Cartilage samples were harvested from 30 individual patients with OA (69.0±14 years; 14 males and 16 females) between March 2015 and March 2016. Patients were admitted to the Department of Orthopaedics, Tianyou Hospital (Wuhan, China) and samples were collected during surgery. An OA diagnosis in these patients was determined according to the criteria of the American College of Rheumatology (17). The present study was performed following approval from the Experimental Animal Ethics Committee of Tianyou Hospital Affiliated to Wuhan University of Science and Technology and informed consent was provided by all participants.

OA cartilage tissue was washed with sterile phosphate-buffered saline (PBS; pH 7.2) and sliced into 1-mm³ sections with ophthalmic scissors. OA chondrocytes were acquired via incubation at 37°C in 0.25% trypsin for 30 min and 0.2% type II collagenase (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 2 h. Chondrocytes were filtered, centrifuged at 37°C at 1,500 × g for 10 min and resuspended in Dulbecco's modified Eagle's medium (DMEM)/F-12 supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37°C and 5% CO₂. Chondrocytes began to converge following 4–5 h incubation. Second generation chondrocytes were used in the present experiment.

Log phase chondrocytes were randomly divided into three groups: Control group without any stimulus, the RES group administered the ultimate concentration of 10 µM RES, and the NAM group administered the ultimate concentration of 20 µM NAM. Chondrocytes were cultivated for 48 h separately prior to subsequent experiments.

Following three washes with PBS for 90 sec, total RNA was extracted from OA chondrocytes using a RNAprep pure kit (DP430; Tiangen Biotech, Co., Ltd., Beijing, China) according to the manufacturer's instructions. RNA was quantified using a NanoDrop 2000 (Thermo Fisher Scientific, Inc.). Isolated RNA was transcribed into cDNA using a PrimeScript One Step miRNA cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's instructions. qPCR (2X SYBR Green qPCR kit; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) was subsequently performed with a reaction mixture of 20 µl containing: 10 µl 2X SYBR Fast qPCR Mix, 0.8 µl PCR forward and reverse primers, 0.4 µl 50X ROX Reference Dye II and 2 µl cDNA template. The PCR reaction was performed as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 3 sec and 60°C for 13 sec. The reaction was performed using an Applied Biosystems^® 7500 Fast Dx Real-Time PCR instrument (Thermo Fisher Scientific, Inc.). Expression of Sirt1 was normalized to GAPDH, as a reference control. Primer sequences were as follows: Sirt1 forward, 5′-TGGACTCCACGACGTACT-3′ and reverse, 5′-TCTCCTGGGAGGCATAGACC-3′ (total length, 122 bp); and GAPDH forward, 5′-AGCCACATCGCTCAGACA-3′ and reverse, 5′-TCTCCTGGGAGGCATAGACC-3′ (total length, 314 bp). Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Relative target gene expression was calculated as a fold change of 2^−ΔΔCq value, in which ΔC_t = C_{ttarfet gene} - C_{tendogenous control} (18). The experiments were performed in triplicate on three independent occasions.

The three groups of OA chondrocytes were seeded at a density of 5×10³ cells/ml into 96-well plates. Following 24, 48 and 72 h incubations at 37°C, 20 µl 5 mg/ml cells were combined with 10 µl sterile 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Beyotime Institute of Biotechnology, Haimen, China) for 4 h at 37°C. The solution containing MTT was removed and cells were mixed with 150 µl dimethyl sulfoxide for 10 min. The plate was read spectrophotometrically at a wavelength of 560 nm by a Bio-Rad model 550 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) to detect cell viability.

Apoptosis of OA chondrocytes was detected using fluorescein isothiocyanate (FITC)-labeled Annexin V and propidium iodide (PI) kit (cat. no. C1063; BestBio, Co., Shanghai, China), according to the manufacturer's instructions. Briefly, the chondrocytes were collected following digestion in EDTA-free 0.25% trypsin for 1 h at 37°C. Following centrifugation at room temperature (1,000 × g; 10 min), 8 ml Annexin-FITC and 10 ml PI were added to the cell suspension and incubated for 15 min at room temperature. The rate of apoptosis was analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and inbuilt software.

Protein expression levels of Sirt1, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), procaspase-3, −9, matrix metalloproteinase 1(MMP1), MMP3, MMP13, Wnt3a, Wnt5a, Wnt7a and GAPDH were determined by western blot analysis. Briefly, the proteins were extracted from the cells in each group using an EpiQuik Total Histone Extraction kit (Epigentek, Farmingdale, NY, USA) according to manufacturer instructions and quantified using a BCA Protein Quantification kit (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instructions. Subsequently, ~50 mg total protein extracts were separated by 12% SDS-PAGE prior to being transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked using 5% milk solution for 2 h at room temperature and washed three times with Tris-buffered saline with Tween-20 (TBST; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The membrane was respectively incubated with the primary rabbit monoclonal antibodies of Sirt1 (cat. no. ab110304; Abcam, Cambridge, MA, USA), Bax, Bcl-2, procaspase-3, −9 (cat. nos. 1063-1, 1071-1, 1061-1 and 1084-1; Epitomics; Abcam), MMP1, MMP3, MMP13, Wnt3a, Wnt5a, Wnt7a, β-catenin and GAPDH (cat. nos. EP1247Y, ab53015, ab39012, ab28472, ab72583, ab100792, ab32572 and ab8245; Abcam) at 4°C overnight at a dilution of 1:100. This incubation was followed by a further three TBST washes for 5 min. The membrane was subsequently incubated at room temperature with secondary goat anti-rabbit antibody conjugated with horseradish peroxidase (cat. no. A0208; Beyotime Institute of Biotechnology) at a dilution of 1:500 for 90 min and washed with TBST. Strip gray levels were quantified using Quantity One software (version 4.62; Bio-Rad Laboratories, Inc.).

Experiments were performed in triplicate on three independent occasions. Data were presented as the mean ± standard deviation and analyzed using a two-tailed Student's t-test. Statistical analyses were conducted using SPSS version 17 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Compared with the control, the expression levels of Sirt1 mRNA and protein in the RES group were significantly increased (P<0.01; Fig. 1A and B, respectively), but decreased significantly in the NAM group (P<0.01).

Compared with the control, the cell viabilities were significantly improved at 24, 48 and 72 h in the RES group (P<0.01; Fig. 2), and significantly suppressed in the NAM group despite the slow growth of chondrocytes (P<0.01). The rate of cell apoptosis was reduced from 13.84 to 6.55% in response to 10 µM RES, but increased from 13.84 to 47.33% in response to 20 µM NAM (P<0.01; Fig. 3).

To assess the effect of Sirt1 on apoptosis, ECM degradation and Wnt/β-catenin signaling in RES-treated OA chondrocytes, the protein expression levels of Bax, Bcl-2, procaspase-3, −9, MMP1, MMP3, MMP13, Wnt3a, Wnt5a, Wnt7a and β-catenin were determined. It was observed that the expression levels of Bax, procaspase-3 and −9, MMP1, MMP3, MMP13, Wnt3a, Wnt5a, Wnt7a, and β-catenin were significantly inhibited following treatment with RES (P<0.01; Figs. 4–6). However, Bcl-2 expression levels were significantly increased in RES-treated OA chondrocytes (P<0.01; Fig. 4). These variation tendencies were the opposite in NAM-treated OA chondrocytes; when the expression was significantly increased in the RES group, it was significantly decreased in the NAM group (P<0.01; Figs. 4–6).