The human ovarian granulosa KGN cell line was obtained from the American Type Culture Collection and maintained in DMEM containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. It has been reported that Hcy levels may be a significant triggering factor of PCOS, where high concentrations of Hcy can induce oxidative stress and ferroptosis (12,18). In the present study, KGN cells were treated with various concentrations of Hcy (0.25, 0.5, 1, 2 and 4 mM, Shanghai Bang Jing Industrial Co., Ltd.; cat. no. BJ-S964101) with or without 5, 10 and 20 µM Fer-1 (APExBIO Technology LLC.; cat. no. A4371) for 24 h (24,25) at room temperature. KGN cells treated with 1 mM Hcy only were used as the model group. The HcY-induced cell models were divided into two groups, one group was added with 10 µM fer-1, the other group was added sequentially with both 10 µM Fer-1 and 40 nm final concentration of 10 µM TET1/2 inhibitor Bobcat339 hydrochloride (cat. no. T5198, TargetMol Chemicals Inc.). Both groups were incubated at 37°C for 24 h.

Cell viability was evaluated using a Cell Counting Kit-8 (CCK-8) assay. Cells were seeded into 96-well plates at a density of 6×10³ cells/well. Following treatment with Hcy (0.25, 0.5, 1, 2 and 4 mM) and/or Fer-1 (5, 10 and 20 µM) for 6, 12, 24 and 48 h at 37°C, cells were incubated with CCK-8 reagent (10 µl/well; cat. no. HY-K0301, MedChemExpress LLC.) for 2 h at 37°C with 5% CO₂. Absorbance of samples was examined at 450 nm using a microliter plate reader (Thermomax Molecular Devices).

The TUNEL Staining kit (Beyotime Institute of Biotechnology) was used to detect cell apoptosis. Briefly, cells at a density of 2×10⁶ were incubated with 4% paraformaldehyde for 30 min at room temperature, followed by treatment with 0.5% Triton X-100. Cells were subsequently incubated with 50 µl TUNEL reaction buffer for 1 h at 37°C. Nuclei were stained with DAPI (cat. no. C1005; Beyotime) at a concentration 5 µg/ml for 5 min at room temperature. The apoptosis-positive cells were indicated by green staining. Cells were washed 3 times with PBS for 3–5 min each and blocked with Antifade Mounting Medium (cat. no. C1005, P0126-5 ml; Beyotime). The images from randomly selected 5 fields were visualized and captured using a fluorescence microscope (magnification, ×200; Olympus Corporation).

Following cell treatment, the KGN cell culture supernatant was collected to determine the ROS, malondialdehyde (MDA), lactate dehydrogenase (LDH) and reduced GSH levels using specific commercial assay kits of ROS (cat. no. E004-1-1), MDA (cat. no. A003-1), LDH (cat. no. A020-2) and GSH (cat. no. A006-2-1) purchased from Nanjing Jiancheng Bioengineering Institute as per the procedures of manufacturer.

Cellular Fe²⁺ levels were measured using the Phen Green^™ SK reagent (cat. no. P14313, Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 50 µM according to the manufacturer's protocol. Briefly, Phen Green^™ SK reagent was added to 2 ml cell-containing medium at 37°C for 1 h. The fluorescence response to Fe²⁺ ions was examined using a confocal laser scanning microscope (magnification, ×400; Zeiss LSM510; Carl Zeiss AG). Fluorescence randomly selected 5 fields was determined by an Image-Pro Premier software (Version 9.2; Media Cybemetics) at excitation and emission wavelengths of 488 and 520 nm, respectively.

ELISA was used to examine the secretion levels of TET1 and TET2 in the cell culture supernatant according to the manufacturer's protocols. These ELISA kits of TET1 (JC-E8295) and TET2 (JLC-G4351) were purchased from Gelatins Biological Reagent Co. A microplate reader was used to detect the optical density at 450 nm. DNA samples were isolated by PowerSoil DNA isolation kit (cat. no. 12888-50; Anbiosci Tech, Ltd.) and the the level of DNA methylation was detected by Methylight with a MethylFlash Methylated DNA Quantification kit (cat. no. P-1035, EpiGentek Group Inc.) according to the manufacturer's protocol. The results were normalized with the control group.

Cells were harvested using RIPA buffer (Beyotime Institute of Biotechnology) and protein concentrations were quantified using a Bicinchoninic Acid Protein kit (Beyotime Institute of Biotechnology). Protein separation (20 µg) was performed by 10% SDS-PAGE followed by transfer onto PVDF membranes (EMD Millipore). The membranes were blocked in 5% skim milk at 25°C for 1 h, and then probed with the corresponding primary antibodies overnight at 4°C. The HRP-conjugated secondary antibody (cat. no. 7074P2; 1:2,000; Cell Signaling Technology, Inc.) was added the next day and incubated at room temperature for 1 h. Blot images were captured using Pierce^™ ECL Western Blotting Substrate (cat. no. 32209; Thermo Fisher Scientific, Inc.). GAPDH was the internal control and protein band images were analyzed using the Image J software (Version 1.8.0.172; National Institutes of Health). The relative intensity for cleaved caspase-3/caspase-3 was divided by the cleaved caspase-3 value by the value of caspase-3. The relative intensity for other proteins was divided by the corresponding target value by the value of GAPDH.

Anti-Bax (cat. no. 5023T; 1:1,000), anti-Bcl-2 (cat. no. 4223T; 1:1,000), anti-cleaved caspase-3 (cat. no. 9661T; 1:1,000), anti-caspase-3 (cat. no. 14220T; 1:1,000), anti-solute carrier family 7 member 11 (SLC7A11; cat. no. 12691S; 1:1,000) and anti-GAPDH (cat. no. 5174T; 1:1,000) antibodies were provided by Cell Signaling Technology, Inc. Anti-GPX4 (cat. no. sc-166570; 1:500) and anti-achaete-scute family BHLH transcription factor 4 (ASCL4; cat. no. sc-365230; 1:500) antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-divalent metal transporter 1 (DMT1; cat. no. 20507-1-AP; 1:1,000) antibody was the product of Proteintech Group, Inc.

All statistical analyzes were performed using the GraphPad Prism 8.0 software (GraphPad Software, Inc.). Data from ≥ three independent experiments was used. Data are presented as the mean ± SD. An unpaired t-test and one way ANOVA followed by Tukey's post hoc test were performed to statistically compare two or ≥ three independent groups, respectively. P<0.05 was considered to indicate a statistically significant difference.

KGN cells were first treated with various concentrations of Hcy (0.25, 0.5, 1, 2 and 4 mM) for 6, 12, 24 and 48 h. Results from the CCK-8 assay demonstrated that cell viability was significantly reduced by Hcy treatment in a dose-dependent manner compared with that in the control group (Fig. 1A). At concentrations of Hcy >1 mM, cell viability decreased more potently (Fig. 1A). Therefore, 1 mM Hcy was chosen for subsequent experimentation. As presented in Fig. 1B, Fer-1 reversed the Hcy-induced decrease in cell viability in a dose-dependent manner, reaching significance at 10 and 20 µM. These results suggest that Fer-1 treatment can restore viability in Hcy-induced KGN cells.

Subsequently, cell apoptosis was assessed using TUNEL staining. As demonstrated in Fig. 2A and B, the cell apoptotic rate was significantly increased in the model group compared with that in the control group. However, Fer-1 treatment significantly inhibited Hcy-triggered cell apoptosis compared with that in the model group in a dose-dependent manner. In addition, as demonstrated in Fig. 2C, Hcy treatment resulted in the significant downregulation of Bcl-2 expression and upregulation of Bax and cleaved caspase-3 expression, which was partially reversed following Fer-1 treatment in a dose-dependent manner, reaching significance at 10 and 20 µM. These results suggest that Fer-1 can potentially protect against Hcy-induced apoptosis in KGN cells.

The effects of Fer-1 on oxidative stress and ferroptosis in Hcy-induced KGN cells were next investigated. As demonstrated in Fig. 3A-D, KGN cells treated with Hcy displayed significantly elevated ROS, MDA and LDH levels and reduced GSH levels compared with those in the untreated control group. However, Fer-1 treatment markedly reversed these effects of Hcy addition on oxidative stress marker levels, reaching significance at 10 and 20 µM. Furthermore, as shown in Fig. 3E and F, intracellular Fe²⁺ levels were significantly increased following Hcy exposure, but Fer-1 treatment significantly reduced this Hcy-induced increase in Fe²⁺ levels in a dose-dependent manner. In addition to significantly decreasing GPX4 and SLC7A11 expression levels, significantly increased ASCL4 and DMT1 expression levels were also observed in the Hcy model group compared with those in the control, which were significantly reversed following 10 and 20 µM Fer-1 treatment (Fig. 3G). These results suggest that Fer-1 treatment can alleviate Hcy-induced oxidative stress and suppress ferroptosis in KGN cells.

Overall DNA methylation levels in KGN cells were subsequently measured following Hcy treatment. As displayed in Fig. 4A, the DNA methylation levels were significantly enhanced in the Hcy-induced group compared with those in the control group. However, Fer-1 treatment significantly reversed this Hcy-induced DNA methylation were in a dose-dependent manner. Furthermore, TET1 and TET2 levels were significantly decreased following Hcy induction, which were subsequently restored following Fer-1 treatment in a dose-dependent manner (Fig. 4B and C). These results suggest that Fer-1 treatment can suppress DNA methylation and increase TET1/2 levels in Hcy-induced KGN cells.

To further explore the regulatory mechanism of Fer-1 on Hcy-induced apoptosis, oxidative stress and ferroptosis, the TET1/2 inhibitor Bobcat339 hydrochloride was used to treat Hcy-induced KGN cells. As shown in Fig. 5A-C, the results demonstrated that in the presence of Fer-1, Bobcat339 hydrochloride treatment significantly reduced TET1 and TET2 levels whilst significantly elevating DNA methylation levels. In addition, as presented in Fig. 5D, cell viability was significantly decreased following 48 h treatment with Bobcat339 hydrochloride in Hcy and Fer-1 treated KGN cells. TUNEL and western blotting assays demonstrated that compared with that in the Hcy and Fer-1 treated group, a significant increase in cell apoptosis in the Fer-1 + Bobcat339 hydrochloride group was observed, which is accompanied with significantly downregulated Bcl-2 and upregulated Bax and cleaved caspase-3 protein expression levels (Fig. 5E-G).

Furthermore, as demonstrated in Fig. 6A-D, Bobcat339 hydrochloride significantly enhanced ROS, MDA and LDH levels but reduced GSH levels compared with those in the Fer-1 group in Hcy-stimulated KGN cells. Fe²⁺ levels were also significantly decreased following Bobcat339 hydrochloride treatment, whilst GPX4 and SLC7A11 protein expression levels were significantly decreased (Fig. 6E-G). By contrast, ASCL4 and DMT1 protein expression levels were significantly increased by Bobcat339 hydrochloride treatment compared with those in the Fer-1 group in Hcy-stimulated KGN cells (Fig. 6E-G). These results suggest that Bobcat339 hydrochloride can reverse the effects of Fer-1 on Hcy-induced apoptosis, oxidative stress and ferroptosis in KGN cells.