Experimental animals and medication

Laboratory Animal Center of Harbin Medical University in Heilongjiang Province approved the protocols for animal experiments (No: YJSDW2023-107), and all methods were performed in accordance with ARRIVE guidelines and relevant and regulations.

Ten female Sprague Dawley (SD) rats (3 weeks old) were used in cellular experiments, and female SD rats (6 weeks old) with a normal estrous cycle were used for animal modelling, all animals sourced from the Department of Animal Science at Harbin Medical University. The cellular experiments rats were intraperitoneally injected with pregnant horse serum (80 IU/rat) for 24 h and then sacrificed by cervical dislocation. The bilateral ovaries were quickly removed, after that piercing follicle with a 1 ml syringe needle. Collect GCs, and cells were maintained in DMEM/F12 (GENEVIEW, China) supplemented with 10% FBS (Biological Industries, USA), at 37℃ with 5% CO2 atmosphere (Dong et al. 2023). The cell experiments were organized into six groups: control group (CON), FER-1 group, CDDP group, RSL3 group, RSL3 + CDDP group, and FER-1 + CDDP group. In addition, the FER-1 + RSL3 group was increased when cytotoxic concentrations were detected.

The experimental animals were categorized into three groups (n = 5 per subgroup): CON group, CDDP group, and CDDP + FER-1 group. Initially, the rats were randomly assigned to either the control group (n = 10) or the CDDP group (n = 20). In the control group, five rats were randomly selected to receive a saline intraperitoneal injection for 7 days, followed by an intraperitoneal injection of saline (2% dimethyl sulfoxide (DMSO), i.p.) three times a week for 2 weeks. The POF condition in rats was induced through an intraperitoneal injection of 2 mg/kg/day CDDP for 7 days. After 7 days, 10 rats with successfully induced POF were divided into two subgroups (n = 5 per subgroup): CDDP and CDDP + FER-1 (1 mg/kg, 2% DMSO), receiving injections of either saline (2% DMSO, i.p.) or FER-1 (1 mg/kg, 2% DMSO) three times a week for a total of 2 weeks, following the protocol described by Fang et al. (2019). After the treatment was finished, the experimental rats were anesthetized by i.p. injection of pentobarbital (50 mg/kg). Take blood from the abdominal aorta and collect them in centrifuge tubes. The blood was centrifuged (3000 r/min) for 15 min at RT to obtain the serum, and the ovaries were measured their weights and then stored at − 80℃ for subsequent experiments. All animals were sacrificed with pentobarbital (100 mg/kg) after surgery (Xue et al. 2022). The specific flow chart for animal modeling is shown in Fig. 4A, and the specific dosage formulated is shown in Supplementary Material S1.

CCK8 assay

GCs from rat ovaries were isolated, seeded in 96-well plates, and cultured at 37 °C in a 5% CO2 atmosphere. Drug treatments included CDDP (10—40 μmol/L) for 24 h, RSL3 (1, 2, 3, and 5 μmol/L), FER-1 (0.5, 1, 2, 5, 10, 20, 30, and 40 μmol/L) for 2 h. The CDDP concentration was chosen based on previous research by our group (Dong et al. 2023). For the CCK8 assay, 5 μL of CCK8 solution (ApexBio, USA) was added to each 100 μL of the medium, incubated for 2 h, and the absorbance (Abs) was measured at 450 nm. Cell viability was expressed as a percentage of the control.

Fe2+ detection

Intracellular and mitochondrial iron levels were determined using FerroOrange and Mito-FerroGreen (Dojindo, Japan), respectively. After treatment, GCs were stained with 1 μmol/L FerroOrange or 5 μmol/L Mito-FerroGreen at 37 ℃ for 30 min in the dark. Images were acquired using a confocal laser scanning microscope (Leica TCS-SP5II, Germany), and the experiment was repeated three times. Fluorescence intensity was analyzed using ImageJ software, with the average fluorescence intensity of each group being normalized to that of the control group.

ROS detection

Intracellular and mitochondrial ROS levels were assessed using DCFH-DA (ApexBio, USA) and MitoSox-Red (MKBIO, China), respectively. GCs were treated under specified conditions in confocal Petri dishes, then double-stained with 50 μmol/L DCFH-DA and 5 μmol/L MitoSox-Red for 30 min in the dark. Afterward, cells were stained with Hoechst33342 (Solarbio, USA) for 10 min. Images were captured using a confocal laser scanning microscope (Leica TCS-SP5II, Germany) equipped with an oil immersion objective. Fluorescence intensity was measured with ImageJ and normalized to the average intensity of the control group.

MMP measurement

To visualize mitochondrial membrane potential (MMP), GCs were cultured in confocal petri dishes and exposed to specific conditions. Following these treatments, the cells were stained with 10 μM Rhodamine123 (Solarbio, USA) in accordance with the manufacturer’s instructions. After incubation for 30 min at 37℃ in the dark, the GCs were further stained with Hoechst33342 for 10 min. They were then washed with phosphate-buffered saline and imaged using a confocal laser scanning microscope (Leica TCS-SP5II, Germany) equipped with an oil immersion objective. Images were chosen from random fields of view across three independent experiments.

TEM

Cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at 4℃, followed by post-fixation in 1% osmium tetroxide for 1 h. The samples were dehydrated through a graded ethanol series and embedded in Spurr’s resin. Ultrathin Sects. (60–80 nm) were cut using an ultramicrotome, mounted on copper grids, and stained with uranyl acetate and lead citrate. Transmission electron microscopy (TEM) (Hitachi HT7700, Japan) at 80 kV and images were captured with a CCD camera using for observe the cellular structures and mitochondrial.

Estrous cycle measurement

Rats underwent daily swabbing and were treated with paraformaldehyde and crystal violet solution before microscopic examination for estrous cycle changes.

ELISA

The concentrations of estradiol (E2) and follicle-stimulating hormone (FSH) were measured using enzyme-linked immunosorbent assay (Shanghai Enzyme-linked Biotechnology, China) following the manufacturer's instructions. The absorbance (Abs) for each group was recorded at 450 nm, and the serum levels of E2 and FSH were determined using a standard curve.

Organ index

Rats were monitored, and their body weights were recorded at four intervals: before treatment, after model establishment, after 1 week of treatment, and at the end of the treatment period. The indices for both ovaries and uteri were then calculated.

Tissue iron assay

Tissue iron levels were assessed using the iron assay kit (Leagene, China). Samples were processed, and their absorbance was measured at 562 nm with an enzyme meter (BIOTEK SYNERGY-H1, USA). The results were then calculated.

MDA/SOD/GSH assay

The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were determined using respective assay kits (Beyotime Biotechnology, China), SOD assay kit (Nanjingjiancheng Bioengineering, China) and GSH assay kit (Beyotime Biotechnology, China). After sample processing, absorbance was measured, and the results were calculated according to kit instructions using an enzyme meter (BIOTEK SYNERGY-H1, USA).

Western blot

Lysate preparation and western blot analysis were conducted following established protocols (Zhang et al. 2023a). Ovarian tissues were lysed in radioimmunoprecipitation assay lysis buffer to extract total proteins, which were then transferred onto polyvinylidene difluoride membranes. Initially, membranes were blocked with 1% bovine serum albumin for 90 min, washed thrice with Tris-buffered saline with Tween 20 (TBST), and incubated with primary antibodies overnight at 4℃. The primary antibodies used were as follows: KEAP-1 (1:1500, #wl03285, Wanlei Biotechnology, China), NRF2 (1:1000, #bs1258, Bioworld Technology, USA), HO-1 (1:1000, #WL02400, Wanlei Biotechnology, China), GPX4 (1:1000, #ab125066, Abcam, UK) and GAPDH (1:1000, #WH199424, ABclonal Technology, China). Following TBST washes, the membranes were incubated with secondary antibody HRP(H + L) IgG (#WH184227, ABclonal Technology, China) for 90 min at room temperature. Detection was performed using an automatic electrochemiluminescence analysis system (BIO-RAD AI-600, USA) was used to show the blot bands.

Molecular docking simulation

The three-dimensional structure of NRF2 for molecular docking was obtained from AlphaFold database (https://alphafold.ebi.ac.uk) (UniProt: a hub for protein information 2015) and the structure of GPX4 was obtained from PubChem (http://pubchem.ncbi.nlm.nih.gov/) (Hähnke et al. 2018) and PyMOL2.6.0 (PDB: 2OBI). FER-1 was docked into GPX4 or NRF2 using Molecular Operating Environment 2019 software (Liu et al. 2017) with number of operations selected by 50 times. Thus, molecular docking scores was assessed based on the magnitude of binding energy and the results were visualized by PyMOL and Discovery studio 2019 software.

Data analyses

Statistical analysis of experimental data was conducted using GraphPad Prism 9.4.1 and IBM SPSS Statistics 25 software. Data were presented as mean ± standard deviation. Statistically significant differences between the mean of multiple groups of independent samples were analyzed using one-way analysis of variance, and the LSD method was used for pairwise comparison between groups. P-value < 0.05 was considered statistically significant.