The human chondrocyte cell line (CHON-001; American Type Culture Collection) was cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) with 1% Penicillin-Streptomycin Solution Hybri-Max^™ (Sigma-Aldrich; Merck KGaA) at 37°C in an incubator with 5% CO₂. To mimic an in vitro model of OA, CHON-001 cells were exposed to 10 ng/ml IL-1β (Novoprotein Scientific, Inc.) for 24 h at 37°C (24). The 5′ AMP-activated protein kinase (AMPK) inhibitor compound C, QCT and erastin were purchased from MedChemExpress, and the cells were treated with QCT (100 µM), QCT and erastin (5 µM) or QCT and compound C (5 µM) for 24 h at 37°C, and then exposed to IL-1β for 24 h at 37°C. Control cells were cultured in medium only.

CHON-001 cells were seeded (5×10⁴ cells/well) in 96-well plates overnight. CHON-001 cells were treated with QCT (0, 25, 50, 100 or 200 µM) for 24 h at 37°C. Subsequently, 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) was added to each well and the cells were incubated for a further 2 h at 37°C. Subsequently, a microplate reader (MULTISKAN MK3; Thermo Fisher Scientific, Inc.) was used to detect the absorbance of each well at 450 nm. Similarly, CHON-001 cells were treated with QCT (50 or 100 µM) for 24 h at 37°C, and then exposed to 10 ng/ml IL-1β for 24 h at 37°C. The cell viability was detected with the CCK-8 assay as well. In addition, CHON-001 cells were treated with erastin (0, 1, 2, 5 or 10 µM) for 24 h at 37°C and the cell viability was detected using a CCK-8 assay.

Cell proliferation was detected using an EdU detection kit (Wuhan Servicebio Technology Co., Ltd.). The cells were fixed in 4% paraformaldehyde (Wuhan Servicebio Technology Co., Ltd.) for 2 h at room temperature and then stained with EdU Apollo567 solution for 1 h at 37°C in the dark, followed by staining with 0.1 µg/ml DAPI (Wuhan Servicebio Technology Co., Ltd.) for 15 min at room temperature. Finally, the EdU-positive cells were observed using a fluorescence microscope (Eclipse Ci-L; Nikon Corporation). A total of three random fields were selected and the EdU-positive cells were counted manually.

A TUNEL detection kit (G1504; Wuhan Servicebio Technology Co., Ltd.) was applied to assess CHON-001 cell apoptosis. The cells were fixed in 4% paraformaldehyde for 2 h at room temperature and washed with PBS for 30 min. Subsequently, the cells were treated with 0.2% Triton X-100 for 2 min at room temperature. The cells were stained with the mixed solution (recombinant TdT enzyme:CF488-dUTP labeling mix:equilibration buffer, 1:5:50) for 1 h at 37°C. The nuclei were stained with 0.1 µg/ml DAPI for 30 min in the dark at room temperature. Polyvinyl alcohol mounting medium with DABCO^® (cat. no. 10981; Sigma-Aldrich; Merck KGaA) was used as the mounting medium. TUNEL-positive cells in three random fields were observed using a fluorescence microscope (Eclipse Ci-L; Nikon Corporation).

Human IL-6 [cat. no. ELK1156; Elk (Wuhan) Biotechnology Co., Ltd.], TNF-α [cat. no. ELK1190; Elk (Wuhan) Biotechnology Co., Ltd.], glutathione (GSH; cat. no. A061-1; Nanjing Jiancheng Bioengineering Institute) and malondialdehyde (MDA; cat. no. A003-1; Nanjing Jiancheng Bioengineering Institute) detection kits were used to detect the IL-6, TNF-α, GSH and MDA levels in the supernatant of CHON-001 cells according to the manufacturers' instructions. Furthermore, the Fe²⁺ and lipid reactive oxygen species (ROS) levels in CHON-001 cells were detected using the Cell Ferrous Iron Colorimetric Assay kit (cat. no. E-BC-K881-M; Wuhan Elabscience Biotechnology Co., Ltd.) and BODIPY 581/591 C11 kit (cat. no. HY-D1301; MedChemExpress). All kits were used according to the manufacturers' instructions. The results were analyzed using a microplate reader (SMR16.1; Wuhan USCN Business Co., Ltd.).

Total protein was extracted from CHON-001 cells using RIPA buffer (Beyotime Institute of Biotechnology) and the protein concentration was measured using the BCA detection assay kit (ASPEN Biotechnology Co., Ltd.). Proteins (20 µg/lane) were resolved using 8% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies against phosphorylated (p-)AMPK (cat. no. ab109402), AMPK (cat. no. ab32047), nuclear factor erythroid 2-related factor 2 (Nrf2; cat. no. ab31163), glutathione peroxidase 4 (Gpx4; cat. no. ab125066), Bcl-2 (cat. no. ab182858), cleaved caspase 3 (cat. no. ab214430), caspase 3 (cat. no. ab32351), aggrecan (cat. no. ab315486), collagen II (cat. no. ab34712), MMP13 (cat. no. ab219620), ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5; cat. no. ab41037) and β-actin (cat. no. ab8227) overnight at 4°C. All primary antibodies were purchased from Abcam and the dilution factor was 1:1,000. The membranes were then probed with the secondary antibody (dilution, 1:5,000; cat. no. AS1107) for 2 h at room temperature. The secondary HRP-conjugated antibody was purchased from ASPEN Biotechnology Co., Ltd. Finally, the bands were visualized using ECL reagent (ASPEN Biotechnology Co., Ltd.). Densitometry was performed using ImageJ software (version 1.8.0; National Institutes of Health).

C57BL/6 mice (8 weeks old; 18–22 g; female; n=24; 6 mice/group) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed with a 12-h light/dark cycle at 24°C with 60% humidity, with ad libitum access to food and water. The experimental protocols were approved by the Ethics Committee of Beijing University of Chinese Medicine (approval no. BUCM-2023032701-2103; Beijing, China) and the animal experiments were performed according to the institutional guidelines. The animals were randomly divided into the following four groups: Sham, OA, OA + QCT 20 mg/kg and OA + QCT 40 mg/kg. The OA models were generated with ACLT surgery on the knee joints of the mice as previously described (25). The animals were treated with QCT (20 or 40 mg/kg) via gavage once per day for 4 weeks in the QCT treatment group. Mouse body weight loss >20% was regarded as a humane endpoint in the present study. The Osteoarthritis Research Society International (OARSI) score was determined based on the data of Safranin O/fast green staining to evaluate cartilage degradation (26). Briefly, a 0–6 subjective scoring system (0, 0.5, 1, 2, 3, 4, 5 and 6) was applied to all four quadrants of the joint: Medial femoral condyle, medial tibial plateau, lateral femoral condyle and lateral tibial plateau (Table I).

All animals were sacrificed using CO₂ at a displacement rate of 40% volume/min at 4 weeks following surgery, and the knee joints of each mouse were collected. The toe reaction and heartbeat of the mouse were checked to confirm animal death. The pathological changes of articular tissues were observed using H&E staining. Briefly, the samples were fixed in 4% paraformaldehyde for 24 h at room temperature and then embedded in paraffin. Subsequently, the specimens (4 µm thick) were placed in distilled water and then stained with hematoxylin for 15 min at room temperature. The sections were then rehydrated in alcohol at concentrations of 90 and 70% for 15 min each. Next, the sections were incubated with eosin staining solution for 10 min at room temperature. Afterwards, the samples were dehydrated with 100% alcohol and placed in in an incubator for drying. Images were captured using a light microscope (CS31; Olympus Corporation).

A modified Saffron-O and Fast Green Stain kit (cat. no. G1371; Beijing Solarbio Science & Technology Co., Ltd.) was used to evaluate the proteoglycan contents in the articular tissues. The samples were fixed in 4% paraformaldehyde for 24 h at room temperature and then embedded in paraffin. Paraffin sections of tissue were obtained from the embedded samples. The 4-µm paraffin sections were heated at 60°C for 1 h, dewaxed twice in xylene solutions for 15 min each and rehydrated in a descending alcohol series. Subsequently, the articular tissue sections were stained with Weigert dye (Wuhan Servicebio Technology Co., Ltd.) for 5 min at room temperature. The sections were first stained with fast green solution for 5 min at room temperature and then stained with safranin O solution for 5 min at room temperature. Images were captured using a light microscope (CS31; Olympus Corporation).

The samples were fixed in 4% paraformaldehyde for 24 h at room temperature and then embedded in paraffin. Paraffin sections of tissue (4-µm) were obtained from the embedded samples and heated in a 60°C oven. Subsequently, samples were deparaffinized, rehydrated in a descending alcohol series and boiled in 0.01 mol/l sodium citrate buffer (pH 6.0) in a microwave oven for 10 min for antigen retrieval. Next, samples were blocked with 0.3% hydrogen peroxide for 15 min at room temperature, and washed with distilled water. The sections were blocked for 30 min using 10% normal goat serum (Thermo Fisher Scientific, Inc.) at room temperature, and probed with primary antibodies specific for ADAMTS5 (1:100; cat. no. DF13268; Affinity Biosciences), MMP13 (1:300; cat. no. ab219620; Abcam), collagen II (1:200; cat. no. ab34712; Abcam) and aggrecan (1:150; cat. no. DF7561; Affinity Biosciences) overnight at 4°C, and then incubated with a HRP-conjugated secondary antibody (1:200; cat. no. AS1107; ASPEN Biotechnology Co., Ltd.) for 30 min at 37°C. The sections were stained with 3,3′-diaminodbenzidine solution (Wuhan Servicebio Technology Co., Ltd.). Subsequently, images were captured using a light microscope (CS31; Olympus Corporation). ImageJ software (version 1.8.0; National Institutes of Health) was used for analysis.

Data are presented as the mean ± SD. Multiple comparisons were conducted using one-way ANOVA with Tukey's post hoc test using GraphPad Prism 7 (Dotmatics). All experiments were repeated at least three times. P<0.05 was considered to indicate a statistically significant difference.

In order to determine the cytotoxic effects of QCT on chondrocytes, a CCK-8 assay was conducted. As shown in Fig. 1A, 200 µM QCT significantly suppressed the viability of CHON-001 cells, while 50 or 100 µM QCT had a limited effect on CHON-001 cell viability. In addition, IL-1β markedly reduced the viability and proliferation, and triggered the apoptosis of CHON-001 cells; however, these changes were reversed by 100 µM QCT (Fig. 1B-D). Overall, QCT attenuated the IL-1β-induced injury of chondrocytes.

To explore the effects of QCT on the ferroptosis of chondrocytes, lipid ROS and Fe²⁺ levels in CHON-001 cells were detected. IL-1β significantly enhanced lipid ROS and Fe²⁺ levels in CHON-001 cells; however, these effects were reversed by treatment with QCT (Fig. 2A and B). Additionally, treatment with 100 µM QCT significantly elevated the p-AMPK, Nrf2 and Gpx4 levels in CHON-001 cells exposed to IL-1β (Fig. 2C). In summary, QCT inhibited the IL-1β-induced ferroptosis of chondrocytes by activating the AMPK/Nrf2/Gpx4 signaling pathway.

There is evidence to indicate that ferroptosis participates in the regulation of cell proliferation (27,28). Thus, in the present study, in order to explore whether QCT attenuated IL-1β-induced chondrocyte injury by modulating ferroptosis, erastin (a ferroptosis activator) was used. As shown in Fig. 3A, 5 µM erastin reduced CHON-001 cell viability to ~50%, with a significant difference compared with the 0 µM treatment group. Thus, 5 µM erastin was utilized in the subsequent experiments.

As shown in Fig. 3B-E, QCT markedly elevated the proliferation and prevented the apoptosis of IL-1β-stimulated CHON-001 cells; however, treatment with erastin reversed these effects. Additionally, QCT significantly elevated the Bcl-2 level and decreased the level of cleaved caspase 3 in the IL-1β-stimulated CHON-001 cells; however, the effects on these protein levels were reversed by erastin (Fig. 3F). Collectively, these results indicated that QCT promoted the proliferation of IL-1β-stimulated chondrocytes by inhibiting ferroptosis.

The present study then examined the effects of QCT on ECM degradation in IL-1β-stimulated chondrocytes. IL-1β significantly decreased the levels of ECM proteins (collagen II and aggrecan) and elevated the levels of ECM-degrading enzymes (MMP13 and ADAMTS5) in CHON-001 cells; however, the effects on these protein levels (except for ADAMTS5) were reversed by treatment with QCT (Fig. 4A and B). Conversely, treatment with erastin reversed the QCT-induced upregulation of collagen II and aggrecan and the downregulation of ADAMTS5 in IL-1β-stimulated CHON-001 cells (Fig. 4A and B). Additionally, QCT significantly reduced the IL-6, TNF-α, lipid ROS, Fe²⁺ and MDA levels, and increased the GSH level in CHON-001 cells exposed to IL-1β; however, erastin reversed these effects (Fig. 4C-H). Overall, QCT attenuated ECM degradation and inflammatory responses in IL-1β-stimulated chondrocytes by inhibiting ferroptosis.

AMPK/Nrf2 signaling serves a crucial role in regulating ferroptosis (29). Therefore, the present study explored whether QCT could attenuate ferroptosis in chondrocytes by modulating AMPK/Nrf2 signaling. Treatment with QCT markedly elevated the p-AMPK, Nrf2 and Gpx4 levels in IL-1β-stimulated CHON-001 cells; however, these changes were reversed by treatment with compound C, an AMPK inhibitor (Fig. 5A). Furthermore, treatment with QCT markedly increased the aggrecan and collagen II levels, and reduced the ADAMTS5 level in IL-1β-stimulated CHON-001 cells, while compound C markedly reversed these effects (Fig. 5B). However, QCT had no effect on the MMP13 levels in IL-1β-stimulated CHON-001 cells (Fig. 5B). Collectively, QCT protected against IL-1β-induced ECM degradation in vitro by suppressing ferroptosis via the activation of the AMPK/Nrf2/Gpx4 signaling pathway.

Finally, to evaluate the therapeutic effects of QCT in vivo, a mouse model of OA was established. Disordered chondrocytes, damaged cartilage surface and proteoglycan loss in cartilage tissues were observed in the OA group; however, these effects were reversed by treatment with QCT, suggesting that QCT attenuated cartilage damage in mice with ACLT-induced OA (Fig. 6A). Additionally, the results of IHC staining indicated that the aggrecan and collagen II levels were reduced, and the ADAMTS5 level was markedly elevated in the cartilage tissues of mice with OA, while these changes were reversed by treatment with 40 mg/kg QCT (Fig. 6A). However, QCT had no effect on the MMP13 level in the cartilage tissues of mice with OA (Fig. 6A). Furthermore, treatment with QCT significantly elevated the p-AMPK, Nrf2 and Gpx4 levels in cartilage tissues of mice with OA (Fig. 6B and C). The OARSI scores confirmed that QCT significantly attenuated the progression of OA (Fig. 6D). In summary, QCT ameliorated OA in mice in vivo via the activation of the AMPK/Nrf2/Gpx4 signaling pathway.