Journal List > Korean J Physiol Pharmacol > v.21(2) > 1026161

Kang, Lee, Kim, Hwang, Lee, and Park: Effect of oleanolic acid on the activity, secretion and gene expression of matrix metalloproteinase-3 in articular chondrocytes in vitro and the production of matrix metalloproteinase-3 in vivo

Abstract

In the present study, we tried to examine whether oleanolic acid regulates the activity, secretion and gene expression of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as the production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effect of oleanolic acid. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. In rabbit articular chondrocytes, the effects of oleanolic acid on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of oleanolic acid on in vivo MMP-3 protein production was also examined, after intra-articular injection to the knee joint of rat. The results were as follows: (1) oleanolic acid inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) oleanolic acid reduced the secretion and proteolytic activity of MMP-3; (3) oleanolic acid suppressed the production of MMP-3 protein in vivo. These results suggest that oleanolic acid can regulate the activity, secretion and gene expression of MMP-3, by directly acting on articular chondrocytes.

Introduction

Osteoarthritis has been reported to be the most common degenerative articular disease, from which millions of people suffered, particularly in the elderly. Among the symptoms of osteoarthritis, degeneration of articular cartilage, formation of osteophytes, synovial inflammation, and changes in subchondral bone are the major pathophysiologic features. Although the cause of osteoarthritis is not fully understood, it involves several biochemical and mechanical factors, such as disruption on the equilibrium between physiologic synthesis and degradation of articular cartilage during the progression of osteoarthritis [12].
While the activation of degradative enzymes leads to the loss and degradation of proteoglycans and collagen in articular cartilage, the matrix metalloproteinases (MMP) play a pivotal role in the destruction of articular cartilage in osteoarthritis patients [34]. MMPs can be classified into collagenases (MMP-1, -8 and -13), gelatinases (MMP-2 and -9), and stromelysins (MMP-3, -7, -10 and -11) [56]. Out of these MMPs, MMP-3 degrades proteoglycans and activates procollagenase in articular cartilage [78]. In addition to MMP-3, MMP-1 and MMP-13 were reported to play important roles in the destruction of cartilage in osteoarthritis. MMP-1 is a commonly detected metalloproteinase in synovial fluid from patients suffering from osteoarthritis [9101112131415]. Another degradative enzyme, ADAMTS-4, is a major aggrecanase in cartilage of mouse and ADAMTS-5 has been known to be important in cartilage matrix destruction during osteoarthritis [1617].
Therefore, we suggest it is valuable to find the potential activity of regulating (inhibiting) the expression and activity of MMPs by the compounds derived from various medicinal plants used as arthritis remedies in folk medicine, for development of new therapeutic strategies for osteoarthritis. We have tried to investigate the potential activity of some natural products on the expression and activity of MMP-3 in articular chondrocytes. As a result of our trial, we previously reported that several natural compounds affected the gene expression, secretion (production) and proteolytic activity of MMP-3, in vitro and in vivo [18192021].
As claimed by various reports, oleanolic acid, a natural product derived from Cornus officinalis, a medicinal plant used for controlling various inflammatory diseases in folk medicine, showed the various biological activities including anti-oxidative and anti-inflammatory effects [2223242526]. Jeon and colleagues reported that oleanolic acid could enhance cognitive function [24]. Also, oleanolic acid was reported to attenuate renal ischemia/reperfusion injury through its antioxidant, anti inflammatory and anti apoptotic activities [25] and inhibit rheumatoid arthritis via modulating T cell immune responses [26].
However, to the best of our knowledge, there is no report about the effect of oleanolic acid on the gene expression, secretion, and enzyme activity of MMP-3, an articular cartilage-degradative enzyme that decomposes proteoglycans, in primary cultured rabbit articular chondrocytes, or on in vivo production of MMP-3 in the rat knee joint. Therefore, to evaluate the chondroprotective potential of oleanolic acid, we investigated its effects on IL-1β-induced gene expression, secretion, and enzyme activity of MMP-3 in vitro, and on production of MMP-3 in vivo.

Methods

Materials

All the chemicals and reagents used in this experiment, including oleanolic acid (purity: 98.0%), were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Gibco-BRL (Grand Island, NY, USA) and recombinant human IL-1β was purchased from R&D Systems (Minneapolis, MN, USA).

Primary cultures of chondrocytes from rabbit articular cartilage

Male New Zealand White rabbits were obtained from Daehan Biolink (Seoul, South Korea) at 2 weeks of age. Animals were housed 1 animal per cage, provided with distilled water and food ad libitum, and kept under a 12 h light/dark cycle (lights on from 08:00~20:00) at constant temperature (22.5℃) and humidity (55%). Animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, and care was regulated by Chungnam National University (the approval number of animal experiment: CNU-00555) (Daejeon, Korea). Rabbit articular chondrocytes were isolated from the tibial plateau and femoral condyle in cartilage of the knee joint. Cartilage was washed in phosphate-buffered saline (PBS) and minced into pieces measuring 2 mm3, approximately. Cartilage tissue was digested for 4 h with 0.2% type II collagenase at 37℃. After collection of individual cells by brief centrifugation, the cells were transferred to 100 mm culture dishes (seeding density: 105 cells/cm2) in 12 mL DMEM supplemented with 10% fetal bovine serum (FBS), in the presence of penicillin (100 units/mL) and streptomycin (100 µg/mL). Cells were cultured at 37℃ in a humidified, 5% CO2/95% air, water-jacketed incubator, and medium was replaced every other day [27].

Treatment of cells with oleanolic acid

Chondrocytes were seeded on 6-well culture plates (for RT-PCR) or 60 mm culture dishes (for western blotting) at a density of 105 cells/cm2. After 2 days in monolayer culture, the cells were incubated for 2 h in growth medium with 1, 10, 50, or 100 µM of oleanolic acid followed by incubation in the presence or absence of IL-1β (10 ng/mL) for 24 h. Oleanolic acid was dissolved in dimethylsulfoxide, diluted in PBS, and administered in culture medium (final concentrations of dimethylsulfoxide were 0.5%). The final pH values of these solutions were between 7.0 and 7.4. Culture medium and 0.5% dimethylsulfoxide in medium did not affect the gene expression, secretion, or proteolytic activity of MMP-3 in primary cultured chondrocytes. The supernatant was collected and centrifuged, and cell and supernatant fractions were stored at −80℃ until use.

Cytotoxicity assay

Chondrocytes were seeded at a density of 2×105/mL (0.1 mL/well) in a 96-well microtiter plate, and allowed to attach for 24 h to keep the log phase growth at the time of drug treatment. Oleanolic acid was dissolved in DMSO, and administered in DMEM supplemented with 10% FBS (final concentrations of DMSO were under 0.5%). 0.5% DMSO alone did not affect the proliferation of chondrocytes. After incubation with the indicated drug concentrations for 72 h, cell proliferation was determined using the sulforhodamine B (SRB) assay [28].

Isolation of total RNA and RT-PCR

Total RNA was isolated from chondrocytes using the Easy-BLUE Extraction Kit (INTRON Biotechnology, Inc. Kyung-kido, South Korea), and reverse transcribed using AccuPower RT Premix (BIONEER Corporation, Daejeon, South Korea) according to the manufacturer's instructions. About 2 µg of total RNA was primed with 1 µg of oligo (dT) in a final reaction volume of 30 µL. 2 µL of RT reaction product was amplified in 20 µL using Thermoprime Plus DNA Polymerase (ABgene, Rochester, NY, USA). PCR was performed with the following primers: MMP-3 (5'ATG GAC CTT CTT CAG CAA 3', 5'TCA TTA TGT CAG CCT CTC 3'), MMP-13 (5'AGG AGC ATG GCG ACT TCT AC 3', 5'TAA AAA CAG CTC CGC ATC AA 3'), MMP-1 (5'TCA GTT CGT CCT CAC TCC AG 3', 5'TTG GTC CAC CTG TCA TCT TC 3'), ADAMTS-4 (5'CAA GGT CCC ATG TGC AAC GT 3', 5'CAT CTG CCA CCA CCA GTG TCT 3'), ADAMTS-5 (5'TGT CCT GCCAGC GGATGT 3'; 5'ACG GAA TTA CTG TAC GGC CTA CA 3'), and type II collagen (5'AAC ACT GCC AAC GTC CAG AT 3', 5'CTG ACG CAC GGT ATA GGT GA 3'). GAPDH (5'ACT GGC GTC TTC ACC ACC AT 3'; 5'AAG GCC ATG CCA GTG AGC TT 3') was used as a quantitative control. The PCR products increased as the concentration of RNA increased. The amplified fragment sizes were 350 base pairs (bp) for MMP-3, 458 bp for MMP-13, 300 bp for MMP-1, 90 bp for ADAMTS-4, 110 bp for ADAMTS-5, 220 bp for type II collagen, and 400 bp for GAPDH. After PCR, 15 mL of PCR products were subjected to 2% agarose gel electrophoresis and visualized with ethidium bromide under a transilluminator [27]. The signal intensity of each band was analyzed by GelQuant software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel).

Western blot analysis for measuring secretion level of MMP-3 in culture supernatant

Chondrocytes (confluent in 60 mm culture dish) were incubated for 2 h in growth medium with 1, 10, 50, or 100 µM of oleanolic acid followed by incubation in the presence or absence of IL-1β (10 ng/mL) for 24 h. After the treatment, the supernatant was collected and the cells were harvested using 3 x trypsin-EDTA solution and then centrifuged in a microcentrifuge (1,200 rpm, 3 min, 4℃). The Bradford assay was used to measure protein concentrations in culture supernatants to ensure consistent weight of protein samples subjected to electrophoresis. Culture supernatant samples containing MMP-3 protein (50 µg each) were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a polyvinylidene difluoride (PVDF) membrane. Blots were blocked using 5% skim milk in Tris-buffered saline/Tween 20 (TBS-T), and probed overnight with MMP-3 antibody in blocking buffer at 4℃. Antibody against MMP-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were washed with TBS-T and probed for 1 h with a secondary antibody conjugated with horseradish peroxidase (Calbiochem, La Jolla, CA, USA). After 4 washes with TBS-T, immunoreactive bands were detected using an enhanced chemiluminescence kit (Pierce ECL western blotting substrate, Thermo Scientific, Waltham, MA, USA). The signal intensity of each band was analyzed by GelQuant software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel).

Casein zymography to measure the proteolytic activity of MMP-3

A modified casein-substrate zymography was carried out using culture supernatants from chondrocytes pretreated for 2 h with oleanolic acid and stimulated for 24 h with IL-1β in DMEM containing 0.5% FBS. The Bradford assay was used to measure protein concentrations in culture media to ensure consistency across samples. Samples were electrophoresed at 4℃ in a 10% SDS gel containing 0.1% casein. After electrophoresis, gels were washed with 10 mM Tris-HCl (pH 8.0) containing 2.5% Triton X-100. Next, gels were incubated at 37℃ for 48 h in 50 mM Tris-HCl (pH 8.0) containing 1% Triton X-100, 0.2 M NaCl, and 5 mM CaCl2. Finally, gels were stained with 1% Coomassie Brilliant Blue, destained, and photographs were taken [27]. The signal intensity of each band was analyzed by GelQuant software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel).

In vivo experiments

Male Sprague–Dawley rats (Daehan Biolink, Seoul, South Korea) weighing 200~210 g were used to investigate the effect of oleanolic acid on production of MMP-3 in articular cartilage in vivo. Animals were housed 5 per cage, provided with distilled water and food ad libitum, and kept under a 12 h light/dark cycle (lights on from 08:00~20:00) at constant temperature (22.5℃) and humidity (55%). Animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, and care was regulated by Chungnam National University (the approval number of animal experiment: CNU-00555) (Daejeon, South Korea). Rats were randomly divided into 4 groups (3 rats/group): control, IL-1β only, 50 µM oleanolic acid plus IL-1β, or 100 µM oleanolic acid plus IL-1β. Rats were anesthetized with vaporized diethyl ether, and those from the 50 µM oleanolic acid plus IL-1β and 100 µM oleanolic acid plus IL-1β treatment groups received a 30 µL injection of 50 µM or 100 µM oleanolic acid, respectively, into the right knee joint. After 3 h, rats from the IL-1β only group, the 50 µM oleanolic acid plus IL-1β group, and the 100 µM oleanolic acid plus IL-1β group received a 30 µL injection of 20 ng IL-1β in sterile PBS into the right knee joint. Rats from the control group were injected with 30 µL of sterile PBS. Rats were euthanized via CO2 asphyxiation 72 h after injections. Articular cartilage (tibial plateau and femoral condyle) was isolated from each animal, homogenized, and prepared for measurement of MMP-3 protein by western blot analysis. Tissue lysates from articular cartilage homogenates containing MMP-3 protein (50 µg each) were subjected to 10% SDS-PAGE, and transferred onto a PVDF membrane. Blots were blocked with 5% skim milk in TBS-T, and probed with MMP-3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in blocking buffer overnight at 4℃. Membranes were washed with TBS-T, and probed for 1 h with a secondary antibody conjugated with horseradish peroxidase (Calbiochem, La Jolla, CA, USA). After 4 washes with TBS-T, immunoreactive bands were detected using an enhanced chemiluminescence kit (Pierce ECL western blotting substrate, Thermo Scientific, Waltham, MA, USA). The signal intensity of each band was analyzed by GelQuant software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel).

Statistics

Means of individual group were converted to percent control and expressed as mean±S.E.M. The difference between groups was assessed using one-way ANOVA and Holm-Sidak test as a post-hoc test. p<0.05 was considered as significantly different.

Results

Effect of oleanolic acid on MMP-3 gene expression in rabbit chondrocytes

To investigate the potential activity of oleanolic acid on the gene expression of MMP-3, the key matrix metalloproteinase involved in destruction of articular cartilage, MMP-3 gene expression was measured after pretreatment of oleanolic acid. As can be seen in Fig. 1, oleanolic acid inhibited IL-1β-induced MMP-3 gene expression.

Effect of oleanolic acid on viability of rabbit chondrocytes

To examine the potential cytotoxicity of oleanolic acid to cultured rabbit chondrocytes, effect of oleanolic acid on viability of rabbit chondrocytes using SRB assay was tested. As shown in Fig. 2, oleanolic acid showed no significant cytotoxicity at the concentrations of 1, 10, 50, and 100 µM. The numbers of cells in oleanolic acid-treated cultures were 100±10%, 91±18%, 102±13%, 107±9%, and 95±11% for control, 1, 10, 50, and 100 µM oleanolic acid, respectively.

Effect of oleanolic acid on the gene expression of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5 or type II collagen in rabbit chondrocytes

If oleanolic acid can affect the gene expression of MMP-3, the key matrix metalloproteinase involved in destruction of articular cartilage, it should be investigated whether oleanolic acid affects the gene expression of MMP-1, MMP-13, ADAMTS-4 or ADAMTS-5, the other degradative enzymes related to destruction of articular cartilage, and type II collagen, in rabbit chondrocytes. As can be seen in Fig. 3, oleanolic acid showed the suppression of IL-1β-induced gene expression of MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, in rabbit chondrocytes. Furthermore, oleanolic acid showed an additional chondroprotective effect by restoring the compromised gene expression of type II collagen by IL-1β, in rabbit chondrocytes.

Effect of oleanolic acid on IL-1β-induced secretion of MMP-3 from rabbit articular chondrocytes

If oleanolic acid can affect the MMP-3 gene expression at the transcriptional level, it should be investigated whether oleanolic acid affects IL-1β-induced secretion of MMP-3 proteins from rabbit articular chondrocytes. As can be seen in Fig. 4, stimulation with IL-1β (10 ng/mL) increased secretion of MMP-3 from chondrocytes. However, oleanolic acid reduced the effect of IL-1β on MMP-3 secretion. This result means that oleanolic acid can control the steps of protein synthesis and secretion of MMP-3.

Effect of oleanolic acid on proteolytic activity of MMP-3 in rabbit articular chondrocytes

To examine the effect of oleanolic acid on the enzyme activity of secreted MMP-3, which is known to degrade proteoglycans, one of the two major matrix components of cartilage, culture supernatants from rabbit articular chondrocytes were analyzed for caseinolytic activity by casein zymography, after treatment with IL-1β for 24 h. As shown in Fig. 4, IL-1β increased the caseinolytic activity of MMP-3 in rabbit articular chondrocytes, and this effect was reduced by pretreatment with oleanolic acid.

Effect of oleanolic acid on MMP-3 production in vivo

To examine whether oleanolic acid shows the potential effect in vivo, we examined the effect of intraarticular injection of oleanolic acid into the knee joint of rats on IL-1β-stimulated production of MMP-3 from articular cartilage tissues. As can be seen in Fig. 5, treatment with IL-1β (20 ng/30 µL) increased MMP-3 production in articular cartilage tissues. However, oleanolic acid inhibited IL-1β-induced MMP-3 production, in vivo.

Discussion

In order to restore the broken equilibrium between physiological synthesis and degradation of articular cartilage during the progression of osteoarthritis, discovering a useful and specific pharmacological tool can be a promising approach to the effective control of this condition. Although osteoarthritis can be defined as a non-inflammatory disease, its development and progression have been attributed to low-grade inflammation in intraarticular sites, as well as to various inflammatory cytokines in articular tissues and fluids that are produced by chondrocytes and/or interact with chondrocytes [29303132].
IL-1β is an inflammatory cytokine that is produced by cells in articular tissues, including chondrocytes, and which can increase expression of MMPs and stimulate the progression of osteoarthritis. IL-1β plays an important role in the initiation and progression of destruction of articular cartilage by suppressing synthesis of collagen and stimulating MMP expression [313334]. In particular, MMP-3 has been reported to play a pivotal pathophysiological role in osteoarthritis by degrading components of the extracellular matrix, such as proteoglycans. MMP-3 levels were increased more than MMP-1 levels in patients suffering from osteoarthritis in knee joints compared to the control group [735].
As claimed by several reports, IL-1β-stimulated expression of MMPs is associated with the suppression of the NF-κB signaling pathway [36373839]. It was reported that coptisine, an isoquinoline alkaloid extracted from an anti-inflammatory medicinal plant, also inhibited the expression of MMP-3 and MMP-13 through inhibition of NF-κB activation in human chondrocytes [36]. Another natural product, matrine, inhibited IL-1β-induced expression of MMPs by suppressing the activation NF-κB in human chondrocytes in vitro [37]. Taraxasterol, a natural product with anti-inflammatory effect, suppressed IL-1β-induced expression of MMP-1, MMP-3, MMP-13 and activation of NF-κB [38]. Also, Schisandrae Fructus, an anti-inflammatory medicinal plant used in folk medicine, inhibited IL-1β-induced expression and activity of MMP-1, MMP-3, and MMP-13 and markedly suppressed the nuclear translocation of NF-κB by blocking IκB-alpha degradation in SW1353 human chondrocytes [39].
IL-1β activates several intracellular signal transduction cascades among which the NF-κB pathway is pivotal. NF-κB is a heterodimer composed of p65, p50 and IκBα subunits present in the cytoplasm as an inactive state. In response to various stimuli, the IκBα subunit is phosphorylated and degraded, thereby facilitating the translocation of p50~p65 heterodimer to the nucleus. The p50~p65 acts as a transcription factor regulating the expression of numerous genes including MMP-3 [39]. In the present study, oleanolic acid affected the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes. We found that oleanolic acid inhibited IL-1β-induced gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5 and restored the gene expression of type II collagen that had been inhibited by IL-1β, in rabbit articular chondrocytes (Figs. 1 and 3). Thus, the chondroprotective effect of oleanolic acid are supported by its regulation of the gene expression of diverse proteases involved in the destruction of articular cartilage in osteoarthritis, as well as by its promotion of the gene expression of type II collagen at the transcriptional level. Additionally, IL-1β-stimulated secretion and the proteolytic activity of MMP-3 from articular chondrocytes were reduced by oleanolic acid (Fig. 4). This result means that oleanolic acid can regulate the step of protein synthesis and secretion of MMP-3 and affects the proteolytic activity of overproduced and oversecreted MMP-3 in tissues of osteoarthritic articular cartilage.
The underlying mechanism of action of oleanolic acid on the gene expression, secretion, and proteolytic activity of MMP-3 are not clear at present, although we are investigating whether oleanolic acid act as a potential regulator of NF-κB signaling pathway in articualr chondrocytes, based on a report on the suppressive effect of oleanolic acid on dextran sodium sulfateinduced colitis in mice through inhibition of NF-κB signaling pathway [40].
Lastly, we investigated the effect of intraarticular injection of oleanolic acid into the knee joint of rats on IL-1β-stimulated production of MMP-3 in articular cartilage tissue. As shown in Fig. 5, oleanolic acid inhibited IL-1β-stimulated production of MMP-3 in articular cartilage tissue. This result shows that, in addition to its in vitro effects, oleanolic acid exerts chondroprotective effect in vivo when administered via intraarticular injection.
Taken together, the inhibitory action of oleanolic acid on the gene expression, secretion, and enzyme activity of MMP-3 in articular chondrocytes and production of MMP-3 in the knee joint of rats might explain, at least in part, the traditional use of Cornus officinalis as an anti-inflammatory agent for diverse inflammatory diseases, in folk medicine. We suggest it is valuable to find the natural products that have specific suppressive effects on the gene expression, secretion, and enzyme activity of MMP-3 - in view of both basic and clinical sciences - and the result from this study suggests a possibility of developing oleanolic acid as a candidate for novel agent controlling cartilage damage in osteoarthritis via intraarticular administration, although further studies are essentially required.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A6A1029617).

Notes

Author contributions: D.G.K. and H.J.L. performed in vitro and in vivo experiments. K.T.K. and S.C.H. wrote the manuscript. C.J.L. and J.S.P. supervised and coordinated the study.

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

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Fig. 1

Effect of oleanolic acid on MMP-3 gene expression in rabbit chondrocytes.

Primary cultured rabbit articular chondrocytes were pretreated with varying concentrations (1, 10, 50, and 100 µM) of oleanolic acid for 2 h and then stimulated with IL-1β (10 ng/mL) for 24 h. MMP-3 gene expression level was measured by RT-PCR. Three independent experiments were performed and the representative data were shown. The upper figure is a representative image data. The signal intensity of each band in images was analyzed by GelQuant software and means of individual group from the three independent experiments were converted to percent control and expressed as mean ±S.E.M. Each bar in the lower figure (graph) represents a mean±S.E.M. of three independent experiments in comparison with that of the control set at 100% (Figs. 1, 3, 4 and 5). *Significantly different from control (p<0.05). +Significantly different from IL-1β alone (p<0.05). cont, control; concentration unit is µM.
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Fig. 2

Effect of oleanolic acid on proliferation of rabbit chondrocytes.

Chondrocytes were incubated for 72 h in the presence of varying concentrations of oleanolic acid. Cell viability was determined using SRB assay as described in Materials and Methods. Each bar represents a mean±S.E.M. of three independent experiments in comparison with that of the control set at 100%.
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Fig. 3

Effect of oleanolic acid on the gene expression of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5, or collagen type II in rabbit chondrocytes.

Primary cultured rabbit articular chondrocytes were pretreated with varying concentrations (1, 10, 50, and 100 µM) of oleanolic acid for 2 h and then stimulated with IL-1β (10 ng/mL) for 24 h. The gene expression level of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5, or collagen type II was measured by RT-PCR.
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Fig. 4

Effects of oleanolic acid on IL-1β-induced secretion of MMP-3 and caseinolytic activity of MMP-3 in rabbit articular chondrocytes.

Primary cultured rabbit articular chondrocytes were pretreated with varying concentrations (1, 10, 50, and 100 µM) of oleanolic acid for 2 h and then stimulated with IL-1β (10 ng/mL) for 24h. Culture supernatants were collected for measurement of both the levels of produced and secreted MMP-3 by western blot analysis and the proteolytic activity of MMP-3 by casein zymography.
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Fig. 5

Effect of oleanolic acid on production of MMP-3 in vivo.

The knee joint of rats were pretreated with 50 or 100 µM of oleanolic acid for 3 h and then stimulated with IL-1β (20 ng/30 µL) for 72 h, by intraarticular injection. Tissue lysates from articular cartilage homogenates containing MMP-3 proteins were collected for measurement of the level of produced MMP-3 in vivo, by western blot analysis. Equal protein loading was evaluated by β-actin levels.
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