Six four-week-old male Sprague-Dawley (SD) rats were purchased from Slac Laboratory Animal Co. (Shanghai, China). The care and use of the animals in this study complied with the Guidance Suggestions for the Care and Use of Laboratory Animals administered by the Ministry of Science and Technology, China (14).

Dried BCB powder (100 g) was extracted with petroleum ether twice at 60–70°C. Subsequently, the extracted BCB powder was dried again, extracted with 1,000 ml 80% (v/v) ethanol, filtered, dried in air and extracted three times with boiling distilled water (1:25, w/v) for 5 h. The extraction solution was filtered and concentrated to 5% of the original volume with a rotary evaporator under reduced pressure. Following deproteination using the Sevag method (chloroform:1-butanol, 4:1) (15), the condensed solution was precipitated by gradually adding 100% ethanol until the final concentration reached 80%. Following overnight precipitation, the precipitates were centrifuged and washed with 15–20 ml of ethanol, acetone and ether. The BCBPs were obtained by freeze-drying the precipitates, as previously described (16).

BCBPs (20 mg) were hydrolyzed with 1 ml 4 M trifluoroacetic acid (TFA) at 110°C for 2 h in a small sealed ampoule filled with nitrogen. After being returned to room temperature, the hydrolysate was neutralized with 0.6 M NaOH. A total of 200 μl of stocked standard solution (2 mg/ml) or hydrolyzed BCBP solution was mixed with 200 μl 0.6 M NaOH and 400 μl 0.4 M methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP), and kept in a water bath at 90°C for 70 min. After being cooled to room temperature, 0.3 M HCl (400 μl) were added and allowed to react with the mixture. Finally, each resulting solution was extracted with 1 ml chloroform three times and filtered through a 0.45-μm membrane for high performance liquid chromatography (HPLC).

BCBPs were analyzed on a HPLC system (Agilent 1200; Agilent Technologies, Santa Clara, CA, USA), an Ultimate™ XB-C18 column (4.6×250 mm, 5 μm; Welch Materials Inc., Ellicott City, MD, USA) and a UV detector. The analysis conditions were as follows: mobile phase A:B, 82:18 (A, 0.1 M phosphate buffer; B, acetonitrile); pH 6.7; flow rate, 1.0 ml/min; injection volume, 20 μl; UV detector, WL 245 nm; column temperature, 30°C. D-mannose (Man), rhamnose (Rham), D-(+) glucuronic acid (GluUA), D-(+) galacturonic acid (GalUA), D-glucose anhydrous (Glc), galactose (Gal) and arabinose (Ara) were analyzed as the standard substances, as previously described (17).

The SD rats were sacrificed by cervical dislocation and the bilateral knee joints were peeled off under sterile conditions. The articular cartilage was cut out, and rinsed three times in PBS (HyClone, Logan, UT, USA) containing penicillin and streptomycin. The cartilage was cut into 1 mm³ sections, transferred onto a dish with 0.2% collagenase II (Sigma, St. Louis, MO, USA) and incubated in a 37°C 5% CO₂ incubator. Supernatant fluid was collected every 1.5 h, then centrifuged for 5 min (1,000 rpm), and the precipitate was resuspended in DMEM containing 10% FBS, 50 mg/l vitamin C, 100 U/ml penicillin and 100 μg/ml streptomycin (HyClone). The cells were cultured in a 25 mm² culture flask and placed in a 37°C 5% CO₂ incubator after adjusting the cell density (3×10⁵/ml; termed P0). The cells were subcultured when they reached 80–90% confluence, as determined under a microscope. Subcequently these cells were named P1, P2 and P3, as previously described (18). The P2 chondrocytes were identified by type II collagen immunohistochemistry.

A total of 100 μl 5×10⁴/ml chondrocytes were plated in a 96-well and cultured for 24 h. They were then treated with 0, 50, 100 and 200 μg/ml BCBPs for 48 h. Subsequently, 20 μl 0.5% MTT (Sigma) were added to each well, followed by incubation for 4 h at 37°C. The purple-blue MTT was dissolved in 150 μl DMSO with shaking for 10 min. The absorbance was measured at 490 nm on an ELISA plate reader (Model EXL800; BioTeK, Winooski, VT, USA).

Following treatment with BCBPs, protein was extracted from chondrocytes using lysis buffer on ice, and protein was then measured using the BCA assay. Protein (20 μg) was separated on a 10% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked for 3 h at room temperature in 5% non-fat dried milk, and incubated with primary antibodies against Wnt-4, β-catenin, Frizzled-2 (Santa Cruz Biotechnology, Inc., CA, USA), GSK-3β (Cell Signaling Technology, Inc., Beverly, MA, USA ), cyclin D1, collagen II (Bio-Word Technology, Natong, China) and β-actin (Santa Cruz Biotechnology, Inc.) overnight at 4°C with rocking. The membranes were incubated horseradish peroxidase (HRP)-conjugated secondary antibody (Zhongshan Goldenbridge Biotech, Beijing, China) at room temperature. The ECL method was used to make the signal visible and β-actin was used as the internal control.

Total RNA was isolated using TRIzol reagent (Invitrogen, Grand Island, NY, USA). RNA (1 μg) was reverse transcribed to cDNA according to the instructions provided by the manufacturer. The obtained cDNA was used to determine the expression of Wnt-4, β-catenin, Frizzled-2, GSK-3β, cyclin D1 and collagen II; β-actin was used as an internal control. The primers used for PCR are presented in Table I. The conditions use for PCR were as follows: 94°C for 4 min, 94°C for 30 sec, 57°C/60°C/58°C/60°C/55°C/63°C/60°C for 45 sec, 72°C for 45 sec, for 35 cycles. The samples were analyzed by gel electrophoresis (1.5% agarose) and were examined using a Gel Documentation System (Model Gel Doc 2000; Bio-Rad, Hercules, CA, USA).

Data are the means of three measurements and are expressed as the means ± standard deviation. The data were processed using the PASW package for Windows (version 18.0). Statistical analysis of the data was performed using the Student’s t-test and ANOVA. Values of P<0.05 were considered to indicate statistically significant differences.

By comparing the retention time of unknown peaks to peaks in the standard samples (Fig. 1B), the BCBPs monosaccharide composition was identified (Fig. 1A), and contained at least seven monosaccharides, including Man, Rham, GluUA, GalUA, Glc, Gal and Ara. Among these, the contents of Ara and Gal were higher than those of the other monosaccharides; the content of Ara was the highest, while the separation of Rham, GluUA, GalUA, Glc, Gal and Ara was easier than that of Man.

The primary chondrocytes resembled small balls when first suspended in DMEM. After 24 h, the majority of the cells were nestled against the culture flask, and their volumes became larger (Fig. 2A and B). Three days later, the cells grew to be tufted (Fig. 2C). The cells had typical characteristics of chondrocytes, having a ‘flagstone’ pattern (Fig. 2D) when confluent in a single cell layer. The P2 and P3 chondrocytes grew much more rapidly and became confluent cells within four to five days (Fig. 2E and F). Collagen type II, one of the main secreted proteins in the chondrocyte extracellular matrix, has been commonly used for the identification of chondrocytes by immunohistochemistry. The cytoplasm stained brown represented the positive expression in chondrocytes.

Chondrocyte viability was detected by MTT assay. As shown in Fig. 3, following treatment with 50, 100 and 200 μg/ml of BCBPs, the cell viability increased by 8.96±1.66, 15.75±2.75 and 9.58±1.59, respectively when compared with the untreated group (treated with 0 μg/ml BCBPs) (P<0.01, P<0.05). These results confirmed that treatment with BCBPs promoted chondrocyte proliferation in a time-dependent manner, as shown in our previous study (19).

To further determine the effects of BCBPs on the Wnt/β-catenin signaling pathway in chondrocytes, we examined the protein and mRNA expression of GSK-3β, Wnt-4, β-catenin, Frizzled-2, cyclin D1 and collagen II by western blot analysis and RT-PCR, respectively. Following treatment with BCBPs, the protein levels of Wnt-4, β-catenin, Frizzled-2, cyclin D1 and collagen II significantly increased compared with the control group (P<0.01, P<0.05). Compared with the control group (P<0.01, P<0.05), the protein expression of GSK-3β in the BCBP-treated chondrocytes was significantly downregulated (Fig. 4). The mRNA expression levels of GSK-3β, Wnt-4, β-catenin, Frizzled-2, cyclin D1 and collagen II were similar to their respective protein levels (Fig. 5). Taken together, our results indicate that BCBPs upregulate the protein and mRNA levels of Wnt-4, β-catenin, Frizzled-2, cyclin D1 and collagen II, and downregulate the levels of GSK-3β.