All chemicals and reagents used in this experiment, including prunetin (purity: 98.0%), quercitrin (purity: 95.0%) and glycyrrhizin (purity: 95.0%) 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).

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 [14].

Chondrocytes were seeded on 6-well culture plates 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 prunetin, glycyrrhizin or quercitrin, followed by incubation in the presence or absence of IL-1β (10 ng/mL) for 24 h. Prunetin, glycyrrhizin or quercitrin 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.

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. Prunetin 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 [15].

Total RNA was isolated from chondrocytes using the Easy-BLUE Extraction Kit (INTRON Biotechnology, Inc. Kyungki-do, 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'), and ADAMTS-5 (5'TGT CCT GCCAGC GGATGT 3'; 5'ACG GAA TTA CTG TAC GGC CTA CA 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, and 400 bp for GAPDH. After PCR, 15 µL of PCR products were subjected to 2% agarose gel electrophoresis and visualized with ethidium bromide under a transilluminator [14].

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).

A modified casein-substrate zymography was carried out using culture supernatants from chondrocytes pretreated for 2 h with prunetin 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 [14].

Male Sprague–Dawley rats (Daehan Biolink, Seoul, South Korea) weighing 200~210 g were used to investigate the effect of prunetin 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: control, IL-1β only, 50 µM prunetin plus IL-1β, or 100 µM prunetin plus IL-1β. Rats were anesthetized with vaporized diethyl ether, and those from the 50 µM prunetin plus IL-1β and 100 µM prunetin plus IL-1β treatment groups received a 30 µL injection of 50 µM or 100 µM prunetin, respectively, into the right knee joint. After 3 h, rats from the IL-1β only group, the 50 µM prunetin plus IL-1β group, and the 100 µM prunetin 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).

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.

To compare the potency of activity on the gene expression of MMP-3, the key matrix metalloproteinase involved in destruction of articular cartilage, effect on MMP-3 gene expression was investigated by pretreatment of glycyrrhizin or quercitrin – major steroidal compound derived from Glycyrrhizae Radix or similar flavonoidal compounds with prunetin – in addition to prunetin (Fig. 1). As shown in Fig. 2, prunetin inhibited IL-1β-induced MMP-3 gene expression, at 10 µM, 50 µM, and 100 µM. However, glycyrrhizin or quercitrin did not significantly affect MMP-3 gene expression.

To investigate the potential cytotoxicity of prunetin to cultured rabbit chondrocytes, effect of prunetin on proliferation of rabbit chondrocytes using SRB assay was tested. As can be seen in Fig. 3, prunetin showed no significant cytotoxicity at the concentrations of 1, 10, 50, and 100 µM. The numbers of cells in prunetin-treated cultures were 100±16%, 97±9%, 89±21%, 105±11%, and 92±21% for control, 1, 10, 50, and 100 µM prunetin, respectively.

If prunetin can affect the gene expression of MMP-3, the key matrix metalloproteinase involved in destruction of articular cartilage, it should be investigated whether prunetin affects the gene expression of MMP-1, MMP-13, ADAMTS-4 or ADAMTS-5, the other degradative enzymes related to destruction of articular cartilage, in rabbit chondrocytes. As shown in Fig. 4, prunetin showed the suppression of IL-1β-induced gene expression of MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5.

If prunetin can affect the MMP-3 gene expression at the transcriptional level, it should be investigated whether prunetin affects IL-1β-induced secretion of MMP-3 proteins from rabbit articular chondrocytes. As shown in Fig. 5, stimulation with IL-1β (10 ng/mL) increased secretion of MMP-3 from chondrocytes. However, prunetin inhibited the effects of IL-1β on MMP-3 secretion. This result means that prunetin can control the steps of protein synthesis and secretion of MMP-3.

To investigate the effect of prunetin 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 can be seen in Fig. 5, IL-1β increased the caseinolytic activity of MMP-3 in rabbit articular chondrocytes, and this effect was inhibited by pretreatment with prunetin.

To investigate whether prunetin shows the potential effect in vivo , we examined the effect of intraarticular injection of prunetin into the knee joint of rats on IL-1β-stimulated production of MMP-3 from articular cartilage tissues. As shown in Fig. 6, treatment with IL-1β (20 ng/30 µL) increased MMP-3 production in articular cartilage tissues. However, prunetin inhibited IL-1β-induced MMP-3 production, in vivo.