Materials and reagents

The following list of reagents was mainly utilized in this study: VCD (Sigma, USA); YLZ herbs (Beijing Tong Ren Tang, China); Dulbecco's Modified Eagle Medium (DMEM) (Gibco, USA); D-hanks solution (AQ, China); Fetal Bovine Serum (FBS) (Gibco, USA); follicle-stimulating hormone receptor (FSHR) (Bioss, China); Immunoglobulin G (IgG) (ZSZSGBBIO, China); 4% polyformaldehyde (KeyGEN Biotech, China); Estradiol (E2) (Raybio, USA); follicle-stimulating hormone (FSH), E2, Anti-mullerian hormone (AMH) enzyme-linked immunosorbent assay (ELISA) (Elabscience, China); Seahorse Xfe 24 kit (Alicelligent, China); Adenosine Triphosphate (ATP) test kit (Beyotime, China); Guanidine isothiocyanate (TRIzol) (Vazyme, China); RIPA Lysis Buffer (RIPA) (Beyotime, China); Polyvinylidene Fluoride (PVDF), Enhanced Chemiluminescence Reagent (ECL) (Millipore, USA); CX43 antibody (Servicebio, China); HIF1α antibody (Affinity, USA); Phospho-Thr981 antibody (PEK), Hexokinase1 antibody (HK1) (Proteintech, USA); Lactate Dehydrogenase antibody (LDH) (WANLEIBIO, China); Glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH) (CST, USA).

Preparation of YLZ and drug-containing serum

YLZ herbs were weighed as the Table 1. Firstly, Lu Jiaoshuang (LJS) was boiled 30 min, ten times the amount of distilled water was added, followed by 30 min of soaking, 45 min of boiling, filtering, and second boil with 8 times the amount of distilled water, Ren Shen (RS) was boiled 1 h separately, filtering, the three solutions were finally mixed and the drug concentration of YLZ was 1.1 g/ml, finally, stored at 4℃after it cooled naturally. Mixing the estradiol valerate tablets and distilled water into suspension completely, and stored. After one week of adaptive feeding, thirty female Sprague Dawley (SD) rats (300 ± 50 g, 8–9 weeks old, purchased from Beijing WTLH, China, Experimental Animal Use License No. SCXK (Beijing) 2021–0011) Approval Number of Animal Ethics Committee of Capital Medical University: AEEI-2021–131) were randomly divided into 3 groups (n = 10/ group): normal serum group (given the same volume of distilled water as YLZ), YLZ serum group (10.08 g/kg, twice a day) and estradiol serum group (0.1008 mg/kg, twice a day). All drugs were administered intragastric for 5 days. The doses of YLZ and estradiol valerate tablets were converted from clinical human doses to rat doses and combined with our findings from earlier studies. One hour after the last gavage, the rats were anesthetized with isoflurane, and blood was collected from the rat's abdominal aorta and centrifuged at 4℃ and 3000 rpm for 15 min. The complement was inactivated (56℃, 30 min) and bacteria were removed through a 0.22 μm filtration membrane, and then stored at -80℃ for subsequent experiments.

Table 1 Composition of YLZUltra High Performance Liquid Chromatography-Q Exactive-Mass Spectrometer (UHPLC-QE-MS) analysis of YLZ and its drug-containing serum

The quality standards and stability of YLZ were detected by UHPLC-QE-MS. The composition of YLZ and its drug-containing serum were analyzed as follows: a 3 ml sample of three independently decocted YLZ was thawed on ice. After a 30 s vortex, the dection was centrifuged at 12000 rpm (Relative Centrifugal Force (RCF) = 13,800 (× g), Radius (R) = 8.6 cm) for 15 min at 4℃. 300μL of supernatant was transferred to a fresh tube and 1000μL of extracted solution containing 10 μg/mL of internal standard was added, then the samples were sonicated for 5 min in an ice-water bath. After placing 1 h in -40℃, the samples were centrifuged at 12000 rpm (RCF = 13,800 (× g), R = 8.6 cm)for 15 min at 4℃. The supernatant was carefully filtered through a 0.22 μm microporous membrane, then take 200μL from each sample and pooled as Quality Control (QC) samples. Store at -80℃ until the UHPLC- MS analysis.

Four hundred μL of three independent blank serum and YLZ-containing serum samples were added to 40μL of hydrochloric acid(2 mol/L), then the mixture was vortexed for 1 min and followed by incubated for 15 min at 4℃. The vortex and incubate cycle was repeated for 4 times. Add 1.6 mL acetonitrile, then the mixture was vortexed for 5 min and the samples were centrifuged at 12000 rpm (RCF = 13,800 (× g), R = 8.6 cm) for 5 min at 4℃. 1800μL of supernatant was transferred to a fresh tube and nitrogen-dried. The dried samples were reconstituted in 150μL of 80% methyl alcohol containing 10 μg/mL of the internal standard by vortex for 5 min. The constitution was then centrifuged at 12,000 rpm (RCF = 13,800 (× g), R = 8.6 cm) for 5 min at 4℃, and 120μL of supernatant was transferred to a fresh glass vial for LC/MS analysis.

LC–MS/MS analysis was performed on an UHPLC system (Vanquish, Thermo Fisher Scientific) with a Waters Ultra Performance Liquid Chromatography Ethylene Bridged Hybrid (UPLC BEH) C18 column (1.7 μm 2.1*100 mm). The elution condition of the mobile phase (A: water; B: acetonitrile) was as Table 2. An Orbitrap Exploris 120 mass spectrometer coupled with Xcalibur software was employed to obtain the MS and MS/MS data based on the Ionospheric Dispersion Analysis (IDA) acquisition mode. During each acquisition cycle, the mass range was from 100 to 1500, the top four of every cycle were screened and the corresponding MS/MS data were further acquired. Sheath gas flow rate: 30 Arb, Aux gas flow rate: 10 Arb, Ion Transfer Tube Temp: 350 ℃, Vaporizer Temp: 350 ℃, Full ms resolution: 60,000, MS/MS resolution: 15,000, Collision energy: 16/32/48 in NCE mode, Spray Voltage: 5.5 kV (positive) or -4 kV (negative).

Table 2 The elution condition of the mobile phase (A: water; B: acetonitrile)Animals and experimental protocol

In vitro experiments, twenty-four 8–9-week-old SPF-grade SD female rats (Beijing WTLH, China, SCXK (Beijing) 2021–0011) were housed at the animal experiments building of Capital Medicine University. The environmental conditions were natural light, temperature of 22 ± 2℃, and humidity of 65 ± 5%. The whole rats had free space with enough water and feed. The animal experimental protocol was approved by the Animal Ethics Committee of Capital Medical University (NO.AEEI-2021–131). After seven days of acclimation culture, rats were divided into four groups: Control group, VCD model group (80 mg/kg), E2 valerate group (0.1008 mg/kg), YLZ medium dose group (10.08 g/kg), All groups except the control group were injected intraperitoneally with VCD for 15 days; the control group was replaced with an equal volume of saline. Meanwhile, the YLZ and E2 groups were gavaged with their corresponding drugs for 6 weeks; the control and VCD groups were gavaged with the same volume of saline.

Estrous cycle monitoring

Vaginal smears were made at 9 a.m. each day for 15 days in the process of model building. Small cotton swabs were soaked in saline, inserted into the vagina of a rat approximately 5 min, rotated clockwise 6 times, then rolled and smeared on a clean slide. After the slide was naturally air-dried, stained with Swiss Giemsa staining solution for approximately 5–10 min, rinsed with water, and observed under an ordinary optical microscope.

Hormone detection by ELISA

The serum was determined using ELISA assays for the level of FSH, E2, and AMH, following the instructions of the kit.

Hematoxylin and eosin (HE) staining

The ovaries were isolated and fixed in 4% polyformaldehyde for 48 h and then were processed for paraffin embedding and sectioning. Serial sections of 4 μm thickness were cut with a Leica RM2016 rotator microtome. The sections were dewaxed with xylene, rehydrated with graded concentrations of ethanol, and then stained with hematoxylin and eosin and observed via light microscopy.

Tissue immunofluorescence

Put the slices in 3 changes of xylene, for 10 min each, then dehydrate in 3 changes of pure ethanol for 5 min each, and wash in distilled water. After repairing antigen was completed, it is naturally cooled. Put the slides 5 min Phosphate Buffered Saline (PBS) (PH7.4) and shake it decoloring shaker 3 times, each time for 5 min. Adding 3% Bovine albumin (BSA) into the tissue and cover it evenly to block, non-specific binding at room temperature for 30 min. (The primary antibody is blocked with 10% donkey serum from goat, and the primary antibody from other sources is blocked with 3% BSA). Adding primary antibody and secondary antibody. The 4',6-diamidino-2-phenylindole (DAPI) solution was dripped into the tissue and incubated at room temperature for 10 min in the dark. Add autofluorescence quencher B solution for 5 min and rinse with running water for 10 min. The Anti-fluorescence quenching was used to seal slides, and collecting images by Fluorescent Microscopy.

Primary ovarian GCs culture

Female SD rats (21–25 days old, 50 ± 5 g) were subcutaneously injected with pregnant mare serum gonadotropin (PMSG) 50 IU and were sacrificed 48 h later. Removed ovaries were washed immediately with D-Hanks and placed in DMEM medium. GCs were harvested in the medium by 1 ml needle puncture of ovarian follicles and then purified by filtration with 40 μm disposable cell filter mesh. After the centrifugation at 1000 × g for 5 min, the cells were resuspended in medium and counted in a hemocytometer. The cells were seeded in a T25 cell culture bottle and cultured in DMEM medium supplemented with 10% FBS and 1% green streptomycin mixture at 37 ℃ and 5% CO2 for 48 h to allow the cells to attach. Cells of logarithmic growth stage were taken and divided into control group, model group (0.5 mM VCD), estradiol group (0.5 mM VCD + 5% E2 containing serum), YLZ group (0.5 mM VCD + 5% YLZ containing serum), HIF1α inhabition group (0.5 mM VCD + 0.5 μM echinomycin (Echi)), HIF1α inhabition + YLZ group (0.5 mM VCD + 0.5 μM Echi + 5% YLZ containing serum).

Identification of primary ovarian GCs

Cell immunofluorescence identified the primary ovarian GCs. Cells were inoculated in a 24-well plate with a cell patch, After cells had grown to 60% confluence, slides were washed with PBS, fixed in 4% paraformaldehyde, and then permeabilized with 0.5% TritonX, incubated with FSHR overnight, incubated with IgG the next day, dried and mounted, finally, observed and captured picture under a fluorescence microscope.

Cell proliferation viability determination

Cell Counting Kit-8 (CCK-8) assay measured the cell proliferation viability. After the cells were successfully attached, E2 and YLZ groups were given drugs respectively for 24 h, 48 h, and 72 h, At the end of theculture, cells of the E2 group and YLZ group in 96-well plates were incubated in 100μL DMEM supplemented with 10 μL CCK-8 reagent for 2.5 h at 37℃ and 5% CO2 incubator. The optical density (OD) value of each well was measured at a wavelength of 450 nm by a multiscan spectrum. The cell proliferation viability = (the OD value of the test group well -the mean OD value of the blank group)/(the OD value of the negative control group well-the mean OD value of the blank group). Each group was established in four wells.

ATP level detection

ATP level detection by the chemiluminescence ATP assay kit, according to the manufacturer’s recommendations.

Cell energy metabolism determination by Seahorse XFe24

The glycolytic metabolic rate was measured on a seahorse XFe24 energy Analyzer(Seahorse Bioscience, USA)0.5 × 104 cells were seeded in an XFe24 culture microplate(Alicelligent, China), and detection of the Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR) value according to the operating instructions.

RT-qPCR

The mRNA expression of HIF1α, Cx43, LDH, HK1, and PEK were detected by RT-qPCR. Granulosa cells were cultured in 6-well plates, Extraction of total RNA was performed with TRIzol reagent after the end of cell intervention in each group. Then the RNA was further reverse-transcribed into cDNA. The following conditions were used for reverse transcription: 42 ℃ for 15 min, 85 ℃ for 5 s, cooling to 4℃ for 5 min, and refrigeration at -20 ℃. The reverse transcription reaction mixture was added to the tubes used for real-time fluorescence quantitative reaction, and the amplification reaction system was as follows: pre-denaturation at 95 ℃ for 10 min, followed by 40 cycles (95 ℃ for 5 s and 60 ℃ for 60 s). The reaction results were analyzed by the relative fold change using the 2 −△△CT method. Table 3 shows the primer sequences used for real-time fluorescence quantitative reaction of the genes. GAPDH was used as the internal reference gene.

Table 3 Primer seguencesWestern blot analysis

The protein expression levels of HIF1α, Cx43, LDH, HK1, and PEK were detected by Western blot. In brief, RIPA lysate and protease inhibitor were added to the cells of each group, centrifuged at 4℃, 12000 rpm for 15 min, and the supernatant was obtained. The protein concentration was determined according to the instructions of the Bicinchoninic Acid (BCA) protein quantitative kit. The total protein was separated by 10% Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane on ice. And then, the membrane was blocked with 5% skim milk at room temperature for 2 h. The primary antibody was added and incubated overnight at 4℃ and the horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG antibody for 1 h at room temperature. The membranes were exposured with ECL. The relative optical density was assessed by ImageJ software.

Statistical analysis

Data of the study were showed as mean ± standard deviation (SD), and data were analyzed by SPSS 19.0. One-way Analysis of Variance (ANOVA) was used to analyze statistical differences between different groups. Multiple comparisons were used in post hoc analysis, Least Significant Difference (LSD) was used for homogeneous variances, and Tamhane T2 was used for heterogeneous variances. P < 0.05 was identified as a statistically significant difference.