7,8-DHF (purity >99%) was procured from Sigma-Aldrich; Merck KGaA. Anti-Nrf2 (cat. no. BS1258) and anti-HO-1 (cat. no. BS6626) antibodies were supplied by Bioworld Technology at a dilution of 1:1,000. Anti-actin (cat. no. 4970) and anti-lamin B (cat. no. 13435) antibodies were obtained from Cell Signaling Technology, Inc. at a dilution of 1:5,000. Secondary HRP-conjugated goat anti-rabbit (cat. no. BS13278) antibody was purchased from Bioworld Technology, Inc. and used at a dilution of 1:5,000. The Cell Counting Kit-8 (CCK-8) and nuclear protein extraction kit were purchased from Beyotime Institute of Biotechnology. The superoxidase dismutase (SOD) and malondialdehyde (MDA) assay kits were supplied by Nanjing Jiancheng Bioengineering Institute. The fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was obtained from Molecular Probes; Thermo Fisher Scientific, Inc.

Cell viability was evaluated using a CCK-8 assay. Briefly, chondrocytes were seeded in 96-well plates at a density of 5×10³ cells/well and cultured with various concentrations of 7,8-DHF (0, 1, 2, 4, 6, 8 and 10 µM) for 8 h at 37°C. Following incubation, 10 µl of CCK-8 solution was added to each well and the cells were further incubated for 1 h at 37°C. The absorbance was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

The primary mouse chondrocytes pre-treated with various doses of 7,8-DHF (0,1,3 and 9 µM) for 8 h were cultured in 40 ng/ml H₂O₂ for 24 h at 37°C. The cells were washed in ice-cold phosphate buffer and total proteins were extracted with 100 µl RIPA for 15 min. The lysates were homogenized using an ultrasonic homogenizer and centrifuged at 13,000 × g for 5 min at 4°C. The MDA contents in the supernatant were measured by monitoring thiobarbituric acid reacting substances. Using a spectrophotometer, the activities of SOD were measured at a wavelength of 532 nm using the assay kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocols.

The generation of ROS was detected using the fluorescent probe, H2DCFDA. Following incubation with various concentrations of 7,8-DHF for 8 h, the primary mouse chondrocytes were treated with/without H₂O₂ for 24 h. The cells were incubated with H2DCFDA (10 µM) for 30 min at room temperature in the dark. The production of ROS in the cells was monitored using a flow cytometer (BD Biosciences) with CellQuest Pro software, version 5.0 (BD Biosciences).

The proteins lysed from the primary mouse chondrocytes and the cartilage tissue extracted from the knee joints were measured and normalized for western blot analysis, as previously described (10). Each experimental unit for the mice consisted of a pool of two cartilage compartments. Nuclear extraction was performed according to the manufacturer's protocols of the nuclear protein extraction kit (Beyotime Institute of Biotechnology). Briefly, normalized volumes of samples (20 µg protein) were separated by 10% SDS-PAGE at 100 V for 100 min in Tris-glycine-SDS running buffer (Thermo Fisher Scientific, Inc.). The gel was transferred for 90 min at 300 mA to nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). Membranes were then incubated for 2 h with blocking buffer (5% skimmed milk in TBST) at room temperature, and incubated overnight with the anti-Nrf2 (1:1,000; cat. no. BS1258; Bioworld Technology, Inc.), anti-HO-1 (1:1,000; cat. no. BS6626; Bioworld Technology, Inc.) antibodies and anti-actin (1:5,000; cat. no. 4970; Cell Signaling Technology, Inc.) antibodies at 4°C. Subsequently, the membranes were washed three times in TBST, then incubated with secondary HRP-conjugated goat anti-rabbit (1:5,000 cat. no. BS13278; Bioworld Technology, Inc.) at room temperature for 1 h and finally washed an additional three times in TTBS. The membranes were visualized using the enhanced chemiluminescence assay (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Densitometry of protein bands was performed for western blot analysis. Bands were semi-quantified by reverse image scanning densitometry with Photoshop (version 6.0; Adobe Systems, Inc.).

RNA was extracted from the knee cartilage of mice using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), following homogenization. The cartilage, which was snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction, was ground with a pestle and liquid nitrogen-chilled mortar prior to TRIzol extraction. Each experimental unit consisted of a pool of 1–2 cartilage compartments and, when pooling was performed, the experimental unit was regarded as a single unit. The purity and yield of the RNA was determined using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies; Thermo Fisher Scientific, Inc.). cDNA was synthesized from the RNA with PrimeScript RT Master mix (Takara Bio, Inc.). Gene expression was measured using a 7500 real-time PCR system in the presence of SYBR Premix Ex Taq reagents, according to the manufacturer's protocol. The thermocycling conditions were as follows: Denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 5 sec, followed by an annealing/extension step at 60°C for 30 sec. The primer sequences are presented in Table I. Relative quantification was defined as 2^−ΔΔCq (11). qPCR was performed in triplicate for each sample.

A total of 92 wild-type B6 mice (8–10 weeks old) were used in the experiments and were housed in standard mouse cages (10 animals per cage) in specific-pathogen-free conditions under a light-dark cycle of 12:12 h at 25±2°C with standard mouse food (RM3; Special Dietary Systems) and water ad libitum. Animal health and behavior were monitored every 12 h. When pain/distress were observed, the animals were treated with Buprenex (Reckitt & Colman Pharmaceuticals, Inc.; 0.1–2.0 mg/kg), in addition to crushed or wet food. If pain or distress continued, the mouse was sacrificed regardless of the scheduled endpoints. The criteria that determined discomfort/distress/pain were any three of the following signs: Abnormal posture, a slow, careful or abnormal (waddling) gait, low activity levels, slow eating, cowering or vocalizing on handling, a change in eye or coat appearance, and weight loss. Sacrifice was confirmed by one of the following criteria: Cervical dislocation following no response to tail or toe pinch, no respiration or heartbeat following continuous monitoring for 30 sec, or rigor mortis. All procedures were performed in accordance with the Nanjing Medical University Institutional Animal Care and Use Committee (Nanjing, China) guidelines. The mice were anesthetized by inhalation of ether during OA surgery. OA was induced following sectioning of the medial meniscotibial ligament, also known as the coronary ligament, which connects the medial meniscus to the periphery of the tibial plateau, and mice undergoing sham surgery were used as a control (12,13). 7,8-DHF (Sigma-Aldrich; Merck KGaA) was subsequently dissolved in ethanol and phosphate-buffered saline (PBS). 7,8-DHF was administered by intraperitoneal injections (5 mg/kg) once a week postoperatively. The knee joints of the mice were harvested 4 and 8 weeks postoperatively, and were stained with Safranin O/Fast Green for assessment using the OARSI scoring system for histologic alterations (14). The mounted slides were placed in 70% ethyl alcohol for 15 min and then stained with 0.04% safranin O/sodium acetate buffer (pH 4.0), for 10 min at 22°C.

Murine knee articular cartilage was obtained from 5-day-old B6 mice and digested with collagenase D, as previously described (15). The chondrocytes isolated from articular cartilage of one litter were seeded onto a 10-cm dish at a density of 5×10⁵ cells/per dish. On reaching 80% confluency, the cells were detached and randomly plated in six-well plates (10⁵/well), and cultured in Ham's F-12 (DMEM/F12; Gibco; Thermo Fisher Scientific, Inc.) containing 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA), 100 IU/ml penicillin (Sigma-Aldrich; Merck KGaA), 5% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 2 mM L-glutamine at 37°C. Only the first passage cells were used for 7,8-DHF treatment, as indicated below.

293 cells (Shanghai Institutes for Biological Sciences, China) were maintained in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare, Chicago, IL, USA) + 200 µM geneticin (G-418; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO₂. The HO-1 promoter was amplified by PCR from RAW cell genome DNA using the following primer sequences: HO-1, forward 5′-GGAAGATCTCTGCAGAGCCCCACTGGAG-3′ and reverse 5′-CCCAAGCTTGGAACAGCAACGCTGT-3′. The PCR product was subsequently inserted into the pGL3 vector at the HindIII and Bg1II sites. A non-specific oligo was used to construct a control plasmid. The 293 cells (3×10⁵ cells/well) seeded in 24-well plates were transfected with the HO-1-ARE-promoter-driven luciferase plasmid (Beyotime Institute of Biotechnology). Transfection was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 24 h of transfection, the cells were treated with 1, 3 and 9 µM of 7,8-DHF for 8 h at 37°C. The relative luciferase activity was measured by normalizing HO-1-ARE-promoter-driven firefly luciferase activity to Renilla luciferase activity with a luciferase assay system (Promega Corporation).

All data are expressed as the mean ± standard deviation. Data following Gaussian distribution were further analyzed using Student's t-test and data without Gaussian distribution was analyzed via the Mann-Whitney U test. Differences between groups were analyzed using one-way analysis of variance and Duncan's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All data were analyzed using SPSS 16.0 (SPSS, Inc.) software.

Primary mouse chondrocytes were treated with various doses of 7,8-DHF for 8 h (9). The cytotoxicity of 7,8-DHF was analyzed in primary mouse chondrocytes and non-toxic concentrations were used in the subsequent experiments (Fig. 1A). To examine whether 7,8-DHF can activate Nrf2 signaling in chondrocytes, the effect of 7,8-DHF on the expression of the Nrf2 downstream protein in primary mouse chondrocytes was examined. The results indicated that the expression of HO-1 was upregulated by 7,8-DHF in a dose- and time-dependent manner (Fig. 1E-J). Furthermore, 7,8-DHF treatment caused the nuclear translocation of Nrf2 and activated HO-1 promoter transactivation activity (Fig. 1B-D). Therefore, these findings suggested that 7,8-DHF effectively activated Nrf2 signaling in primary chondrocytes.

To investigate whether 7,8-DHF affects oxidative stress damage, the level of intracellular ROS, the content of MDA and the activity of SOD were measured. The H₂O₂-induced ROS fluorescence intensity was markedly weakened in the chondrocytes pretreated with 7,8-DHF (Fig. 2A). 7,8-DHF treatment suppressed the expression level of MDA (Fig. 2B) and increasing the activity of SOD (Fig. 2C). The results suggested that 7,8-DHF attenuated H₂O₂-induced oxidative stress in primary chondrocytes.

The vehicle-treated OA mice exhibited a loss of knee joint cartilage with exposed subchondral bone, whereas the 7,8-DHF-treated group exhibited a greater extent of cartilage damage alleviation (Fig. 3A). The 7,8-DHF-treated group exhibited a lower staining score of 1.70±1.10 for the tibia (P<0.05) 4 weeks following surgery (Fig. 3B). In the 7,8-DHF-treatment group, the stained sections of the joints harvested at 8 weeks had lower scores of 2.20±0.93 for the femur and 2.45±1.40 for the tibia, indicating that 7,8-DHF significantly protected the cartilage from damage in the femoral condyle and medial tibial plateau (Fig. 4A and B). Therefore, 7,8-DHF had a protective effect on the cartilage of the OA mouse model.

The cartilage of the knee joints was used to examine changes in the OA-associated mRNA expression levels via RT-qPCR analysis. Consistent with the histopathological results, the cartilage of the OA group exhibited significantly higher mRNA expression levels of TNF-α, IL-1β, IL-6, MMP-1, MMP-3 and MMP-13 compared with that in the sham group, and the upregulated gene expression levels were suppressed by 7,8-DHF (Fig. 5A-F).

The expression of HO-1 and Nrf2 in the knee cartilage obtained from mice was further examined, in order to investigate whether Nrf2-HO-1 was substantially upregulated in the 7,8-DHF-treatment group. The results of the present study confirmed that the protein and gene expression levels of HO-1 and Nrf2 increased following 7,8-DHF treatment (Fig. 6A-E). This was consistent with the data in vitro, indicating that 7,8-DHF may serve a protective role in cartilage erosion in OA mice via Nrf2 signaling pathway activation.