Quercetin attenuates the symptoms of osteoarthritis in vitro and in vivo by suppressing ferroptosis via activation of AMPK/Nrf2/Gpx4 signaling

Introduction

Osteoarthritis (OA) is a common type of arthritis, which is characterized by degenerative lesions of articular cartilage or other joint tissues (1–3). The incidence of OA is closely related to age (4). Prieto-Alhambra et al (4) reported that the incidence rate of knee and hip OA continues to increase with age. Turkiewicz et al (5) reported that the proportion of patients with OA aged ≥45 years may increase to ~30% by 2032. The increasing prevalence of OA will lead to a marked social and economic burden. Clinically, drug therapy, such as treatment with non-steroidal anti-inflammatory drugs and glucocorticoids, surgery, such as total hip arthroplasty, and physical therapy are commonly used methods for the treatment of OA (6–9). However, evidence has indicated that the long-term use of drugs used to treat OA is associated with a number of negative side effects, including gastrointestinal discomfort and liver function impairment (10,11). Thus, the development of promising novel treatment methods for OA is urgently required.

Traditional Chinese medicine (TCM) has been practiced in China for >5,000 years (12). TCM has been shown to exert a therapeutic effect on multiple diseases (13,14). Quercetin (QCT) is a bioactive compound that can be isolated from various TCM formulas, such as Panax notoginseng and Ginkgo biloba (15,16). QCT exhibits various pharmacological properties, including antioxidant, anti-inflammatory and anti-bacterial activities (17,18). Furthermore, QCT has been demonstrated to exert chondroprotective effects in murine models of OA (19,20). However, to the best of our knowledge, the mechanisms through which QCT attenuates the symptoms of OA remain largely unclear.

Ferroptosis is a type of iron-dependent cell death that is induced by iron accumulation and lipid peroxidation (21,22). Ferroptosis serves a crucial role in human diseases, including OA (23). Activation of ferroptosis is able to elevate the MMP13 levels and reduce the type II collagen (collagen II) levels in chondrocytes, suggesting that ferroptosis can contribute to the progression of OA (23). However, whether QCT can attenuate the development of OA by affecting ferroptosis remains largely elusive.

In the present study, IL-1β-stimulated chondrocytes and a mouse model of anterior cruciate ligament transection (ACLT)-induced OA were established in order to explore the role of QCT in the treatment of OA disease.

Materials and methods

Cell culture

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.

Cell counting kit-8 (CCK-8) assay

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.

5-Ethynyl-2′-deoxyuridine (EdU) staining 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.

TUNEL assay

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).

ELISA

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.).

Western blot analysis

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).

Animal experiments

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).

Table I. Semi-quantitative scoring system.

Table I.

Semi-quantitative scoring system.

Grade	Osteoarthritic damage
0	Normal
0.5	Loss of Safranin-O without structural changes
1	Small fibrillations without loss of cartilage
2	Vertical clefts down to the layer immediately below the superficial layer and some loss of surface lamina
3	Vertical clefts/erosion to the calcified cartilage extending to <25% of the articular surface
4	Vertical clefts/erosion to the calcified cartilage extending to 25–50% of the articular surface
5	Vertical clefts/erosion to the calcified cartilage extending to 51–75% of the articular surface
6	Vertical clefts/erosion to the calcified cartilage extending >75% of the articular surface

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).

Safranin O/fast green staining assay

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).

Immunohistochemistry (IHC)

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.

Statistical 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.

Results

QCT attenuates the IL-1β-induced apoptosis of chondrocytes

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.

Figure 1. QCT attenuates IL-1β-induced apoptosis in chondrocytes. (A) CHON-001 cells were treated with QCT (0, 25, 50, 100 or 200 µM) for 24 h at 37°C. Cell viability was detected using a CCK-8 assay. (B) CHON-001 cells were treated with QCT (50 or 100 µM) for 24 h at 37°C, and then exposed to IL-1β for 24 h at 37°C. Cell viability was detected using a CCK-8 assay. (C) Cell proliferation was evaluated using an EdU staining assay. Magnification, ×200. Scale bar, 100 µm. (D) Cell apoptosis was assessed using a TUNEL assay. Magnification, ×200. Scale bar, 100 µm. **P<0.01 vs. no QCT treatment or control group; ##P<0.01 vs. IL-1β group. CCK-8, Cell Counting Kit-8; EdU, 5-ethynyl-2′-deoxyuridine; QCT, quercetin.

QCT inhibits the IL-1β-induced ferroptosis of chondrocytes by activating the AMPK/Nrf2/Gpx4 signaling pathway

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.

Figure 2. QCT inhibits IL-1β-induced ferroptosis in chondrocytes by activating AMPK/Nrf2/Gpx4 signaling. CHON-001 cells were treated with QCT (50 or 100 µM) for 24 h, and then exposed to IL-1β for 24 h. (A) Lipid ROS and (B) Fe2+ levels in CHON-001 cells were evaluated using ELISAs. (C) Western blotting was used to detect p-AMPK, AMPK, Nrf2 and Gpx4 levels in CHON-001 cells. The level of p-AMPK was normalized to that of AMPK. **P<0.01 vs. control group; ##P<0.01 vs. IL-1β group. AMPK, 5′ AMP-activated protein kinase; Gpx4, glutathione peroxidase 4; Nrf2, nuclear factor erythroid 2-related factor 2; p-, phosphorylated; QCT, quercetin; ROS, reactive oxygen species.

QCT promotes the proliferation of IL-1β-stimulated chondrocytes by inhibiting ferroptosis

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.

Figure 3. QCT promotes proliferation and inhibits apoptosis in IL-1β-treated chondrocytes by inhibiting ferroptosis. (A) CHON-001 cells were treated with erastin (0, 1, 2, 5 or 10 μM) for 24 h at 37°C. Cell viability was detected using a CCK-8 assay. **P<0.01 vs. erastin (0 µM) group. (B) CHON-001 cells were treated with QCT (100 µM) or QCT and erastin (5 µM) for 24 h at 37°C, and then exposed to IL-1β for 24 h at 37°C. Cell viability was detected using a CCK-8 assay. (C) Cell proliferation was evaluated using an EdU staining assay. (D) EdU-positive cells were counted and quantified. Magnification, ×200. Scale bar, 100 µm. (E) Cell apoptosis was assessed using a TUNEL assay. Magnification, ×200. Scale bar, 100 µm. (F) Western blotting was used to detect Bcl-2, cleaved caspase 3 and caspase 3 levels in CHON-001 cells. **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. IL-1β group; ^P<0.05, ^^P<0.01 vs. IL-1β + QCT group. CCK-8, Cell Counting Kit-8; EdU, 5-ethynyl-2′-deoxyuridine; QCT, quercetin.

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.

QCT attenuates extracellular matrix (ECM) degradation and inflammatory responses in 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.

Figure 4. QCT attenuates extracellular matrix degradation and inflammatory responses in IL-1β-treated chondrocytes by inhibiting ferroptosis. (A) CHON-001 cells were treated with QCT (100 µM) or QCT and erastin (5 µM) for 24 h at 37°C, and then exposed to IL-1β for 24 h. Western blotting was used to detect aggrecan, collagen II, MMP13 and ADAMTS5 levels in CHON-001 cells. (B) Relative expression levels of aggrecan, collagen II, MMP13 and ADAMTS5 were semi-quantified. CHON-001 cells were treated with QCT (100 µM) or QCT and erastin (5 µM) for 24 h at 37°C, and then exposed to IL-1β for 24 h at 37°C. (C) IL-6, (D) TNF-α, (E) lipid ROS, (F) GSH, (G) Fe2+ and (H) MDA levels in CHON-001 cells were evaluated using ELISAs. **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. IL-1β group; ^P<0.05, ^^P<0.01 vs. IL-1β + QCT group. ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif 5; collagen II, type II collagen; GSH, glutathione; MDA, malondialdehyde; QCT, quercetin; ROS, reactive oxygen species.

QCT protects against IL-1β-induced ECM degradation in vitro by suppressing ferroptosis via the activation of the AMPK/Nrf2/Gpx4 signaling pathway

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.

Figure 5. QCT protects against IL-1β-induced chondrocyte injury in vitro by suppressing ferroptosis via the activation of AMPK/Nrf2/Gpx4 signaling. CHON-001 cells were treated with QCT (100 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. (A) Western blotting was used to detect p-AMPK, AMPK, Nrf2 and Gpx4 levels in CHON-001 cells. The level of p-AMPK was normalized to that of AMPK. (B) Western blotting was used to detect aggrecan, collagen II, MMP13 and ADAMTS5 levels in CHON-001 cells. **P<0.01 vs. control group; ##P<0.01 vs. IL-1β group; ^P<0.05 ^^P<0.01 vs. IL-1β + QCT group. ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif 5; AMPK, 5′ AMP-activated protein kinase; collagen II, type II collagen; Gpx4, glutathione peroxidase 4; Nrf2, nuclear factor erythroid 2-related factor 2; p-, phosphorylated; QCT, quercetin.

QCT ameliorates OA in mice in vivo via 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.

Figure 6. QCT ameliorates OA in mice in vivo via activation of AMPK/Nrf2/Gpx4 signaling. (A) Pathologic changes of mouse cartilage tissue were evaluated using H&E staining and safranin O/fast green assays. Immunohistochemistry was used to assess aggrecan, collagen II, MMP13 and ADAMTS5 levels in mouse cartilage tissues. Magnification, ×200. Scale bar, 100 µm. (B) Western blotting was used to detect p-AMPK, AMPK, Nrf2 and Gpx4 levels in mouse cartilage tissues. (C) The level of p-AMPK was normalized to that of AMPK and the other proteins were normalized to β-actin. (D) OARSI score in each group. **P<0.01 vs. sham group; #P<0.05, ##P<0.01 vs. OA group. ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif 5; AMPK, 5′ AMP-activated protein kinase; collagen II, type II collagen; Gpx4, glutathione peroxidase 4; Nrf2, nuclear factor erythroid 2-related factor 2; OA, osteoarthritis; OARSI, Osteoarthritis Research Society International; p-, phosphorylated; QCT, quercetin.

Discussion

It has been demonstrated that QCT exerts beneficial effects against multiple diseases, including diabetes mellitus, Alzheimer's and other neurodegenerative diseases and OA (30–32). Hu et al (19) found that QCT prevented the progression of OA in rats by attenuating cartilage degradation and suppressing chondrocyte apoptosis. Feng et al (33) indicated that QCT attenuated damage to rat chondrocytes by suppressing oxidative stress, endoplasmic reticulum stress and cell apoptosis. Qiu et al (20) found that QCT restored mitochondrial dysfunction and inhibited ECM degradation in rats with OA by activating AMPK/sirtuin 1 signaling, thereby attenuating OA progression. These findings demonstrate the critical roles of QCT in the development of OA. The present study revealed a novel mechanism that underlies the chondroprotective effects of QCT in OA. In the present study, QCT markedly attenuated articular cartilage injury in mice with OA. Additionally, 100 µM QCT significantly enhanced the proliferation and reduced the apoptosis of IL-1β-stimulated chondrocytes. Furthermore, to the best of our knowledge, the present study was the first to demonstrate that QCT notably suppressed the ferroptosis of IL-1β-stimulated chondrocytes, as demonstrated by the reduced lipid ROS and Fe2+ levels. These results demonstrated that QCT attenuated the symptoms of OA by suppressing ferroptosis.

Chondrocyte ferroptosis has been found to aggravate the progression of OA (34). Ferroptosis is characterized by iron overload and the accumulation of lipid ROS (35). Furthermore, iron overload and lipid peroxidation are key pathological characteristics of OA (36,37). Iron overload is often observed in the tissues of elderly individuals, including knee joint tissues (38,39). Iron overload has been found to elevate the expression levels of the matrix-degrading enzymes MMP13 and ADAMTS5 in cartilage tissues (40,41). In the present study, lipid ROS production and the Fe2+ levels were markedly elevated in IL-1β-stimulated chondrocytes, suggesting that IL-1β induced ferroptosis in chondrocytes. However, treatment with QCT significantly reversed these effects. These data suggested that QCT possesses anti-ferroptosis properties in OA. Notably, the inhibition of chondrocyte ferroptosis has been found to attenuate the development of OA. For example, Zhou et al (42) found that curcumin exerted protective effects against erastin-induced ferroptosis in chondrocytes through the upregulation of Nrf2. He et al (43) indicated that biochanin A markedly attenuated articular cartilage injury in a mouse model of iron overload-associated OA by suppressing iron levels and activating Nrf2/Gpx4 signaling. Xu et al (44) found that tanshinone IIA was able to inhibit ECM degeneration in chondrocytes by inhibiting ferroptosis. The results of the present study demonstrated that treatment with QCT notably increased the aggrecan and collagen II levels in IL-1β-stimulated chondrocytes; however, the activation of ferroptosis evidently reversed these phenomena. Furthermore, the inhibitory effects of QCT on the apoptosis and inflammatory responses of IL-1β-stimulated chondrocytes were reversed by the activation of ferroptosis. Thereby, the present study indicated that QCT could prevent apoptosis, inflammation and ECM degradation in OA by suppressing ferroptosis.

AMPK can serve as an energy sensor that participates in a number of signal transduction pathways, including ferroptosis (45,46). The activation of AMPK signaling can inhibit ferroptotic cell death (46). Furthermore, the activation of AMPK signaling has been found to inhibit the development of OA in a mouse model of OA (47,48). There is evidence to indicate that AMPK can act as an activator of Nrf2 and Gpx4, two negative regulators of ferroptosis (49–51), suggesting that the activation of AMPK/Nrf2/Gpx4 signaling can suppress ferroptosis (52). Wan et al (53) revealed that baicalein was able to reduce chondrocyte ferroptosis by activating AMPK/Nrf2 signaling. Consistent with these previous findings, the present study demonstrated that treatment with QCT elevated the p-AMPK, Nrf2 and Gpx4 levels in the cartilage tissues of mice with OA. Similarly, QCT significantly elevated the p-AMPK, Nrf2 and Gpx4 levels in IL-1β-stimulated chondrocytes; however, these changes were reversed by treatment with compound C, an AMPK inhibitor. Collectively, QCT suppressed ferroptosis in vitro and in vivo by activating the AMPK/Nrf2/Gpx4 signaling pathway.

However, whether QCT activation of AMPK/Nrf2/Gpx4 signaling was direct or indirect in the current study remains unclear. In addition, only one cell line was used in the present study, and the results should be validated using primary chondrocytes. Furthermore, the treatment duration in the animal experiment was relatively short (4 weeks), and it should be explored whether long-term effects could be observed. Although increasing evidence suggests that QCT protects against OA in vitro and in vivo, clinical testing of QCT is rare (54,55). Thus, it is difficult to suggest the advantages of QCT compared with other established anti-OA drugs such as oral non-steroidal anti-inflammatory drugs at present (6,7).

In conclusion, the findings of the present study demonstrated that QCT prevented the development of OA in vitro and in vivo by suppressing ferroptosis via the activation of AMPK/Nrf2/Gpx4 signaling. The findings illustrated that QCT could suppress chondrocyte ferroptosis via the activation of the AMPK/Nrf2/Gpx4 signaling pathway, providing novel insights into the regulatory mechanisms of QCT in OA. Additionally, these findings further support the potential use of QCT in the treatment of OA.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

SD designed the overall study with contributions from XL. SD designed and carried out experiments, collected data and wrote the draft. XL, GX and LC carried out experiments and analyzed the data. SD, LC and JZ discussed and edited the paper. JZ supervised the study, designed experiments and cowrote the paper. SD and JZ confirm the authenticity of all the raw data. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

The protocols for animal care and use of laboratory animals were approved by the Ethics Committee of Beijing University of Chinese Medicine (approval no. BUCM-2023032701-2103; Beijing, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References