Protective Effect of Ergothioneine against Oxidative Stress-Induced Chondrocyte Death

Reactive oxygen species (ROS) induce oxidative stress in cells and are associated with various diseases, including autoimmune diseases. Ergothioneine (EGT) is a natural amino acid derivative derived from the ergot fungus and has been reported to exhibit an effective antioxidant function in many models of oxidative stress-related diseases. Recently, mutations in OCTN1, a membrane transporter of EGT, have been reported to be associated with rheumatoid arthritis. Therefore, we investigated the chondrocyte-protective function of EGT using a model of oxidative stress-induced injury of chondrocytes by hydrogen peroxide (H2O2). Human chondrocytes were subjected to oxidative stress induced by H2O2 treatment, and cell viability, the activity of lactate dehydrogenase (LDH) released into the medium, dead cell ratio, intracellular ROS production, and mitochondrial morphology were assessed. EGT improved chondrocyte viability and LDH activity in the medium and strongly suppressed the dead cell ratio. EGT also exerted protective effects on intracellular ROS production and mitochondrial morphology. These results provide evidence to support the protective effects of EGT on chondrocytes induced by oxidative stress.


Introduction
Reactive oxygen species (ROS) are a group of highly oxidatively active molecules that are produced during oxidative phosphorylation in mitochondria, and ROS are associated with the various physiological functions of cells [1].ROS can be produced by many factors such as ultraviolet (UV) light, radiation, smoking, aging, food, and inflammation.Cells have ROS scavenging systems such as superoxide dismutase, catalase, and glutathione peroxidase, which maintain the homeostasis of intracellular ROS concentrations.However, when the production of ROS exceeds the antioxidant capacity of the cell and the homeostasis of intracellular ROS concentration is disrupted, intracellular lipids, enzymes, and nucleic acids are peroxidized, disrupting the maintenance of vital functions [2].This condition is called oxidative stress, and oxidative stress caused by the excessive accumulation of ROS has been reported to be involved in many diseases, including cancer [3], hypertension [4], atherosclerosis [5], neurodegenerative diseases [6], diabetes [7], and autoimmune diseases [8].
Recently, oxidative stress has been reported to be involved in rheumatoid arthritis (RA) and osteoarthritis (OA) [9,10].These diseases are caused by the chronic inflammation of the synovial membrane, which leads to the degeneration of bone, cartilage, and other joint tissues [11].The excessive accumulation of ROS in chondrocytes induces mitochondrial dysfunction associated with membrane lipid peroxidation, leading to chondrocyte death [12].Since articular cartilage has low proliferative capacity and lacks the ability to self-repair, chondrocyte death is considered an important factor in the pathological progression of joint-destructive diseases [13].Therefore, the reduction of excess intracellular ROS accumulation and the protection of cellular physiological functions by supplementation with antioxidants could be an effective therapeutic strategy against oxidative stress-related diseases.The standard treatment for OA and RA is the oral administration of anti-rheumatic drugs, non-steroidal anti-inflammatory drugs, and steroids, or the intra-articular injection of hyaluronic acids; however, these treatments have certain side effects [14,15].Therefore, the development of a safe supplement therapy that complements the patient's antioxidant capacity through the intake of dietary antioxidants is necessary [16].
Ergothioneine (EGT) is a unique amino acid derivative derived from the ergot fungus and has a strong antioxidant capacity and high chemical stability [17].The potential efficacy of EGT as a therapeutic agent is becoming clear, with many reports that EGT is effective in neurodegenerative diseases such as Parkinson's disease caused by neuronal oxidative stress [18,19].Recently, OCTN1, a plasma membrane transporter of EGT, has been reported to be associated with RA [20].These reports suggest that investigating the effects of EGT on oxidative stress in chondrocytes is important for establishing new therapeutic strategies for OA and RA.Therefore, this study aimed to examine the potential effectiveness of EGT for degenerative diseases of articular cartilage by evaluating the protective effect of EGT against the cytotoxicity induced by oxidative stress.
Cell cytotoxicity was examined using a Cytotoxicity LDH Assay Kit-WST (Dojindo).Chondrocytes were seeded with 10,000 cells in a 24-well plate, and after 24 h incubation, the cells were treated with H 2 O 2 (1 mM) or with the addition of EGT (0.1, 0.25, 0.5, and 1 mM).After an additional 24 h incubation, the activity of lactate dehydrogenase (LDH) in the supernatant was measured following the manufacturer's instructions.Cell morphology and confluency were analyzed by using CELLCYTE X (CYTENA, Freiburg, Germany).

Live/Dead-Cell Assay
To detect H 2 O 2 -induced chondrocyte death, Diyo-1 (AAT Bioquest, Sunnyvale, CA, USA) and SYTO-59 (Invitrogen, Carlsbad, CA, USA) double-staining was performed.Diyo-1 is a membrane-impermeable nuclear-staining dye that penetrates dead cell membranes and emits green fluorescence.By contrast, SYTO-59 is a membrane-permeable nuclearstaining dye that can penetrate the cell membrane of both dead and living cells and emits red fluorescence.Therefore, the ratio of dead cells to total cells can be calculated by dividing the number of green fluorescence-stained cells by the number of red fluorescencestained cells.These fluorescent dyes were dissolved at a concentration of 1 µM each in chondrocyte basal medium containing FBS and supplements.Chondrocytes were seeded with 10,000 cells in a 24-well plate, and after 24 h incubation, the cells were treated with H 2 O 2 (1 mM) or with the addition of EGT (1 mM) at 37 • C for 3, 6, and 12 h.The control group was treated with PBS.Following treatments, fluorescence images were obtained by using CELLCYTE X (CYTENA).

ROS Assay
Intracellular ROS production was evaluated using dichloro-dihydro-fluorescein diacetate (DCFH-DA) dye (ROS Assay Kit Photo-oxidation Resistant DCFH-DA, Dojindo).The chondrocytes were treated with H 2 O 2 (1 mM) and EGT (1 mM) at 37 • C for 3, 6, and 12 h in the presence of DCFH-DA dye according to the manufacturer's instructions.The control group was treated with PBS.Live-cell fluorescence images were obtained and analyzed by using CELLCYTE X (CYTENA).

Mitochondrial Staining
Mitochondrial visualization was performed using MitoView Green (Biotium, San Francisco, CA, USA).This probe is independent of mitochondrial membrane potential and fluoresces when fractionated into the mitochondrial membrane.Therefore, mitochondrial mass can be evaluated.Chondrocytes were treated with H 2 O 2 (1 mM) and EGT (1 mM) at 37 • C for 2 h in the presence of MitoView Green (200 nM), and fluorescence images were obtained using CELLCYTE X (CYTENA).

Statistical Analysis
The results are shown as mean ± standard deviation (SD).Statistical analysis was performed using JMP Pro version 17 software (JMP Statistical Discovery LlC, Cary, NC, USA) and statistically significant differences between the groups were determined using one-way analysis of variance followed by Dunnett's test or Tukey's test.The statistical significance was set at p < 0.05.

Effect of EGT and H 2 O 2 on Collagen Gene Expression and Cell Viability
An increase in the gene expression of COL2A1 in chondrocytes treated with EGT was observed compared to untreated chondrocytes (Figure 1B).When treating the chondrocytes with 0.1, 0.25, 0.5, and 1 mM of EGT for 24 h, no significant difference was observed (Figure 1C).On the other hand, on application of 0.1, 0.25, 0.5, and 1 mM of H 2 O 2 to chondrocytes for 24 h, a significant decrease in cell viability of approximately 20% was observed at 0.5 mM H 2 O 2 , and a significant decrease of approximately 95% was observed at 1 mM (Figure 1D).cytes with 0.1, 0.25, 0.5, and 1 mM of EGT for 24 h, no significant difference was observed (Figure 1C).On the other hand, on application of 0.1, 0.25, 0.5, and 1 mM of H2O2 to chondrocytes for 24 h, a significant decrease in cell viability of approximately 20% was observed at 0.5 mM H2O2, and a significant decrease of approximately 95% was observed at 1 mM (Figure 1D).

EGT Ameliorates H2O2-Induced Chondrocyte Damage
In comparison, when treating the chondrocytes with 0.1, 0.25, 0.5, and 1 mM of EGT in addition to 1 mM H2O2, the addition of 0.5 and 1 mM of EGT improved the morphological changes in chondrocytes and decreased the viability caused by H2O2 (Figure 2A,B).Similarly, the activity of extracellularly released LDH, which was increased by H2O2 treatment, was significantly decreased by the addition of 0.5 and 1 mM of EGT (Figure 2C).

EGT Ameliorates H 2 O 2 -Induced Chondrocyte Damage
In comparison, when treating the chondrocytes with 0.1, 0.25, 0.5, and 1 mM of EGT in addition to 1 mM H 2 O 2 , the addition of 0.5 and 1 mM of EGT improved the morphological changes in chondrocytes and decreased the viability caused by H 2 O 2 (Figure 2A,B).Similarly, the activity of extracellularly released LDH, which was increased by H 2 O 2 treatment, was significantly decreased by the addition of 0.5 and 1 mM of EGT (Figure 2C).

Inhibitory Effect of EGT on H2O2-Induced Chondrocyte Death
The essential mechanism of cell death by treatment with H2O2 is the peroxidation of polyunsaturated fatty acids by excess intracellular ROS accumulation and subsequent disruption of cell membrane continuity [21,22].Therefore, the double-staining assay for live and dead cells using the difference in cell membrane permeability of nuclear-staining dyes was performed.No dead cells were observed at 3 h after the addition of 1 mM H2O2 and 1 mM EGT, but a significant increase in the dead cell ratio was observed in the H2O2treated group at 6 h after treatment (Figure 3A,B).After 12 h of treatment, most of the cells were identified as dead cells in the H2O2-treated group.On the other hand, in the H2O2 + EGT-treated group, these cell deaths were strongly inhibited and no significant increase in the dead cell ratio was observed at 12 h after treatment.No significant change in the dead cell ratio was observed in the group treated with EGT compared to the control group.

Inhibitory Effect of EGT on H 2 O 2 -Induced Chondrocyte Death
The essential mechanism of cell death by treatment with H 2 O 2 is the peroxidation of polyunsaturated fatty acids by excess intracellular ROS accumulation and subsequent disruption of cell membrane continuity [21,22].Therefore, the double-staining assay for live and dead cells using the difference in cell membrane permeability of nuclear-staining dyes was performed.No dead cells were observed at 3 h after the addition of 1 mM H 2 O 2 and 1 mM EGT, but a significant increase in the dead cell ratio was observed in the H 2 O 2 -treated group at 6 h after treatment (Figure 3A,B).After 12 h of treatment, most of the cells were identified as dead cells in the H 2 O 2 -treated group.On the other hand, in the H 2 O 2 + EGT-treated group, these cell deaths were strongly inhibited and no significant increase in the dead cell ratio was observed at 12 h after treatment.No significant change in the dead cell ratio was observed in the group treated with EGT compared to the control group.

Inhibitory Effect of EGT on H 2 O 2 -Induced Chondrocyte Death
The essential mechanism of cell death by treatment with H 2 O 2 is the peroxidation of polyunsaturated fatty acids by excess intracellular ROS accumulation and subsequent disruption of cell membrane continuity [21,22].Therefore, the double-staining assay for live and dead cells using the difference in cell membrane permeability of nuclear-staining dyes was performed.No dead cells were observed at 3 h after the addition of 1 mM H 2 O 2 and 1 mM EGT, but a significant increase in the dead cell ratio was observed in the H 2 O 2 -treated group at 6 h after treatment (Figure 3A,B).After 12 h of treatment, most of the cells were identified as dead cells in the H 2 O 2 -treated group.On the other hand, in the H 2 O 2 + EGTtreated group, these cell deaths were strongly inhibited and no significant increase in the dead cell ratio was observed at 12 h after treatment.No significant change in the dead cell ratio was observed in the group treated with EGT compared to the control group.

Effect of EGT on ROS Production in H2O2-Treated Chondrocytes
Cells treated with H2O2 have increased intracellular ROS concentrations.Therefore, intracellular ROS accumulation was monitored using DCFH-DA dye, a fluorescent probe for intracellular ROS.Consequently, significant increases in intracellular ROS concentrations were observed at 3, 6, and 12 h after H2O2 treatment, but the addition of EGT significantly suppressed these increases (Figure 4A,B).No significant changes in intracellular ROS concentrations were observed after treatment with EGT.No changes in cell counts were observed (Figure 4C).

Effect of EGT on ROS Production in H 2 O 2 -Treated Chondrocytes
Cells treated with H 2 O 2 have increased intracellular ROS concentrations.Therefore, intracellular ROS accumulation was monitored using DCFH-DA dye, a fluorescent probe for intracellular ROS.Consequently, significant increases in intracellular ROS concentrations were observed at 3, 6, and 12 h after H 2 O 2 treatment, but the addition of EGT significantly suppressed these increases (Figure 4A,B).No significant changes in intracellular ROS concentrations were observed after treatment with EGT.No changes in cell counts were observed (Figure 4C).

Effect of EGT on Mitochondrial Morphology of H2O2-Treated Chondrocytes
Cell death due to oxidative stress causes mitochondrial dysfunction in its early stages [23].Therefore, mitochondrial staining using MitoView Green dye was performed to observe mitochondrial morphology in the pre-membrane disruption phase caused by H2O2.In chondrocytes treated with H2O2 for 2 h, mitochondrial condensation was observed prior to plasma membrane damage, with a significant increase in fluorescence intensity (Figure

Effect of EGT on Mitochondrial Morphology of H 2 O 2 -Treated Chondrocytes
Cell death due to oxidative stress causes mitochondrial dysfunction in its early stages [23].Therefore, mitochondrial staining using MitoView Green dye was performed to observe mitochondrial morphology in the pre-membrane disruption phase caused by H 2 O 2 .In chondrocytes treated with H 2 O 2 for 2 h, mitochondrial condensation was observed prior to plasma membrane damage, with a significant increase in fluorescence intensity (Figure 5A,B).However, no significant change in fluorescence intensity was observed in the H 2 O 2 + EGT-and EGT-treated groups compared to the control group.

Discussion
EGT is a naturally occurring amino acid derivative with safe and potent antioxidant properties that may be a potential therapeutic agent for oxidative stress-related diseases.In this study, to examine the chondrocyte-protective effects of EGT, an oxidative stressinduced chondrocyte injury model was generated using H2O2.The results showed that 1 mM EGT potently inhibited H2O2-induced chondrocyte death, demonstrating its efficacy in a model of oxidative stress-induced cartilage damage in vitro.
Firstly, the cytotoxicity of EGT on chondrocytes was investigated.The results of the CCK-8 assay showed that EGT has no cytotoxicity to chondrocytes up to a concentration of 1 mM.Previous studies have reported no cytotoxicity after 24 h of exposure to 1 mM EGT [24][25][26], which is consistent with our results.However, some reports have suggested that even concentrations not reaching 1 mM may be cytotoxic [27,28], possibly due to differences in the expression of the EGT transporter (OCTN1) in different cell types.OCTN1 has been found to be widely distributed in many mammalian organs and cells [29].Accordingly, EGT, the substrate of OCTN1, is absorbed from the small intestine and then transported to all tissues in the body and retained for a long period of time [30].Therefore, when administrating EGT to the human body, the appropriate EGT-application concentration should be carefully considered.
The protective effect of EGT on chondrocytes against H2O2-induced oxidative stress was evaluated using the LDH-release assay and the cell nuclear double-staining assay.In this study, chondrocyte damage caused by 1 mM H2O2 was inhibited in a dose-dependent manner by the addition of EGT.In addition, 1 mM EGT markedly inhibited chondrocyte death.
H2O2 is most commonly used as an oxidant for oxidative stress-induced cellular damage [31][32][33][34].Several studies have reported on the protective effects of EGT against H2O2induced oxidative stress, including reports on phaeochromocytoma [35] and hippocampal

Discussion
EGT is a naturally occurring amino acid derivative with safe and potent antioxidant properties that may be a potential therapeutic agent for oxidative stress-related diseases.In this study, to examine the chondrocyte-protective effects of EGT, an oxidative stressinduced chondrocyte injury model was generated using H 2 O 2 .The results showed that 1 mM EGT potently inhibited H 2 O 2 -induced chondrocyte death, demonstrating its efficacy in a model of oxidative stress-induced cartilage damage in vitro.
Firstly, the cytotoxicity of EGT on chondrocytes was investigated.The results of the CCK-8 assay showed that EGT has no cytotoxicity to chondrocytes up to a concentration of 1 mM.Previous studies have reported no cytotoxicity after 24 h of exposure to 1 mM EGT [24][25][26], which is consistent with our results.However, some reports have suggested that even concentrations not reaching 1 mM may be cytotoxic [27,28], possibly due to differences in the expression of the EGT transporter (OCTN1) in different cell types.OCTN1 has been found to be widely distributed in many mammalian organs and cells [29].Accordingly, EGT, the substrate of OCTN1, is absorbed from the small intestine and then transported to all tissues in the body and retained for a long period of time [30].Therefore, when administrating EGT to the human body, the appropriate EGT-application concentration should be carefully considered.
The protective effect of EGT on chondrocytes against H 2 O 2 -induced oxidative stress was evaluated using the LDH-release assay and the cell nuclear double-staining assay.In this study, chondrocyte damage caused by 1 mM H 2 O 2 was inhibited in a dose-dependent manner by the addition of EGT.In addition, 1 mM EGT markedly inhibited chondrocyte death.
H 2 O 2 is most commonly used as an oxidant for oxidative stress-induced cellular damage [31][32][33][34].Several studies have reported on the protective effects of EGT against H 2 O 2 -induced oxidative stress, including reports on phaeochromocytoma [35] and hippocampal neural cells [36].Similar to these studies, the results of this study showed that EGT had a cytoprotective effect against H 2 O 2 -induced oxidative stress.
On monitoring intracellular ROS using a fluorescence probe, EGT significantly inhibited the H 2 O 2 -induced increase in intracellular ROS concentration.This is similar to previous studies [37].EGT did not reduce intracellular ROS concentration exclusively.
H 2 O 2 , a metabolite of superoxide, is produced mainly by leukocytes and macrophages and has harmful effects on normal tissue in autoimmune diseases such as RA [38].Under physiological conditions, the extracellular concentration of H 2 O 2 , the most stable ROS, is up to approximately 10 µM, whereas under pathological conditions, the concentration of ROS reaches as high as 1 mM, nearly 100 times higher than that in natural conditions [39,40].
ROS are not only harmful to the organism but are also involved in physiological activities [41].EGT reacts strongly with free radicals, including hydroxyl radicals, whereas it is less directly reactive with H 2 O 2 [42,43].This property implies that only the most dangerous hydroxyl radicals can be scavenged without excessively scavenging the ROS necessary for physiological activity.In the present study, EGT treatment alone was able to potently inhibit cell death without showing cytotoxicity, which may be due to this property.Further, this property may be responsible for EGT not reducing the intracellular ROS production exclusively.
Finally, mitochondrial morphology was observed using MitoView Green.Hydroxyl radicals produced in mitochondria peroxidize mitochondrial membranes, leading to mitochondrial dysfunction [44,45].In the present study, EGT suppressed the changes in mitochondrial morphology caused by H 2 O 2 .This indicates that EGT protects mitochondria from oxidative stress damage caused by H 2 O 2 .
Several studies have tested the efficacy of antioxidant therapy for RA [46][47][48].EGT, similar to these antioxidants, is possibly safer because it does not violate the body's natural ROS homeostasis, which is expected to be effective against RA.Further, drug therapy for RA may elevate oxidative stress in patients [49,50].EGT is also expected to serve as an adjunctive supplement to reduce these side effects of conventional drug therapy.
The present study investigated H 2 O 2 -induced chondrocyte death, and the findings are consistent with previous findings conducted with the naturally occurring antioxidants vitamin C and vitamin E [51,52].Compared to these antioxidants, however, EGT has pharmacokinetic properties more suitable for supplement therapy as its blood levels are maintained for long periods of time after a single administration.Hence, EGT could be a better antioxidant supplement.
However, the future application of antioxidant therapy with EGT for RA requires the further evaluation of the efficacy and safety of EGT in vivo.In addition, since EGT is transported to organs throughout the body via the ubiquitously expressed OCTN1, its systemic effects must also be carefully investigated.
This study has a few limitations.First, the results of this study did not directly demonstrate efficacy against RA.Further studies in disease models using rheumatoid cells from patients are needed to further evaluate efficacy.Furthermore, the pathogenesis of RA is complex, and, in addition to oxidative stress, many factors contribute, including chronic inflammatory conditions and cartilage matrix destruction.Therefore, further detailed studies using cellular and animal models of inflammation and matrix destruction are needed to explore the potential therapeutic effects of EGT for RA.

Conclusions
In this study, EGT potently inhibited chondrocyte death induced by H 2 O 2 without harming chondrocytes.EGT also improved ROS production and the mitochondrial morphological changes caused by H 2 O 2 .This result suggests the efficacy of EGT in oxidative stress-induced chondrocyte damage.Thus, EGT may be a candidate therapeutic agent for degenerative diseases caused by oxidative stress.

3. 4 .
Effect of EGT on ROS Production in H 2 O 2 -Treated Chondrocytes Cells treated with H 2 O 2 have increased intracellular ROS concentrations.Therefore, intracellular ROS accumulation was monitored using DCFH-DA dye, a fluorescent probe for intracellular ROS.Consequently, significant increases in intracellular ROS concentrations were observed at 3, 6, and 12 h after H 2 O 2 treatment, but the addition of EGT significantly suppressed these increases (Figure ??A,B).No significant changes in intracellular ROS concentrations were observed after treatment with EGT.No changes in cell counts were observed (Figure ??C).

Figure 4 .
Figure 4. EGT inhibits H 2 O 2 -induced intracellular ROS production in chondrocytes.(A) Fluorescence images of chondrocytes treated with H 2 O 2 (1 mM) and EGT (1 mM) for 3, 6, and 12 h in the presence of DCFH-DA dye (scale bar: 100 µm).(B) Quantitative data on the average fluorescence intensity of chondrocytes obtained from fluorescence images.Values are presented as mean ± SD (n = 3).(C) Changes in cell count over time.The values are expressed as 100% of the cell count in each group