Male Sprague-Dawley (SD) rats at 6 weeks of age were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Animal experiments were strictly conducted in accordance with the Guide for the Care and Use of Laboratory Animals of Fujian University of Traditional Chinese Medicine (Fuzhou, China). The study protocol was approved by the Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine. The SD rats were sacrificed using carbon dioxide (cage size, 7×11×5 inches; flow rate, 1.3 l/min), according to the Guide for the Care and Use of Animals.

PSO (psoralen, NIFDC, batch no. 110739-201115, 99.3% pure) was first dissolved in phosphate-buffered-saline (PBS; HyClone Laboratories, Inc., Logan, UT, USA) to a concentration of 10⁻³ mol/l, and stored at −20°C. The PSO-conditioned culture medium was prepared by diluting the stock solution in low-Dulbecco's modified Eagle's medium (Low-DMEM) containing 10% fetal bovine serum (FBS) (both from HyClone Laboratories, Inc.), filtering through a 0.22-µm filter and stored at 4°C before use.

Articular chondrocytes were isolated and cultured as previously described (16). The morphological changes and growth feature of chondrocytes were recorded under a phase-contrast microscope (Olympus, Tokyo, Japan). The primary chondrocytes were termed passage 0 (P0); the P2 chondrocytes were identified by immunohistochemical staining of type II collagen. P2 chondrocytes approximately at 50% were used in this study.

Chondrocytes were seeded at the 96-well culture plates with a density of 2×10⁴ cells/ml and incubated (100 µl/well) for 24 h. The cells were then treated with PSO-conditioned culture medium at different concentrations (0, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵ and 10⁻⁴ mol/l) for different periods (24, 48 and 72 h). At the end of intervention, the supernatant was then removed and 100 µl 1% MTT (Sigma-Aldrich) was added to each well. After 4 h of incubation at 37°C, the supernatant was replaced with 150 µl/well of DMSO (Hengxing Chemical Preparation Co., Ltd., Tianjin, China). The absorbance was measured at 490 nm using an enzyme labeling instrument (model EXL800; BioTek, Winooski, VT, USA). The optimum conditions of intervention were selected for further examination. To further investigate whether PSO promotes chondrocyte proliferation via the canonical Wnt-β/catenin signaling pathway, the chondrocytes were treated with PSO (10⁻⁶ mol/l) in the absence or presence of 0.2 µg/ml Dickkopf-1 (DKK-1; R&D Systems, Minneapolis, MN, USA) for 48 h, as previously described (17).

After treatment, total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Grand Island, NY, USA). RNA (1 µg) was reverse transcribed into cDNA using a reverse transcription kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA) according to the manufacturer's instructions. Then DNA bands were analyzed via gel electrophoresis (1.5% agarose) using the Gel Documentation System (Bio-Rad, Hercules, CA, USA) and normalized to that of β-actin. The PCR primers were as follows: β-actin forward, 5′-GAG AGG GAA ATC GTG CGT GAC-3′ and reverse, 5′-CAT CTG CTG GAA GGT GGA CA-3′; Wnt-4 forward, 5′-TCA GCC CAC AGG GTT TCC A-3′ and reverse, 5′-CGC TCG CCA GCA TGT CTT T-3′; β-catenin forward, 5′-AAG GAA GCT TCC AGA CAT GC-3′ and reverse, 5′-AGC TTG CTC TCT TGA TTG CC-3′; Frizzled-2 forward, 5′-TCG AGG CCA ATT CGC AGT A-3′ and reverse, 5′-CAG GAA GGA TGT GCC GAT G-3′; glycogen synthase kinase-3β (GSK-3β) forward, 5′-AAA GTG CAT CGC TGG CTT A-3′ and reverse, 5′-GTC GAC GGT TTG TTT CCA AT-3′; cyclin D1 forward, 5′-AAT GCC AGA GGC GGA TGA GA-3′ and reverse, 5′-GCT TGT GCG GTA GCA GGA GA-3′; Col-II forward, 5′-CCA GAG TGG AAG AGC GGA GAC-3′ and reverse, 5′-CAG TGG ACA GTA GAC GGA GGA AAG-3′; and β-actin forward, 5′-CAC CCG CGA GTA CAA CCT TC-3′ and reverse, 5′-CCC ATA CCC ACC ATC ACA CC-3′.

After treatment, total proteins were extracted from cells using radioimmunoprecipitation assay lysis buffer (RIPA) with 1 mM phenylmethanesulfony fluoride (PMSF) (both from Beyotime Biotechnology, Shanghai, China) and quantified using a bicinchoninic acid (BCA) assay. Twenty micrograms of proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (12%) and transferred onto a PVDF membrane. Subsequently, the membrane was blocked with 5% non-fat milk in TBST solution for 2 h at room temperature (RT). After incubation with the primary antibodies against Wnt-4 (sc-5214), Frizzled-2 (sc-68327; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), β-catenin (9582s), GSK-3β (9315; Cell Signaling Technology, Inc., Beverly, MA, USA), cyclin D1 (BS6532), Col-II (BS1071; Bioworld Technology, Natong, China) overnight at 4°C, and the HRP-conjugated secondary antibodies (ZB-2301; Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 1 h at RT, the blots were detected using a Bio-Rad Chemi Doc XRS+ (Bio-Rad), and β-actin was used as the control.

After treatment with PSO at the concentration of 10⁻⁶ mol/l, chondrocytes were fixed with ice-cold methanol at 4°C for 30 min, permeabilized with 0.5% Triton X-100 for 10 min, and blocked in 5% bovine serum albumin (BSA) for 1 h at room temperature. Then the cells were incubated with rabbit anti-β-catenin antibody overnight at 4°C, and TRITC-conjugated secondary antibody (Zymed Laboratories, San Francisco, CA, USA) was applied for 1 h at room temperature in the dark. Following DAPI staining, confocal images were acquired with a fluorescence microscope (Olympus, Tokyo, Japan).

All the experiments were repeated three times independently and all the data were analyzed by one way analysis of variance (ANOVA) or Student's t-test using SPSS 19.0 software (SPSS Inc, Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The cultured chondrocytes in this study exhibited typical morphology with a spherical, fusiform and slab stone shape, as described in previous studies (17,18) (Fig. 1). The P2 chondrocytes exhibited a typical polygonal or spherical shape, as shown by the identification of type II collagen immunohistochemical staining. The cytoplasm of positive chondrocytes was stained brown, whereas the negative control failed to stain in the cytoplasm (Fig. 2).

The effect of PSO on cell viability was measured by the MTT assay. As shown by the results of the MTT assay, the viability of the chondrocytes was increased following treatment with different PSO dosages of 10⁻⁸–10⁻⁴ mol/l and for increasing time periods as compared with the viability noted in the untreated cells. The cell viability of the 10⁻⁶ mol/l PSO concentration treatment group was slightly higher than that in the other PSO concentration treatment groups. However, there was no significant difference among the 10⁻⁸–10⁻⁴ mol/l PSO concentration treatment groups. Furthermore, treatments with 10⁻⁷, 10⁻⁶ and 10⁻⁵ mol/l of PSO were more effective on the third day compared with the first day, and all the comparisons showed significant difference (P<0.01 or P<0.05) (Fig. 3). These results preliminarily indicate that PSO promotes the viability of chondrocytes in a time- and concentration-dependent manner. Thus, the concentrations of 10⁻⁷, 10⁻⁶ and 10⁻⁵ mol/l (low, medium and high doses, respectively) of PSO for a 48-h incubation were used for further examination.

The morphology of the PSO-treated chondrocytes at 48 h was observed by phase-contrast microscope. Compared with the untreated cells, the PSO-treated chondrocytes exhibited changes in cell size and shape, and in particular an increase in cell number (Fig. 4).

To evaluate the effect of PSO on the Wnt/β-catenin signaling pathway in chondrocytes, RT-PCR and western blot analysis were performed to determine the expression levels of Wnt-4, Frizzled-2, β-catenin, cyclin D1 and GSK-3β in chondrocytes. Compared with the control group, PSO efficiently upregulated the mRNA expression of Wnt-4, Frizzled-2, β-catenin, cyclin D1 (P<0.01 or P<0.05), but downregulated the mRNA expression of GSK-3β (P<0.01 or P<0.05) (Fig. 5). Similarly, the protein levels, respectively, corresponded to the mRNA expression (P<0.01 or P<0.05) (Fig. 6).

To gain insight into the effect of PSO on the promotion of β-catenin nuclear translocation, immunofluorescence staining was applied. According to the staining results, we found that PSO markedly promoted the translocation of β-catenin into the nucleus (P<0.01 or P<0.05) (Fig. 7). β-catenin is a protein that mainly localizes in the membrane with minimal localization in the cytoplasm. It can enter into the nucleus to transactivate target gene expression upon activation in the cytoplasm (19). These results indicate that PSO activates the Wnt/β-catenin signaling pathway by promoting β-catenin nuclear translocation.

The loss of type II collagen is a characteristic of articular cartilage degradation. Thus, we investigated the influence of PSO on Col-II expression. The results revealed that the mRNA expression and protein level of Col-II were markedly enhanced in the PSO-treated chondrocytes (P<0.01 or P<0.05) (Fig. 8), compared with levels in the untreated cells, indicating that PSO upregulates the expression of Col-II to promote cartilage ECM synthesis, and confer a positive effect on chondrocyte proliferation.

To further confirm the effects of PSO on chondrocyte proliferation by activating the Wnt/β-catenin signaling pathway, we also investigated the influences of Wnt/β-catenin signaling inhibition on β-catenin and cyclin D1 expression. The expression of β-catenin and cyclin D1 was decreased in cells pre-treated with DKK-1 compared with that without DKK-1 treatment (P<0.01 or P<0.05), implying that PSO is involved in the regulation of chondrocyte proliferation by activating the Wnt/β-catenin signaling pathway (Fig. 9).