Curcumin and collagenase II were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and a 10 mM stock solution was prepared in dimethyl sulfoxide, and diluted with cell culture medium immediately prior to use.

Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; ThermoFisher Scientific, Inc.) and 100 IU/ml penicillin, 100 mg/ml streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

Recombinant human IL-1β was supplied by PeproTech, Inc. (Rocky Hill, NJ, USA). Primary antibodies against type II collagen (AB746) and MMP-13 (AB39012) were purchased from Abcam (Cambridge, UK). Primary antibodies against NF-κB inhibitor α (IκBα; 4814S), phosphorylated (p)-IκBα (9246S) and NF-κB p65/RelA (8242S) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). The selective NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), primary antibodies against GAPDH (AB8245) and lamin B1 (AB16048), and horseradish peroxidase (HRP)-conjugated secondary antibody (AB7097) were obtained from Abcam. Cell culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc.).

A total of eight 5-week-old male rats (300–410 g) were purchased from Shanghai SLAC Laboratory Animal, Co., Ltd. and kept in common rabbit cages and fed a standard diet with tap water ad libitum. All the rat procedures were approved by the Institutional Care and Use Committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. The rats were anaesthetized with pentobarbital sodium (40–45 mg/kg) and the articular cartilage was isolated from femoral and tibial articular joints of by aseptic dissection following air injection into the ear vein. Following two washes with PBS, the cartilage sections were treated with 2 mg/ml pronase (EMD Millipore, Billerica, MA, USA) in serum-free DMEM at 37°C for 1 h in 5% CO₂, followed by overnight digestion with 0.25 mg/ml collagenase II dissolved in serum-free DMEM. Isolated chondrocytes were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. All experiments were performed on chondrocytes between passage 1 and 3. Fibroblasts were used as control group, according to the manufacturer's protocol for VECTASTAIN® ABC kit (AK-5000) and toluidine blue staining kit (H-5000) were provided by Vector Laboratories (Southfield, MI, USA). The chondrocytic phenotype of the cultured cells was confirmed by toluidine blue staining of glycosaminoglycan and immunocytochemical staining.

To exclude stimulation that may be caused by other cytokines or growth factors present in the FBS, chondrocytes were serum-starved and exposed to 10 ng/ml IL-1β for 24 h. Experiments were designed to mimic the cellular events that occur in OA. To acquire the optimum timepoint and concentration of curcumin for the treatment of chondrocytes, chondrocytes were co-treated with either of the following: i) 10 ng/ml IL-1β and 50 µM curcumin for various time periods (0, 12, 24, 36 or 48 h); or ii) 10 ng/ml IL-1β and various concentrations of curcumin (0, 25, 50, 75, or 100 µM) for 36 h. To investigate p65/RelA translocation and IκBα phosphorylation, chondrocytes were pretreated with 10 ng/ml IL-1β in the presence or absence of 50 µM curcumin for 0, 10, 20, 30, 40 or 60 min, after which nuclear and cytoplasmic extracts were prepared using a nuclear and cytoplasmic protein extraction kit (P0028; Beyotime Institute of Biotechnology) following the manufacturer's protocol. The experiments were performed in triplicate.

Total RNA was isolated from cells (1.2×10⁶ cells/well) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and 2 µg was reverse-transcribed into cDNA using an Advantage® RT-for-PCR kit (639506; Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. qPCR was performed using SYBR Green PCR master mix (Takara Bio, Inc., Otsu, Japan), in accordance with the manufacturer's protocols. The primer sequences used are presented in Table I. The PCR conditions were set as follows: Initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 30 sec with a final extension at 72°C for 3 min. mRNA levels were normalized to levels of the endogenous control GAPDH, and the relative expression levels were calculated using the 2^−ΔΔCq method (19). Data were obtained from three independent experiments performed in triplicate.

Chondrocytes (1.2×10⁶ cells/well) were washed twice with ice-cold PBS, lysed with Radioimmunoprecipitation Assay buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1 mM phenylmethylsulfonyl fluoride on ice for 30 min, and centrifuged at 16,992 × g at 4°C for 20 min. Protein concentrations were determined using the Bicinchoninic Acid method (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts (60 µg) of protein were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked in 1% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 1 h, and subsequently incubated with the following primary antibodies: anti-type II collagen (1:200), anti-MMP-13 (1:200), anti-IκBα (1:200), anti-p-IκBα (1:200), anti-NF-κB p65/RelA (1:200), anti-GAPDH (1:1,000) and anti-lamin B1 (1:1,000) at 4°C overnight. Following three washes with Tris-buffered saline containing 0.05% Tween-20, the membranes were incubated with goat anti-rat IgG H&L HRP-conjugated secondary antibody (1:2,000; AB7097) at room temperature for 1 h. The immunoblots were analyzed by Enhanced Chemiluminescence (ECL) with an ECL Plus kit (Beyotime Institute of Biotechnology), and the bands were quantified by ImageJ software version 1.48 (National Institutes of Health, Bethesda, MD, USA) and normalized to GAPDH expression. Data were obtained from three independent experiments performed in triplicate.

Cells (1.2×10⁶ cells/well) were seeded onto glass coverslips in 24-well plates, cultured for 24 h at 37°C, pre-incubated with serum-starved DMEM for 1 h at 37°C, and subsequently stimulated with IL-1β (10 ng/ml) alone, IL-1β (10 ng/ml) and curcumin (50 µM), or IL-1β (10 ng/ml) and PDTC (0.1 mmol/l) for 30 min at 37°C. The cells were washed three times with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton X-100/PBS for 15 min. Following blocking in PBS containing 10% normal goat serum (Gibco; Thermo Fisher Scientific, Inc.) for 1 h, the cells were incubated with anti-p65/RelA (1:50) overnight at 4°C, followed by incubation with a fluorescein isothiocyanate-conjugated goat anti-rat IgG H&L (PE) secondary antibody (1:50; AB7010; Abcam, Cambridge, UK) for 1 h at room temperature. Finally, cells were rinsed in PBS three times and covered with Fluoromount mountant (Gallard-Schlesinger Industries, Garden City, NY, USA). Fluorescence signals were examined and imaged under a Zeiss Axioskop 40 fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) by using Image-J software version 1.45 (National Institutes of Health).

Cell proliferation was evaluated using a Cell Counting kit-8 (CCK-8; Beyotime Institute of Biotechnology) in accordance with the manufacturer's protocol. Chondrocytes were treated with IL-1β for 24 h prior to curcumin, and subsequently co-treated with either curcumin or PDTC, a combination of curcumin plus PDTC, or were untreated. Cells (6×10³ cells/well) were seeded into 96-well plates and incubated for 12, 24, 48 or 72 h at 37°C. Following this, 10 µl of CCK-8 was added to each well (90 µl medium was mixed with 10 µl CCK-8) and the plates were incubated at 37°C for 2 h. Following incubation, the absorbance was measured at a wavelength of 450 nm using an automated plate reader. The cell viability was calculated as a percentage of the viable cells in the curcumin-treated group compared with the untreated control. Each experiment was repeated three times independently.

All experiments were carried out at least three times, and the data are presented as the mean ± standard deviation. Statistical analyses were performed using SPSS software version 17.0 (SPSS Inc., Chicago, IL, USA). Comparisons between experimental and control data were evaluated by Student's t-test or analysis of variance. Multiple groups were compared using analysis of variance, the comparison between groups using LSD test. P<0.05 was considered to indicate a statistically significant difference.

The chondrocytic phenotype of the isolated rat chondrocytes was confirmed by toluidine blue staining of glycosaminoglycan and immunocytochemical staining of type II collagen (Fig. 1). RT-qPCR and western blot analyses were performed to assess the regulation of MMP-13 and type II collagen mRNA and protein expression by IL-1β using chondrocytes that were cultured in the presence or absence of IL-1β for 24 h. IL-1β treatment significantly decreased the expression of type II collagen (Fig. 2A) and markedly increased the expression of MMP-13 (Fig. 2B). These results indicated that IL-1β induces MMP-13 expression, but inhibits type II collagen expression in rat chondrocytes.

To determine the optimum conditions of curcumin, serum-starved rat chondrocytes were treated with IL-1β (10 ng/ml) for 24 h and subsequently cultured in medium containing 50 µM curcumin. The chondrocytes were harvested after 0, 12, 24, 36 or 48 h and the expression levels of MMP-13 and type II collagen proteins were examined by western blot analysis. As shown in Fig. 3, MMP-13 protein expression was significantly decreased, whereas type II collagen was markedly increased in chondrocytes following treatment with curcumin compared with the untreated group (0 h). Moreover, the decrease in MMP-13 expression was lowest at 36 h, and the increase in type II collagen peaked at 48 h.

Following this, the optimum concentration of curcumin for treatment of IL-1β-treated chondrocytes was determined. Rat chondrocytes were treated 10 ng/ml IL-1β and various concentrations of curcumin (0, 25, 50, 75, or 100 µM) for 36 h. As presented in Fig. 4, curcumin markedly upregulated the expression of type II collagen, and significantly inhibited the expression of MMP-13 in IL-1β-pretreated chondrocytes at each concentration compared with the untreated group (0 µM). The optimum concentration by which curcumin had the strongest effect on type II collagen and MMP-13 expression levels was determined to be 50 µM.

To investigate the effect of curcumin on IL-1β-induced NF-κB activation, serum-starved chondrocytes were treated with IL-1β in the presence or absence of curcumin for 0–60 min and then subjected to cell component fractionation. The resultant cytoplasmic and nuclear extracts were used for western blot analysis to examine the protein expression of p65/RelA. The results demonstrated that IL-1β treatment led to a decrease in the cytoplasmic expression and an increase in the nuclear translocation of p65/RelA, which accumulated in the nucleus in a time-dependent manner (Fig. 5A). However, upon co-treatment with curcumin, neither the cytoplasmic nor the nuclear expression of p65/RelA exhibited a significant difference at any of the indicated time points compared with IL-1β-treated chondrocytes (Fig. 5B). Protein expression levels of p65/RelA decreased in the cytoplasm (Fig. 5C) and gradually increased in the nucleus (Fig. 5D) over time with IL-1β treatment.

As curcumin may stabilize p65/RelA in the cytoplasm (20), the effect of curcumin on the IL-1β-induced nuclear translocation of p65/RelA was further investigated by immunofluorescence staining. IL-1β-stimulated chondrocytes exhibited clear nuclear translocation of p65/RelA from the cytoplasm (Fig. 6), whereas co-treatment with curcumin and PDTC notably blocked nuclear translocation of p65/RelA. Taken together, these findings suggested that curcumin inhibits IL-1β-induced nuclear translocation of p65/RelA and suppresses IL-1β-induced NF-κB activation in chondrocytes.

The phosphorylation and subsequent degradation of IκBα are essential for the nuclear translocation of p65/RelA and the activation of NF-κB signaling. Therefore, the ability of curcumin to inhibit IL-1β-induced phosphorylation and degradation of IκBα was examined. Chondrocytes were serum-starved and then treated with IL-1β in the presence or absence of curcumin for 0–60 min. The phosphorylation of IκBα in IL-1β-treated chondrocytes increased markedly and peaked at 40 min (Fig. 7A). However, upon co-treatment with IL-1β and curcumin, no significant differences in IκBα phosphorylation were observed compared with IL-1β-treated chondrocytes (Fig. 7B). These findings suggested that curcumin may block IL-1β-induced IκBα phosphorylation in rat chondrocytes.

To elucidate the potential pharmacological effects of curcumin on OA, CCK-8 assays were performed to evaluate cell proliferation. Proliferation of IL-1β-treated chondrocytes was demonstrated to gradually increase following any of the three treatments compared with the control, whereas proliferation gradually decreased in untreated cells. Furthermore, although the proliferation activity of each pharmacologic intervention group increased significantly compared with IL-1β-treated chondrocytes, there were no significant differences in the cell proliferation activity among the groups treated with curcumin, PDTC or curcumin plus PDTC (Fig. 8).

The effects of curcumin and PDTC on the phosphorylation of IκBα were further examined. Consistent with the previous data (Fig. 2B), curcumin and PDTC inhibited the expression of p-IκBα and MMP-13 proteins, but upregulated type II collagen protein expression levels compared with the control (Fig. 9A); no significant changes to IκBα expression were observed. Curcumin and PDTC treatments also led to the downregulated expression of MMP-13 and p65/RelA mRNA, and upregulated type II collagen mRNA expression levels compared with the control; no significant changes to IκBα expression were observed compared with the control. (Fig. 9B). Co-treatment of IL-1β-stimulated chondrocytes with curcumin and PDTC did not cause further inhibition of MMP-13 expression or upregulation of type II collagen expression beyond that caused by treatment with curcumin or PDTC alone. Notably, no significant differences were observed in cell proliferation or the expression levels of MMP-13 and type II collagen in the pharmacological intervention groups, and chondrocytes co-treated with curcumin plus PDTC did not exhibit enhanced effects in proliferation or MMP-13 and type II collagen expression levels compared with cells treated with curcumin or PDTC alone, suggesting that the regulation of chondrocytes by curcumin and PDTC may follow the same underlying molecular mechanism.