AS101 (catalog number: A872502) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Doxorubicin hydrochloride (catalog number: MB1087) was obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Enzyme-linked immunosorbent assay (ELISA) kits for E2 (catalog number: CEA441Ra96T), FSH (catalog number: CEA830Ra96T), AMH (catalog number: CEA228Ra96T), were acquired from Wuhan Saver Biological Technology Co., Ltd. (Wuhan, China). MDA (catalog number: BC0025) and SOD (catalog number: BC5165) were acquired from Beijing Solarbio Science and Technology Co., Ltd. (Beijing); ROS (catalog number: MA0219) was obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China).

Tissue processing and embedding were performed using a KD-BMIV automatic tissue dehydrator and a BLIV tissue embedding machine (Wuhan Servicebio Technology Co., Ltd., Wuhan, China). Centrifugation was carried out with a room temperature tabletop high-speed centrifuge (model: 5424R; Eppendorf AG, Hamburg, Germany). Histological observations were conducted using a ci-1 biological microscope (Nikon Corporation, Tokyo, Japan) and a CKX-41 inverted microscope (Olympus Corporation, Tokyo, Japan). Absorbance measurements were recorded on a RT-6100 microplate reader (Rayto Life and Analytical Sciences Co., Ltd., Shenzhen, China).

We procured 18 female SD rats, each 8 weeks old and weighing approximately 210 ± 10 g, from Beijing Huafukang Biotechnology Co., Ltd. The rats were housed in our institution’s experimental animal center, with each group accommodated in identically sized cages. The rats were maintained under standard conditions, including a 12-hour light/dark cycle, a temperature of 25℃, adequate ventilation, and dryness. They were provided with ample and equal quantities of food and water. Regular replacement and disinfection of the rat cages and bedding were ensured. All animal experiments and procedures involved in this study received approval from our institution’s Animal Welfare Ethics Review Committee. This study was approved by the Experimental Animal Welfare Ethics Committee of Hebei University (Ref:202328SR).

All experimental animals underwent a one-week acclimatization period in our institution’s animal experiment room. The 18 rats were randomly allocated into three groups, each containing six individuals. Group A, designated as the blank control group, received intraperitoneal injections of 0.9% saline (0.1mL) for 21 consecutive days. Group B, referred to as the modeling group, was pre-treated with intraperitoneal injections of 0.9% saline (0.1mL) for five days, followed by an intraperitoneal injection of doxorubicin (10 mg/kg) on the sixth day, and continued saline injections for the subsequent 15 days. Group C, the treatment group, followed the same initial protocol as Group B but received AS101 (prepared in PBS, pH = 7.4) (3 mg/kg) injections every other day after the doxorubicin injection, for a total of two times. Notably, no fatalities were observed among the rats in any of the three groups.

The rats are weighed every two days, with the data duly recorded.Each rat undergoes a vaginal smear procedure every morning between 7 and 8 o’clock. Specific steps include inserting a sterilized cotton swab, moistened with physiological saline, approximately 0.5 cm into the rat’s vagina and gently rotating it 2–3 times. (1) The proestrus phase typically spans 12–14 h, during which most cells in the rat vaginal smear are nucleated epithelial cells, interspersed occasionally with keratinocytes and leukocytes. (2) The estrus phase generally endures for 25–27 h, with all cells in the rat vaginal smear being anucleated keratinized epithelial cells, and a small number of oval epithelial cells visible occasionally. (3) The metestrus phase typically lasts for 55–57 h, characterized predominantly by the presence of numerous leukocytes in the vaginal smear, along with a few nucleated epithelial cells. (4) The diestrus phase generally persists for 6–8 h, during which the vaginal smear exhibits a variety of cells concurrently, including leukocytes, keratinocytes, and nucleated epithelial cells [14]. Following the removal of secretions, they are smeared on a glass slide, stained with HE, and observed under an optical microscope to ascertain the rat’s estrous cycle.

Upon completion of the observation period, tail blood samples were collected from each rat group for testing serum levels of E2, FSH, and AMH. Concurrently, blood samples were drawn from the rats’ femoral arteries to assess the serum levels of SOD, MDA, and ROS. The collected blood was transferred into a 5 ml centrifuge tube, centrifuged at 3000 r/min for 20 min to separate the serum, and subsequently stored in a refrigerator at -4℃. Following this, an enzyme-linked immunosorbent assay (ELISA) was employed to quantify the serum hormone levels as well as the serum concentrations of SOD, MDA, and ROS. The specific procedures were meticulously executed in accordance with the instructions provided with the reagent kit.

Following the observation period, the rats were euthanized via carbon dioxide asphyxiation. The skin and subcutaneous tissue were dissected sequentially to locate both ovaries, which were then completely excised for histological analysis. The intact ovarian tissue was fixed in a tissue fixative, dehydrated, embedded in paraffin, and cooled. Finally, 5-µm-thick tissue sections were prepared on slides. One in every four consecutive sections was selected, with an average of five sections per group subjected to deparaffinization and HE staining. The slides were sealed with neutral balsam, and follicular development was evaluated under a light microscope. Under the microscope, the total number of primordial and atretic follicles in five fields of view was counted from HE-stained sections of the entire fragment, and the average number was determined.

After a 14-day observation period, rats were euthanized by carbon dioxide asphyxiation. The skin and subcutaneous tissues were sequentially incised to expose and completely dissect both ovaries for histological evaluation. The intact ovarian tissues were fixed in fixative solution, dehydrated, embedded in paraffin, cooled, and then sectioned into 5 μm-thick slices, which were mounted on glass slides. One section was selected from every four consecutive slices, with an average of five sections per group. These sections were deparaffinized, stained with HE, mounted with neutral balsam, and assessed for follicular development under an optical microscope. Primordial follicles throughout the entire section were counted on HE-stained slides under the microscope. The total numbers of primordial and atretic follicles were recorded across five fields of view, and the averages were calculated.

The specific steps are as follows: (1) Check the carbon dioxide cylinder pressure to ensure an adequate gas supply. (2) Adjust the CO₂ flow rate to the recommended level. 3) Place the rats in the euthanasia chamber. (4) Open the CO₂ cylinder valve and introduce CO₂ into the chamber at the predetermined flow rate. (5) Monitor the rats until respiration ceases, pupils are dilated, and no muscular movement is observed. (6) After confirming death, continue CO₂ infusion for at least one additional minute to ensure complete euthanasia.