Open Access

Calcium chelator BAPTA‑AM protects against iron overload‑induced chondrocyte mitochondrial dysfunction and cartilage degeneration

  • Authors:
    • Xingzhi Jing
    • Qiang Wang
    • Ting Du
    • Weimin Zhang
    • Xiaoyang Liu
    • Qiang Liu
    • Tao Li
    • Guodong Wang
    • Feifei Chen
    • Xingang Cui
  • View Affiliations

  • Published online on: August 31, 2021     https://doi.org/10.3892/ijmm.2021.5029
  • Article Number: 196
  • Copyright: © Jing et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Osteoarthritis (OA) is a common joint disease that is characterized by cartilage degradation. Iron deposition in the joints is common during the pathogenic progression of OA and recent studies have indicated that iron overload is an important contributor to OA progression. Calcium chelators have been reported to inhibit iron influx via modulating transferrin receptor protein 1 internalization, and they have been identified as a potential approach to the treatment of iron overload‑induced diseases. The aim of the present study was to investigate the effect of calcium chelators on the progression of iron overload‑induced OA. Primary chondrocytes were treated with various concentrations of ferric ammonium citrate (FAC) to mimic iron overload in vitro, followed by co‑treatment with the calcium chelator BAPTA acetoxymethyl ester (BAPTA‑AM). Subsequently, intracellular iron levels, cell viability, reactive oxygen species (ROS) levels, mitochondrial function and morphological changes, as well as MMP levels, were detected using commercial kits. It was demonstrated that FAC treatment significantly promoted chondrocyte apoptosis and the expression of MMPs, and these effects were reversed by co‑treatment with BAPTA‑AM. Moreover, BAPTA‑AM suppressed iron influx into chondrocytes and inhibited iron overload‑induced ROS production and mitochondrial dysfunction. These results indicated that calcium chelators may be of value in the treatment of iron metabolism‑related diseases and iron overload‑induced OA progression.

Introduction

Our previous studies have demonstrated the detrimental effect of iron on osteoarthritis (OA) chondrocytes (1), and that arresting iron influx or decreasing iron concentration using iron chelators may inhibit iron deposition-induced OA progression (2). Iron chelators are drugs that may be beneficial for patients with iron overload (3). However, considering the severe side effects of iron chelators, such as renal damage and neurological dysfunction, new strategies for inhibiting the iron influx into cells must be investigated (4). As the most abundant trace metal in the human body, iron is crucial for the majority of cellular metabolic processes (5). Iron homeostasis is delicately regulated in humans, and iron overload may damage cells by promoting the production of reactive oxygen species (ROS) (6). Iron overload can generate highly toxic hydroxyl radicals and result in ferroptosis (7). As the human body lacks effective ways for excreting excess iron, iron overload is common among elderly individuals and has been reported to be implicated in several diseases or pathological conditions (8). OA is one of the most common complications in a variety of diseases characterized by abnormal intracartilaginous hemorrhage or iron deposition, such as hereditary hemochromatosis, hemophilic arthropathy and rheumatoid arthritis, in which cartilage degeneration and OA progression are common. Moreover, iron deposition has also been observed in traumatic and age-related OA (9). Iron is considered to be the main cause of continual degeneration of the knee joint in traumatic arthritis (10). Iron has also been reported to induce crystal deposition in the supersaturated synovial fluid through the process of nucleation and cartilage damage in OA (11). Moreover, recent findings suggested that cellular iron overload-induced oxidative stress and mitochondrial dysfunction play pivotal roles in OA progression (12,13). Oxidative stress and mitochondrial dysfunction were also reported to play important roles during OA development (14). Iron metabolism has been demonstrated to be an important contributor to normal mitochondrial function. By converting from Fe2+ to Fe3+ and vice versa, iron can participate in the mitochondrial oxidative respiratory chain and exchange a single electron with other substrates. Excess iron can lead to increased ROS production and destroy mitochondrial structure; mitochondrial dysfunction, in turn, can produce excess ROS and result in MMP activation and decreased collagen synthesis (15).

Iron in the diet is taken up by enterocytes through the divalent metal transporter 1 (DMT1) and exported via ferroportin to the general circulation. Then, plasma iron is delivered into tissues or organs via transferrin (TF) receptor 1 (TfR1)-mediated endocytosis after binding to TF in most types of cells (16). As Ca2+ and Fe2+ have equally positive charges, it is possible that Ca2+ may affect Fe2+ influx. It was recently demonstrated that the L-type and T-type calcium channels are involved in iron uptake into the heart under conditions of iron overload (17). Sripetchwandee et al (18) reported that blockade of the mitochondrial calcium uniporter prevents cardiac mitochondrial dysfunction caused by iron overload. Kumfu et al (19) found that combining an iron chelator and a T-type calcium channel blocker exhibited greater cardioprotective efficacy compared with iron chelator alone in an iron overload-induced thalassemia mouse model. Moreover, Zhang et al (20) reported that calcium channel blockers (CCBs) ameliorated iron overload-associated hepatic fibrosis by altering iron transport, and indicated that CCBs are potential therapeutic agents for iron overload-induced diseases. Calcium was reported to modulate receptor-mediated endocytosis and, as TfR1 modulates iron influx through this pathway, it was further demonstrated that calcium could antagonize iron-TF-TfR1 complex internalization and, thus, modulate iron uptake (21).

The present study was undertaken to investigate the effect of the calcium chelator BAPTA acetoxymethyl ester (BAPTA-AM) on iron overload-induced chondrocyte apoptosis and MMP expression. Primary chondrocytes were treated with various concentrations of ferric ammonium citrate (FAC) to mimic iron overload in vitro, and the effects of BAPTA-AM on iron influx into chondrocytes, intracellular iron concentration and ROS production were investigated. Moreover, mitochondrial depolarization and mitochondrial morphology were examined to determine the mitochondrial function using immunofluorescence and flow cytometry assays. The aim was to determine whether calcium chelators may be potential therapeutic agents in treating iron metabolism diseases and iron overload-induced OA progression.

Materials and methods

Cell isolation and culture

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.

Cell Counting Kit-8 (CCK-8) assay

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.

Western blot analysis

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).

Reverse transcription-quantitative (RT-q)PCR analysis

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′.

Immunofluorescence staining

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.).

Measurement of intracellular iron levels

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).

Evaluation of intracellular ROS

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.

Measurement of mitochondrial membrane potential

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).

Mitochondrial-specific fluorescence staining

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.).

Evaluation of apoptosis

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.

Statistical analysis

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.

Results

Iron overload promotes chondrocyte apoptosis and MMP expression

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).

Iron overload impairs mitochondrial dynamics in chondrocytes

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 decreases intracellular iron concentration

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.

Calcium chelator inhibits ROS production and protects against collapse of mitochondrial membrane potential induced by iron overload

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.

Calcium chelator protects chondrocytes against iron overload-induced mitochondrial damage

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.

Calcium chelator protects chondrocytes against iron overload-induced apoptosis and lowers the expression of MMPs

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.

Discussion

As the most abundant trace metal in the body, iron participates in a variety of vital functions, including DNA synthesis, oxygen transport, energy metabolism and cellular respiration. However, excess iron accumulation is common in certain tissues or organs, such as the bone marrow, heart and brain, and may lead to the development of pathological conditions or diseases (12,27). By participating in the mitochondrial respiratory chain, iron leads to the generation of ROS through exchanging single electrons with substrates. This triggers oxidative stress, lipid peroxidation and DNA damage, which may result in genomic instability and DNA repair defects, ultimately compromising cell viability and promoting programmed cell death (28). Our previous studies have demonstrated a link between iron overload and OA. Iron overload is common in OA cartilage, as indicated by the presence of hemosiderin deposition. Abnormal cartilage iron accumulation is not only found in hemophilic arthropathy and hereditary hemochromatosis, but also in traumatic arthritis and rheumatoid arthritis (29,30). A potential cause of increased iron deposition in the afflicted joints of patients with OA may be the microbleeding due to the compromised vasculature, which may result in intra-articular iron loading. Iron chelation is the main treatment of patients with systemic iron overload. Therefore, it is important to investigate new treatment strategies to inhibit iron overload-induced detrimental effects.

Cellular iron homeostasis is delicately regulated by several iron metabolism-related proteins, among which TfR1 plays the most important role in controlling cellular iron levels (31). Chondrocytes take up iron mainly through TfR1 mediated iron-TF-TfR1 complex endocytosis. Ca2+ was reported to regulate iron-TF-tfR1 complex internalization and promote iron influx (21,32). In the present study, the calcium chelator BAPTA-AM was used to test the efficacy of calcium chelator treatment in iron overload-induced chondrocyte damage. BAPTA-AM significantly reduced iron levels in chondrocytes and inhibited iron overload-induced cell apoptosis and the expression of MMPs, thus providing new insights into the treatment of iron overload-induced diseases.

Recently, mitochondria have been shown to play a pivotal role in the progression of OA (33,34). However, the role of mitochondrial dysfunction in iron overload-induced chondrocyte apoptosis and MMP expression remains to be fully elucidated. In the present study, the immunofluorescence results demonstrated that mitochondrial destruction increased along with leakage of cytochrome c from the mitochondria to the cytoplasm and an increase in the expression of BAX. These results indicated that excess iron may lead to disrupted mitochondrial function and morphology. Mitochondrial fission and fusion play important roles in the adaptation of mitochondria to harmful stimuli (35). Mitochondrial fission occurs at the early stages of mitochondrial dysfunction and morphology disruption (36). The results of the present study demonstrated that FAC promoted the expression of mitochondrial fission-related proteins in a time-dependent manner. Mitochondrial morphological assays produced similar results. Following treatment with 100 µm FAC for 24 h, the mitochondrial shape changed from a long wire-like to a short, granular shape, while the detrimental effects of iron overload were reversed and the number of wire-like shaped mitochondria was increased following calcium chelator treatment. These results indicated that calcium chelators may protect chondrocytes against iron overload-induced mitochondrial dysfunction.

Iron is required for mitochondria to synthesize heme and iron-sulfur clusters, which are essential for mitochondrial function (37). However, excess iron accumulation in mitochondria may catalyze the generation of highly reactive OH-radicals by reacting with H2O2 and eventually causing ROS overproduction. Mitochondria are major organelles that generate ROS, but excessive ROS production induces mitochondrial dysfunction. Elevated ROS levels could further destroy mitochondrial structure and function, subsequently activating the mitochondrial apoptotic pathway (38). The results of the present study demonstrated that ROS production was markedly reduced by calcium chelator treatment. The results also revealed that FAC treatment decreased mitochondrial membrane potential and lead to mitochondrial membrane depolarization, whereas these effects were notably attenuated by BAPTA-AM treatment.

Oxidative stress has been identified as an important contributor to OA progression. Increased ROS generation does not only activate the mitochondrial apoptotic pathway, but also promotes the expression of MMPs (39). The present study demonstrated that iron reduced cell viability and increased chondrocyte apoptotic rate, which was also reversed by calcium chelator treatment. Our previous study demonstrated that pro-inflammatory cytokines could disrupt iron homeostasis and promote iron influx via promoting the expression of the iron influx regulators TfR1 and DMT1 (26). In the present study, it was observed that the pro-inflammatory cytokine IL-1β promoted mitochondrial fission protein and MMP expression, which was induced by iron overload. This effect was also shown to be inhibited by calcium chelator treatment.

In conclusion, using an in vitro iron overload model, the present study demonstrated that excess iron is toxic to chondrocytes. Iron overload did not only promote chondrocyte apoptosis, but also promoted MMP expression via catalyzing ROS production and mitochondrial dysfunction. Calcium chelator treatment decreased intracellular iron concentration and iron overload-induced ROS overproduction and mitochondrial dysfunction, thus protecting against iron overload-induced chondrocyte apoptosis and MMP expression. These findings indicate that calcium chelators may hold promise in the prevention or treatment of iron overload-related OA progression. This was a preliminary study designed to demonstrate whether a calcium chelator could protect against iron overload-induced chondrocyte degeneration, as compared to iron chelators. There are numerous calcium chelator agents that have been approved by FDA; in addition, the present study is potentially limited by the lack of in vivo experiments, and further studies are required to fully elucidate the exact mechanism underlying the role of the calcium chelator BAPTA-AM in modulating chondrocyte iron influx.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Authors' contributions

XJ designed the experiments. XJ and QW wrote the manuscript and analyzed the data. TD, WZ, XL, TL, GW and FC carried out the experiments. QL, TD and QW made substantial contributions to analysis and interpretation of data, supervised the present study, searched the literature and revised the manuscript. XC and TD confirm the authenticity of all the raw data. All the authors have read and approved the final manuscript.

Ethics approval and consent to participate

All animal protocols were approved by the Institutional Animal Care of the Shandong Provincial Hospital Affiliated to Shandong First Medical University (no. 2020-526).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

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October-2021
Volume 48 Issue 4

Print ISSN: 1107-3756
Online ISSN:1791-244X

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Copy and paste a formatted citation
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Spandidos Publications style
Jing X, Wang Q, Du T, Zhang W, Liu X, Liu Q, Li T, Wang G, Chen F, Cui X, Cui X, et al: Calcium chelator BAPTA‑AM protects against iron overload‑induced chondrocyte mitochondrial dysfunction and cartilage degeneration. Int J Mol Med 48: 196, 2021
APA
Jing, X., Wang, Q., Du, T., Zhang, W., Liu, X., Liu, Q. ... Cui, X. (2021). Calcium chelator BAPTA‑AM protects against iron overload‑induced chondrocyte mitochondrial dysfunction and cartilage degeneration. International Journal of Molecular Medicine, 48, 196. https://doi.org/10.3892/ijmm.2021.5029
MLA
Jing, X., Wang, Q., Du, T., Zhang, W., Liu, X., Liu, Q., Li, T., Wang, G., Chen, F., Cui, X."Calcium chelator BAPTA‑AM protects against iron overload‑induced chondrocyte mitochondrial dysfunction and cartilage degeneration". International Journal of Molecular Medicine 48.4 (2021): 196.
Chicago
Jing, X., Wang, Q., Du, T., Zhang, W., Liu, X., Liu, Q., Li, T., Wang, G., Chen, F., Cui, X."Calcium chelator BAPTA‑AM protects against iron overload‑induced chondrocyte mitochondrial dysfunction and cartilage degeneration". International Journal of Molecular Medicine 48, no. 4 (2021): 196. https://doi.org/10.3892/ijmm.2021.5029