Biological Effects of the Herbal Plant-Derived Phytoestrogen Bavachin in Primary Rat Chondrocytes

Abstract

The aim of this study was to examine the anabolic and anticatabolic functions of bavachin in primary rat chondrocytes. With bavachin treatment, chondrocytes survived for 21 d without cell proliferation, and the proteoglycan content and extracellular matrix increased. Short-term monolayer culture of chondrocytes showed that gene induction of both aggrecan and collagen type II, major extracellular matrix components, was significantly upregulated by bavachin. The expression and activities of cartilage-degrading enzymes such as matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs were inhibited significantly by bavachin, while tissue inhibitors of metalloprotease were significantly upregulated. Bavachin inhibits the expression of inducible nitric oxide synthase, a representative catabolic factor, and downregulated the expression of nitric oxide, cyclooxygenase-2, and prostaglandin E₂ in a dose-dependent manner in chondrocytes. Our results suggest that the bavachin has anabolic and potent anticatabolic biological effects on chondrocytes, which may have considerable promise in treating articular cartilage degeneration in the future.

Osteoarthritis (OA) is a representative degenerative disease associated with aging and is the most common type of arthritis.¹⁾ The clinical symptoms of OA are defined as chronic joint pain, stiffness, and loss of mechanical joint function caused by progressive cartilage degeneration.²⁾ Moreover, the prevalence of OA is largely age-dependent and is increased significantly in people over age 65.¹⁾ Although the prevalence of OA is less in those under 40, prevalence is increasing in these populations because of obesity, individual heredity, mechanical injuries, and surgical trauma of joints.¹⁾ The socioeconomic burden associated with OA is increased by both direct medical expenses and indirect costs caused by decreased productivity in the workplace.²⁾ Although various studies on the pathophysiological etiologies of OA are ongoing, many of the factors causing this disease are still unknown. Hence, clinical treatments of OA have been mainly focused on relieving chronic joint pain by suppressing inflammation in the degenerative joint more so than on cartilage regeneration.

Homeostasis of articular cartilage is precisely regulated by the balance between synthesis (anabolism) and degradation (catabolism) of the extracellular matrix (ECM), which is mainly composed of collagen Type II and proteoglycan synthesized from chondrocytes to maintain mechanical function in response to a compressive overload on joints.³⁾ However, perturbations in chondrocytes with normal metabolic properties induce the progressive destruction of ECM through the up-regulation of cartilage-degrading enzymes such as matrix metalloproteinase (MMP)-13, MMP-3, MMP-1, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5.³⁾ Recent research has focused on cartilage regeneration and prevention of cartilage degeneration through conversion to anabolic and anti-catabolic processes from the catabolic microenvironment of degenerative joints using a potent natural compound derived from herbal plants.^4,5)

The prevalence of OA is approximately 10% in men and 14% in women aged 60 years or older.⁶⁾ It has been suggested that the depletion or altered metabolism of estrogen may be a risk factor in increasing the prevalence of OA. Although etiological links between depletion or altered metabolism of estrogen and the increase in OA prevalence remain largely unknown, estrogen replacement therapy, without specific side effects such as increased risk of breast cancer, myocardial infraction, and stroke, can be considered as a promising treatment for OA patients. Recent studies have suggested that phytoestrogens might mimic effects of estrogen in OA by selective binding of estrogen receptors and modulators.

Bavachin is a phytoestrogen derived from the herbal plant Psoralea corylifolia.⁷⁾ The aim of the present study was to evaluate bavachin-induced anabolic and anti-catabolic effects associated with the increase of extracellular matrix and the suppression of cartilage-degrading enzyme expression in primary rat chondrocytes.

MATERIALS AND METHODS

Biological Safety Assay of Bavachin

To observe the biological safety of bavachin (CAS 9879-32-4; Synonym: 7-hydroxy-2-(4-hydroxyphenyl)-6-(3-methylbut-2-enyl)-2,3-dihydroch romen-4-one); SC-202489) that was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, U.S.A.), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using dimethyl thiazolyl diphenyl tetrazolium salt was performed in normal human oral keratinocytes (NHOKs) used as normal cells. NHOKs were purchased from ScienCell Research Laboratories (Carlsbad, CA, U.S.A.). The NHOKs were maintained in Dulbeco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. NHOKs were seeded at a density of 5×10⁴ cells/well in 96-well plates, and allowed to attach to the well overnight. After incubation, the cultured cells were treated with various concentrations of bavachin in triplicate and incubated at 37°C in a 5% humidified CO₂ incubator for 24 h. Dimethyl thiazolyl diphenyl tetrazolium salt was then added to each well and incubation was continued for a further 4 h at 37°C. In order to dissolve the resulting formazan, the cells were resuspended in 200 µL dimethyl sulfoxide (DMSO) and the optical density (OD) of the solution was determined using a spectrometer at an incident wavelength of 570 nm. The experiments were repeated three times, independently. The mean OD±standard deviation (S.D.) for each group of replicates was calculated. The entire procedure was repeated three times. The inhibitory rate of cell growth was calculated using the equation, % Growth inhibition=([1−OD extract treated]/[OD negative control])×100.

Isolation and Culture of Primary Rat Chondrocytes

The procedures used in the present study were in compliance with the guidelines of the Institutional Animal Care and Use Committee in Chosun University, Gwangju, Republic of Korea. Chondrocytes were obtained from knee joint cartilage of 5-d-old Sprague-Dawley rats by enzymatic digestion in DMEM/Ham’s F-12 (1 : 1) culture medium with sequential treatments of 0.2% pronase and 0.025% collagenase P, as previously described.^8,9) Alginate beads and monolayers were prepared for short- and long-term studies.

For alginate bead cultures, isolated primary rat chondrocytes were re-suspended in 1.2% alginate, and beads were formed by drop-wise addition into a 105 mmol CaCl₂ solution. Chondrocytes embedded within alginated beads were cultured in DMEM/Ham’s F-12 (1 : 1) culture medium containing either 0.5 or 1 µmol bavachin (Santa Cruz Biotechnology Inc.). In addition, 100 ng/mL of bone morphogenetic protein (BMP)-7 (Stryker Biotech, Hopkinton, MA, U.S.A.), a well-known anabolic and anti-catabolic growth factor in articular cartilage matrix and chondrocyte homeostasis,⁹⁾ was treated as a positive control to compare with the anabolic and anti-catabolic effects of bavachin. Triplicate wells were used for each condition. Culture media were changed every other day for 21 d.

For monolayer cultures, isolated primary rat chondrocytes were counted and plated onto a 25 cm² culture flask at 1.5×10⁵ cells/mL. The chondrocytes were treated in DMEM/Ham’s F-12 (1 : 1) culture medium containing either 5 or 10 µmol bavachin (Santa Cruz Biotechnology Inc.) for 24 h. As well as alginate beads culture, 100 ng/mL BMP-7 used for a positive control. The supernatant was collected 24 h after the initiation of each treatment and subjected to Western blot to extract total RNA and total proteins.

Cell Survival Assay

To assess the survival rate of primary rat chondrocytes during long-term (21 d) alginate bead culture, assays (Molecular Probes, Carlsbad, CA, U.S.A.) using calcein AM to stain live cells and ethidium bromide homodimer-1 to stain dead cells were performed following the manufacturer’s protocol. Survival was measured every week (7, 14, and 21 d) during the culture period. At least 100 cells were counted in triplicate for each data point.

Assessment of Proteoglycan Content

At day 21 of culture, the alignate-bead-embedded primary rat chondrocytes were collected to assess the proteoglycan content using the dimethylmethylene blue (DMMB) assay. The proteoglycan contents measured in the cell-associated matrix were quantified by DNA measurement to determine the total amount of proteoglycan produced and retained in the alginate beads per cell. Using PicoGreen (Molecular Probes), cell numbers were determined by assay of total DNA in the cell pellets as previously described.^8,9)

Particle Exclusion Assay for Matrix Assessment

The primary rat chondrocytes and their pericellular matrix were visualized using a particle exclusion assay, as previously described.^8,9) At the end of the culture period, chondrocytes embedded in alginate were released by 55 mmol sodium citrate (pH 6.8). The chondrocytes were pelleted by centrifugation, resuspended in DMEM/F-12 culture media, and then plated on a multi-well cell culture dish. They were allowed to settle and attach to the plates for 12 h. Formalin-fixed erythrocytes were then added and allowed to settle for 15 min. Cells were then observed and photographed with an inverted phase-contrast microscope (Eclipse 2000, Nikon, Melville, NY, U.S.A.).

Quantitative Polymerase Chain Reaction (PCR) and Quantitative Real-Time PCR

Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, U.S.A.) following the manufacturer’s instructions. After isolation of total RNA from primary rat chondrocytes, reverse transcription was performed with 1 µg of total RNA using the ThermoScript reverse transcriptase (RT)-PCR system (Invitrogen) for first strand cDNA synthesis according to the manufacturer’s instructions.

For quantitative PCR (qPCR), cDNA was amplified by 2X TOPsimple™ DyeMIX-nTaq (Enzynomics, Seoul, Republic of Korea) using a SureCycler 8800 (Agilent Technologies, Santa Clara, CA, U.S.A.). Gene induction was determined using agarose gel electrophoresis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control in the reactions for normalization. Primers used for qPCR were as follows: for aggrecan (NM_022190.1), forward primer, 5′-TGA GTG TGA GCA TCC CTC AAC CAT-3′ and reverse primer, 5′-ATG CTG TTC ACT CGA ACC TGT CCT-3′ (PCR product size: 211 bp); for collagen type II (NM_0212929.1), forward primer, 5′-ATG TCA GCC TTT GCT GGC TTA GGA-3′ and reverse primer, 5′-AGT CAT CTG GAC GTT AGC GGT GTT-3′ (PCR product size: 471 bp); for GAPDH (NG_028301.1), forward primer, 5′-TGA CTC TAC CCA CGG CAA GTT CAA-3′ and reverse primer, 5′-TCT CGT GGT TCA CAC CCA TCA CAA-3′ (PCR product size: 269 bp).

For quantitative real-time PCR (qRT-PCR), cDNA was amplified using the MyiQ Real-Time PCR Detection System (Bio-Rad, Life Science Research, Hercules, CA, U.S.A.). A Ct value was obtained from each amplification curve using iQ5 Optical System Software provided by the manufacturer (Bio-Rad, Life Science Research, Hercules). Relative mRNA expression was determined using the ^ΔΔCt method, as detailed by the manufacturer. GAPDH was used as the internal control. Primers used for qRT-PCR were as follows: for aggrecan (NM_022190.1), forward primer, 5′-TCC ACA CGC TAC ACA CTG GAC TTT-3′ and reverse primer, 5′-TCT CGT TGG TGT CTC GGA TTC CAT-3′; for collagen type II (NM_0212929.1), forward primer, 5′-AGG TGA CAA AGG AGA AGC TGG AGA-3′ and reverse primer, 5′-TTA GAG CCA TCT TTG CCA GAG GGA-3′; for GAPDH (NG_028301.1), forward primer, 5′-TGA CTC TAC CCA CGG CAA GTT CAA-3′ and reverse primer, 5′-ACG ACA TAC TCA GCA C CAG CAT CA-3′.

Western Blot

After treatment with bavachin for 24 h, primary rat chondrocytes were then harvested, lysed using lysis buffer (Cell Signaling Technology, Danvers, MA, U.S.A.) containing protease and phosphatase inhibitor cocktails, and incubated for 1 h at 4°C. Lysates were centrifuged at 14000×g for 10 min at 4°C. Total protein concentrations of the cell lysates were determined by the bicinchoninic acid protein assay (Thermo Scientific, Rockford, IL, U.S.A.). In addition, conditioned media were collected to detect cartilage-degrading enzymes secreted from the chondrocytes treated with bavachin. Then, 5× loading buffer was added to equal amounts of protein and conditioned media, and the mixture was boiled at 90°C for 10 min. Both proteins and conditioned media were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking for 2 h with 5% bovine serum albumin in Tris-buffered saline containing Tween-20 at room temperature, membranes were incubated with primary antibodies at 4°C overnight and then incubated with horseradish peroxidase-conjugated secondary antibodies. The following antibodies were used: antibodies against MMP-13, MMP-1, MMP-3, ADAMTS-4, ADAMTS-5, tissue inhibitor of metalloprotease (TIMP)-1, TIMP-2, TIMP-3, TIMP-4, nitric oxide synthase (NOS)-2, cyclooxygenase (COX)-2, prostaglandin E2 (PGE2), and GAPDH. The immunoreactive bands were visualized using the ECL System (Amersham Biosciences, Piscataway, NJ, U.S.A.) and were exposed on radiographic film.

Gelatin Zymography

Gelatin zymography was performed to assess the activity of cartilage-degrading enzymes secreted from primary rat chondrocytes treated with bavachin. An equal volume of conditioned media was mixed with non-reducing sample buffer (4% SDS, 0.15 mol Tris (pH 6.8), and 20% (v/v) glycerol containing 0.05% (w/v) bromophenol blue) and resolved on a 10% polyacrylamide gel containing copolymerized 0.2% (1 mg/mL) swine skin gelatin. After electrophoresis of the conditioned media samples, gels were washed with cold phosphate buffered saline (PBS) containing 2.5% (v/v) triton X-100 for 30 min and washed twice with cold PBS for 15 min. After washing, gels were incubated in the zymogram renaturing buffer (50 mmol Tris–HCl (pH 7.6), 10 mmol CaCl₂, 50 mmol NaCl, and 0.05% Brij-35) at 37°C for 72 h. After renaturation of cartilage-degrading enzymes, gels were stained with 0.1% Coomassie brilliant blue R250. The gelatinolytic activity, revealed as a clear band on a background of uniform light blue staining, was photographed with an inverted phase-contrast microscope (Eclipse 2000, Nikon, Melville, NY, U.S.A.).

Measurement of Total Nitric Oxide Production

Nitric oxide (NO) production was assessed spectrophotometrically as formed nitrites (NO₂). Primary rat chondrocytes were plated in a 6-well plate and treated with 0.5 or 1 µmol bavachin for 24 h. Then 50 µL of the culture medium was reacted with 50 µL of sulfanilamide and 50 µL of N-1-napthylethylenediamine dihydrochloride (NED). Then the absorbance was measured at 540 nm using a spectrophotometer (Epoch Spectrophotometer, BioTek, Winooski, VT, U.S.A.).

Statistical Analysis

Statistical analysis was performed by Student’s t-test using StatView 5.0 software (SAS Institute, Cary, NC, U.S.A.), with p-values less than 0.05 considered to be statistically significant in each test

RESULTS

Bavachin Does Not Affect the Viability and Proliferation of NHOK Used as Normal Cells

To determine the biological safety of bavachin, NHOKs were treated with various concentrations (0.5, 1 and 10 µmol) of bavachin for 24 h. An MTT assay was then performed to assess the cell cytotoxicity. As shown in Fig. 1A, relative cell cytotoxicity of bavachin with 0.5, 1 and 10 µmol bavachin for 24 h were revealed as 104%, 101%, and 104.7%, respectively. Therefore, these are suggesting that bavachin did not affect the survival of NHOKs used as the normal cells. To confirm the biological safety of bavachin in NHOKs, microscopy was used to visualize the live and dead cells stained with Calcein AM (green fluorescent) and ethidium homodimer-1 (red fluorescence), respectively. As shown in Fig. 1B, the HNOKs incubated with 0.5, 1 and 10 µmol bavachin for 24 h were stained green due to the cleavage of the membrane permeable Calcein AM by the cytosolic esterase in living cells. Taken together, bavachin has an herbal plant-derived materials with biological safety.

Fig. 1. Bavachin with Biological Safety Maintains the Viability of Primary Rat Chondrocytes without Proliferation for 21 d

A: Human normal oral keratinocytes were treated with different doses of bavachin (0.1, 1 and 10 µmol) for 24 h. After the indicated stimulating condition, cell cytotoxicity was measured using an MTT assay and was quantified by measuring the optical density at 570 nm. The data represent triplicate results of 3 independent experiments. B: Cell survival assay using green calcein AM for live cells (green color) and ethidium bromide homodimer-1 for dead cell (red color). C: The survival rate of primary rat chondrocytes treated with 0.5 and 1 µmol bavachin and 100 ng/mL BMP-7 was measured after 7, 14, and 21 d of culture. At least 100 cells were counted in triplicate for each data point. More than 80% of chondrocytes treated with bavachin (0.5 and 1 µmol) and 100 ng/mL BMP-7 survived at each data point. D: At 21 d, the survival of chondrocytes was visualized using calcein AM to stain live cells (stained as green fluorescence) and ethidium homodimer-1 to stain dead cells (stained as red fluorescence). Both bavachin (0.5 and 1 µmol) and 100 ng/mL BMP-7 did not alter the survival of primary rat chondrocytes. E: At 21 d, the proliferation of chondrocytes was assessed using PicoGreen. Cell numbers at the end of 21 d of culture were measured in triplicate samples using a DNA assay and are expressed as a percentage of the control cultures. Both bavachin and BMP-7 did not induce proliferation in alginate bead cultures.

Bavachin Does Not Alter the Viability and Proliferation of Primary Rat Chondrocytes for 21 d in Alginate Bead Culture

To verify whether bavachin is cytotoxic in primary rat chondrocytes cultured for 21 d, assays of live and dead cells were performed once per week (at 7, 14, and 21 d) during the culture period. As shown in Fig. 1C, more than 80% of chondrocytes embedded in 1% alginate beads treated with either 0.5 or 1 µmol of bavachin were viable at each assessment point. As well as the survival rates of chondrocytes stimulated with bavachin for 21 d, chondrocytes stimulated with 100 ng/mL of BMP-7 for 21 d maintained 80% over of cell survival rates. To confirm viability, primary chondrocytes stained by either Calcein AM or ethidium bromide homodimer-1 were photographed using an inverted phase-contrast microscope. In the chondrocytes stimulated with either bavachin or 100 ng/mL of BMP-7 for 21 d, the live cells stained by Calcein AM appeared as green fluorescence in significantly greater numbers than did dead cells stained by ethidium bromide homodimer-1 appearing as red fluorescence as shown in Fig. 1D. These results clearly show that bavachin is biologically safe in primary rat chondrocytes. DNA assays using PicoGreen were performed to elucidate whether bavachin increases the proliferation of chondrocytes. As shown in Fig. 1E, neither bavachin (0.5 and 1 µmol) nor BMP-7 (100 ng/mL) increased the proliferation of primary rat chondrocytes during the culture period.

Bavachin Stimulates Matrix Formation and Proteoglycan Production in Primary Rat Chondrocytes

To verify whether bavachin increases proteoglycan production in chondrocytes, alginate beads containing primary rat chondrocytes were cultured for 21 d in the presence of bavachin (0.5 and 1 µmol). Proteoglycan content was then assessed using the DMMB assay. As shown in Fig. 2A, 100 ng/mL BMP-7 used as positive control increased significantly the proteoglycan content (by approximately 3 folds) more than control (p<0.01). However, 0.5 and 1 µmol of bavachin increased the proteoglycan accumulation per chondrocyte by approximately 22±8.2% (p<0.05) and 64±10.3% (p<0.01), respectively, as compared to controls, in a dose-dependent manner (Fig. 2A). Furthermore, the particle exclusion assay showed that the pericellular matrix of the chondrocytes was significantly increased by bavachin, even less than chondrocytes stimulated with 100 ng/mL BMP-7 as shown in Fig. 2B. However, to verify whether bavachin up-regulates the matrix components such as aggrecan and collagen type II, we performed qPCR and qRT-PCR as shown in Figs. 2C and D. As well as the results of particle exclusion assay, the induction of aggrecan mRNA was increased to approximately 30±8.9% (p<0.05) and 47±5.7% (p<0.01) by 5 and 10 µmol of bavachin, respectively, as a dose-dependent manner. Furthermore, the induction of collagen type II mRNA, a major component of extracellular matrix, was significantly increased to approximately 34±10.7% and 58±12.6% by 5 and 10 µmol of bavachin, respectively. Taken together, these results suggest that bavachin exerts an anabolic effect through increasing the formation of the extracellular matrix and its major components in primary rat chondrocytes.

Fig. 2. Bavachin Increases the Formation of Extracellular Matrix through Accumulation of Proteoglycan and Induction of Anabolic Genes Such as Aggrecan and Collagen Type II in Primary Rat Chondrocytes

A: Primary rat chondrocytes in alginate beads were treated with 0.5 and 1 µmol bavachin and 100 ng/mL BMP-7 for 21 d. At the end of the culture period, the amount of proteoglycan in the cell-associated matrix was measured by DMMB assay and normalized using DNA content. The accumulation of proteoglycan was increased in a dose-dependent manner. B: The matrix accumulation of primary rat chondrocytes after 21 d culture was visualized in an exclusion assay. A representative chondrocyte in each treatment condition was photographed using an inverted phase-contrast microscope. The pericellular matrix increased in primary rat chondrocytes treated with bavachin in a dose-dependent manner. C and D: Monolayers of primary rat chondrocytes were treated with bavachin (5 and 10 µmol) and 100 ng/mL BMP-7 for 24 h in a serum-free medium. To assess the induction of anabolic genes such as those expressing aggrecan (C) and collagen type II (D) by qPCR and qRT-PCR, cDNA was synthesized using total RNA extracted from chondrocytes treated with bavachin and BMP-7. Anabolic genes were up-regulated in chondrocytes in a dose-dependent manner. The values are expressed as mean±S.D.

Bavachin Suppresses the Expression of Cartilage-Degrading Enzymes in Primary Rat Chondrocytes

Next, to verify whether bavachin can suppress the expression of cartilage-degrading enzymes in primary rat chondrocytes, we performed a Western blot using specific antibodies such as MMP-13 (collagenase 3, 50 kDa), MMP-3 (stromelysin-1, 43 kDa), MMP-1 (interstitial collagenase, 55 kDa), ADMATS-4 (90 kDa), and ADAMTS-5 (73 kDa). As shown in Fig. 3A, expressions of cartilage-degrading enzymes including metalloproteinases such as MMP-13, MMP-1 and MMP-3, aggrecanses such as ADAMTS-4 and ADAMTS-5 were down-regulated gradually in chondrocytes treated with bavachin as a dose-dependent manner. As well as the bavachin-induced down-regulation of cartilage-degrading enzymes, BMP-7 suppressed the expression of cartilage degrading enzymes in chondrocytes. To elucidate whether bavachin can inhibit the gelatinolytic activities induced by activation of cartilage-degrading enzymes, we performed the gelatin zymography. As shown in Fig. 3B, gelatinolytic activities were significantly reduced by bavachin as a dose-dependent manner compared to control. 100 ng/mL of BMP-7 used as positive control suppressed the gelatinolytic activities as similar with the chondrocytes stimulated with bavachin. These results indicate that bavachin suppresses the activation of cartilage-degrading enzymes and that bavachin might exert an anti-catabolic effect through suppressing both expression and activation of cartilage-degrading enzymes in primary rat chondrocytes.

Fig. 3. Bavachin Suppresses the Expression and Activation of Cartilage-Degrading Enzymes in Primary Rat Chondrocytes

A: Monolayers of primary rat chondrocytes were treated with bavachin (5 and 10 µmol) and 100 ng/mL BMP-7 for 24 h in serum-free medium. To assess the expression of cartilage-degrading enzymes, conditioned media were collected and were analyzed by Western blot. Cartilage-degrading enzymes such as MMP-13, MMP-3, MMP-1, ADAMTS-4, and ADAMTS-5 were decreased in a dose-dependent manner. B: To assess the activation of cartilage-degrading enzymes, gelatin zymography was performed by loading equal volumes of the conditioned media samples harvested from the defined treatment conditions onto polyacrylamide gel containing swine skin gelatin. The activity of cartilage-degrading enzymes was gradually decreased by bavachin in a dose-dependent manner. C: To verify the bavachin-induced TIMPs expression as an anti-catabolic effects, Western blot using specific TIMP-1, -2, -3 and -4 antibodies were performed using conditioned media harvested from the monolayer culture of primary rat chondrocytes stimulated with bavachin (5 and 10 µmol) and 100 ng/mL BMP-7 for 24 h. The expression of TIMPs were gradually increased by bavachin as a dose-dependent manner.

The Specific Endogenous TIMPs Were Up-Regulated in Primary Rat Chondrocytes Treated with Bavachin

As an anti-catabolic effect, the up-regulation of TIMPs can counteract the catabolic effects of cartilage-degrading enzymes. Therefore, we examined whether bavachin antagonizes the catabolic activity of MMPs in primary rat chondrocytes by stimulating expression of TIMPs. As shown in Fig. 3C, the expression of multiple TIMPs such as TIMP-1 (50 kDa), TIMP-2 (55 kDa), TIMP-3 (43 kDa) and TIMP-4 (90 kDa) were significantly up-regulated in a dose-dependent manner in conditioned media collected from primary rat chondrocytes treated with bavachin. Whereas, the 100 ng/mL BMP-7 used as positive control did not affect the expression of TIMPs. Hence, the simultaneous inhibition of cartilage-degrading enzymes and stimulation of multiple TIMPs suggests that bavachin has potent anti-catabolic and pro-anabolic effects in primary rat chondrocytes.

Bavachin Suppresses Oxidative Stress in Primary Rat Chondrocytes

Oxidative stress is closely associated with cartilage destruction in various synovial joints, so reducing oxidative stress might also reduce the tissue levels of multiple cartilage-degrading enzymes. Therefore, to verify whether bavachin suppresses the synthesis of NO, an oxidative stressor, the NO content in primary rat chondrocytes treated with bavachin was assessed. As shown in Fig. 4A, bavachin gradually suppressed NO production in a dose-dependent manner. In particular, 10 µmol bavachin reduced NO induction by approximately 20% (p<0.01) as compared with non-treated controls. Furthermore, the expression of NOS2, a family of enzymes catalyzing the production of NO from L-arginine, was significantly down-regulated as shown as Fig. 4B. The basal level of COX-2 (prostaglandin-endoperoxide synthase 2), an enzyme that plays a key role in the inflammatory cascade, was also gradually down-regulated and the basal expression level of PGE2, a down-stream target of COX-2, was significantly down-regulated. These findings suggest that bavachin might induce an anti-catabolic effect by suppressing inflammation in primary rat chondrocytes.

Fig. 4. Bavachin Suppresses NO Production and the Expression of Catabolic Factors Such as NOS2, COX-2, and PGE2 in Primary Rat Chondrocytes

A: Monolayers of primary rat chondrocytes were treated with bavachin (5 and 10 µmol) for 24 h in serum-free medium. To assess NO production, conditioned media were collected and analyzed by NO assay. NO production was decreased in a dose-dependent manner in chondrocytes treated with bavachin. B: To assess the expression of catabolic factors such as NOS2, COX-2, and PGE2, Western blot was performed using total proteins extracted from primary rat chondrocytes cultured in defined culture conditions. The expression of catabolic factors such as NOS2, COX-2, and PGE2 was suppressed in a dose-dependent manner by treatment with bavachin.

DISCUSSION

Estrogen deficiency has been linked to the increase of OA prevalence and severity in menopausal women.¹⁰⁾ In addition, the prevalence of OA has been estimated to be approximately 1.7 times higher in elderly women than in elderly men.¹¹⁾ Furthermore, the incidence of OA increased significantly in an animal model of osteoporosis following ovariectomy.¹²⁾ These studies suggest that estrogen deficiency might be in part a pathophysiological etiology of OA and that estrogen replacement might decrease the prevalence of OA in menopausal women. Estrogen replacement therapy has been investigated to verify its anabolic and anti-catabolic effects in both ovariectomized animal models prior to clinical trials^12,13) and in menopausal patients with OA.^14,15) Although estrogen replacement therapy in both animal models and human studies reduced the incidence and prevalence of OA, it has also had undesirable side effects such as headache, fluid retention, weight gain, swollen breasts, increased risk of breast or uterine cancer, and heart failure.^16,17) Therefore, to overcome the side effects of estrogen therapy, safer estrogen analogues with similar physiological functions are needed for clinical treatment of OA.

Phytoestrogen is a plant-derived natural compound that exists in a wide variety of foods.¹⁶⁾ Its structure and physiological functions are similar to those of estrogen. Previous studies have reported anabolic and anti-catabolic effects of phytoestrogens such as daidzein, genistein, and resveratrol in articular cartilage and intervertebral discs.^5,18,19) Dong et al.²⁰⁾ have reported that bavachin, a phytoestrogen purified from natural herbal plants such as Psoraleae corylifoliae²¹⁾ and Piper longum,²²⁾ has estrogen-like activity in estrogen-receptor-positive human breast adenocarcinoma MCF-7/BOS cells. Therefore, we hypothesized that bavachin may have chondroprotective effects as a result of anabolic or anti-catabolic activity in articular cartilage.

In articular cartilage, chondrocytes are embedded within an extracellular matrix composed mainly of aggrecan and collagen type II. Therefore, one of the strategies to repair the mechanical function of joints in OA, the regeneration of articular cartilage by increasing the extracellular matrix, is being studied in clinical trials.⁴⁾ For example, BMP-7, a member of transforming growth factor-β (TGF-β)/BMP superfamily, is a representative anabolic growth factor to repair the articular cartilage through the stimulation of cartilage-specific extracellular proteins such as collagen type II, aggrecan, decorin, fibronectin and hyaluronan.^23–25) Recently, Hunter et al., performed the phase 1 safety and tolerability study of BMP-7 in symptomatic knee OA and reported that patients received the BMP-7 were a trend toward symptomatic improvement without toxicity.²⁶⁾ Therefore, BMP-7 was used as a positive control to evaluate the bavachin-induced anabolic and anti-catabolic effects in primary rat chondrocytes.

To assess the cell cytotoxicity of bavachin, we performed a cell cytotoxicity assay using MTT in primary human oral keratinocytes. Cell cytotoxicity did not observed in primary human oral keratinocytes treated with 0.1–10 µmol bavachin for 24 h. Furthermore, the survival rate of primary rat chondrocytes in presence with 0.5 and 1 µmol bavachin and 100 ng/mL BMP-7 for 21 d was approximately 80% over as compared with that of non-treated controls. These results indicate that bavachin may have acceptable cytotoxicity levels in normal human cells and chondrocytes.

Next, to verify the anabolic effects of bavachin in primary rat chondrocytes, we demonstrated that bavachin not only significantly increased the accumulation of proteoglycan but also increased the pericellular matrix. In addition, the mRNA of integral matrix components such as aggrecan and collagen type II was significantly up-regulated in the treated chondrocytes. Unfortunately, the anabolic effects of bavachin were verified as lower than that of 100 ng/mL BMP-7 used as positive control in the results of proteoglycan content, matrix formation and the induction of extracellular matrix component genes. Recently, Im et al., showed that the proteoglycan production in adult human primary articular chondrocytes stimulated with 100 ng/mL BMP-7 for 21 d was greater than at least 3 folds more compared with non-treated control as same as our results⁵⁾ (Fig. 2A). Furthermore, 100 µmol resveratrol, a phytoestrogen derived from grapes and red wines, increased the proteoglycan production as similar with 100 ng/mL BMP-7 in adult human primary articular chondrocytes.⁵⁾ Therefore, bavachin may need to be verified the comparative anabolic effects with BMP-7 in adult human primary articular chondrocyte to use as therapeutic reagent for OA as our further studies. However, our results are suggesting that bavachin has potent anabolic effects that increase the extracellular matrix by up-regulating integral components in chondrocytes.

A central feature of OA involves the progressive destruction of the extracellular matrix on the surfaces of various joints.²⁾ In arthritic cartilage, the progressive destruction of extracellular matrix is mediated by various cartilage-degrading enzymes such as MMPs and aggrecanases.²⁾ Hence, antagonizing these proteases may potentially prevent and retard progressive cartilage destruction.²⁷⁾ Recently, Zhang et al., reported that β-ecdysterone, an estrogen analog purified from Chinese herbal medicines, had an anti-catabolic effects through the expressional down-regulation of MMPs such as MMP-3 and MMP-9 in primary rat chondrocytes.²⁸⁾ In present study, we demonstrated that bavachin had an anti-catabolic effect that not only gradually suppressed the expression of cartilage-degrading enzymes such as MMP-13, MMP-1, MMP-3, ADAMTS-4 and ADAMTS-5, but also induced the up-regulation of multiple TIMPs, the endogenous inhibitors of MMPs, in primary rat chondrocytes. Therefore, our results suggest that bavachin has potent anti-catabolic and pro-anabolic effects in preventing or retarding cartilage destruction in OA. Similar to the effects of bavachin in chondrocytes, recent studies have shown that antioxidants such as lactoferricin and resveratrol have pro-anabolic and anti-catabolic effects in primary chondrocytes isolated from bovine intervertebral discs.^3,19)

Although the biological linkage between OA and aging is not fully clarified, previous studies have shown that the one of the possible pathophysiological etiologies associated with the aging process is low-grade inflammation.²⁹⁾ It has been reported that the progressive accumulation of glycation end products, resulting from non-enzymatic glycation of proteins, results in stiffness and fragility of articular cartilage through up-regulation of matrix-degrading enzymes.^30,31) Up-regulation of both NOS2 and COX-2 has been considered a key factor associated with the accumulation of glycation end products in OA.³²⁾ Furthermore, the up-regulation of the NOS2–NO pathway leads to chondrocyte apoptosis and induces the progressive destruction of articular cartilage in an animal OA model.³³⁾ On the other hand, the suppression of NO production resulted in anti-catabolic effects such as down-regulation of cartilage-degrading enzymes by inhibiting low-grade inflammation.³⁴⁾ In addition, Abramson et al. have reported that the activation of the COX-2–PGE2 pathway causes progressive cartilage destruction by inducing inflammation in synovial joints.³⁵⁾ It has also been shown that selective COX-2 inhibition prevents cytokine-induced inflammatory cartilage damage.³⁶⁾ Inhibiting inflammation inducers by down-regulating inflammatory cytokine-mediated cartilage-degrading enzymes may therefore prevent or retard progressive cartilage destruction.^37,38) In the present study, we demonstrated that bavachin suppressed inflammation inducers such as NOS2, NO, COX-2, and PGE2 in primary rat chondrocytes in a dose-dependent manner. Therefore, our results clearly suggest that Bavachin may prevent cartilage destruction by suppressing catabolic factors in chondrocytes.

In summary, this study demonstrates the potent anabolic and anti-catabolic effects of bavachin in primary rat chondrocytes. Treatment with bavachin not only increased proteoglycan accumulation and the formation of extracellular matrix through an anabolic effect but also inhibited the expression of cartilage-degrading enzymes such as MMP-13, MMP-1, MMP-3, ADAMTS-4, and ADAMTS-5 by an anti-catabolic effect. Furthermore, as another potent anti-catabolic and pro-anabolic effect, bavachin not only increased the level of TIMPs but also suppressed the level of inflammatory factors such as NO, NOS2, COX-2, and PGE2. Indeed, bavachin may play an important role in the future prevention and treatment of degenerative joint diseases, and its potent anabolic and anti-catabolic properties may warrant future application in tissue engineering.

Acknowledgment

This study was supported by research fund from Chosun University, 2014.

Conflict of Interest

The authors declare no conflict of interest.

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