Ethics statement

The study was authorized by the academic ethics committee of The Second Affiliated Hospital of Hunan University of Chinese Medicine[NO.2023-45]. All procedures were strictly implemented according to the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals. Informed consent was obtained from all parents or legal representatives after they were fully informed of the study objectives. All laboratory procedures were used to minimize the pain of mice.

Study subjects

A total of 161 patients with DOR who received treatment at The Second Affiliated Hospital of Hunan University of Chinese Medicine from January 2021 to December 2022 were selected, among which 30 were excluded as per the inclusion and exclusion criteria, 11 were unwilling to participate, 10 withdrew, and 5 had incomplete data. Finally, 105 patients were included as the study subjects.

The inclusion criteria were as follows [22]: (1) age between 18 and 40 years; (2) 0.5 ng/mL < anti-Mullerian hormone (AMH) < 1.1 ng/mL; (3) antral follicle count (AFC) < 7; (4) 10 IU/L < follicle-stimulating hormone (FSH) < 25 IU/L; (5) clinical manifestations of irregular menstruation, infertility, oligomenorrhea, amenorrhea, sexual dysfunction, dyspareunia and vaginal dryness; (6) DOR treatment with YJZYD.

The exclusion criteria were as below [22, 23]: (1) previous history of unilateral or bilateral oophorectomy; (2) administration of sex hormones including estrogen, progesterone, and gonadotropins within one week before receiving treatment; (3) diagnosis of corpus luteum cyst, residual follicle, or a space-occupying lesion in the ovary, PCOS, or pelvic inflammatory disease; (4) pregnant or receiving hormone replacement therapy, dehydroepiandrosterone, and other treatment methods; (5) with severe respiratory diseases, cardiovascular diseases, liver and kidney diseases, hematopoietic system diseases or mental illness.

Treatment method

All participants avoided the 1st-4th day of the menstrual period and began to take YJZYD from the 5th day of the menstrual cycle for 21 consecutive days. YJZYD is composed of cooked Rehmannia glutinosa (30 g), Angelica sinensis (15 g), Paeonia lactiflora (15 g), and Cornus officinalis (15 g). In the specific compatibility, clinical addition and subtraction should be carried out according to the specific symptoms of the patients, and the prescription should be adjusted accordingly. Each dose of the traditional Chinese medicine was added with 500 mL water, decocted (150 mL/time), and administrated to the patients three times a day for consecutive 6 months. Of these, cooked Rehmannia glutinosa, Angelica sinensis, Paeonia lactiflora, and cornus officinalis were all purchased from TongRenTang (Beijing, China).

Sample and data collection

Before and after treatment, 5 mL fasting elbow vein blood was gathered from the patients on the 2nd to 3rd day of the menstrual cycle, centrifuged at 2000 r/min for 20 min, and stored in a refrigerator at 4 °C. Serum hormone testing was performed on the same day.

Ultrasound measurement

Ovarian volume (OV), AFC, and endometrial thickness (EMT) pre- and post-treatment were evaluated using ultrasound Voluson E6 (GE, Healthcare, Milwaukee, WI, USA). AFC was the sum of follicles observed in both ovaries using ultrasound in the early follicular phase (on days 2–4) of the menstrual cycle. OV and EMT were documented on days 12–21 of the menstrual cycle.

Experimental animals

Female C57BL/6 mice (n = 36, 25 ± 2 g, 6 weeks old) were purchased from the experimental animal resource platform of the Chinese Academy of Sciences. Mice were reared in a specific pathogen-free standard animal room and 12-h light/dark cycle at 23 ± 2 °C, with a relative humidity of 50-60% and free access to food and water.

Establishment of DOR mouse models

After one week of continuous vaginal smear examination, all 36 female mice exhibited regular estrous cycles following a 1-week continuous vaginal smear examination. DOR mouse models were established based on a previous study [24]. Briefly, mice were intraperitoneally injected with cyclophosphamide (Cy) (75 mg/kg, 500 µL; Endoxan, Shionogi & Co., Osaka, Japan) once a week on the first day of weeks 2–5, with the injection of 0.9% normal saline instead of Cy as the control. Following the treatment, vaginal exfoliated cell smears were conducted to observe the estrous cycle [25]. When the model mice exhibited estrous cycle disorder, it was determined that the DOR mouse models were successfully established.

The mice were randomly divided into the following 9 groups: the Control group: without any treatment; the model group (DOR): intraperitoneal injection of Cy (75 mg/kg, 500 µL) [24]; the Sal group: intraperitoneal injection of an equal amount of 0.9% normal saline solution; the DOR + YJZYD group: intraperitoneal injection of Cy (75 mg/kg, 500 µL) and gavage of YJZYD solution (10 mL/kg, 3 mg/µL) [9]; the DOR + PW group: intraperitoneal injection of Cy (75 mg/kg, 500 µL) and gavage of purified water (PW) equivalent to YJZYD solution; the DOR + YJZYD + PD98059 group: intraperitoneal injection of Cy (75 mg/kg, 500 µL) and PD98059 (1 mg/kg; an ERK inhibitor) [26]; and gavage of YJZYD solution (10 mL/kg)], the DOR + YJZYD + Vehicle group: intraperitoneal injection of Cy (75 mg/kg, 500 µL) and Vehicle equivalent to PD98059 [10% dimethyl sulfoxide (DMSO) + 90% (20% SBE-β-CD in Saline)], and gavage of YJZYD solution (10 mL/kg); the DOR + TPA group: intraperitoneal injection of 75 mg/kg (500 µL) of Cy and 150 µg/kg 12-O-tetradecanoylphorbol-13-acetate (TPA; an ERK activator) [27]; the DOR + Vehicle 2 group: intraperitoneal injection of 75 mg/kg (500 µL) of Cy and the same amount of Vehicle (94 mg/mL Ethanol) as TPA. Among them, YJZYD solution was concentrated before vacuum freeze-dried into powder, resuspended in 200 µL pure water at a concentration of 3 mg/µL, and stored at 4 °C for subsequent animal and cell experiments. PD98059 (MedChemExpress, Monmouth Junction, NJ, USA) is an inhibitor of the MAPK/ERK pathway. TPA (Adooq Bioscience, CA, USA) is an activator of the MAPK/ERK pathway. After 5 weeks, all mice were euthanized with 150 mg/kg pentobarbital sodium, with ovarian and serum samples collected.

Ovarian morphology analysis and AFC

Referring to previous research [25], ovarian morphology analysis and AFC were conducted. Simply put, after fixation with 4% paraformaldehyde, dehydration, and paraffin embedding, the ovaries were cut into Sect. (5 μm) and subjected to hematoxylin and eosin (HE) staining, with the ovarian morphology observed under an optical microscope (Olympus Corporation, Tokyo, Japan), and the follicle number counted, as reported previously [28]. A primitive follicle is characterized by the presence of a central oogonia that is enveloped by a layer of flat follicular cells. Primary follicles are composed of oocytes and a layer of cubic granulosa cells around them. Secondary follicles are composed of oocytes and their surrounding multi-layer cubic follicular cells. A mature follicle is one in which the follicular lumen is large, the ovarian mound is distinct, the follicular endothelial cells are immediately adjacent to the follicular granulosa and separated from the granulosa cells by a basement membrane, and the endothelial cells are polygonal, with clear cytoplasm and rounded nuclei. Follicular atresia is distinguished by the unclear or complete disappearance of the egg cell’s structure, shrinkage of the zona pellucida, and collapse of the follicular wall [2].

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and fluorescence double labeling

Ovarian GCs were identified by the FSHR [29]. After xylene dewaxing and gradient ethanol hydration, mouse ovarian tissue sections were stained with the TUNEL apoptosis detection kit (Elabscience Biotechnology, Wuhan, Hubei, China), followed by 3 washes with tris-buffered saline (TBS) for 5 min and an incubation with rabbit anti-FSHR (1:500, GTX64391, GeneTex, Irvine, CA, USA). Later, the sections were rinsed in TBS before 1-h incubation with secondary antibodies (1:200, GTX26721, GeneTex). A fluorescence microscope (Zeiss, Oberkochen, Germany) was employed to observe the percentage of apoptotic positive cells in each field, and the average was calculated.

Enzyme-linked immunosorbent assay (ELISA)

The levels of serum AMH, FSH, luteinizing hormone (LH), and estradiol (E2) were determined as per the instructions of the ELISA kits. The human AMH ELISA kit, human or mouse FSH ELISA kit, human or mouse LH ELISA kit, and human or mouse E2 ELISA kit were all acquired from Xinfan Biological Technology (Shanghai, China), and the mouse AMH ELISA kit was procured from Fusheng Industrial (Shanghai, China).

Cell culture

Human ovarian GCs (KGN) were acquired from Yubo Biological Technology (Shanghai, China) and identified using the short tandem repeat method. KGN cells were cultured in the Dulbecco’s modified Eagle’s medium/F12 (Gibco, Grand Island, NY, USA) comprising 10% fetal bovine serum (Gibco) and 1% antibiotic (Gibco) in an incubator at 37 °C containing 5% CO2, with the medium changed every 2 days. Cells at a confluence of about 80% in P3 generation were selected for the subsequent experiments. Because Cy was inert in vitro, we used 4-Hydroperoxy-Cyclophosphamide (4-HC) (8 μm) (Anjiekai Biomedical Science and Technology, Wuhan, Hubei, China) as an in vitro intervention. 4-HC is a precursor of Cy in an activated form [24].

KGN cells were divided into the following 8 groups: the KGN group (blank control without any treatment), the KGN + 4-HC group [cells treated with 4-HC (8 μm) for 24 h], the KGN + Vehicle 1 group [cells were treated with an equal amount of Vehicle (30 mg/mL in DMSO) to 4-HC for 24 h], the KGN + 4-HC + YJZYD group [cells were treated with 4-HC (8 μm) [30] and YJZYD solution (1 mg/mL) [8] for 24 h], the KGN + 4-HC + YJZYD + PD98059 group [cells were cultured with 4-HC (8 μm), YJZYD solution (1 mg/mL), and PD98059 (5 μm) [19] for 24 h], the KGN + 4-HC + YJZYD + Vehicle group [cells were treated with 4-HC (8 μm), YJZYD solution (1 mg/mL), Vehicle [10% DMSO + 90% (20% SBE-β-CD in Saline)] equivalent to PD98059 for 24 h], the KGN + 4-HC + TPA group [cells were subjected to treatment with 8 μm 4-HC and 200 nM TPA [31] for 24 h], and the KGN + 4-HC + Vehicle 2 group (cells were treated with 8 μm 4-HC and Vehicle equal to TPA for 24 h).

Cell counting kit-8 (CCK-8) assay

KGN cell suspensions (100 µL/well) were seeded onto a 96-well plate in an incubator at 37 °C containing 5% CO2 for pre-cultivation. Cell proliferative ability was assessed using the CCK-8 kit (Beyotime, Shanghai, China). Cells were added with 20 µL/well of CCK-8 solution and incubated at 37 °C for 1 h. The absorbance of 24 h, 48 h, and 72 h was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Shanghai, China).

Flow cytometry

Fluorescein isothiocyanate-Annexin V Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA) was used to evaluate the apoptosis of KGN cells treated for 24 h. Cells were harvested by trypsin detachment and cultured in the presence of AnnexinV/propidium iodide at 20–25 °C for 20 min. Cell apoptosis was analyzed using a flow cytometer (FACScan, BD Biosciences) equipped with Cell Quest 3.0 software.

Western blot

The total protein solution was extracted by lysis in radioimmunoprecipitation assay buffer (Beyotime). The protein concentration was ascertained using the bicinchoninic acid protein assay kit (Beyotime). An equal amount of protein (50 µg) onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific) for the separation of the target protein. The separated protein was then shifted to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). Thereafter, the membrane was interacted with primary antibodies Anti-Bcl-2 (1:1000, ab32124, Abcam, Cambridge, UK), Anti-Bax (1:1000, ab32503, Abcam), Anti-Cleaved-caspase-3 (1:500, ab32042, Abcam), Anti-phospho (p)-Drp1 Ser616 (1:1000, ab314755, Abcam), Anti-Drp1 (1:1000, ab184247, Abcam), Anti-MFN1 (1:1000, ab191853, Abcam), Anti-MFN2 (1:2000, ab205236, Abcam), Anti-ERK1/2 (1:10000, ab184699, Abcam), Anti-p-ERK1/2 (1:1000, ab201015, Abcam) overnight at 4 °C. Subsequently, the membrane was washed before being interacted with horseradish peroxidase-labeled goat-anti-rabbit secondary antibody (IgG, 1:2000, ab205718, Abcam) at room temperature for 1 h, and then detected using enhanced chemiluminescence. Image J software (National Institutes of Health, Bethesda, MD, USA) was applied to perform grayscale analysis on the bands, with GAPDH as the internal reference. Each experiment was repeated thrice.

Mitochondrial staining

MitoTracker Green reagent (Beyotime) was used to determine the number of mitochondria in each group of KGN cell treated for 24 h. After various treatments, cells were washed with 1 × phosphate-buffered saline (PBS) three times and incubated with MitoTracker Green reagent for 30 min. Finally, after 3 washes with 1 × PBS, cells were subjected to Hoechst staining for 10 min and imaging under a fluorescence microscope (Zeiss). The mitochondria number was counted using Image J (National Institutes of Health), and the average number of mitochondria in each cell was analyzed.

Statistical analysis

All data were statistically analyzed and plotted using GraphPad Prism 8.01 (GraphPad Software, San Diego, CA, USA). The Shapiro-Wilk test method was used to test the normal distribution. The measurement data of the normal distribution were expressed as mean ± standard deviation (SD), and the paired t test was used for inter-group comparisons. Non-normal distribution measurement data were expressed in quartiles, and inter-group comparisons were conducted using the Wilcoxon matched-pairs signed rank test. One-way analysis of variance (ANOVA) was adopted for multi-inter group comparisons, and Tukey’s multiple comparisons test was utilized for post hoc testing. The difference was statistically significant with P < 0.05.