Rosmarinic acid induces rabbit articular chondrocyte differentiation by decreases matrix metalloproteinase-13 and inflammation by upregulating cyclooxygenase-2 expression
Eo and Kim Journal of Biomedical Science
Rosmarinic acid induces rabbit articular chondrocyte differentiation by decreases matrix metalloproteinase-13 and inflammation by upregulating cyclooxygenase-2 expression
Seong-Hui Eo 0
Song Ja Kim 0
0 Department of Biological Sciences, College of Natural Sciences, Kongju National University , Gongju 32588 , Republic of Korea
Background: Matrix metalloproteinases (MMPs) are known to play an important role in the degradation of the extracellular matrix and the pathological progression of osteoarthritis (OA). The natural polyphenolic compound rosmarinic acid (Ros. A) has been shown to suppress the inhibitory activity of matrix metalloproteinases (MMPs). However, the effects of Ros. A on OA have not been investigated. Methods: In the current study, primary articular chondrocytes were cultured from rabbit articular cartilage and treated with Ros. A. Phenotypic characterization was performed by western blotting to assess specific markers, prostaglandin E2 (PGE2) assays, and alcian blue staining to measure sulfated-proteoglycan production. Results: We report that in rabbit articular chondrocytes, Ros. A increased type II collagen, sulfated-proteoglycan, cyclooxygenase-2 (COX-2), and PGE2 production in a dose- and time-dependent manner. Furthermore, Ros. A suppressed the expression of MMP-13. In addition, treatment with Ros A activated extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase signaling pathways. Inhibition of MMP-13 enhanced Ros. A-induced type II collagen expression and sulfated-proteoglycan synthesis but COX-2 and PGE2 production were unchanged. Ros. A-mediated up-regulation of ERK phosphorylation was abolished by the MEK inhibitor, PD98059, which prevented induction of the associated inflammatory response. Inhibition of p38 kinase with SB203580 enhanced the increase in type II collagen expression via Ros. A-mediated down-regulation of MMP-13. Conclusions: Results suggest that ERK-1/2 regulates Ros. A-induced inflammation and that p38 regulates differentiation by inhibiting MMP-13 in rabbit articular chondrocytes.
Rosmarinic acid (Ros; A); Matrix metalloproteinase-13 (MMP-13); Type II collagen; COX-2; Chondrocytes; ERK pathway; p38 Kinase pathway
Osteoarthritis (OA), the most common chronic and
degenerative articular disease, is most prevalent in the
]. The main feature of this disease is cartilage
degradation, formation of osteophytes, and synovial
inflammation, among other alterations. The cause of OA
is still unclear, and there is no disease-modifying
treatment available except for joint replacement surgery .
Articular chondrocytes are the only resident cell type
in articular cartilage, which is composed of dense
collagenous extracellular matrix (predominantly type II) and
proteoglycans (importantly aggrecan) [
]. In articular
cartilage, the loss of type II collagen and proteoglycans
is due to the activation of extracellular matrix
(ECM)degrading enzymes such as matrix metalloproteinases
(MMPs) and ADAMTS [
]. In particular, MMPs play a
central role in articular cartilage destruction in OA and
rheumatoid arthritis patients. Among them, MMP-13
was reported to play important roles in the degradation
of proteoglycans and the activation of pro-collagenase in
the cartilage of individuals with OA [
In addition to MMPs, cyclooxygenase-2 (COX-2) and
inducible nitric oxide synthases also play important roles
in the pathogenesis of OA [
]. COX enzymes metabolize
arachidonic acid promoting the formation of prostaglandin
H2, which leads to increased production of prostaglandin
E2 (PGE2). There are two isoforms of the COX enzyme:
COX-1 is found in most tissues and is constitutively
expressed in normal cells, whereas COX-2 is not expressed
in healthy tissue but is stimulated by inflammatory
cytokines (interleukin-1 beta (IL-1β) and tumor necrosis factor
(TNF-α)), growth factors, and tumor promoters [
Moreover, COX-2 and PGE2 are important mediators of
inflammation and are implicated in bone resorption and
joint pain .
Rosmarinic acid (Ros. A;
α-o-caffeoyl-3,4-dihydroxyphenyl lactic acid) a naturally phenolic compound that is
mainly found in species of the Boraginaceae, Labiate, and
Anthocerotaceae families of herbs [
], and was first
isolated from rosemary (Rosmarinus officinalis). Many
studies have reported that Ros. A has various biological
and pharmacological activities, such as anti-tumor,
antioxidant, anti-inflammatory, anti-fibrosis, anti-mutagenic,
and hepatoprotective effects [
]. Recently, studies
have examined the inhibitory effect of Ros. A on MMP-2
and MMP-13 activity [
]. Hence, Ros. A is used to
treat bronchial asthma, peptic ulcers, cataracts, arthritis,
cancer, and rheumatoid arthritis [
]. However, the
molecular mechanism underlying Ros. A-induced
inflammation and differentiation, mediated by MMP-13 expression,
has not been elucidated.
The mitogen-activated protein kinase (MAPK) is a
signaling pathway that is can be activated in articular
cartilage; it is well documented that this pathway regulates
the production of MMPs, which are devoted to the
degeneration of chondrocytes [
]. MAPK family members
include extracellular signal-regulated kinase (ERK)-1/2,
p38, and c-Jun N-terminal kinase (JNK). ERK-1/2 and p38
play major roles in mediating chondrocyte proliferation
and differentiation and have been found to be associated
with inflammation [
In this study, we investigated if Ros. A can regulate
differentiation and inflammation in chondrocytes. Our
findings indicate that Ros. A promotes the inflammatory
response of rabbit articular chondrocytes by activating
ERK and suppressing MMP-regulated differentiation via
the p38 kinase pathway.
Reagents and antibodies
Rosmarinic acid (Ros. A), with purity greater than 98%,
was purchased from Cayman chemical Co. (Ann Arbor,
MI, USA). Primary antibodies specific for MMP-13,
type II collagen, and actin were obtained from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and
phosphorylated ERK and phosphorylated p38 MAP kinase
antibodies were purchased from Cell Signaling Technology
Inc. (Danvers, MA, USA). A COX-2 antibody was obtained
from Cayman chemical Co. Anti-rabbit IgG antibody and
anti-goat lgG were obtained from Sigma-Aldrich (St. Louis,
MO, USA) and anti-mouse IgG was purchased from Enzo
Life Sciences International, Inc. (New York, NY, USA).
Anti-mouse IgG-FITC, Anti-mouse IgG-TRITC and alcian
blue solution were purchased from Sigma-Aldrich.
Primary culture of chondrocytes from rabbit articular cartilage
Rabbit articular chondrocytes were isolated from
two-weekold New Zealand White rabbits (Koatech, Pyeongtaek,
Republic of Korea) as described previously [
slices were digested with 0.2% collagenase type II for 8 h in
a 37 °C CO2 incubator. Isolated chondrocytes (2 × 105 cells/
dish) were seeded in Dulbecco’s modified Eagle’s medium
(DMEM; Invitrogen, Carlsbad, CA, USA) containing 10%
(v/v) fetal bovine serum (Tissue Culture Biologicals, Los
Alamitos, CA, USA), penicillin (50 unit/mL, Sigma-Aldrich),
and streptomycin (50 μg/mL, Sigma-Aldrich), and
maintained as monolayers in a 5% CO2 incubator at
37 °C. The medium was replaced with fresh medium 2 d
after seeding. This study protocol was approved by the
Ethics Committee of the Kongju National University.
Treatment of cells with Ros. A
Ros. A was first dissolved in dimethyl sulfoxide (DMSO,
Sigma-Aldrich, St. Louis, USA) and then dissolved with
specific culture medium to the desired final
concentration; for this, the overall DMSO concentration was less
than 0.1% (v/v). After 3 d, the cell cultures were treated
with various concentrations (0, 25, 50, 75, and 100 μg/mL)
of Ros. A for 3 or 24 h. Alternatively, cells were treated
with 75 μg/mL Ros. A for various time periods. PD98059
(PD; Calbiochem, San Diego, CA, USA) and SB203580
(SB; Biomol, Plymouth Meeting, PA, USA), used to inhibit
MMP and MMP-13, were added 2 h before treatment
with Ros A. These compounds were used to inhibit
ERK1/2 and p38, respectively. The differentiation status and
inflammation responses of articular chondrocytes was
determined by examining the expression of type II collagen
and COX-2 by western blot analysis.
Western blot analysis
After the indicated treatment, cell samples were washed
once with cold phosphate-buffered saline (PBS) and
lysed using a buffer containing 50 mM tris-HCl
(pH 7.4), 150 mM NaCl, 1% nonidet P-40, and 0.1%
SDS supplemented with protease inhibitors and
phosphatase inhibitors on ice for 30 min. Proteins were
extracted and cell debris was removed by centrifugation
at 13,000 rpm for 10 min at 4 °C. Protein
concentrations were determined using the bicinchoninic acid
(BCA) assay. Equal amounts of the extracted proteins
(30 μg) were separated by 8% sodium dodecyl sulfate
polyacrylamide gel electrophoresis and transferred to a
nitrocellulose membrane. The membrane was blocked
with 5% non-fat dry milk in tris-buffered saline (TBS)
for 1 h at room temperature and washed three times
with TBS containing 0.05% tween-20 (TBS-T buffer).
The membrane was incubated with primary antibodies
overnight at 4 °C. Membranes were then washed three
times with TBS-T buffer and then incubated with
peroxidase-conjugated secondary antibody for 2 h at
room temperature. The enhanced chemiluminescence
(ECL) reagent was used to identify reactive bands.
Finally, the bands were quantified using the LAS4000
(Fuji Film, Tokyo, Japan). The bands were quantified by
densitometric analysis using the ImageJ software package.
Alcian blue staining
The cells were fixed with 3.5% paraformaldehyde in
PBS at room temperature for 20 min and stained with
0.1% alcian blue in 0.1 M HCl overnight. The
chondrocytes were washed three times with PBS buffer
and then incubated in 6 M guanidine HCl for 6 h.
Production of sulfated proteoglycan was measured at
595 nm using a microplate reader.
Cells (2 × 104 cells/well) were seeded in 96-well plates.
After 24 h of treatment, conditioned medium was
harvested and PGE2 concentrations were determined
using an ELISA assay kit according to instructions
supplied by the manufacturer (Assay Designs, Ann Arbor, MI,
USA). Samples were assayed in triplicate for each of three
independent experiments. PGE2 levels were calculated by
comparing values to a standard curve.
The expression of type II collagen and COX-2 at the
protein level in rabbit chondrocytes was analyzed by
Immunofluorescence microscopy. Cells were fixed with
3.5% paraformaldehyde in PBS for 20 min at room
temperature and permeabilized with 0.1% triton X-100
in PBS for 15 min. The cells were then blocked with 5%
skim milk to prevent non-specific reactions. Then, fixed
cells were incubated with antibodies against type II
collagen (1:100) and COX-2 (1:100) for 2 h at room
temperature. Cells were washed three times with PBS
and incubated with secondary antibodies (1:50) at room
temperature for 1 h. Then cells were counterstained with
4′6′-diamidi-no-2-phenylindole dihydrochloride (DAPI;
Invitrogen, Burlington, ON, Canada). Fluorescence images
were recorded using a BX51 fluorescence microscope
(Olympus, Tokyo, Japan).
All experimental data were replicated at least three
times. Data are presented as the mean ± standard
deviation (SD). Results were analyzed using a one-way
analysis of variance (ANOVA), and all pairwise
comparisons between groups were conducted using the
Turkey post hoc test; p values ≤0.05 were considered
Effect of Ros. A on rabbit chondrocyte differentiation
We performed western blot analysis and alcian blue
staining to identify the effects of Ros. A on the
differentiation of rabbit articular chondrocytes; we examined
type II collagen (a marker of chondrocyte
differentiation) expression and sulfated-proteoglycan
(cartilagespecific marker molecule) production after exposure to
Ros. A. As shown in Fig. 1, western blot analysis
showed that Ros. A increased the expression of type II
collagen in a dose- and time-dependent manner (Fig. 1a
and b, upper panel). Densitometric evaluation of
western blots was performed in triplicate (Fig. 1a and b,
lower panel). The synthesis of sulfate proteoglycans was
also examined. Consistent with the expression patterns
of type II collagen, alcian blue staining revealed that
Ros. A induced the production of sulfated proteoglycan
in a dose- and time- dependent manner (Fig. 1c and d).
These results indicate that Ros. A promotes the
differentiation of chondrocytes.
Effect of Ros. A on inflammation in rabbit chondrocytes
To determine whether Ros. A affects inflammation in
chondrocytes, these cells were treated with various
concentrations of Ros. A for 3 h or with 75 μg/ml of Ros. A for
various time periods (Fig. 2). A concentration-dependent
increase in COX-2 expression was observed (Fig. 2b, upper
panel). Stimulation of cells resulted in a marked increase in
COX-2 expression, which reached a maximum at 3 h, and
decreased thereafter (Fig. 2a, upper panel). Densitometric
evaluation of representative western blots was performed in
triplicate (Fig. 2a and b, lower panel).
To assess the effect of Ros. A on COX-2 activity,
we quantified the production of PGE2 in untreated
rabbit chondrocytes or those treated with Ros. A
(Fig. 2c and d). A significant increase in PGE2
synthesis was verified in treated rabbit chondrocytes.
Increases in PGE2 production and COX-2 expression
induced by Ros. A were similar (Fig. 2c and d). These
data suggest that Ros. A induces inflammation by
increasing COX-2 expression and PGE2 production in
rabbit articular chondrocytes.
Effect of Ros. A on MMP-13 expression in rabbit chondrocytes
Because previous studies have showed that Ros. A
modulates the expression of MMP-13 [
], we performed
western blotting to evaluate this (Fig. 3). Treatment with
Ros.A resulted in a significant decrease in MMP-13 in a
dose- and time- dependent manner (Fig. 3a and b, upper
panel). Densitometric evaluation of representative
western blots was performed in triplicate (Fig. 3a and b,
Many studies have shown that MMPs are involved in
both physiological collagen turnover in articular cartilage
and matrix degradation in OA cartilage. In addition,
previous studies have suggested that inflammatory cytokines
stimulate the expression of MMPs [
]. Hence, we
evaluated if the aforementioned effects of Ros. A on type II
collagen and COX-2 expression were because of
MMP13 activity. Chondrocytes were treated with 75 μg/mL
Ros. A in the absence or presence of 10 μM MMP
inhibitor (MMPI) or 113 nM MMP-13 inhibitor for 24 h
(Fig. 4). Treatment with MMPI and MMP-13 inhibitors
enhanced Ros. A-induced differentiation and suppressed
MMP-13 expression. However, this did not affect Ros
Ainduced COX-2, ERK-1/2, and p38 expression (Fig. 4a,
left panel). Densitometric evaluation of representative
western blots was performed in triplicate (Fig. 4a,
right panel). This was confirmed by alcian blue
staining, PGE2 assays, and immunofluorescence staining
(Fig. 4b and c). These results indicate that Ros. A
suppresses MMP-13-regulated type II collagen expression
and sulfate proteoglycan production in rabbit articular
chondrocytes. Taken together, our results suggest that
RosA reduces-MMP13 regulated differentiation in rabbit
Effect of ERK-1/2 and p38 on Ros. A-induced differentiation and inflammation in rabbit chondrocytes
We next examined whether Ros. A-mediated MAPK
activation is associated with the differentiation and
inflammation of chondrocytes (Fig. 5). Several
studies have indicated that the MAPK pathway is
involved in regulating these processes [
Western blot analysis indicated a
concentrationindependent increase in ERK-1/2 and p38
phosphorylation levels, reaching a maximum at 75 μg/mL
(Fig. 5a, upper panel). Ros. A treatment resulted in
the phosphorylation of p38 and ERK-1/2 in
timedependent manner (Fig. 5b, upper panel).
Densitometric evaluation of representative western blots was
performed in triplicate (Fig. 5a and b, lower panel).
To examine whether ERK-1/−2 and p38 play crucial
roles in the regulation of type II collagen and
COX2, we treated Ros. A chondrocytes with the specific
inhibitors, PD or SB (Fig. 6). Treatment with the
ERK-1/2 inhibitor PD restored COX-2 expression
and PGE2 production in Ros.A-treated chondrocytes
(Fig. 6a and b (lower panel)). Inhibition of p38 with
SB promoted Ros. A-induced differentiation via
MMP-13 (Fig. 6a and b (upper panel)). Moreover,
consistent with western blotting data,
immunofluorescence analysis showed that treatment with SB
enhances the Ros. A-mediated increase in type II
collagen, and that PD abolishes the increase in
COX-2 expression (Fig. 6c).
OA is the most prevalent disease of joints in elderly
patients, affecting many millions of individuals
worldwide and resulting in knee pain and locomotor system
]. OA is characterized by intra-articular
inflammation, cartilage degeneration, and subchondral
bone remodeling. In normal physiological conditions,
homeostasis is maintained in the ECM of articular
chondrocytes through the regulation of synthesis and
degradation . However, this cartilage homeostasis
is disrupted in OA and RA, causing continual loss of
cartilaginous tissues [
]. Much is known about
how cartilage is formed during skeletal development
and a number of factors are known to contribute to
the development of OA. However, the exact causes of
this disease are still unclear and very little is known
about how to successful apply this knowledge to
control the OA disease process [
plant-derived compounds that protect or stimulate
healing of the cartilage have been investigated as ideal
drugs for OA, due to their few side effects and
antiinflammatory activities [
Type II collagen is the main protein component of
and is specific for cartilage, forming up to 50% of its
ECM. Particularly, this marker can be used as a factor
to predict the response to therapy in OA. Collagen
and proteoglycans are degradation and lost due to
excessive secretion of MMPs and other proteolytic
22, 28, 29
]. The MMP family contains
approximately 28 members, which can be divided into
subgroups such as collagenases, gelatinases, and
stromelysins. Among them, MMP-1 and MMP-13 are
considered to be the major collagenases in
chondrocytes during conditions of low inflammation [
Notably, MMP-13 has 5- to 10-fold greater activity than
collagen type II and gelatinase activity that is greater than
44-fold higher than that of MMP-1 . Therefore,
MMP-13 has been considered a key regulator of cartilage
degradation and has become a valid target for OA
therapy. In this study, we found that Ros. A can suppress
MMP-13 protein expression (Fig. 3) and the
upregulation of type II collagen and sulfated proteoglycan
(Fig. 1). In addition, MMPI and MMP-13 inhibitors
selectively reduced MMP-13 levels and stimulated Ros
A-induced differentiation (Fig. 4).
Previous studies have provided clear evidence that OA
is associated with increased production of IL-1β, which
plays a key role in chondrocyte damage through the
upregulation of pro-inflammatory factors including MMPs
and COX-2 [
]. Excess induction of COX-2 expression
leads to elevated production of PGE2. Although OA
tissues can be damaged by very low levels of
proinflammatory cytokines, through increased production
of MMP-13, which induces proteoglycan degradation,
this was shown to further enhance the loss of type II
collagen in OA joints [
]. In this study, our data
demonstrated Ros. A-induced expression of COX-2 in rabbit
articular chondrocytes (Fig. 2); however, degradation of
type II collagen and MMP-13 expression were not
elevated by COX-2. In addition, inhibition of MMP-13
by MMPI or an MMP-13 inhibitor had no effect of
COX-2 expression (Fig. 4).
Ros. A was isolated for the first time from
Rosmarinus officinalis L. by Scarpati and Oriente in 1958
]. This compound was structurally characterized
as an ester of caffeic acid and
3,4-dihydroxyphenyllactic acid. It is known to exhibit various
pharmacological activities, notably anti-oxidant, anti-microbial,
and anti-inflammatory activities, and thus has been
used to treat peptic ulcers, arthritis, cataracts,
cancer, and bronchial asthma, among other illnesses
]. Hur et al. reported that Ros. A induces the
preferential apoptotic activity of activated and
effector T-cells via the mitochondrial pathway [
Furthermore, Han et al. investigated the effect of RA
on MKN45 human gastric cancer cells and found
that it exerted an anti-cancer effect via the inhibition
of pro-inflammatory cytokines and the inactivation
of inflammatory pathways [
]. Moon et al.
reported that Ros. A treatment sensitizes human
leukemia U937 cells to TNF-α-induced apoptosis
through the suppression of nuclear factor-κB and
reactive oxygen species . In previous investigations,
pretreatment with Ros. A was shown to reduce
COX-2 mRNA expression in a TPA-challenged skin
mouse model [
]. In addition, in a murine collagen
induced arthritis model, Ros. A was shown to
remarkably reduce the frequency of COX-2-expressing
cells, when compared to that in untreated mice [
However, strikingly, Ros. A did not reduce COX-2
expression, but rather upregulated type II collagen
and sulfated proteoglycan in chondrocytes.
The MAPK signal transduction pathway promotes cell
proliferation, differentiation, and apoptosis, which could
account for the effects observed in some degenerative
diseases such as OA [
]. It also serves as the
predominant system that regulates the production of
MMPs, which promote the degeneration of
chondrocytes. p38 and ERK play major roles in mediating
chondrocyte proliferation, dedifferentiation, inflammation,
and related gene expression . To investigate the
involvement of the MAPK cascade in the Ros. A-induced
differentiation and inflammation of chondrocytes, the
phosphorylation patterns of the ERK1−/2 and p38
were assessed by western blotting after Ros A
treatment. Chondrocytes treated with Ros A displayed
enhanced ERK-1/2 and p38 kinase activity (Fig. 5).
Additionally, whereas inhibition of ERK, through
treatment with PD, abolished Ros. A-induced COX-2
expression, suppression of p38 through treatment with
SB accelerated MMP-13-induced type II collagen
expression (Fig. 6). Thus, in rabbit articular
chondrocytes, Ros. A enhances inflammation through ERK-1/2
signaling and MMP-regulated differentiation via MMP-13
inhibition and downstream p38 kinase signaling. A
graphical pathway summarizing the underlying mechanisms is
shown in Fig. 7.
Our results, using Ros. A, demonstrate that that p38
regulates differentiation by inhibiting MMP-13 and
that ERK-1/2 regulates Ros. A-induced inflammation
in rabbit articular chondrocytes. This information is
useful to understanding the molecular mechanism of
OA and Ros. A may be a potential candidate for
further investigation for future use in the treatment
or cartilage-related disorders including OA.
Fig. 7 A graphical depiction of the effects of rosmarinic acid (Ros. A)
on the regulation of inflammation and differentiation in rabbit
COX-2: Cyclooxygenase-2; EMC: Extracellular matrix; ERK: Extracellular
signalregulated kinase; MAPK: Mitogen-activated protein kinase; MMPI: Matrix
metalloproteinases inhibitor; MMPs: Matrix metalloproteinases;
OA: Osteoarthritis; PD: PD98059; PGE2: Prostaglandin E2; Ros. A: Rosmarinic
acid; SB: SB203580
This work was supported by a grant from the National Research Foundation
of Korea (NRF) funded by the Korea Government (MEST)
Availability of data and materials
All data generated or analyzed during this study are included in this
SHE was designed experiments, conducted research, and wrote manuscript.
SJK contributed in the data analysis, designed experiments, conducted
research, and wrote manuscript. Both authors read and approved the final
Ethics approval and consent to participate
The study was approved by the Ethics Committee of Kongju National
University, Gongju, Korea (Gongju, Republic of Korea; IRB no. 2011–2).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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