Normal human articular cartilage was obtained from eight patients (4 men, 4 women; age 24–41 years) after death or from trauma in Tianjin Hospital (Tianjin, China). The normal donors were significantly younger whose articular cartilage had no degenerative changes. Written informed consent was obtained from the patients according to the terms of the Ethics Committee of Tianjin Hospital. Chondrocytes were isolated from cartilage as previously described (17). Briefly, cartilage fragments were digested with 0.25% trypsin for 15 min and incubated with 0.2% (v/v) collagenase for 4 h at 37°C. The resulting cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (both from HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C and 5% CO₂ in a humidified incubator.

Human chondrocytes (1×10⁵ cells/well) were pretreated with various concentrations of carvacrol (0, 1, 5 and 10 µg/ml; Sigma-Aldrich; Merck KGaA) for 2 h and then co-incubated in the absence or presence of IL-1β (10 ng/ml; Sigma-Aldrich; Merck KGaA) for 24 h at 37°C.

Cell viability was measured by the MTT assay. Briefly, following treatments, human chondrocytes (1×10⁵ cells/well) were incubated with MTT solution (5 mg/ml; Sigma-Aldrich; Merck KGaA) at 37°C for 4 h; subsequently, the purple formazan crystals were dissolved using dimethyl sulfoxide by shaking at room temperature for 10 min. Spectrophotometric absorbance was measured at 570 nm using a multifunctional microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were repeated at least three times.

Nitrite levels in the culture medium were detected by the Griess reaction as previously described (18). The level of PGE2 was evaluated using a ELISA kit (cat no. KHL1701; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacture's protocol. All experiments were repeated at least three times.

Total RNA was extracted from human chondrocytes (1×10⁶ cells/well) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. First-strand cDNA was synthesized from total RNA (1 µg) using the PrimeScript RT Reagent kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan). Subsequently, a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to conduct RT-qPCR using SYBR Premix Ex Taq II (Takara Bio, Inc.). Primers used in qPCR are listed as follows: iNOS forward, 5′-TTTCCAAGACACACTTCACCA-3′ and reverse, 5′-ATCTCCTTTGTTACCGCTTCC−3′; COX-2 forward, 5′-GAGAGATGTATCCTCCCACAGTCA-3′ and reverse, 5′-GACCAGGCACCAGACCAAAG-3′; MMP-3 forward, 5′-GCATTGGCTGAGTGAAAGAGACTGTATC-3′ and reverse, 5′-ATGATGAACGATGGACAGATGA-3′; MMP-13 forward, 5′-AGTAGTTCCAAAGGCTACAACTTGTTT-3′ and reverse, 5′-GGAGTGGTCAAGCCCTAAGGA-3′; GAPDH forward, 5′-CTGGGCTACACTGAGCA-3′ and reverse, 5′-AAGTGGTCGTTGAGGGCAATG−3′. GAPDH was used as a reference gene. The PCR amplification cycles were performed as follows: 30 sec at 95°C, followed by 40 cycles of 5 sec at 95°C and 30 sec at 60°C. The 2^−ΔΔCt method (19) was used to calculate relative changes in gene expression. All experiments were repeated at least three times.

Human chondrocytes (1×10⁶ cells/well) were lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitor mixtures (Sigma-Aldrich; Merck KGaA). Protein concentrations were determined using a Bradford assay (Bio-Rad Laboratories, Inc.). Protein lysates (30 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (both from Bio-Rad Laboratories, Inc.). Membranes were blocked in 5% non-fat milk for 2 h at room temperature and then incubated with rabbit anti-human iNOS (1:500; ab3523), COX-2 (1:500; ab52237), MMP-3 (1:500; ab53015), MMP-13 (1:3,000; ab39012), NF-κB p65 (1:50,000; ab32536) and IκBα primary antibodies (1:1,000; ab32518; all from Abcam, Cambridge, UK) overnight at 4°C. Following washing with TBS containing 0.1% Tween-20 (TBST; Sigma-Aldrich; Merck KGaA), membranes were incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit immunoglobulin G antibodies (1:1,000; sc-2922; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. Membranes were washed with TBST buffer, and immunoreactivity was detected with Enhanced Chemiluminescence reagent (GE Healthcare Life Sciences) and quantified by the Quantity One (Bio-Rad Laboratories, Inc.) version 5.2 software. β-actin was used as the internal control. All experiments were repeated at least three times.

Statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. One-way analysis of variance followed by Newman-Keuls post-hoc test was used for the statistical comparison of multiple groups. Results from two groups were evaluated using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Carvacrol cytotoxicity on chondrocyte viability was examined by MTT assay. Compared with untreated chondrocytes, the various treatments with carvacrol at concentrations between 1 and 10 µg/ml did not significantly affect cell viability (Fig. 1A). Treatment with IL-1β (10 ng/ml) significantly reduced cell viability (Fig. 1B); whereas, pretreatment with carvacrol reversed the effects of IL-1β in a concentration-dependent manner (Fig. 1B). The highest inhibition was observed with 10 µg/ml carvacrol treatment. This concentration of carvacrol was used in the following experiment.

The effects of carvacrol on NO and PGE2 production in IL-1β-induced chondrocytes were also investigated. IL-1β treatment significantly induced the production of NO and PGE2 in OA chondrocytes (Fig. 2), and these increased levels of expression were significantly inhibited in cells co-treated with carvacrol.

Western blot analysis was used to determine the effects of carvacrol on iNOS and COX-2 expression in human chondrocytes stimulated with IL-1β. The mRNA and protein expression levels of iNOS and COX-2 were markedly increased following IL-1β incubation compared with untreated controls (Fig. 3). By contrast, chondrocytes that were co-treated with carvacrol exhibited a notable decrease in iNOS and COX-2 expressions compared with IL-1β-treated OA chondrocytes.

A number of studies have demonstrated that MMPs serve crucial roles in the initiation and progression of OA (20–22). Therefore, the effects of carvacrol on MMP-3 and MMP-13 expression in IL-1β-induced chondrocytes were examined. RT-qPCR analysis results demonstrated that the mRNA expression levels of MMP-13 and MMP-3 generated in IL-1β-induced chondrocytes increased significantly compared with controls (Fig. 4A and B, respectively). However, pretreatment with carvacrol greatly inhibited these IL-1β-induced effects. Similarly, western blot analysis demonstrated that carvacrol was able to suppress IL-1β-induced MMP-3 and MMP-13 protein expression in OA chondrocytes (Fig. 4C).

Activation of NF-κB signaling pathway has been reported to participate in inflammation in OA (23). Therefore, the effects of carvacrol on NF-κB activation in human chondrocytes stimulated with IL-1β were investigated. Western blot analysis data revealed that IL-1β treatment significantly increased the protein expression level of phosphorylated-NF-κB p65 and reduced the protein expression level of IκBα in chondrocytes compared with untreated chondrocytes (Fig. 5). Notably, co-treatment with carvacrol significantly decreased the IL-1β-induced expression of NF-κB in chondrocytes and increased the protein expression level of IκBα.