Molecular docking prediction

mTOR structure was determined using the Protein Data Bank (https://www.rcsb.org). The Traditional Chinese Medicine Systems Pharmacology database was used to obtain dhBBR structure data file. Docking results of mTOR/dhBBR were visualized using the Pymol 2.4.0 software (Schrödinger, USA).

POI mouse modeling and drug treatment

Sixty female BALB/c mice (7–8 weeks old; 18–22 g) were used in this study (Chengdu Dashuo Biotechnology Co., Ltd, China). A mouse model of POI was established via subcutaneous multisite injection of mouse zona pellucida glycoprotein 3 (pZP3) (Chinese Peptide Co., Ltd, China). Briefly, pZP3 lyophilized powder was dissolved in double-distilled water and mixed with an equal volume of Freund’s complete adjuvant (Sigma, USA) to achieve a working concentration of 50 nmol/L. Each mouse received 0.1 mL of pZP3 via subcutaneous injection at multiple sites, including the toes and abdomen. Two weeks after initial immunization, the same method was used for second subcutaneous injection of 0.1 mL pZP3 per mouse with Freund’s incomplete adjuvant. Vaginal cytology smears were performed 5–6 weeks after first immunization. Two consecutive disrupted estrous cycles and significantly increased anti-zona pellucida antibody levels confirmed successful establishment of the POI model. A normal estrous cycle involved a regular series of pre-estrus, estrus, and post-estrus phases separated by four or five days. Any pattern outside this regular cycle was considered abnormal (Carolino et al. 2019). This study was approved by the Animal Ethics Committee of Fujian Medical University (No. IACUC FJMU 2023 − 0214).

In the 8th week after initial immunization, sixty mice were randomly divided into the following groups described below. Group 1: Control, POI, POI + L-dhBBR, POI + M-dhBBR, and POI + H-dhBBR; mice in the POI group received daily oral administration of 12.5, 25, and 50 mg/kg dhBBR. Group 2: Control, POI, POI + dhBBR, POI + dhBBR + recombinant IL-17 (rIL-17), and POI + dhBBR + anti-CD25 monoclonal antibody (aCD25mAb); mice in the POI group received daily oral administration of 50 mg/kg dhBBR. Additionally, treatment groups received intraperitoneal injections of 100 ng/mice IgG, 100 ng/mice rIL-17, and 250 µg/mice aCD25mAb thrice a week. Treatment continued for four weeks. At the end of the experiment, the mice were anesthetized with isoflurane, their blood was collected, and they were euthanized. Their spleens and ovaries were harvested for analysis.

CD4 + T cell differentiation and drug treatment

Primary CD4 + T cells were purchased from Biobw (Bio-53558; Changsha, China) and cultured in the Roswell Park Memorial Institute-1640 medium (R8758, Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, and 50 µM 2-mercaptoethanol. After reaching the logarithmic growth phase, CD4 + T cells were differentiated into Treg and Th17 cells under conditions described below. For the Treg cell differentiation, CD4 + T cells were mixed with the anti-CD3 (2 µg/mL), anti-CD28 (1 µg/mL), anti-IL-2 (10 ng/mL), and anti-transforming growth factor (TGF)-β (8 ng/mL) antibodies and cultured for three days. For Th17 cell differentiation, CD4 + T cells were mixed with the anti-CD3 (2 µg/mL), anti-CD28 (1 µg/mL), anti-IL-23 (20 ng/mL), anti-IL-6 (20 ng/mL), anti-TGF-β (2 ng/mL), anti-interferon (IFN)-γ (5 µg/mL), and anti-IL-4 (5 µg/mL) antibodies and cultured for four days.

After differentiation, the cells were treated with different doses of dhBBR or MHY1485 (mTOR agonist; 5 μm). The cells were divided into groups as described below. Group 1: Control, Treg-induced, Treg-induced + L-dhBBR (0.25 µM), Treg-induced + M-dhBBR (0.5 µM), and Treg-induced + H-dhBBR (1 µM). Group 2: Control, Th17-induced, Th17-induced + L-dhBBR (0.25 µM), Th17-induced + M-dhBBR (0.5 µM), and Th17-induced + H-dhBBR (1 µM). Group 3: Control, dhBBR (1 µM), Treg-induced, Treg-induced + dhBBR, Th17-induced, and Th17-induced + dhBBR. Group 4: Th17-induced, Th17-induced + dhBBR, and Th17-induced + dhBBR + mTOR agonist. Group 5: Treg-induced, Treg-induced + dhBBR, and Treg-induced + dhBBR + mTOR agonist.

Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining

Ovaries and spleens were removed from mice and fixed with 4% paraformaldehyde (158127, Sigma-Aldrich, USA), dehydrated, and cut in 5-um sections. Ovarian samples were used for H&E and IHC staining of cleaved-caspase 3 and Ki-67. Spleen samples were subjected to GTP-Rheb IHC staining. hematoxylin (ST2067, Beyotime, China) and eosin (YE2080, Hefei Bomei Biotechnology Co., Ltd, China) were used to perform H&E staining, according to the manufacturer’s instructions. For IHC staining, tissue sections were incubated with 3% H2O2 for 10 min at room temperature. After blocking with bovine serum, the samples were incubated with primary antibodies against cleaved-caspase 3 (1:100; GB11532; ServicebioChina), Ki-67 (1:400; Cat. HA721115; HUABIOChina ), and GTP-Rheb (1:100; Cat. 15924-1-AP; ProteintechUSA) at 4 °C overnight. After washing thrice with phosphate-buffered saline (PBS), the samples were incubated with the secondary antibodies (1:100; Cat. GB23303; ServicebioChina ) at 37 °C for 30 min. Finally, changes were visualized using the DAB kit (1:20; Cat. ZLI-9018; ZsbioChina). Images were analyzed using an image analysis system (Halo 101-WL-HALO-1; Indica Labs) and Digital Trinocular Camera Microscope.

Enzyme-Linked Immunosorbent Assay (ELISA)

Mouse aZP (ZC-38389; ZCIBIO, China), mouse IFN-γ (ZC-37905; ZCIBIO), mouse TGF-β1 (ZC-39043; ZCIBIOChina ), mouse IL-17 A (ZC-37971; ZCIBIO), mouse tumor necrosis factor (TNF)-α (ZC-39024; ZCIBIO), mouse IL-10 (ZC-37962; ZCIBIOChina ), Mouse E2 (ZC-38046; ZCIBIO China), mouse FSH (ZC-38059; ZCIBIOChina), mouse IL-23 (ZC-37979; ZCIBIOChina), mouse IL-35 (ZC-37985, ZCIBIOChina ), and mouse anti-Mullerian hormone (AMH; ZC-38503; ZCIBIO, China ) ELISA kits were used to determine the protein expression levels in the serum samples of each mouse group. SpectraMAX Plus384 enzyme marker (Molecular Devices, Santa Clara, CA) was used to measure the optical density at 450 nm.

Quantitative Polymerase Chain Reaction (qPCR) assay

Next, mRNA levels of cytochrome P450 (Cyp)-17a1, Cyp19a1, Cyp11a1, steroidogenic acute regulatory protein (Star), and luteinizing hormone receptor (Lhr) in mouse ovaries and Foxp3, TGF-β1, IL-10, retinoic acid-related orphan receptor (ROR)-γt, IL-17, IL-22, and Rheb in CD4 + T cells were quantified via qPCR. Total RNAs in ovarian tissues and CD4 + T cells were extracted using the Molpure Cell/Tissue Total RNA Kit (19221ES50; YEASEN, Shanghai, China), and reverse-transcribed using the PrimeScript RT reagent Kit (RR047A; Takara, Japan). TB Green Premix Ex Taq II (RR820A; Takara) was used to perform qPCR, and QuantStudio Design & Analysis SE Software (Thermo Fisher, USA) was used to analyze the CT value; fold change was normalized using the 2−△△CT method. All primers are listed in Table 1. PCR conditions were set as follows: 95 °C for 30 s, followed by 45 cycles at 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s.

Table 1 Primers in this studyCell Counting Kit (CCK)-8 assay

Log-phase CD4 + T cells were collected and washed with PBS. CD4 + T cells (5 × 103) were seeded in a 96-well-plate and cultured at 37 °C and 5% CO2. Then, dhBBR (0, 0.01, 0.1, 1, 10, and 100 µM) was added for 24 h, followed by the addition of CCK-8 working solution (no. BS350B; Biosharp, China) for 2 h at 37 °C and 5% CO2. Optical density was measured at 450 nm.

Flow Cytometry (FCM) assay

Ovarian and spleen tissues were washed with PBS and ground. The cell suspension was collected, filtered through a 200-mesh cell sieve, centrifuged, and washed. Then, log phase CD4 + T cells were collected, and cultured at 2 × 105 cells/well with drugs for Treg or Th17 induction. After mixing with the cell activation cocktail, the cells were cultured for 4 h at 37 °C and centrifuged at 350 × g, and the supernatant was discarded. Cells mixed with 100 µL PBS were incubated with anti-CD4 and anti-CD25 antibodies for 30 min at 4 °C in the dark. Then, 500 µL 1× True Nuclear Fixation Concentrate was added to each well and incubated for 50 min. After washing, FOXP3 and IL-17 A labeling was performed for 30 min at 4 °C. Finally, data were analyzed using the Flow Analyzer (Cytoflex; Beckman, USA).

Western Blotting (WB) assay

Spleens from each POI mouse group were ground, and CD4 + T cells were collected to assess the Rheb/mTOR pathway. Tissues and cells were lysed using the radioimmunoprecipitation assay lysis buffer. Protein concentration was determined using the BCA Protein Assay Kit (P0009; Beyotime, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was conducted to separate the protein sample (20 µg), which was transferred to the PDGF membrane. After incubating with primary antibodies against TOR (1:5000; 66888-1-Ig; Proteintech, USA), p-mTORSer2448 (1:2000; 67778-1-Ig; Proteintech, USA), p70S6K (1:1000; A2190; Abclonal, China), p-p70S6K (1:1000; AP0502; Abclonal), 4E-BP1 (1:1000; A24691; Abclonal, China), p-4E-BP1 (1:2000; AP1363; Abclonal), Rheb (1:1000; 13879; Cell Signaling Technology), RORγt (1:1000; bs-23109R; Bioss, China), and β-actin (1:5000; AC026; Abclonal, China) 4 °C overnight, the membrane was washed with PBS and incubated with HRP goat anti-mouse IgG (H + L) (1:5000; AS003; Abcam) and goat anti-aabbit IgG (H + L) HRP (1:5000; S0001; Affbiotech, China) secondary antibodies at room temperature for 2 h. Densitometric analysis was performed using a Gel Imaging System (Analytik Jena US LLC).

Co-Immunoprecipitation (Co-IP) assay

Co-IP was performed to determine Rheb protein enrichment in CD4 + T cells and binding between Rheb and mTOR in 293T cells. For CD4 + T cells, IP was performed using antibody-conjugated agarose beads against GTPγS and GDP. The cells were divided into seven groups: GDP, GTPγS, GTPγS + 1 µM dhBBR, GTPγS + Treg-induced, GTPγS + Treg-induced + dhBBR, GTPγS + Th17-induced, and GTPγS + Th17-induced + dhBBR groups. Then, 293T cells were transfected with Myc-tagged Rheb expression plasmid (Myc-Rheb) for 24 h and co-incubated with 0.25, 0.5, and 1 µM dhBBR for 24 h. CD4 + T or 293T cells (CL-0005; Wuhan) from each group were collected and washed thrice. Then, 106 cells from each group were mixed with the 100 µL IP lysis buffer (including protease inhibitor) to lyse the cells on ice for 30 min. A co-IP kit (PK10007; Proteintech) was used for analysis. Subseqeuntly, 3 mg whole-cell lysates was incubated with specific antibodies against Rheb (13879; Cell Signaling Technology) and rabbit IgG (A7016; Beyotime) overnight at 4 °C. Protein A Sepharose bead slurry was used to precipitate the immune complexes, and spin columns were used to elute the products. Then, products (10 µL) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the proteins. WB was performed using HRP-conjugated protein A (PK10007; Proteintech), anti-mTOR (66888-1-Ig; Proteintech), and anti-Myc (ab32; Abcam) antibodies, according to the manufacturers’ protocol.

Immunofluorescence (IF) assay

IF was performed to determine GTP-Rheb levels in CD4 + T cells treated with dhBBR with Treg or Th17 induction. After 20-min serum bloking, the cells in each group were incubated with the GTP-Rheb antibody (1:100; 15924-1-AP; Proteintech) overnight at 4 °C. The sections were washed thrice and incubated with the FITC-labeled goat anti-rabbit antibody (1:100; GB22303; Servicebio), followed by incubation with 4’,6-diamidino-2-phenylindole (G1012; Servicebio) for 10 min. Digital Scanning Viewing Software (ImageJ; National Institutes of Health) and Data Image Analysis System (OlyVIA; OLYMPUS) were used to analyze GTP-Rheb expression.

Statistical analyses

GraphPad Prism 8.0 (GraphPad Software, Inc. USA) was used to analyze the data. All data are represented as the mean ± standard deviation. Statistical significance was set at P < 0.05. One-way analysis of variance, followed by Tukey’s multiple comparisons test was used to compare multiple groups, whereas an unpaired two-tailed Student’s t-test was used to compare two groups.