Human chondrocytes were purchased from Procell Life Science & Technology Co., Ltd. (cat. no. CP-H107). Chondrocytes were cultured in human chondrocyte complete culture medium (cat. no. CM-H107; Procell Life Science & Technology Co., Ltd.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), streptomycin (100 mg/ml) and penicillin (100 U/ml) at 37˚C in a humidified atmosphere with 5% CO₂. Human chondrocytes were treated with ACR (ACR group; cat. no. V900845; Sigma-Aldrich; Merck KGaA) or an equal volume of sterilized water (Con group) for 24 h at 37˚C.

Human chondrocytes were seeded into a 96-well plate at a density of 5.0x10³ cells/well. Following exposure to 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 or 6.4 µg/ml ACR for 24 h at 37˚C, chondrocyte viability was determined using a CCK-8 assay kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. In brief, human chondrocytes were incubated with 10 µl CCK-8 regent at 37^˚C for 1 h. Next, cell viability was calculated from the optical density measured at 450 nm using a microplate reader and PBS without any cells was used as a blank control (Thermo Fisher Scientific, Inc.). The half-maximal inhibitory concentration (IC₅₀) values were calculated via the Reed-Muench method (18).

Human chondrocytes were also seeded in a 96-well plate at a density of 5,000 cells/well. To validate which type of cell death could be induced by ACR, human chondrocytes were preincubated with ferrostatin-1 (Fer-1) (1 µM; a ferroptosis inhibitor), benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone (Z-VAD-FMK) (20 µM; a caspase-3 inhibitor), necrostatin-1 (Nec-1) (20 µM; a necroptosis inhibitor) and 3-MA (20 µM; an autophagy inhibitor) for 1 h. The human chondrocytes were then further treated with 0.35 µg/ml ACR at 37^˚C for 24 h. Chondrocyte viability was determined using a CCK-8 assay kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. The absorbance values were determined at OD450 nm using a microplate reader (Thermo Fisher Scientific, Inc.). Dead cells (%)=100-OD experimental group/OD control group x100.

Human chondrocytes were seeded in a 96-well plate at a density of 5.0x10³ cells/well and treated with 0.35 µg/ml ACR for 24 h at 37˚C. EdU-positive cells were stained with a Cell-Light^™ EdU Apollo In Vitro kit (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. A total of 100 µl of 50 µM EdU medium was added to each well and incubated at 37^˚C for 2 h. The medium was then discarded. Afterwards, the cells were washed twice with PBS for 5 min each time. Next, 100 µl 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well and incubated for 30 min at room temperature, and 100 µl of 1X DAPI (cat. no. LI-9557; OriGene Technologies, Inc.) was added to each well and incubated for 30 min at room temperature, avoiding light and then discarded. Once the staining reaction solution was discarded, 100 µl PBS was added to each well. After washing three times, the slides were observed under a fluorescence microscope (Keyence Corporation).

EdU is a thymidine nucleoside analogue that is incorporated into replicating DNA molecules instead of thymine during cell proliferation and rapidly labels the replication activity of DNA in cells by the specific reaction of EdU with fluorescent dyes (19). EdU-positive cells indicate active replication.

Human chondrocytes were seeded into a six-well plate at a density of 1x10⁶ cells/well and treated with 0.35 µg/ml ACR for 24 h at 37˚C. Subsequently, chondrocytes were stained with Senescence β-Galactosidase Staining kit (cat. no. C0602; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 10 min at room temperature. The cells were washed three times with PBS (5 min/time). After that, the PBS was discarded, and 1 ml β-gal staining solution (cat. no. C0602; Beyotime Institute of Biotechnology) was added to each well and incubated at 37˚C for 1 h followed by three washes with PBS (5 min/time). Aging cells were observed under a light microscope and determined by counting the number of blue-stained cells. The cytoplasm of β-galactosidase-containing cells was blue or dark blue. A total of five fields of view were randomly selected from each section, the number of blue or dark blue cells was counted manually, and the average was taken for statistical analysis.

Human chondrocytes (1x10⁶ cells/well) were seeded on coverslips, fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) at room temperature for 10 min and permeabilized with 1% Triton X-100 (Beyotime Institute of Biotechnology) for 15 min at room temperature. Slides were blocked with 8% BSA (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 2 h and then incubated with γ-H2A histone family member X primary antibody (γ-H2AX; cat. no. C2035S; 1;100; DNA Damage Assay kit; Beyotime Institute of Biotechnology) or anti-AMP-activated protein kinase primary antibody (cat. no. 5832, anti-AMPK; 1:50; CST Biological Reagents Co., Ltd.) at 4˚C overnight. Then, slides were incubated with FITC-conjugated secondary antibody (cat. no. ZF-0314; OriGene Technologies, Inc.) at room temperature for 2 h in the dark and mounted with fluorescent mounting medium with DAPI (cat. no. LI-9557; OriGene Technologies, Inc.). The slides were observed under a light microscope (Keyence Corporation). The density of relative fluorescence intensity was analyzed using ImageJ 1.43b software (National Institutes of Health).

Human chondrocytes (2x10⁶ cells in total for each sample) were lysed with RIPA buffer (cat. no. R0010, Beijing Solarbio Science & Technology Co., Ltd.) containing protease and phosphatase inhibitors (Sigma-Aldrich; Merck KGaA) for 30 min on ice. The lysates were centrifuged at 11,000 x g for 30 min at 4˚C and protein concentration was determined using a BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of proteins (20 µg/lane) were isolated by SDS-PAGE on a 12% gel and transferred onto polyvinylidene fluoride membranes (MilliporeSigma). The membranes were blocked with 6% non-fat milk (MilliporeSigma) at room temperature for 2 h and incubated with primary antibodies as follows: p53 (cat. no. 2527; 1:1,000), cyclin-dependent kinase inhibitor 1 (p21; cat. no. 2947; 1:1,000), cyclin-dependent kinase inhibitor protein (p16; cat. no. 80772; 1:1,000), light chain (LC) 3I/II (cat. no. 3868; 1:1,000), sequestosome 1 (p62; cat. no. 88588; 1:1,000), GPX4 (cat. no. 59735, 1:1,000), SLC7A11 (cat. no. 12691; 1:1,000; SLC7A11); transferrin receptor protein 1 (TfR1; cat. no. 13113; 1:1,000; SLC7A11), FTH1 (cat. no. 4393, 1:1,000), AMPK (cat. no. 5832, 1:1,000), phosphorylated (p)-AMPK (cat. no. 2537, 1:1,000), serine/threonine-protein kinase ULK1 (cat. no. 8054, 1:1,000), p-ULK1 (cat. no. 14202, 1:1,000), p-mTOR (cat. no. 5536, 1:1,000); mTOR (cat. no. 2983, 1:1,000) and GAPDH (cat. no. 5174, 1:1,000) (all CST Biological Reagents Co., Ltd.) at 4˚C overnight. After washing with PBST (0.1% Tween-20 in PBS; Beijing Solarbio Science & Technology Co., Ltd.) three times, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (cat. no. ZB-2301; 1:5,000; OriGene Technologies, Inc.) for 2 h at room temperature. The protein signal was determined using Immobilon Western Chemiluminescent HRP Substrate (cat. no. WBKLS0500; MilliporeSigma). Relative protein expression was normalized to that of GAPDH. The density of each band was analyzed using ImageJ 1.43b software (National Institutes of Health).

Specific small interfering (si)RNA targeting AMPK (siRNA-AMPK; 5'-AAAGTGAAGGTTGGCAAACATGA-3') or negative control (NC; 5'-CCGAUAGGUUUACUGCCAATT-3') was purchased from Guangzhou RiboBio Co., Ltd. and transfected using HiperFect Transfection Reagent (Qiagen GmbH) according to the manufacturer's instructions, and subsequent experiment was performed after 48 h of transfection. In brief, human chondrocytes were seeded into a six-well plate at a density of 1x10⁶ cells/well for 24 h and then 10 µl HiperFect Transfection Reagent was mixed with 100 µl serum-free culture medium. Subsequently, 4 µl siRNA-AMPK (10 µM) or NC (10 µM) was added and it was incubated at room temperature for 10 min. Subsequently, the mixture was added to each well of a six-well plate at a final concentration of 20 nM and incubated at 37˚C for 24 h before cells were collected for further study.

Human chondrocytes were seeded into a 6-well plate at a density of 1x10⁶ cells/well and lipid ROS levels were determined following incubation with 5 µM C11-BODIPY581/591 fluorescent probe (MedChemExpress) for 20 min at 37˚C in the dark. Then, fluorescence was observed under a light microscope (Keyence Corporation).

MMP was determined using the JC-1 MMP Assay kit (cat. no. ab113850; Abcam) according to the manufacturer's instructions. Human chondrocytes were seeded into a six-well plate at a density of 1x10⁶ cells/well and treated with 0.35 µg/ml ACR for 24 h at 37˚C. Subsequently, cells were incubated with 1X JC-1 staining solution for 20 min in the dark at 37˚C and observed under a fluorescence microscope (Keyence Corporation). Normal MMP was shown in red with JC-1 dimers whereas depolarized membrane potential was shown in green with JC-1 monomers.

Human chondrocytes were stained with 1 µM FerroOrange (Dojindo Laboratories, Inc.) in Hanks' balanced salt solution (cat. no. H1045; Beijing Solarbio Science & Technology Co., Ltd.) at 37˚C for 30 min in the dark and immediately observed under a fluorescence microscope (Keyence Corporation).

ROS and MDA content were measured using a ROS Assay kit (cat. no. S0033S; Beyotime Institute of Biotechnology) and Lipid Peroxidation MDA Assay kit (cat. no. S0131S; Beyotime Institute of Biotechnology), respectively, according to the manufacturer's instructions. Human chondrocytes were seeded into a 6-well plate at a density of 1x10⁶ cells/well and were treated with 0.35 µg/ml ACR for 24 h at 37˚C. After that, the cells were collected for further determination of intracellular ROS and MDA contents.

For ROS quantification, human chondrocytes were incubated with 10 µM DCFH-DA for 20 min at 37^˚C in the dark and immediately observed under a fluorescence microscope (Keyence Corporation). The density of relative fluorescence intensity was analyzed using ImageJ 1.43b software (National Institutes of Health).

For MDA quantification, 0.1 ml of sample or PBS (blank control) was added to the centrifuge tube, followed by 0.2 ml of MDA assay working solution. The mixture was heated at 100˚C for 15 min. The water bath was cooled to room temperature and centrifuged at 1,000 x g for 10 min at room temperature. After that, 200 µl of supernatant was added to a 96-well plate, followed by determination of absorbance at 532 nm using a microplate reader (Thermo Fisher Scientific, Inc.). The relative MDA content in the sample solution was calculated from the standard curve.

Human chondrocytes were fixed in 2.5% glutaraldehyde solution at room temperature for 2 h, post-fixed in 1% aqueous osmium tetroxide at room temperature for 2 h and dehydrated in an ethanol gradient (50, 70 and 90%) at room temperature for 15 min separately and 100% acetone at room temperature for 15 min. The samples were prepared by gradient infiltration of anhydrous acetone and epoxy resin overnight, embedded in resin and polymerized at 60˚C for 48 h. The samples were cut into 50 nm ultrathin sections using an ultramicrotome (Leica Microsystems GmbH), stained with 3% uranium acetate-lead citrate (Beijing Zhongxing Bairui Technology Co., Ltd.) at room temperature for 15 min and examined by 120 keV transmission electron microscopy (Tecnai G2 20 TWIN; Thermo Fisher Scientific, Inc.).

Human chondrocytes were seeded into a six-well plate at a density of 1x10⁶ cells/well and treated with 0.35 µg/ml ACR for 24 h at 37˚C. Subsequently, the cells were collected for cell death and cell cycle analysis using an Annexin V-PE/7-AAD Apoptosis Detection (cat. no. 40310ES50; Shanghai Yeasen Biotechnology Co., Ltd.) and Cell Cycle Assay Kit (Red Fluorescence; E-CK-A351; Elabscience Biotechnology Co., Ltd.) according to the manufacturer's instructions.

The cells were centrifuged at 300 x g at 4˚C for 5 min to collect the cells. The cells were washed twice with PBS precooled at 4˚C and centrifuged at 300 x g at 4˚C for 5 min. Subsequently, 250 µl 1X binding buffer was added to resuspend the cells and the concentration was adjusted to 1x10⁶ cells/ml. Then, 100 µl cell suspension was transferred into a 5 ml flow tube and incubated with 5 µl annexin V/PE and 10 µl 7-AAD in the dark at room temperature for 15 min. The cells were tested within 1 h using a flow cytometer (BD LSRFortessa^™; BD Biosciences). The data was analyzed using FlowJo 10.8.1 software (FlowJo LLC).

Approximately 5x10⁵ human chondrocytes were collected. The cells were centrifuged at 300 x g for 5 min and the supernatant was discarded. Next, 0.3 ml PBS was added to resuspend the cells. Afterwards, 1.2 ml of-20˚C anhydrous ethanol was added, mixed thoroughly and the samples were placed at -20˚C for 1 h. Next, the samples were centrifuged at 300 x g for 5 min, the supernatant was discarded and 1 ml PBS was added to resuspend the cells for 15 min at room temperature. Afterwards, the mixture was centrifuged at 300 x g for 5 min, the supernatant was discarded and 100 µl of RNase A was added. Finally, 400 µl PI Reagent (50 µg/ml) was added and mixed through thoroughly, and the cells were incubated for 30 min at 4˚C under low light. The red fluorescence at 488 nm was recorded immediately on a BD LSRFortessa flow cytometer (BD Biosciences). The data was analyzed using FlowJo 10.8.1 software (FlowJo LLC).

Human chondrocytes were seeded into a six-well plate at a density of 1x10⁶ cells/well and were transfected with GFP-mCherry-LC3 adenovirus at 100 multiplicity of infection (MOI) for 24 h at 37˚C. Following treatment with 0.35 µg/ml ACR for 24 h at 37˚C, cells were fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) at room temperature for 10 min and immediately observed under a microscope (Keyence Corporation). GFP degraded in the acidic environment. After the red-green fluorescence was merged, the yellow spots that appeared after the merge were only autophagosomes. The red spots indicated autolysosomes. If the autophagic flow was unblocked, the red fluorescence was more than the yellow fluorescence. If the autophagic flow was blocked, it was mainly yellow fluorescence. Autophagic flux was determined by counting the cells with GFP+/mCherry+ (yellow) or GFP-/mCherry+ (red) puncta manually using a fluorescence microscope (Keyence Corporation).

The data are expressed as the mean ± standard deviation of three independent experimental repeats. Unpaired Student's t test was used for comparisons between two groups. Comparisons between three or more groups were performed via one-way ANOVA followed by Bonferroni's post hoc test. All analyses were performed using GraphPad Prism 8 statistical software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

To analyze the effects of ACR on human chondrocyte viability, cells were treated with ACR at 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 µg/ml ACR for 24 h. Compared with the control, the cell survival rate of human chondrocytes decreased significantly in a dose-dependent manner (P<0.05; Fig. 1). The IC₅₀ of ACR in human chondrocytes was 0.35 µg/ml.

To determine the effect of ACR on cell proliferation, human chondrocytes were incubated with 0.35 µg/ml ACR at 37˚C for 24 h and EdU staining was performed. EdU-positive cells/field were significantly decreased in chondrocytes treated with ACR compared with Con chondrocytes (P<0.001; Fig. 2A). In addition, cell cycle arrest was observed in human chondrocytes treated with ACR, as evidenced by elevation in the G₀-G₁ phase cell population and downregulation of the S phase cell population compared with Con (P<0.001; Fig. 2B).

Compared with Con, 0.35 µg/ml ACR significantly increased the number of SA-β-gal-positive cells after 24 h (P<0.001; Fig. 3A). In addition, relative fluorescence of γ-H2AX (a biomarker of DNA damage) was increased in human chondrocytes treated with ACR compared with Con (Fig. 3B). Moreover, flow cytometry analysis showed that ACR increased the number of dead cells to ~28% compared with that of Con (11%; P<0.001; Fig. 3C). Western blotting showed that the expression levels of cell arrest-associated proteins, including p53, p21 and p16, were significantly enhanced in human chondrocytes treated with ACR compared with those in Con chondrocytes (P<0.05; Fig. 3D). These observations indicated that ACR contributed to the senescence of human chondrocytes.

It was investigated whether 0.35 µg/ml ACR decreased human chondrocyte cell death in a time-dependent manner. CCK-8 assay showed that cell viability was significantly decreased in human chondrocytes treated with 0.35 µg/ml ACR at 24, 48 and 72 h (P<0.01; Fig. 4A). To validate which type of cell death was induced by ACR, human chondrocytes were preincubated with Fer-1, Z-VAD-FMK, Nec-1 and 3-MA for 1 h. Human chondrocytes were further treated with 0.35 µg/ml ACR at 37˚C for 24 h. Fer-1 and 3-MA significantly reversed ACR-induced cell death (P<0.01; Fig. 4B). These findings demonstrated that ACR promoted chondrocyte cell death by inducing ferroptosis and autophagy.

Ultrastructural analysis indicated a higher level of outer mitochondrial membrane rupture in chondrocytes treated with 0.35 µg/ml ACR at 37˚C for 24 h compared with that in Con chondrocytes (Fig. 5A). Meanwhile, the mitochondrial ridge was decreased or disappeared in human chondrocytes treated with ACR (Fig. 5A). By contrast, preincubation with 3-MA at 37˚C for 1 h reversed these effects (Fig. 5A). Autophagic flux was activated in human chondrocytes treated with ACR, as evidenced by the elevation in red puncta, whereas 3-MA preincubation significantly decreased the red puncta in human chondrocytes (P<0.001; Fig. 5B). Western blotting of autophagic flux showed that the LC3II/LC3I ratio was increased, whereas expression of p62 was decreased in human chondrocytes treated with ACR (P<0.01; Fig. 5C). By contrast, 3-MA preincubation decreased the LC3II/LC3I ratio and increased the protein expression of p62 compared with the ACR treatment alone (P<0.01; Fig. 5C). These results indicate that ACR activated autophagic flux in human chondrocytes.

Changes in mitochondrial function are a hallmark of ferroptosis (20). Hence, cellular MMP was evaluated in human chondrocytes treated with 0.35 µg/ml ACR at 37˚C for 24 h. When human chondrocytes were treated with ACR, poly JC-1 molecules were dissociated into mono JC-1 molecules, suggesting a reduction or loss of MMP (Fig. 6A). In the Fer-1 group, poly JC-1 was enhanced whereas mono JC-1 was decreased in human chondrocytes (Fig. 6A). FerroOrange staining showed an increase in red fluorescence density after ACR treatment whereas Fer-1 preincubation decreased the red fluorescence density in chondrocytes (Fig. 6B). A C11 BODIPY fluorescent probe was used to detect intracellular lipid ROS content. The data showed that ACR increased lipid-ROS accumulation in chondrocytes whereas preincubation with Fer-1 decreased intracellular lipid-ROS levels (Fig. 6C). Intracellular ROS and MDA levels were investigated. Fer-1 attenuated ACR-induced upregulation of ROS and MDA contents (P<0.01; Fig. 6D). Western blotting of ferroptosis-associated proteins demonstrated that ACR reduced expression of GPX4, SLC7A11, TfR1 and FTH1 in chondrocytes whereas Fer-1 abolished these effects (P<0.05; Fig. 6E). These observations indicated that ACR induced chondrocyte ferroptosis.

Some studies have demonstrated an important role of AMPK/ULK1 in autophagy and ferroptosis (21,22); thus, the present study investigated the effects of ACR on AMPK signaling. ACR increased the phosphorylation of AMPK and ULK1 but suppressed activation of mTOR in human chondrocytes at 24 and 48 h (P<0.05: Fig. 7A). To elucidate if ACR induced autophagy and ferroptosis by activating AMPK signaling, a specific siRNA targeting AMPK was used. Western blotting showed that transfection with siRNA AMPK significantly reduced expression of AMPK compared with NC (Fig. 7B). IF staining showed that siRNA AMPK notably suppressed the fluorescence density of AMPK in human chondrocytes even in the presence of ACR (Fig. 7C). Furthermore, ACR-induced mitochondrial membrane rupture was reversed by silencing AMPK in human chondrocytes (Fig. 7D). Moreover, the upregulated levels of lipid ROS and Fe²⁺ were blocked by transfection with siRNA-AMPK (Fig. 7E and F). These data showed that ACR-induced ferroptosis and autophagy were achieved by activating AMPK/ULK1 signaling in human chondrocytes.