A total of 30 male C57/BL6 mice (aged 5 days and weighing <5 g) were used in the present study. The mice were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (35 mg/kg body weight) and sacrificed via cervical dislocation. All animal protocols were approved by the Institutional Animal Care of the Shandong Provincial Hospital Affiliated to Shandong First Medical University (no. 2020-526). Briefly, cartilage was obtained from bilateral knee joints and minced into pieces. The cartilage pieces were then washed with cold PBS, followed by the addition of 0.25% trypsin-EDTA for 30 min at 37°C. After washing with PBS, samples were digested with 0.25% collagenase solution at 37°C for 7 h. After collection and centrifugation at 100 × g for 10 min at 37°C, chondrocytes were resuspended in DMEM/F12 (HyClone; Cytiva) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and cultured in a humidified atmosphere of 5% CO₂ at 37°C. Chondrocytes at passages 1 or 2 were used in the following experiments.

CCK-8 was obtained from Wuhan Boster Biological Technology, Ltd. and used to detect cell viability according to the manufacturer's instructions. Briefly, cells were collected and resuspended in DMEM/F12 supplemented with 10% FBS at a density of 5×10⁴ cells/ml, and 200 µl medium containing 1×10⁴ cells were added into each well of a 96-well plate. Increasing concentrations of FAC (1-200 µm) were then added and cultured for different time periods (0-5 days). The culture medium was changed every other day. CCK-8 solution (10 µl) was added into each well on days 0, 1, 3 and 5 and incubated at 37°C in the dark for 1 h. The absorbance at 450 nm was recorded using spectral IMAX190 absorbance microplate reader (Molecular Devices, LLC). Optical density values at 450 nm were recorded to evaluate cell viability.

Chondrocytes were seeded in 6-well plates at a density of 1×10⁵ cells per well. After being subjected to various treatments, chondrocytes were collected and lysed with 100 µl RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.) on ice for 1 h. The lysates were then collected and centrifuged at 300 × g for 20 min at 4°C, the supernatant was collected and the protein concentration was detected using the BCA assay method. Equal amounts of protein (25 µg/lane) were electrophoresed using 12% SDS-PAGE and transferred to PVDF membranes (MilliporeSigma). The PVDF membranes were then blocked with 5% skimmed milk for 1 h at room temperature and incubated with targeted primary antibodies against MMP3 (1:1,000; cat. no. 17873-1-AP; ProteinTech Group, Inc.), MMP13 (1:1,000; cat. no. ab39012; Abcam), mitochondrial fission 1 protein (FIS1; 1:1,000; cat. no. 10956-1-AP; ProteinTech Group, Inc.), dynamin-related protein 1 (DRP1; 1:1,000; cat. no. 8570; Cell Signaling Technology, Inc.), mitochondrial fission factor (MFF; 1:1,000; cat. no. 84580; Cell Signaling Technology, Inc.), BAX (1:1,000; cat. no. 50599-2-Ig; ProteinTech Group, Inc.), cytochrome c (1:1,000; cat. no. ab13575; Abcam) and β-actin (1:500; cat. no. BM0627; Wuhan Boster Biological Technology, Ltd.) at 4°C overnight. After washing with Tris-buffered saline with Tween-20 [50 mM Tris (pH 7.6), 150 mm Nacl and 0.1% Tween-20] three times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. BA1055; Wuhan Boster Biological Technology, Ltd.) at room temperature for 1 h. The protein bands were visualized with enhanced chemiluminescence reagent (Wuhan Boster Biological Technology, Ltd.) and images were captured. The density of each band was quantified by ImageJ software (version 1.8.0; National Institutes of Health).

Chondrocytes were seeded in 12-well plates at a density of 5×10⁴ cells per well and treated with 100 µm FAC (MilliporeSigma) for 24 h. Chondrocytes were then treated with TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) and total RNA was extracted using the Toyobo Total RNA Extraction kit (Toyobo Life Science) following the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from RNA using the First Strand cDNA Synthesis kit (Toyobo Life Science) and amplified using SYBR Green Real-time PCR Master Mix (Toyobo Life Science) under the following cycling conditions: 30 sec of polymerase activation at 95°C, followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec. Relative expression levels were calculated using the 2^−ΔΔcq method (22). GAPDH was used as an internal control. Each cDNA sample was assayed in triplicate. The sequences of the primers for the genes of interest are listed as follows: A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5): Forward, 5′-TAT GAC AAG TGC GGA GTA TG-3′ and reverse, 5′-TTC AGG GCT AAA TAG GCA GT-3′; MMP3: Forward, 5′-ATG CCC ACT TTG ATG ATG ATG AAC-3′ and reverse, 5′-CCA CGC CTG AAG GAA GAG ATG-3′; MMP13: Forward, 5′-GCT GGA CTC CCT GTT G-3′ and reverse, 5′-TCG GAG CCT GTC AAC T-3′; and GAPDH: Forward, 5′-CTC CCA CTC TTC CAC CTT CG-3′ and reverse, 5′-TTG CTG TAG CCG TAT TCA TT-3′.

Chondrocytes at passage 1 or 2 were seeded in 24-well plates at a density of 1×10⁴ cells per well. Following treatment with 100 µm FAC (MilliporeSigma) for 24 h, cells were washed with serum-free DMEM/F12 (HyClone; Cytiva) and incubated with diluted Mito-Tracker Red solution (1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min at 37°C in the dark. Cells were then washed with PBS three times and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then washed with PBS three times and treated with 0.1% Triton X-100 for 15 min. After blocking with 10% FBS (Wuhan Boster Biological Technology, Ltd.) for 30 min at room temperature, the cells were then incubated with mixed rabbit polyclonal BAX primary antibody (1:1,000; cat. no. 50599-2-Ig; ProteinTech Group, Inc.) and mouse monoclonal cytochrome c antibody (1:1,000; cat. no. ab13575; Abcam) at 4°C overnight. Following rinsing with PBS three times, mixed goat anti-rabbit FITC-conjugated secondary antibody (1:200; cat. no. BA1105; Wuhan Boster Biological Technology, Ltd.) and goat anti-mouse Dylight 405 secondary antibody (1:400; Beyotime Institute of Biotechnology) was added and incubated for 1 h at room temperature in the dark. Finally, cells were washed with PBS and analyzed at a magnification of ×200 under a fluorescence microscope (EVOS™ FL Auto; Thermo Fisher Scientific, Inc.).

Briefly, primary chondrocytes were treated with 100 µm FAC, with or without 10 µm BAPTA-AM (Selleck Chemicals), for 6 h. Then, cells were loaded with 0.5 µmol/l calcein-AM for 30 min at 37°C. Chondrocytes were then washed with PBS three times. Free calcein (non-metal-bound) was measured for fluorescence (excitation: 488 nm; emission: 517 nm) using a fluorescence microscope (EVOS™ FL Auto; Thermo Fisher Scientific, Inc.) at a magnification of ×200. To quantify the fluorescence values, four separate fields monitored were randomly selected and the mean fluorescence signal was processed with Image Pro Plus software (version 6.0; National Institutes of Health).

Chondrocytes were seeded in 12-well plates at a density of 1×10⁴ cells per well and treated with 100 µm FAC, with or without 10 µm BAPTA-AM, for 24 h. After treatment, the intracellular ROS level was determined using the Reactive Oxygen Species Assay kit (cat. no. S0033; Beyotime Institute of Biotechnology) according to manufacturer's instructions. Briefly, chondrocytes were washed with serum-free DMEM/F12 (HyClone; Cytiva) three times, then 10 µm dichloro-dihydro-fluorescein diacetate (DCFH-DA) was added to the culture medium and incubated in the dark for 20 min. Chondrocytes were then washed with DMEM/F12 (HyClone; Cytiva) and examined at a magnification of ×100 under a fluorescence microscope (EVOS™ FL Auto; Thermo Fisher Scientific, Inc.) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Cells were seeded in 12-well plates at a density of 1×10⁴ per well and treated with 100 µm FAC, with or without 10 µm BAPTA-AM, for 24 h. After washing with DMEM/F12 three times, chondrocytes were incubated with JC-1 staining solution (Beyotime Institute of Biotechnology) for 30 min at room temperature. After incubation, the culture medium was changed and the chondrocytes were washed with washing buffer. The fluorescence was examined using a fluorescence microscope. Aggregated JC-1 in mitochondria with high mitochondrial membrane potential levels produced red fluorescence, whereas when mitochondrial membrane potential decreased, JC-1 was found in its monomer form and produced green fluorescence.

To quantify mitochondrial membrane potential in chondrocytes, cells were incubated with JC-1 staining solution as described above. After incubation, cells were re-suspended in 500 ml DMEM/F12 and the fluorescence intensity was recorded with a FACSCalibur flow cytometer (BD Biosciences).

Mito-Tracker Green (Beyotime Institute of Biotechnology) was used to evaluate the morphological changes of mitochondria. Mito-Tracker Green is an mitochondrial membrane potential-independent mitochondrial staining reagent. Briefly, chondrocytes were treated with FAC (100 µm), with or without 10 µm BAPTA-AM, for 24 h; subsequently, cells were washed twice with FBS-free DMEM/F12 and incubated with diluted Mito-Tracker Green solution (1:1,000) for 30 min at 37°C in the dark. The shapes of the mitochondria were imaged at a magnification of ×400 under a fluorescence microscope (EVOS™ FL Auto; Thermo Fisher Scientific, Inc.).

An Annexin V-FITC/PI Apoptosis Detection kit (cat. no. C1063; Beyotime Institute of Biotechnology) was used to detect the cell apoptosis rate according to the manufacturer's instructions. Chondrocytes were seeded in 6-well plates at a density of 1×10⁵ per well and treated with 100 µm FAC, with or without 10 µm BAPTA-AM, for 24 h. After the aforementioned treatment, cells were washed with PBS and then stained with Annexin V-FITC/PI for 20 min in the dark. A FACSCalibur flow cytometer (BD Biosciences) was used to analyze the results. Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺ cells were considered as early and late apoptotic cells, respectively.

The results are presented as mean ± SD. All experiments were repeated three times independently. Unpaired Student's t-test was used to evaluate differences between two groups. Differences among multiple groups were determined using one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate statistically significant differences.

First, primary chondrocytes were treated with various concentrations of FAC to mimic iron overload in vitro, and then CCK-8 assay was performed to verify the cytotoxic effects of iron on chondrocytes. As shown in Fig. 1A, FAC decreased chondrocyte viability in a dose-dependent manner. FAC at a concentration of 1 µm slightly enhanced chondrocyte proliferation, while FAC at concentrations >50 µm significantly decreased chondrocyte viability. Western blotting demonstrated that FAC promoted the expression of the cell apoptosis marker cleaved caspase-3 in a dose-dependent manner, and FAC at 100 µm was associated with significantly elevated cleaved caspase-3 expression. Next, the expression of the OA-related markers, MMP3 and MMP13, was examined using western blot and RT-qPCR analyses. As shown in Fig. 1C and D, FAC promoted MMP3 and MMP13 protein expression in a dose-dependent manner, with the protein levels peaking at 100 µm FAC. Similar results were obtained using RT-qPCR analysis, with 100 µm FAC significantly promoting ADAMTS5, MMP3 and MMP13 mRNA expression (Fig. 1B).

Mitochondrial function plays an important role in OA progression (15). To further investigate the mechanisms underlying iron overload-induced accelerated progression of OA, chondrocytes at passage 1 or 2 were stimulated with FAC to mimic iron overload in vitro. Immunofluorescence staining was then performed to examine the localization of cytochrome c and BAX proteins. It was observed that FAC promoted mitochondrial BAX expression and translocation of cytochrome c from the mitochondria to the cytoplasm, indicating that mitochondrial structure was destroyed after FAC treatment (Fig. 2A). Mitochondrial fission and fusion play important roles in maintaining the function and morphology of mitochondria (23). The results of the present study demonstrated that the expression of mitochondrial fission proteins (DRP1, MFF and FIS1) was upregulated following treatment with 100 µm FAC in a time-dependent manner (Fig. 2B and C). In addition, the protein expression of BAX and cytochrome c was upregulated following treatment with 100 µm FAC. These results indicated that FAC promoted mitochondrial fission and caused destruction of mitochondrial morphology and leakage of cytochrome c, a classic caspase-dependent apoptosis inducer in mitochondria, further promoting mitochondria-dependent apoptosis.

Calcium chelator was reported to inhibit iron influx (21). To test this hypothesis, chondrocytes were treated with 100 µm FAC, with or without BAPTA-AM. The fluorescence dye calcein-AM was used to assess the ferrous iron uptake and outflow in chondrocytes. As shown in Fig. 3, the fluorescence intensity significantly decreased in the FAC treatment group, indicating elevated iron content in chondrocytes. By contrast, the fluorescence intensity significantly increased in the BAPTA-AM co-treatment group, indicating that the BAPTA-AM co-treatment decreased iron concentration in chondrocytes.

It was reported that excess iron in mitochondria could generate ROS via the Fenton reaction (5). As shown in Fig. 4A, DCFH-DA staining demonstrated that FAC promoted ROS production and this effect was inhibited by the calcium chelator BAPTA-AM. Iron overload impairs normal mitochondrial morphology and function, whereas mitochondrial dysfunction, in turn, affects cellular iron influx and increases ROS production (24). Next, JC-1 staining was utilized to investigate whether calcium chelator could inhibit the collapse of the mitochondrial membrane potential. As shown in Fig. 4B-D, the ratio of green JC-1 monomers to red JC-1 aggregates was increased in the FAC treatment group, while BAPTA-AM treatment suppressed the changes in mitochondrial membrane potential induced by iron. These results indicated that calcium chelators may protect against iron overload-induced ROS production and mitochondrial dysfunction.

Mitochondria in healthy chondrocytes display a wire-like shape. As shown in Fig. 5A, FAC treatment (100 µm) significantly increased the number of granulated mitochondria in chondrocytes, while BAPTA-AM restored the normal mitochondrial shape in chondrocytes. Mitochondrial fission and fusion are known to regulate the function and morphology of mitochondria. A number of studies have shown that mitochondrial fission is an early event during the process of cell death (25). As shown in Fig. 5A, the expression of the mitochondrial fission proteins, DRP1, MFF and FIS1, was found to be upregulated by treatment with 100 µm FAC, and it was decreased by treatment with 10 µm BAPTA-AM. Moreover, it was observed that the pro-inflammatory cytokine IL-1β increased FAC-induced mitochondrial fission protein expression, whereas this effect was also reversed by BAPTA-AM. In conclusion, the aforementioned results indicated that iron overload significantly promoted mitochondrial destruction and dysfunction, and calcium chelator treatment could effectively preserve the normal mitochondrial morphology and function.

Our previous findings demonstrated the association between iron overload and OA progression (26). In the present study, it was investigated whether calcium chelator treatment could protect chondrocytes against iron overload-induced apoptosis and suppress the expression of MMPs. As shown in Fig. 6A and B, FAC treatment significantly promoted chondrocyte apoptosis, whereas BAPTA-AM decreased the chondrocyte apoptosis rate induced by 100 µm FAC. A hallmark of OA chondrocytes is the increased production of MMPs. As shown in Fig. 6C and D, BAPTA-AM treatment decreased chondrocyte MMP3 and MMP13 expression, which was induced by 100 µm FAC. Moreover, it was observed that FAC and IL-1β co-treatment significantly promoted MMP3 expression compared with the FAC group, and this effect was also inhibited by BAPTA-AM. In summary, these results indicated that calcium chelator treatment may protect chondrocytes against iron overload-induced apoptosis and lower OA-related marker expression.