drug interventions could also be investigated with the proposed modeling platform. However, rigorous validation with large patient populations will be needed before this enters clinical use.
... Curcumin also exerts potent anti-apoptotic and anti-catabolic effects on IL-1β-stimulated cultures of articular chondrocyte. 32 Further study by Huang and his colleagues (2013) showed that curcumin dramatically mitigate the progression and severity of collagen-induced arthritis in mice and inhibits the production of the B-cellactivating factor that belongs to the TNF family. 33 Curcumin also inhibits COX-2, LOX, and inducible NOS enzymes, which are important in the inflammatory process, thereby acting as an anti-inflammatory agent. ...
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Background: Osteoarthritis (OA) is one of the most prevalent chronic degenerative arthritis diseases and a major cause of pain and physical disability among elderly patients. It can affect any joint in the body but most commonly, hip and knee joints. The etiology of the disease is multifactorial, OA affected by a range of mechanical and biochemical factors. Various studies provided compelling evidence that low-grade inflammation and synovitis are playing a pivotal role in its pathogenesis along with oxidative stress. Unfortunately, there is no cure for the disease; thus, most current treatments are prescribed for alleviating symptoms only. Curcumin, a natural polyphenolic compound, has been used for centuries in ayurvedic medicine that gained an increasing surge of interest to explore its potential properties. Many in vitro and in vivo studies reported powerful anti-inflammatory and antioxidant capacity for treating various pathological conditions, including OA, curcumin has shown chondroprotective potential on osteoarthritis disease. Aim of the Study: This study was designed to evaluate the anti-inflammatory effect of curcumin as an additive therapy to a non-steroidal anti-inflammatory drug, meloxicam, in the management of knee osteoarthritis. Patients and Method: This prospective open-labeled randomized controlled trial was conducted among patients with mild to moderate knee OA. Sixty-two patients were enrolled in this study; only 42 patients completed the study. Patients were assigned randomly into two groups; group (A) 21 patients treated with meloxicam alone (15 mg/day), group (B) 21 patients treated with a combination of meloxicam (15 mg/day), and curcumin (1600 mg/day) for 12 weeks. Inflammatory biomarkers (IL-1β, IL-6, and TNF-α) serum levels were evaluated at the time of enrolment and after 12 weeks of treatment. Results: Results gained from this study showed that treatment of knee OA patients with a combination of meloxicam and curcumin has a better effect on overall pain and physical function in addition to a remarkable decrease in serum pro-inflammatory biomarkers (IL-1β, IL-6, TNF-α) level (-39%,-24%,-30%) respectively after 12 weeks of treatment in respect to baseline levels. However, this reduction was significant only for IL-6. While those patients treated with meloxicam alone demonstrated no significant reduction. Conclusion: Curcumin represents a safe and effective anti-inflammatory product that exhibits a synergistic effect when used in combination with meloxicam, resulting in pain and physical activity improvement, which its anti-inflammatory effect may reflect.
... An in vitro study reported that curcumin antagonizes glycosaminoglycans release in cartilage explants (Clutterbuck, Mobasheri, Shakibaei, Allaway, & Harris, 2009). Another in vitro trial has demonstrated that curcumin inhibited the production of catabolic and inflammatory mediators by chondrocytes (Mathy-Hartert et al., 2009). ...
Turmeric (Curcuma longa) and its constituent, curcumin, have been used for their therapeutic properties for a long time. Most of the medicinal impacts of turmeric and curcumin might be attributed to their anti‐inflammatory, antinociceptive, and antioxidant effects. In the present review, the preventive and therapeutic potentials of turmeric and its active constituent, curcumin, on inflammatory disorders and pain as well as patents related to their analgesic and anti‐inflammatory effects, have been summarized to highlight their value on human health. A literature review was accomplished in Google Scholar, PubMed, Scopus, Google Patent, Patentscope, and US Patent. Several documents and patents disclosed the significance of turmeric and curcumin to apply in several therapeutic, medicinal, and pharmaceutical fields. These phytocompounds could be applied as a supplementary therapy in phytotherapy, inflammatory disorders such as arthritis, inflammatory bowel diseases, osteoarthritis, psoriasis, dermatitis, and different types of pain including neuropathic pain. However, because of inadequate clinical trials, further high‐quality studies are needed to firmly establish the clinical efficacy of the plant. Consistent with the human tendency to the usage of phytocompounds rather than synthetic drugs, particular consideration must be dedicated to bond the worth of turmeric and curcumin from basic sciences to clinical applications.
... Furthermore, lower aggrecan loss in IL-1β-stimulated articular cartilage explants has been reported to occur at a curcumin concentration no lower than 100 μM [49]. Additionally, it was reported that probucol would also bring side effects, such as ventricular arrhythmia, torsades de pointes, and syncope [50]. ...
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Osteoarthritis (OA) is a chronic joint disease characterized by cholesterol accumulation in chondrocytes, cartilage degeneration, as well as extracellular matrix (ECM) destruction, and joint dysfunction. Curcumin, a chemical that can reduce cholesterol levels in OA patients, also can inhibit the progression of OA. However, a high concentration of curcumin may also trigger apoptosis in normal chondrocytes. Besides curcumin, probucol that is found can also effectively decrease the cholesterol level in OA patients. Considering that high cholesterol is a risk factor of OA, it is speculated that the combination treatment of curcumin and probucol may be effective in the prevention of OA. To investigate the possible effects of such two chemicals on OA pathophysiology, chondrocyte apoptosis and autophagy behavior under inflammatory cytokine stress were studied, and specifically, the PI3K-Akt-mTOR signaling pathway was studied. Methods. Cell proliferation, colony formation, and EdU assay were performed to identify the cytotoxicity of curcumin and probucol on chondrocytes. Transwell assay was conducted to evaluate chondrocyte migration under TNF-α inflammation stress. Immunofluorescence, JC-1, flow cytometry, RT-PCR, and western blot were used to investigate the signal variations related to autophagy and apoptosis in chondrocytes and cartilage. A histological study was carried out on OA cartilage. Glycosaminoglycan (GAG) release was determined to evaluate the ECM degradation under stress. Results. Compared with a single intervention with curcumin or probucol, a combined treatment of these two chemicals is more effective in terms of protecting chondrocytes from stress injury induced by inflammatory cytokines. The promoted protection may be attributed to the inhibition of apoptosis and the blockage of the autophagy-related PI3K/Akt/mTOR pathway. Such results were also verified in vitro by immunofluorescence staining of OA chondrocytes and in vivo by immunohistochemistry staining of cartilage. Besides, in vivo studies also showed that when applied in combination, curcumin and probucol could block the PI3K-AKT-mTOR signaling pathway; promote COL-II expression; suppress P62, MMP-3, and MMP-13 expression; and inhibit TNF-α-stimulated cartilage degradation. Moreover, the combined medication could help reduce the release of ECM GAGs in OA cartilage and alleviate the severity of OA. Conclusion. A combined treatment of curcumin and probucol could be used to protect chondrocytes from inflammatory cytokine stress via inhibition of the autophagy-related PI3K/Akt/mTOR pathway both in vitro and in vivo, which might be of potential pharmaceutical value for OA prevention and therapy. 1. Introduction OA is a chronic inflammatory disease closely related to cartilage degeneration. Researchers have found that a high level of total cholesterol is related to the OA process. Specifically, in a prospective cohort study, total cholesterol and triglycerides are verified to be associated with new bone marrow lesion formation in asymptomatic middle-aged women [1] and result in cartilage defect and OA eventually. Another possible explanation could be lipid embolism caused by serum cholesterol, which may cause osteonecrosis leading to OA. Hypertension, obesity, abnormal blood lipids, and high cholesterol, such conditions known as “metabolic syndrome” [2] are common among OA patients. The interrelationship between high cholesterol levels and increased risk of OA has been studied extensively in recent years [3, 4], and previous reports have shown that inhibition of de novo cholesterol synthesis may provide better OA remiment outcome [5, 6]. In this context, OA should be considered as a syndrome rather than merely a joint disease. Autophagy is an important self-maintenance mechanism by which a cell protects itself when facing harmful stress [7]. Active autophagy is related to cholesterol effluent, and it can delay disease progress to a certain alleviated extent. Specifically, nitro-oleic acid, a ligand of CD36, reduces cholesterol accumulation by modulating fluidized LDL uptake and cholesterol efflux in RAW264.7 macrophages, and FGF21 induces autophagy-mediated cholesterol efflux to inhibit atherogenesis via the upregulation of RACK1 [8]. However, autophagy activity tends to drop in several cells and tissues with age. In OA chondrocytes, autophagy markers decrease significantly [9], accompanying with dysfunctional autophagy, enhanced apoptosis, and less migration [10]. Therefore, chemicals that can regulate autophagy in chondrocytes and stabilize the cholesterol level may be of potential medication value for OA prevention and therapy. Curcumin, a diferuloylmethane, is extracted from the root of Curcuma longa [11]. In China and India, Curcuma longa has been used as a medicinal herb with a long history. Recent research indicated that curcumin can function to reduce cholesterol levels [12]. Additionally, previous authors have verified that curcumin can promote autophagy and reduce apoptosis in several cells [13]. Moreover, probucol, another cholesterol regulator, can also activate autophagy and inhibit apoptosis in nerve cells by blocking PI3K/Akt/mTOR signals [14]. Since autophagy takes an important role in chondrocyte physiology, and the PI3K/Akt/mTOR pathway is essential in regulating autophagy in OA patients [15], we speculated that curcumin and probucol may be of potential value for OA prevention. In the present study, these two chemicals were applied together to investigate their effects on chondrocytes in vitro and on cartilage in vivo. 2. Materials and Methods 2.1. Animals Healthy male Sprague Dawley rats from the Animal Experimental Center of Wuhan University (Wuhan, China) were involved in this study. The rats were fed under specific pathogen-free conditions at a constant room temperature (24°C) and relative humidity (45%–55%). All rats had free access to sterile food and water and lived under a light/dark cycle of 12 h. The present study was approved by the Laboratory Animal Welfare & Ethics Committee, Renmin Hospital of Wuhan University. Efforts were made to minimize animal suffering in the study. 2.2. Reagents The reagents included DMEM/F12 high glucose (Hyclone, Utah, USA), penicillin (Hyclone, Utah, USA), streptomycin (Hyclone, Utah, USA), curcumin (Bellancom, Beijing, China), trypsin (Google Biotechnology, Wuhan, China), collagenase-II, bovine serum albumin (BSA), probucol (Sigma-Aldrich, St. Louis, MO, USA), KeyFluor488 Click-iT EdU kits, DAPI, (KeyGEN BioTECH, Nanjing, China), AnnexV-PI kits (BD, USA), Counting Kit-8 (CCK-8) reagents, goat serum (Beyotime Institute of Biotechnology, Shanghai, China), TNF-α (Peprotech, Inc., Suzhou, China), Caspase-3, Bcl-2, Lc3, Bax, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, GADPH, Berclin-1, COL-II, P62, FITC, Cy3, MMP-3, JC-1 assay kits (Abcam, USA), TRIzol reagents (Invitrogen, Thermo Fisher Scientific, Inc. USA), a RevertAid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc. USA), MMP detection kits (Solarbio Science & Technology, Beijing, China), and chemiluminescent luminol reagent (Santa Cruz Biotechnology, Texas, USA). 2.3. Chondrocyte Culture and Identification Briefly, cartilage was extracted from the knee joints of 35 male Sprague-Dawley rats (4 weeks, weighing ). Cartilage samples were minced into thin slices (1 mm³) and digested with 3 ml of 0.25% trypsin for 40 min followed by further treatments with type II collagenase for another 6 h. Chondrocytes were then been centrifuged and collected. Subsequently, the isolated chondrocytes were cultured in 5 ml of DMEM/F12 with 20% fetal bovine serum and incubated at 37°C in 5% CO2. 2.4. CCK-8 Assay To determine the appropriate study concentration of probucol and curcumin for further investigation in the subsequent experiments, cell viability was detected by the CCK-8 test. The chondrocytes were first cultured in a 96-well plate, and CCK-8 reagents were added, which was incubated at 37°C for another 2 h. The chondrocyte viability was detected by OD 450 nm with an automatic microplate reader. All studies were conducted in triplicate. 2.5. Cell Groups Based on the above CCK-8 results, cells were randomly divided into five groups (): control, TNF-ɑ, TNF-ɑ + curcumin (50 μM), TNF-ɑ + probucol (100 μM), and TNF-ɑ + probucol (50 μM) + curcumin (25 μM). After excluding other cytokines or growth factors, TNF-α aqueous solution (20 ng/ml) was mixed with normal chondrocytes to mimic the inflammatory cytokine environment in OA [16]. 36 h later, curcumin, probucol, or both of them were added, and the chondrocytes were further incubated for another 24 h. 2.6. Flow Cytometry of Annexin V-FITC-PI Staining The apoptosis rates of chondrocytes were measured with an AnnixV-PI apoptosis detection kit. In short, the chondrocytes were held at 25°C for 15 min and treated with PI solution (5 μl) and FITC-labeled annexin V (5 μl) for 10 min in the dark. The apoptosis rates were evaluated with a flow cytometer (BD Biosciences, USA). 2.7. Colony Formation Assay The chondrocytes were placed on a six-well plate and mixed with curcumin and probucol at predescribed concentrations. After that, the cells were incubated for another two weeks without curcumin and probucol. Subsequently, the colonies were fixed with methanol, stained with Wright-Giemsa solution, and counted for their numbers [17]. 2.8. Transwell Migration Assay Transwell assays were used to evaluate cell migration. First, the transwell chambers were washed with serum-free medium, and chondrocytes were cultured in DMEM medium with 10% FBS as the chemical attractant. After incubation for 48 h, cells attached to the membrane were discarded, and those entering the lower membrane were fixed with methanol and stained with 0.2% crystal violet. Under a microscope (×200), the cells invaded by the matrix gel in 5 random fields of view were photographed. 2.9. JC-1 for Mitochondrial Membrane Permeability (MMP) Assessment An MMP detection kit was used to evaluate the MMP in chondrocytes. After the chondrocytes were washed with PBS, 800 μl of JC-1 working fluid was mixed with the chondrocytes and stained at 37°C for 25 minutes. Subsequently, 2 ml of medium containing serum was added to the working fluid after staining. The red-green fluorescence ratio was measured by a FACS Caliber flow cytometer (Becton, Dickinson, and Company) and an Olympus fluorescence microscope (Olympus Corporation, Japan). 2.10. EdU Incorporation Assay Chondrocyte proliferation was assessed by a keyFluor488 Click-iT-EdU kit. First, the chondrocytes were placed in a six-well plate, and 100 μl of EdU was added into the plate, followed by incubation at 37°C for 2 h. Second, the cells were fixed with 4% paraformaldehyde at room temperature, washed with BSA containing 3% glycine, and incubated with 0.5% TritonX-100 and 1× click-it reaction solution in the dark at room temperature. Last, Hoechst 33342 was added to the six-well plate, and the whole plate was placed in a dark environment for 20 minutes and then washed three times with PBS. The stained cells were observed with a fluorescence microscope. 2.11. Reverse Transcription Quantitative Polymerase Chain Reaction (RT-PCR) TRIzol reagents were used to isolate the total RNA from chondrocytes. To determine the expression levels of inflammation-related genes, first-strand complementary cDNA chains were synthesized using the RevertAid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.). Quantitative PCR was performed for 40 cycles in a StepOnePlus device (Applied Biosystems; Thermo Fisher Scientific, Inc.), and each cycle contained temperature at 95°C for 10 secs, followed by 5 seconds at 95°C and 20 seconds at 60°C. The additional primers were as follows: COL2, 5-CTTAGGACAGAGAGAGAAGG-3; Rev, 5-ACTCTGGGTGGCAGAGTTTC-3; MMP-3, 5-TTTGGCCGTCTCTTCCATCC-3; Rev, 5-GGAGGCCCAGAGTGTGAATG-3; MMP-13, 5-GG AGCATGGCGACTTCTAC-3; Rev, 5-GAGTGCTCCAGGGTCCTT; GADPH, 5-CTCAACTACATGGTCTACATGTTCCA-3; and Rev, 5-CTTCCCATTCTCAGCCTTGACT-3. GADPH was used as an internal reference. Moreover, the 2-ΔΔCq method was employed to calculate the relative levels of mRNA expression. 2.12. Western Blot To extract the total proteins from the chondrocytes, organophosphorus inhibitors, protease inhibitors, and RIPA lysates were mixed at a ratio of 1 : 1 : 50. The proteins were separated by electrophoresis and transferred to polyvinylidene fluoride membranes, which were sealed for one hour. After that, a primary antibody was added to the membranes, which were then washed three times with TBST and incubated with horseradish peroxidase-labeled anti-rabbit goat IgG for 1 hour. Subsequently, the membranes were washed with TBST again, and the protein bands were observed with chemiluminescent luminol reagent (Santa Cruz Biotechnology, Inc.) and an Image Lab quantitative analysis system (Bio-Rad Laboratories Inc.). The relative protein levels were compared by normalizing to GADPH. The primary antibodies were as follows: Bcl-2, Bax, Beclin-1, LC3, mTOR, PI3K, Akt, p-Akt, p-PI3K, p-mTOR, and GAPDH. 2.13. OA Animal Model In Vivo Study SD rats (8 weeks old, weighing 250-280 g) were randomly divided into five groups, which are denoted as control (), OA (), OA+50 mg/kg curcumin (), OA+100 mg/kg probucol (), and OA+75 mg/kg curcumin-probucol (). The specific dosages were determined according to the earlier literature [18]. A rat OA model was created by excising the medial meniscus and the anterior cruciate ligament of the rats’ right knee. Four weeks later, the groups with medications were treated with curcumin and probucol intramuscular injections once every three days for a total of 8 weeks, while the OA and the control groups were injected with normal saline. All rats were sacrificed after 3 months. 2.14. Immunofluorescence and Immunohistochemistry After washed with PBS, the cartilage tissues and chondrocytes were fixed with paraformaldehyde for 12 h at 4°C and then dehydrated in 30% sucrose solution. Next, the tissues were sliced into pieces of 10 μM and incubated with P62 and COL-II at room temperature for 1 h. Subsequently, the section slices were then immunostained with FITC or Cy3-labeled secondary antibodies for 1 h, and DAPI was applied to counterstain the nuclei for 5 min. The sections were then incubated overnight with the primary antibodies for MMP-3 or MMP-13 at 4°C, and they were then incubated with biotinylated secondary antibodies. All sections were observed under an Olympus fluorescence microscope mentioned above. The proportions of stain-positive cells in the samples were analyzed by Image Pro Plus 6.0 (Media Cybernetics, Inc., USA). 2.15. Glycosaminoglycan Release Assay Papain-digested cartilage explants and their defrosted supernatants were examined in 96-well plates using the dimethyl methylene blue (DMMB) method [16]. Briefly, each sample was diluted in distilled water to a total volume of 40 μl per well in triplicate. Shark chondroitin sulfate (Sigma-Aldrich) was used as a standard (0-70 ng). DMMB solution (200 μl) was added to each well, and the whole plate was immediately transferred to a Multiskan Ascent Scanner (Thermo Labsystems, Basingstoke, UK) with Ascent Software (version 2.6, Thermo Labsystems, Finland). Total GAG release was observed from a spectrophotometric reading of the digested cartilage and its supernatants at 540 nm. For each well, the percentage of GAG release was calculated by dividing the GAG readings from the supernatants by the total GAG release. 2.16. Statistical Analysis For each group, the data are expressed as . Intragroup differences were assessed with Student’s -test and one-way analysis of variance by SPSS 16.0 (SPSS, Inc., USA) followed by a Bonferroni posthoc correction for multiple testing with GraphPad Prism (version 7.04; GraphPad Software, Inc., USA). Specifically, differences with were considered statistically significant. 3. Results 3.1. Effects of Probucol and Curcumin on Chondrocyte Proliferation CCK-8 was used to detect chondrocyte activity. The most appropriate concentrations of probucol and curcumin to counteract inflammatory cytokine stress were found to be 100 μM and 50 μM, respectively (Figures 1(a) and 1(b)). It is noteworthy that both these substances could promote chondrocyte proliferation in a dose-dependent manner. Here, we chose these substances at optimal concentrations of 12.5%, 25%, 50%, and 100% to the most appropriate concentration for combinations [10]. Considering the possible reported side effects of such substances [19], a combination of curcumin 25 μM + probucol 50 μM was used in this study, and the results suggest that such a combination can promote chondrocyte proliferation (Figures 1(c)). Colony formation assays further confirmed that they play a promotive role in chondrocyte proliferation (Figures 1(d) and 1(e)), and such effect is in a synergistic way by the two chemicals. In the EdU assays with TNF-α treatment, the chondrocytes showed a low proliferation ratio. However, after treating with 50 μM curcumin or 100 μM probucol, the proliferation ratio got increased; and with the combined treatment, such increasement became more significant (Figures 1(f) and 1(g)). (a)
... Stimulation of osteochondral slices with biological stimuli showed that chondrocytes in slice cultures responded adequately to external stimulation with catabolic molecules. TNF-α is known to be a proinflammatory cytokine in the joint and to induce cartilage matrix degradation both in vivo and in vitro [25,26], but other explant models either took significantly longer for a similarly strong reduction in matrix expression and degradation of proteoglycans or required additional catabolic stimuli such as oncostatin-M or interleukin-1β [11,27,28]. Here, the short perfusion distance between the medium and cells could be an advantage for pharmacological testing, since a shorter diffusion distance results in a higher and faster penetration by the corresponding factors than in thicker explant cultures. ...
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For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-β3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-β3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.
Injurious overloading and inflammation perturbate homeostasis of articular cartilage, leading to abnormal tissue-level loading during post-traumatic osteoarthritis. Our objective was to gain time- and cartilage depth-dependent insights into the early-stage disease progression with an in vitro model incorporating for the first time the coaction of (1) mechanical injury, (2) pro-inflammatory interleukin-1 challenge, and (3) cyclic loading mimicking walking and considered beneficial for cartilage health. Cartilage plugs (n=406) were harvested from the patellofemoral grooves of young calves (N=6) and subjected to injurious compression (50% strain, rate 100%/s; INJ), interleukin-1α-challenge (1 ng/ml; IL), and cyclic loading (intermittent 1h loading periods, 15% strain, 1 Hz; CL). Plugs were assigned to six groups (control, INJ, IL, INJ-IL, IL-CL, INJ-IL-CL). Bulk and localized glycosaminoglycan (GAG) content (DMMB assay, digital densitometry), aggrecan biosynthesis (³⁵S-sulfate incorporation), and chondrocyte viability (fluorescence microscopy) were assessed on days 3–12. The INJ, IL, and INJ-IL groups exhibited rapid early (days 2–4) GAG loss in contrast to CL groups. On day 3, deep cartilage of INJ-IL-CL group had higher GAG content than INJ group (p<0.05). On day 12, INJ-IL-CL group showed more accumulated GAG loss (normalized with control) than INJ-IL group (average fold changes 1.97 [95%CI: 1.23–2.70]; 1.66 [1.42–1.89]; p=0.007). Aggrecan biosynthesis increased in CL groups on day 12 compared to day 0. Despite promoting aggrecan biosynthesis, this cyclic loading protocol seems to be beneficial early-on to deep cartilage, but later becoming incapable of restricting further degradation triggered by marked but non-destructive injury and cytokine transport.
The Chemistry inside Spices & Herbs: Research and Development brings comprehensive information about the chemistry of spices and herbs with a focus on recent research in this field. The book is an extensive 2-part collection of 20 chapters contributed by experts in phytochemistry with the aim to give the reader deep knowledge about phytochemical constituents in herbal plants and their benefits. The contents include reviews on the biochemistry and biotechnology of spices and herbs, herbal medicines, biologically active compounds and their role in therapeutics among other topics. Chapters which highlight natural drugs and their role in different diseases and special plants of clinical significance are also included. Part II continues from the previous part with chapters on the treatment of skin diseases and oral problems. This part focuses on clinically important herbs such as turmeric, fenugreek, ashwagandha (Indian winter cherry), basil, Terminalia chebula (black myrobalan). In terms of phytochemicals, this part presents chapters that cover resveratrol, piperine and circumin. Audience: This book is an ideal resource for scholars (in life sciences, phytomedicine and natural product chemistry) and general readers who want to understand the importance of herbs, spices and traditional medicine in pharmaceutical and clinical research.
Herbal supplements containing curcumin and other ingredients are used for pain management in horses with osteoarthritis (OA). To test the effects of a herbal supplement containing curcumin and other ingredients in horses with lameness due to naturally occurring OA. Two‐period randomised crossover design. Ten Thoroughbreds with naturally occurring chronic OA were randomly assigned to the treatment (BLP; Buteless® Performance) or control (CTR, no supplement) groups and fed daily for 30 days. On Days −1 (before treatment), 15 and 30, lameness examination, range of motion, pain on palpation and force platform data were collected. Plasma curcumin concentration and its metabolites were measured on Days 1 and 14. Gastroscopy, a complete blood count and a serum biochemistry panel were performed on each horse before treatment Day −1 and Day 31. Gastric lesions (ulcers) were scored in real time by a masked investigator. Mean peak vertical force (PVF), measured by the force platform, significantly increased in the lame limb of the BLP‐treated horses on Days 15 (0.40 ± 0.13 N/kg, (p = 0.0025) and 30 (0.63 ± 0.14 N/kg, p < 0.0001) compared to the CTR group. In addition, mean normalised PVF was higher in the BLP group on Day 15 (p = 0.0438) and on day 30 (p = 0.0003) when compared to CTR horses for the same days. The PVF significantly improved (≥5%; range, 5.2–33%) in six of nine individual BLP‐treated horses and did not improve (<5%; range, 0–3.4%) in three of nine BLP‐treated horses. Also, PVF improved (≥5%; range, 7.6–15.4%) in three of nine horses in the CRT group. Squamous gastric lesion scores significantly decreased in both groups by Day 31. Plasma curcumin‐O‐sulphate concentrations (1.2–3.3 ng/ml) were present in 9/10 BLP‐treated horses by Day 14. Small sample size and absence of a positive treatment (non‐steroidal anti‐inflammatory drug) control. The BLP supplement containing curcumin achieved plasma concentrations and improved weight bearing in some treated horses with naturally occurring OA.
Objective To gain insight into Treg interactions with synovial tissues in early OA, an equine tri-culture model of OA was used to test the hypothesis that Tregs, in the absence of T Helper 17 cells, are sufficient to resolve inflammation elicited by IL-1β. Methods To model normal and OA joints, synoviocytes were co-cultured with chondrocytes in a transwell system and +/- stimulated with IL-1β. Tregs were activated and enriched, then added to co-cultures, creating tri-cultures. At culture end, synoviocytes and chondrocytes were analyzed for gene expression, Treg Foxp3 expression was reexamined by flow cytometry, and conditioned media were evaluated by ELISA. Results Tregs increased IL-10 and IL-4 in tri-culture media and increased TIMP1 gene expression in synoviocytes and chondrocytes. Tregs increased IL-6 in conditioned media and Il6 gene expression in synoviocytes, which was additive with IL-1β. In chondrocytes, addition of Tregs decreased Col2b gene expression while Acan gene expression was decreased by IL-1β and addition of Tregs. IL-17A was detected in tri-cultures. CCL2 and CCL5 were increased in tri-cultures. Conclusions In a tri-culture model of OA, addition of Tregs resulted in conditions conducive to chondroprotection including increased concentration of IL-10 and IL-4 in conditioned media and increased gene expression of TIMP1 in both chondrocytes and synoviocytes. However, there was increased concentration of the catabolic cytokine IL-6, and decreased gene expression of Col2b and Acan in IL-1β-stimulated chondrocytes. These results suggest that blocking IL-6 could enhance Treg function in mitigating OA progression.
The aim of this systematic review was to evaluate the efficacy and safety of all types of Curcuma longa extract versus placebo for knee osteoarthritis (OA) treatment. The research was conducted by using the databases of PubMed, Embase, Scopus, and Cochrane Library through April 2021. Randomized controlled trials (RCTs) that compared the effect of Curcuma longa extract with placebo for patients with knee OA were considered eligible. The pooled results were expressed as mean differences or relative risks with 95% confidence intervals. A total of 10 RCTs with 783 patients were eligible for this meta-analysis. The pooled analysis showed that Curcuma longa extract was associated with significantly better pain relief and functional improvement compared with placebo for knee OA. Moreover, the smallest effect sizes of VAS for pain and WOMAC total score exceeded the minimum clinically important differences (MCIDs). Current evidence indicates that, compared with placebo, Curcuma longa extract has more benefit in pain relief and functional improvement for symptomatic knee OA. However, considering the potential heterogeneity in the included studies, more future high-quality RCTs with large sample sizes are necessary to confirm the benefits of Curcuma longa extract on knee OA.
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Interleukin 1β (IL-1β) is a pleiotropic pro-inflammatory cytokine that plays a key role in mediating cartilage degradation in osteoarticular disorders such as osteoarthritis (OA) and rheumatoid arthritis (RA). At the cellular level, IL-1β activates matrix degrading enzymes, down-regulates expression of matrix components and induces chondrocyte apoptosis. Curcumin (diferuloylmethane) is an anti-inflammatory phytochemical agent that has recently been shown to antagonize the pro-inflammatory effects of cytokines in chondrocytes and other cells. To test the hypothesis that curcumin also protects chondrocytes from morphological alterations induced by IL-1β, we investigated its in vitro effects on apoptotic signalling proteins and key cartilage-specific matrix components in IL-1β-stimulated chondrocytes. Human articular chondrocytes were pre-treated with 10 ng/ml IL-1β alone for 30 min before being co-treated with IL-1β and 50 μM curcumin for 5, 15 or 30 min, respectively. The ultrastructural morphology of chondrocytes was investigated by transmission electron microscopy. The production of collagen type II, the adhesion and signal transduction receptor β1-integrin, the apoptosis marker activated caspase-3 was analysed by immunohistochemistry, immunoelectron microscopy and Western blotting. Transmission electron microscopy of chondrocytes stimulated with IL-1β revealed early degenerative changes which were relieved by curcumin co-treatment. The suppression of collagen type II and β1-integrin synthesis by IL-1β was inhibited by curcumin. Additionally, curcumin antagonized IL-1β-induced caspase-3 activation in a time-dependent manner. This study clearly demonstrates that curcumin exerts anti-apoptotic and anti-catabolic effects on IL-1β-stimulated articular chondrocytes. Therefore curcumin may have novel therapeutic potential as an adjunct nutraceutical chondroprotective agent for treating OA and related osteoarticular disorders.
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The transcription factor NF-κB is a pivotal regulator of inflammatory responses. While the activation of NF-κB in the arthritic joint has been associated with rheumatoid arthritis (RA), its significance is poorly understood. Here, we examine the role of NF-κB in animal models of RA. We demonstrate that in vitro, NF-κB controlled expression of numerous inflammatory molecules in synoviocytes and protected cells against tumor necrosis factor α (TNFα) and Fas ligand (FasL) cytotoxicity. Similar to that observed in human RA, NF-κB was found to be activated in the synovium of rats with streptococcal cell wall (SCW)-induced arthritis. In vivo suppression of NF-κB by either proteasomal inhibitors or intraarticular adenoviral gene transfer of super-repressor IκBα profoundly enhanced apoptosis in the synovium of rats with SCW- and pristane-induced arthritis. This indicated that the activation of NF-κB protected the cells in the synovium against apoptosis and thus provided the potential link between inflammation and hyperplasia. Intraarticular administration of NF-kB decoys prevented the recurrence of SCW arthritis in treated joints. Unexpectedly, the severity of arthritis also was inhibited significantly in the contralateral, untreated joints, indicating beneficial systemic effects of local suppression of NF-κB. These results establish a mechanism regulating apoptosis in the arthritic joint and indicate the feasibility of therapeutic approaches to RA based on the specific suppression of NF-κB.
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When activated, NF-κB, a ubiquitous transcription factor, binds DNA as a heterodimeric complex composed of members of the Rel/NF-κB family of polypeptides. Because of its intimate involvement in host defense against disease, this transcription factor is an important target for therapeutic intervention. In the present report we demonstrate that curcumin (diferuloylmethane), a known anti-inflammatory and anticarcinogenic agent, is a potent inhibitor of NF-κB activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor (TNF) rapidly activated NF-κB, which consists of p50 and p65 subunits, and this activation was inhibited by curcumin. AP-1 binding factors were also found to be down-modulated by curcumin, whereas the Sp1 binding factor was unaffected. Besides TNF, curcumin also blocked phorbol ester- and hydrogen peroxide-mediated activation of NF-κB. The TNF-dependent phosphorylation and degradation of IκBα was not observed in curcumin-treated cells; the translocation of p65 subunit to the nucleus was inhibited at the same time. The mechanism of action of curcumin was found to be different from that of protein tyrosine phosphatase inhibitors. Our results indicate that curcumin inhibits NF-κB activation pathway at a step before IκBα phosphorylation but after the convergence of various stimuli.
Honors thesis (Biological Sciences)--Cornell University, May 2005. Includes bibliographical references (leaves 16-19).
Prostaglandin E2 (PGE2) and stromelysin are produced by equine chondrocytes and synovial cells in vitro in response to recombinant human (rh) interleukin-1 (IL-1) alpha and beta, and equine mononuclear cell supernatants (MCS) containing IL-1. However, culture conditions are important. PGE2 concentrations increase in proportion to the concentration of fetal calf serum (FCS) in the culture medium, whereas stromelysin concentrations are inversely proportional to the concentration of FCS. Equine MCS, containing a lower concentration of IL-1 than the concentration of rhIL-1 used in these experiments, stimulated production of much higher levels of PGE2 than rhIL-1. In addition, equine MCS induced the production of broadly similar levels of PGE2 by both chondrocytes and synovial cells, whereas rhIL-1 was more active on equine synovial cells than equine chondrocytes. Although equine MCS induced both stromelysin and PGE2 production by equine articular cells, on the whole rhIL-1 failed to induce stromelysin production. This supports previous observations of species restrictions in the activity of human IL-1 on equine cells. Therefore, experiments using mammalian cells and heterologous IL-1 should be interpreted with caution.
Articular cartilage covers the ends of long bones in synovial joints, providing smooth articulation and cushioning of the underlying bone during joint movement. The tissue can be viewed as a viscoelastic, composite material composed of collagen type II (and smaller amounts of other collagens) entrapping compressed (underhydrated) proteoglycan aggregates which generate a high osmotic/swelling pressure. This abundant extracellular matrix (ECM) is synthesized and turned over by relatively few cells, the chondrocytes. These cells produce a compartmentalized ECM, the components of which are heterogeneous and vary with anatomical location. They also undergo changes with age and altered functional requirements. Articular cartilage contains no separating basement membranes, nerves, lymphatics, or blood vessels. Access to nutrients and elimination of waste products occur via diffusion through the extracellular matrix. The turnover of collagen is much slower than that of proteoglycans. Products of the metabolic turnover of the matrix macromolecules are released continuously into the synovial cavity and ultimately reach the blood circulation where they can be measured as "markers" of metabolic changes.
Horse articular cartilage glycosaminoglycans (GAGs) were measured in synovial fluids from 48 joints affected with osteoarthritis (OA), 22 normal joints, four joints with osteochondritis, three joints with traumatic arthritis and seven joints infected with bacteria. Serum and urine from individual horses were also examined for the presence of GAGs. High levels of GAGs were found in synovial fluids (SF) from horses with OA. In each case, the level was higher in the synovial fluid than in the serum or urine from the same horse. Horses with OA showed high GAG levels in SF, serum and urine compared to horses with normal and infected joints. High levels were also found in horses with osteochondritis and traumatic arthritis. Levels of synovial fluid GAG reflect cartilage destruction in arthritis and may be useful for monitoring disease progression in the equine species.
The turnover of proteoglycans was investigated in articular cartilage in explant culture by analysing the components released into the culture medium. The effect of IL-1 alpha on the release of fragments derived from different proteoglycan domains and hyaluronan (HA) was determined over 4 days in culture. The effect of IGF-1 (100 ng/ml) on matrix degradation of proteoglycan and its ability to inhibit the effects of IL-1 (10 ng/ml) was also assessed. The rate of release of G1 and G2 globular domains of proteoglycans into the culture medium was determined by radioimmunoassay. In unstimulated control cartilage there was a greater release of proteoglycan G2 domain than of G1 domain suggesting that cleavage occurred between them and that some G1 was preferentially retained bound in the matrix. Compared with control cartilage IL-1 stimulated the release of all proteoglycan components and hyaluronan. IL-1 had a greater effect on the release of G1 than on G2 domain, but also resulted in some net loss of these proteins (approximately 45% as detected in the immunoassays). In explants treated with both IL-1 and IGF-1 there was much less release of proteoglycan fragments and evidence for less extensive degradation. IGF-1 was particularly affective in preventing any increase in HA release and also preventing the apparent loss of G1 and G2 domains. It also partially inhibited the release of G1 and G2 domains and the sulphated glycosaminoglycan fragments. IGF-1 was therefore an effective antagonist of IL-1 action on cartilage. It is not known at what level it blocks the chondrocyte response to IL-1, but it clearly results in the suppression of matrix degradative activity.
A model system of explanted cartilage has been used in vitro to determine whether insulin-like growth factor 1 (IGF 1), which promotes matrix formation is effective in the presence of cytokines such as interleukin 1 (IL1) and tumour necrosis factor (TNF), which induce net matrix depletion. IGF 1 induced a dose-dependent 2.5-fold stimulation of proteoglycan synthesis, with a half-maximal dose of 25 ng/ml. A similar relative increase occurred in response to IGF 1 (10-100 ng/ml) in cartilage cultured also with IL1 or TNF (5-500 pM). There was no detectable qualitative change in the average molecular size or charge of the aggregating proteoglycan synthesized by explants exposed to IGF 1 alone or with IL1 or TNF. The increased production of prostaglandin E2, which is initiated when IL1 or TNF bind to the chondrocytes, was the same in the presence or absence of IGF 1. The time taken for 50% of pre-labelled proteoglycan to be released from the explants (t1/2) increased in the presence of IGF 1 (100 ng/ml) from 21 to 32 days in control cultures and from 8 to 26 days in cartilage cultured with IL1 (50 pM). It is concluded that IGF 1 enhances the synthesis of aggregating proteoglycan in cartilage exposed to cytokines and can directly decrease both the basal and the cytokine-stimulated degradation of proteoglycan in cartilage.