In Vivo ExperimentConstruction of POF Rat Model and KTC Treatment

This experiment was approved by the Experimental Animal Ethics Committee of Guangzhou Miers Biotechnology Co., LTD. (IACUC-MIS20230069). SD female rats (6–8 weeks old, 200 ± 5 g) were purchased from Guangdong Medical Laboratory Animal Center. The POF rat model was constructed according to the previous studies [19, 20]. Cyclophosphamide (CTX, Cat.HY-17420, MCE, USA) was injected intraperitoneally for 14 days with an injection dose of 50 mg/kg on the first day and 8 mg/kg on the subsequent day. KTC (Guiyang Xintian Pharmaceutical, China) suspension (2.0 mL) was intragastically administered with doses of 0.6 g/kg/d for the low dose group and 1.8 g/kg/d for the high dose group. Dehydroepiandrosterone (DHEA, Cat.HY-14650, MCE, USA) was used as a positive control and was intragastically administered with a dose of 13.5 mg/kg. The rats were weighed daily and their estrus cycles were recorded. After the experiment, the rats were euthanized, and the ovaries were removed and weighed immediately.

Autophagy Modulation Experiments

To elucidate the role of AMPK-mTOR signaling in KTC-mediated POF alleviation, autophagy inhibitor 3-Methyladenine (3-MA, Cat.HY-19312, MCE, USA) and mTOR-specific inhibitor Rapamycin (RAPA, Cat.HY-10219, MCE, USA) were administered to KTC-treated POF rats. The inhibitor intervention groups were designed as follows: KTC + 3-MA group (rats received intraperitoneal injections of 15 mg/kg 3-MA one hour prior to KTC-H (1.8 g/kg/d) intragastric administration twice a week), KTC + RAPA group (rats received intraperitoneal injections of 2 mg/kg RAPA one hour prior to KTC-H (1.8 g/kg/d) intragastric administration per day for two weeks). Inhibitor dosing regimens were based on established protocols for ovarian autophagy modulation [21, 22]. All inhibitors were dissolved in dimethyl sulfoxide (DMSO) and diluted with saline (final DMSO concentration < 0.1%). Control groups received vehicle solution (0.1% DMSO in saline) at equivalent volumes. The intervention lasted 14 days concurrent with KTC treatment.

Histopathology

Fresh ovarian tissue was removed and fixed in 4% paraformaldehyde solution (Cat.P0099, Beyotime, China). After paraffin (Cat.39601095, Leica Biosystems, Germany) embedding, the tissue was sectioned to 4-μm-thick slices, hematoxylin and eosin (H&E, Cat.C0105S, Beyotime, China) staining were used to observe the ovary morphology and the number and morphology of follicles at all development stages. Follicles were classified and counted by two independent blinded observers as described [23]. Every 10th section was chosen to analyze and the raw count was multipled by ten to estimate the total number of follicles. Follicles without clear oocyte nuclei were excluded. Primordial follicles are characterized by an oocyte surrounded by one layer of flattened granulosa cells. Primary follicles have one to two complete layers of cuboidal granulosa cells surrounding the oocyte. Secondary follicles have more than two layers of cuboidal granulosa cells. Mature follicles have multiple layers of cuboidal granulosa cells with a cumulus oophorus and antral spaces.

For immunohistochemistry, paraffin sections were immersed in xylene (Cat.B83606, Sigma-Aldrich, USA) for dewaxing, and then immersed in gradient ethanol solution (Cat.459828, Sigma-Aldrich, USA) to remove xylene. To inactivate endogenous peroxidase, an appropriate amount of peroxidase blocker (Cat. P0100A, Beyotime, China) was employed. The sections were soaked in citric acid buffers (Cat.P0083, Beyotime, China) to heat repair the antigens. The antibody was added and incubated at 4℃ overnight. On the second day, the second antibody was added for incubation, hematoxylin was re-stained after DAB (Cat.P0202, Beyotime, China) color development, and 1% hydrochloric acid alcohol was returned to blue. Primary antibodies were as follows: Rabbit polyclonal anti-BMP15 (Cat.18982–1-AP, Proteintech, USA, 1:200), Rabbit polyclonal anti-BMP7 (Cat.CL488-12,221, Proteintech, USA, 1:200), Rabbit polyclonal anti-P16 (Cat.10883–1-AP, Proteintech, USA, 1:200), Rabbit monoclonal anti-γH2AX (Cat.ab229914, Abcam, UK, 1:200).

ELISA Analysis

Serum samples were collected after centrifuging blood at 3000 rpm for 10 min. The ELISA kits used were as listed: Rat estradiol (E2) ELISA kit (Cat.KT-59814, Kamiya, USA), Rat anti-Mullerian hormone (AMH) ELISA kit (Cat.CSB-E11162r, Cusabio, China), Rat follicle-stimulating hormone (FSH) ELISA Kit (Cat.CSB-E06869r, Cusabio, China), Rat Malondialdehyde (MDA) ELISA Kit (Cat.AE20805R, A&E bio, China), Rat reactive oxygen species (ROS) ELISA Kit (Cat.J23513, GILED, Wuhan, China), and Rat Super Oxidase Dimutase (SOD) ELISA Kit (Cat.JN4055, Jining Bio, China).

The analysis was conducted according to the manufacturers’ instructions. In brief, antibody working solution and avidin-peroxidase (ABC) working solution were prepared, and 100 µl of standard product was added. The enzyme label plate was covered with sealing plate membrane and reacted at 37℃ for 90 min. The liquid in the enzyme label plate was removed and 100 µl of biotin anti-antibody working solution was added to each well, and washed with buffer solution for 3 times after incubation. OD values were measured at 450 nm by ELx800 Absorbance Microplate Reader (Bio-Tek, USA).

Western Blot Analysis

The total protein was extracted with RIPA lysate (Cat. P0013C, Beyotime, China), separated with sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membrane (Cat.IPVH00010, Millipore, USA) and sealed with 5% skim milk powder solution. The film was incubated with the corresponding primary antibody at 4℃ overnight, and after cleaning with PBS, the film was incubated with the corresponding secondary antibody at room temperature for 2 h, and the ECL (Cat.P0018S, Beyotime, China) method was developed. The signal was detected with the iBright 1500 Image system (Thermo Fisher Scientific, Pittsburgh PA). Semi-quantitative analysis was performed on the western blot bands and intensities of the bands were quantified using image J. Primary antibodies were used as Table 1.

Table 1 Antibodies for Western blot analysisApoptosis Detection

The TUNEL Assay Kit (Cat.ab206386, Abcam, UK) was used to detect apoptosis. In brief, soak the slices in xylene and ethanol solutions of different concentrations for dewaxing hydration, rinse ddH2O, add protease K (Cat.P1005, Beyotime, China) for repair, and inactivate endogenous peroxidase with 3% H2O2. It was incubated in the working liquid labeled by HRP at 37℃ and the color was developed by DAB method. Hematoxylin was re-dyed, 1% hydrochloric acid alcohol was differentiated into anti-blue, ddH2O was washed and dried naturally.

For flow cytometry assay, the ovarian samples were prepared into single-cell suspension, then they were stained according to the manufacturer’s instruction of the Annexin V-FITC Apoptosis Detection Kit (Cat.CA1020, Solarbio, China) and detected by the BD FACSCalibur™ Flow Cytometer (BD Biosciences, USA).

Network Pharmacological Analysis

The active ingredients of KTC were retrieved from the TCMSP database (https://old.tcmsp-e.com/tcmsp.php). Targets for active ingredients were then searched through the TCMSP database and the target names were converted to gene names through the uniprot database (https://www.uniprot.org/). Differentially expressed genes (DEGs) were screened in GEO database. Therapeutic targets obtained from CTD, DisGeNet, genecards, NCBI, OMIM, and PharmGKB databases were combined with genes from GEO databases to screen for targets that appeared in at least two databases. These targets were then crossed with KTC therapeutic targets to identify KTC therapeutic targets for PCOS. Finally, Cytoscape 3.7.2 software was used to construct the relationship network between KTC active components and target genes, and identify the active components related to the core target of KTC treatment of POF. Through cross-analysis of therapeutic targets of KTC and POF in bioinformatics database, potential therapeutic targets of KTC for POF were screened. PPI network was constructed using potential therapeutic targets, and core targets were further screened.

Statistical Analysis

Data were presented as mean ± SD and analyzed using GraphPad PRISM 9.5.1 software. Differences were compared using ANOVA with the Bonferroni test. Statistical significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001.