SFN (purity ≥98%), Safranin O and toluidine blue stain were obtained from Beyotime Institute of Biotechnology; collagenase type II, H₂O₂ and DMSO were bought from Sigma-Aldrich (Merck KGaA). BCA protein assay kit was obtained from Beyotime Institute of Biotechnology; primary antibodies against GRP78, CHOP and GAPDH were obtained from Wuhan Sanying Biotechnology; primary antibodies against Bax, Bcl-2, Cleaved caspase-3 and SIRT1 were purchased from Cell Signaling Technology, Inc.; In Situ Cell Death Detection kit was obtained from Roche Diagnostics; secondary antibodies (cat. no. SA00001-14; Wuhan Sanying Biotechnology), including Goat Anti-Rabbit IgG (cat. no. B900210; Wuhan Sanying Biotechnology) and Alexa Fluor^® 488-labeled goat anti-rabbit IgG (H+L) secondary antibody (cat. no. SA00009-3; Wuhan Sanying Biotechnology), were obtained from Jackson ImmunoResearch Laboratories, Inc.; Cell-Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. DMEM-F12 was purchased from Gibco (Thermo Fisher Scientific, Inc.).

A total of 10 C57BL/6 mice (5 males and 5 females; age, 10 days; weight, 4 g; the Animal Center of the Chinese Academy of Sciences) were euthanized by 100 mg/kg sodium pentobarbital. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Wenzhou Medical University (35) and experiments were approved by the Animal Ethics Committee of Wenzhou Medical University. The knee cartilage of mice were collected carefully under aseptic conditions using a dissecting microscope, and tissue was treated with 2 mg/ml (0.1%) collagenase II for 4 h at 37°C. The digested tissue was centrifuged (800 × g) for 5 min at 37°C. Then, chondrocytes (5×10⁴ cells/cm²) were seeded into culture flasks. Cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin/streptomycin) and incubated in an atmosphere of 5% CO₂ at 37°C. The culture medium was replaced every 2–3 days. When 80–90% confluency was attained, cells were fused and 0.25% trypsin-EDTA solution was used to subculture the chondrocytes. In order to avoid phenotypic loss, chondrocytes from the first and second channels were used.

CCK-8 assay was used to evaluate chondrocyte viability according to the manufacturer's instructions. First, chondrocytes were incubated in 96-well plates (8,000 cells/well) for 24 h. Then, they were treated with a concentration gradient (0.0, 12.5, 25.0, 50.0, 100.0 and 200.0 µM) of SFN for 24 h and 48 h. Finally, 10 mol/l CCK-8 solution was added to each well for 2 h at 37°C, after which optical density was spectrophotometrically measured at 450 nm (Thermo Fisherr Scientific, Inc.).

Intracellular ROS levels were detected using the HDCFDA Probe Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Intracellular ROS generation was measured at 485 nm (excitation) and 535 nm (emission) using a microplate reader (Bio-Rad Laboratories, Inc.).

In order to obtain total proteins from chondrocytes, RIPA lysis buffer (Beyotime Institute of Biotechnology) was added to 1 mM PMSF and kept on ice for 10 min, after which the mixture was centrifuged at 1,000 × g for 15 min at 4°C. BCA protein detection kit was used to estimate protein concentration. Then, 40 ng protein was separated by SDS-PAGE (10%) and transferred to polyvinylidene fluoride membrane. Membranes were blocked using 5% skimmed milk for 2 h at 37°C and detected overnight at 4°C with the following antibodies: CHOP (1:1,000; cat. no. 15204-1-AP; Wuhan Sanying Biotechnology), GRP78 (1:1,000; cat. no. 11587-1-AP; Wuhan Sanying Biotechnology), Bcl-2 (1:1,000; cat. no. 15071; Cell Signaling Technology, Inc.), Bax (1:1,000; cat. no. 5023; Cell Signaling Technology, Inc.), Cleaved caspase-3 (1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), SIRT1 (1:500; cat. no. 2493; Cell Signaling Technology, Inc.) and GAPDH (1:1,000; cat. no. 10494-1-AP; Wuhan Sanying Biotechnology).

The membrane was washed using TBS-Tween-20 (TBST; 5% Tween-20) and incubated with the corresponding secondary antibodies (1:1,000; cat. no. SA00001-14; Wuhan Sanying Biotechnology) for 2 h at room temperature. After being washed three times using TBST, the blots were visualized using electrochemiluminescence plus reagent (Invitrogen; Thermo Fisherr Scientific, Inc.). Image Lab 3.0 software (Bio-Rad Laboratories, Inc.) was used to measure the intensity of each band.

Apoptotic chondrocytes were measured by TUNEL staining using an In Situ Cell Death Detection kit, according to the manufacturer's instructions. The chondrocytes (1×10⁵) were seeded in a 6-well plate and treated with SFN (50 µM) or H₂O₂ (50 µM) for 24 h at 37°C, fixed in 4% paraformaldehyde for 45 min at 37°C, incubated with 0.5% Triton X-100 for 15 min at 37°C and washed using PBS for 5 min. Finally, cells were stained using the In Situ Cell Death Detection kit for 60 min at 65°C, after which the nuclei were stained with DAPI for 1 min at 37°C (Beijing Solarbio Science & Technology Co., Ltd.). In total, 25 fields of each slide were randomly selected and images were observed using a fluorescence microscope (magnification, ×100; scale bar, 50 µm; Olympus Corporation).

Double-stranded siRNA for mouse SIRT1 gene silencing was purchased from Abcam. The SIRT1 siRNA sequence was as follows: 5′-UUGGGAUUCACGCAUAGGAGCACUG-3′. Cells (1×10⁵) were seeded into a 6-well plate, incubated for 24 h at 37°C and transfected with negative control [non-specific non-targeting siRNA (scramble); Abcam] or SIRT1 siRNA duplexes for 36 h at 50 nM using Lipofectamine^® 2000 siRNA transfection reagent (Thermo Fisherr Scientific, Inc.), according to the manufacturer's instructions. The chondrocytes were serum-starved overnight followed by incubation at 37°C with SFN (50 µM) for 24 h. After 24 h, the interference efficiency of the siRNA was evaluated by reverse transcription-quantitative PCR (RT-qPCR).

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisherr Scientific, Inc.). The concentration and purity of RNA was evaluated by spectrometry at 260 and 280 nm. The synthesis of cDNA was performed using the PrimeScript™ RT reagent kit [cat. no. 2312, Hangzhou Multisciences (Lianke) Biotech Co., Ltd.]. qPCR was performed using SYBR Green Real-Time PCR Master mix (Thermo Fisherr Scientific, Inc.) according to the manufacturer's protocol. The amplification conditions were as follows: SIRT1, 45 sec at 94°C followed by 30 cycles of 30 sec at 94°C, 30 sec at 52°C and 60 sec at 72°C; GAPDH, 45 sec at 94°C followed by 30 cycles of 30 sec at 94°C, 30 sec at 58°C and 60 sec at 72°C. Primers sequences, designed by Primer-Express V3.0 (Thermo Fisherr Scientific, Inc.), were as follows: SIRT1 forward, 5′-CTCTGAAAGTGAGACCAGTAGC-3′ and reverse, 5′-TGTAGATGAGGCAAAGGTTCC-3′ (product, 213 bp); and GAPDH forward, 5′-TCTTGCTCAGTGTCCTTGC-3′ and reverse, 5′-CTTTGTCAAGCTCATTTCCTGG-3′ (product, 457 bp). Relative gene expression was analyzed using the 2^−ΔΔCq method (36).

Cells were rinsed using PBS and fixed in 4% paraformaldehyde for 15 min at 37°C after which they were washed three times using PBS. Then, they were treated with 0.1% Triton X-100 diluted in PBS for 15 min at room temperature. Next, chondrocytes were blocked using 10% goat serum (cat. no. SL038; Beijing Solarbio Science & Technology Co., Ltd.; dissolved in PBS) for 4 h at 37°C and incubated at 4°C overnight in the presence of primary antibodies against collagen II (1:300; cat. no. 28459-1-AP; Wuhan Sanying Biotechnology). Following incubation, cells were exposed to Alexa Fluor^® 594-labelled conjugated secondary antibodies (1:400; cat. no. IC1051T; R&D Systems, Inc.) for 1.5 h. Finally, they were exposed to DAPI (10 µg/ml; 37°C) (Beyotime Institute of Biotechnology) for 1 min and observed using a fluorescence microscope (magnification, ×200; scale bar, 20 µm; Olympus Corporation). Fluorescence intensity was assessed using ImageJ software 6.0 (National Institutes of Health).

The paraffin-embedded sections (6 µm) were deparaffinized in xylene for 15 min at 37°C and rehydrated by incubation in series of graded ethanol (100, 95, 85, 75 and 0%) for 5 min at 37°C and endogenous peroxidase. Then, sections were treated with 3% (v/v) hydrogen peroxide for 10 min at 37°C. Antigen retrieval of the sections was performed using 0.4% pepsin (Sangon Biotech Co., Ltd.) in 5 mM HCl at 37°C for 20 min. Then, sections were incubated in the presence of 10% BSA (cat. no. ST025; Beyotime Institute of Biotechnology) for 45 min at room temperature, after which they were treated with primary antibodies against SIRT1 (1:500; cat. no. 2493; Cell Signaling Technology, Inc.) overnight at 4°C. Finally, they were treated with horseradish peroxidase-conjugated secondary antibodies (1:1,000; cat. no. SA00001-14; Wuhan Sanying Biotechnology) for 3 h at 4°C. Following incubation at room temperature for 1 h, the slices were rinsed (5 min, 37°C), stained using a Metal Enhanced DAB Substrate kit (2 min, 37°C; cat. no. DA1015; Beijing Solarbio Science & Technology Co., Ltd.), dehydrated (5 min, 37°C), mounted and examined under a microscope (magnification, ×40; CX41; Olympus Corporation). Quantitative analysis was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

A total of 45 C57BL/6 male wild-type mice (age, 10 weeks; weight, 40 g) were obtained from the Animal Center of the Chinese Academy of Sciences (Shanghai, China). The protocol for animal care and use conformed to The Guide for the Care and Use of Laboratory Animals of the National Institutes of Health published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996) (37) and was approved by the Animal Care and Use Committee of Wenzhou Medical University. Experimental OA mice were established by destabilization of the medial meniscus (DMM) as previously described (38). Mice were housed under 12-h light/dark cycles and constant temperature (21–23°C) and humidity (50–60%). Water and food were provided ad libitum. Anesthesia was performed via intraperitoneal injection of 2% (w/v) pentobarbital (40 mg/kg). Faint breathing, myasthenia, lack of independent reaction, cyanosis or coma were considered to indicate that mice were close to death; mice were euthanized by cervical dislocation. The joint capsule was carved followed by transection of the medial meniscotibial ligament of the right knee using microsurgical scissors. During surgery, the lateral meniscotibial ligament was always protected. As a control, the medial meniscotibial ligament was not transected while arthrotomy was performed in the left knee. Mice were randomly separated into three groups (n=15/group): Control (sham-operated), DMM and DMM + SFN [mice were intraperitoneally administered with SFN at 20 mg/kg body weight (39,40)]. Parameters indicating the condition of mice were observed daily, including fur brightness, food and water intake, defecation and behavior. Furthermore, body weight was measured each week.

The extent of cartilage degeneration and synovitis in stained sections was assessed using light microscopy with the Osteoarthritis Research Society International (OARSI) scoring system, as previously described (41).

The data are presented as mean ± SD (n≥3). Statistical analysis was performed using GraphPad Prism version 5.0 software (GraphPad Software, Inc.). Comparisons between groups were performed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Safranin O and toluidine blue staining were used to characterize mouse chondrocytes. Chondrocytes were stained red by Safranin O and the cytoplasm was stained purple by toluidine blue (Fig. 1A and B). In addition, collagen II in chondrocyte cytoplasm was stained red by immunofluorescence with no positive staining observed in the nucleus (Fig. 1C). These results imply that the cells isolated from mouse articular cartilage were chondrocytes.

The chemical structure of SFN is shown in Fig. 1D. In order to determine the cytotoxic effects of SFN, CCK-8 assay was performed. Cells were incubated with increasing concentrations (0.0, 12.5, 25.0, 50.0, 100.0 and 200.0 µM) of SFN for 24 and 48 h. SFN exhibited cytotoxic effects at ≥100 µM after 24 or 48 h but not <100 µM (Fig. 1E and F). Therefore, 50 µM was selected for use in subsequent experiments.

In order to determine the anti-apoptotic effect of SFN on H₂O₂-induced chondrocytes, the expression levels of Bax, Cleaved caspase-3 and Bcl-2 were evaluated by western blot analysis. H₂O₂ elevated levels of Bax and Cleaved caspase-3, whereas Bcl-2 levels were downregulated (Fig. 2A-D). SFN downregulated levels of Bax and Cleaved caspase-3. Moreover, SFN significantly activated SIRT1 (Fig. 2A and E). These data indicate that SFN exerted an anti-apoptotic effect in mouse chondrocytes.

In order to determine whether ERS in chondrocytes was associated with the cytoprotective effects of SFN on cartilage, cells were treated with H₂O₂, a typical ERS inducer. Expression levels of ERS-associated factors GRP78 and CHOP were measured. Western blotting assay exhibited increased expression levels of these proteins in response to H₂O₂; these effects were stopped by SFN (Fig. 3A-C). Moreover, apoptosis of cells in the H₂O₂ group was high compared with the control group. SFN treatment decreased cell apoptosis (Fig. 3D and E). Furthermore, H₂O₂ induced ROS production, which was inhibited by SFN (Fig. 3F).

In order to confirm the aforementioned results, SIRT1 siRNA was used to detect the potential mechanism of SFN on H₂O₂-stimulated chondrocytes. The mRNA expression levels of SIRT1 were significantly lower in SIRT1-silenced cells than in control cells (Fig. 4F). SIRT1 levels were elevated in the H₂O₂ + SFN group but suppressed in the SIRT1 siRNA transfection group (Fig. 4A and E). Moreover, Bcl-2 levels were suppressed in the SIRT1 siRNA transfection group whereas the expression levels of Bax and Cleaved caspase-3 increased significantly (Fig. 4A-D). These results demonstrated the anti-apoptotic effect of SFN, which was mediated by activation of SIRT1.

The chondroprotective effect of SFN on OA was investigated in vivo. A DMM mouse model was established, followed by intraperitoneal injection of 20 mg/kg SFN dissolved in DMSO every 2 days for 8 weeks. Safranin O staining was used to evaluate the effect of SFN on the development of OA. Compared with the sham group, the surface of the cartilage was notably destroyed in the DMM group (Fig. 5A). The degradation level of the cartilage matrix in the SFN group was lower than the OA but higher than the sham group. The OARSI scores in the SFN group were lower than those of the DMM group (Fig. 5B). Moreover, synovitis score in the DMM group was higher than in the sham group; SFN reversed this effect (Fig. 5A and C). In order to investigate the effect of SFN on SIRT1 expression in vivo, immunohistochemical staining for SIRT1 was performed (Fig. 5D and E). Compared with the DMM group, significantly more SIRT1-positive chondrocytes were detected in the DMM + SFN group. These results imply that SFN exhibited chondrocyte-protective effects during the progression of OA.