Cell lines and cell culture

Procell Life Science & Technology in China provided mouse BMSC and the human granulosa-like tumor cell line KGN. The Translational Medicine Research Center at Zhujiang Hospital in China provided the HEK293T cells. The culture media used for BMSC was Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Gibco, USA), which contained 1% penicillin–streptomycin and 10% fetal bovine serum (FBS). KGN cells were grown in DMEM/F12 media (produced in China by KeyGEN BioTECH), which was enhanced with 1% penicillin–streptomycin and 10% foetal bovine serum. 10% FBS and 1% penicillin–streptomycin were added to high-glucose DMEM (KeyGEN BioTECH, China) for the cultivation of HEK293T cells. The incubation was conducted at a temperature of 37 °C in a controlled environment with 95% oxygen and 5% carbon dioxide.

To prepare KGN cells, culture them in dishes and incubate at 37 °C with 5% CO2 for 24 h. Once the adhesion rate reaches 50–70%, randomly assign the cells into four experimental groups: the control group (NC group), the cisplatin-induced damage model group (CDDP group), the group treated with Exo (CDDP + Exo group), and the group treated with miR-21-Exo (CDDP + miR-21-Exo group). The NC group receives no treatment. The CDDP, CDDP + Exo, and CDDP + miR-21-Exo groups are administered 5 mg/L of cisplatin (Jiangsu Haosen Pharmaceutical Group Co., Ltd., China). After 24 h, the cisplatin-containing medium is replaced with fresh medium. The CDDP + Exo and CDDP + miR-21-Exo groups are then incubated for an additional 24 h with 30 μg/mL of Exosomes or miR-21-Exosomes. The CDDP group is given an equivalent volume of phosphate-buffered saline (PBS).

Transfection of cells

A lentiviral vector (LV-hsa-mir-21) from GeneChem (Shanghai) was used to add miR-21 to both KGN cells and BMSC. We used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to measure the levels of miR-21 in BMSC and their Exo after infection. In the experiment, granulosa cells overexpressing miR-21 (LV-miR-21-KGN) and control granulosa cells (LV-NC-KGN) were treated with or without cisplatin. The experiment was divided into four groups: the empty vector group (LV-NC), the miR-21 virus transfection group (LV-miR-21), the empty vector plus cisplatin group (LV-NC + CDDP), and the miR-21 virus transfection plus cisplatin group (LV-miR-21 + CDDP).

Bioinformatics research showed which genes miR-21 targets and suggested that miR-21 would bind to the 3' untranslated region (UTR) of MALT1 mRNA. There were four separate groups in the rescue tests. These were the NC group, the CDDP group, the CDDP + miR-21-Exo group, and the CDDP + miR-21-Exo + OE-MALT1 group. Before adding the exosomes, transfection was done. The MALT1 overexpression plasmid was created by IGE Biotechnology in China and was made to contain only the coding region. This plasmid was transfected into the CDDP + miR-21-Exo + OE-MALT1 group. Following the manufacturer's instructions, the transfection process was carried out using LipoMax transfection reagent (IGE Biotechnology, China). Using an empty vector plasmid, the NC group, CDDP group, and CDDP + miR-21-Exo group were transfected.

For the 293T cells, the experimental group was transfected with miR-21 mimic, while the control group was transfected with miR-21 NC. The miR-21 mimic and miR-21 NC from IGE Biotechnology in China were used for transfection, along with LipoMax transfection reagent, following the manufacturer's instructions.

The processes of isolating, identifying, labeling, and detecting Exo and miR-21-Exo are performed

To isolate miR-21-Exo or Exo from the serum-free culture supernatants of miR-21-BMSC or BMSC, ultracentrifugation is employed. First, the collected supernatant is centrifuged at 300 g for 10 min, followed by 2000 g for 10 min, and then 10,000 g for 30 min to remove dead cells and debris. The resulting supernatant is then centrifuged at 100,000 g for 70 min to pellet the exosomes. The pellet is resuspended in PBS for further analysis. The morphology of the exosomes is examined using transmission electron microscopy (TEM), their size is measured with a Flow NanoAnalyzer from NanoFCM, and their surface markers are analyzed via western blotting.

The exosomes were labeled with the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR; Molecular Probes, USA). The labeled exosomes were washed with PBS at 100,000 g for 70 min. They were then co-incubated with CDDP-stimulated KGN cells for 24 h. Afterwards, the KGN cells were treated with a 4% solution of paraformaldehyde to preserve them, and their nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Solarbio, China). Nikon produced a confocal microscope that was used to visualize exosome uptake.

Creation of a rat model with chemotherapy-induced POI and administration of Exo or miR-21-Exo by injection

Female Sprague–Dawley (SD) rats, aged 7–8 weeks and weighing 180-220 g, were acquired from the Experimental Animal Center of Southern Medical University. The rats were housed in a regulated environment with a temperature of 30 ± 2 °C and a 12-h cycle of light and darkness. They had unrestricted access to food and water. This study was approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University, accepted this study (Approval No. LAEC-2022–208) and ensured compliance with all ethical criteria.

A total of thirty-two female rats with regular estrous cycles were randomly assigned to four groups: the NC group (n = 8), the POI group (n = 8), the POI + Exo group (n = 8), and the POI + miR-21-Exo group (n = 8). The chemotherapy-induced POI model was established by administering cisplatin solution intraperitoneally at a dosage of 1 mg/kg for a duration of 14 consecutive days (Qu et al. 2022). The rats in the NC group were given an equivalent amount of saline at the identical location.

Successful establishment of the chemotherapy-induced POI model was anticipated 14 days after the completion of cisplatin injections. After receiving cisplatin injections, rats in the Exo and miR-21-Exo groups were administered injections of 150 μg of Exo or miR-21-Exo, which were dispersed into 1 ml of PBS every other day for a total of 7 injections. In contrast, rats in the POI group were given injections of PBS. After a period of two weeks following the administration of Exo or miR-21-Exo, half of the rats were sacrificed. Samples of serum, ovarian tissue, and other organs including the heart, liver, spleen, lung, kidney, and uterine were obtained. The remaining female SD rats were co-housed in a ratio of two females to one male for 10 days, ensuring that they had enough water and food available. After the co-housing period, female and male rats were separated based on gender. Female rats were monitored for signs of pregnancy and litter size.

Animal imaging conducted in vivo

Following the injection of exosomes for in vivo monitoring, the rats were seen and monitored using the PerkinElmer IVIS Spectrum in vivo imaging system (China) at predetermined intervals of time (1 h, 3 h, 24 h, 48 h, 21 days, and 28 days). Fluorescent pictures showing the movement and distribution of DiR-miR-21-Exo were captured using light with a wavelength of 740 nm to excite the molecules and a wavelength of 790 nm to detect their emission. The images were then analyzed using PerkinElmer's Living Image 4.5.5 program.

Examination of the estrous cycle

Regular vaginal cytology checks were exams were performed to track the estrous cycles on a daily basis. Vaginal secretions were obtained by utilizing a small cotton swab, which was then treated with trypan blue dye (Biosharp, China) and observed using a bright-field upright microscope (Leica, Germany).

The structure and number of ovarian follicles

The ovarian tissue was preserved in 4% PFA for a period of 24 to 48 h and then encased in paraffin. Consecutive sections with a thickness of 5 μm were acquired, subjected to hematoxylin and eosin (H&E) staining, and examined using a 3D scanner.

Follicle counting involved consecutive sectioning with counting every fifth slice to tally the overall quantity of each type of follicle, resulting in a final count multiplied by 5 (Tilly 2003). Each sample group consisted of at least 3 rats for statistical analysis. Subsequently, using 3D scanner software, primordial, primary, secondary, and mature follicles were identified and quantified based on morphological features under 100 × magnification.

TUNEL analysis of ovarian

Follicle apoptosis was assessed using the TUNEL assay kit (Elabscience, China). The tissue was sectioned into 5 mm pieces and fixed in paraffin. Images were obtained using a fluorescence microscope after staining with fluorescein isothiocyanate (FITC) and DAPI. The data analysis was conducted using the ImageJ software.

Immunohistochemistry

The paraffin-embedded sections underwent treatment using rabbit polyclonal antibodies against Proliferating Cell Nuclear Antigen (PCNA), NACHT, LRR and PYD domains-containing protein 3 (NLRP3), and GSDMD, all at a dilution of 1:1000. The sections were then stained using secondary antibodies and diaminobenzidine (DAB) reagent. After being submerged in a solution of hematoxylin dye for a duration of 3 min, the sections were rinsed with water and then subjected to a 1-s process of differentiation in hydrochloric acid alcohol. Finally, the sections were incubated in PBS for a period of 1 min.

The proportion of cells that exhibited positive staining was assessed using a 3DHISTECH brightfield scanner from Hungary.

Enzyme-Linked Immunosorbent Assay (ELISA)

In order to identify the existence of estradiol (E2) in the fluids surrounding the cells, it is more effective to provide the testosterone substrate (Huang et al., 2020). After 48 h, the liquid portion of the cells was gathered, and the levels of estradiol were determined using ELISA kits from R&D Systems, USA. The ELISA kits were utilized to quantify the levels of E2, Follicle Stimulating Hormone (FSH), and anti-Müllerian hormone (AMH) in serum samples obtained on the 0th day, 28th day, and 42nd day after the initiation of cisplatin injection. A spectrophotometric analysis was performed using a BioTek microplate reader from the United States to measure the absorbance at a wavelength of 450 nm.

Cell proliferation experiment

We employed the Cell Counting Kit 8 (CCK8; APExBIO, USA) to assess cellular proliferation. The KGN cells were initially placed in a 96-well plastic plate. A 100 μL cell supernatant and a 10 μL CCK8 solution were added to every well. The plate was then kept in an incubator set at a temperature of 37 °C for a pperiod of 1 h. We assessed cell proliferation at 0, 24, 48, 72, and 96 h by measuring absorbance at 450 nm using a microplate reader.

Analysis using fluorescence-activated cell sorting

To detect apoptosis, cells were stained with V-FITC and propidium iodide (V-FITC/PI) using the apoptosis detection kit from Beyotime Biotechnology, China, following the instructions provided by the manufacturer. Fluorescence-activated cell sorting (FACS) was used to sort cells that had been stained.

qRT-PCR Analysis

RNA extraction was performed on cells or half of each mouse ovary using RNAex Pro reagent (Agbio, USA). The process of reverse transcription was conducted using Evo M-MLV RT Premix (Agbio, USA) and 500 ng of RNA. The qRT-PCR analysis was conducted using SYBR Pro Taq HS Premix (Agbio, USA) on a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). U6 was used as the internal control for miR-21, while GAPDH was used as the internal control for Follicle Stimulating Hormone Receptor (FSHR), Cytochrome P450 Family 11 Subfamily A Member 1 (CYP11A1), Cytochrome P450 Family 19 Subfamily A Member 1 (CYP19A1), Hydroxysteroid (17-beta) dehydrogenase 1 (HSD17B1), Steroidogenic Acute Regulatory Protein (STAR), NLRP3, CASP1, GSDMD, and MALT1. The data analysis was performed with the software provided by Bio-Rad. The primer sequences can be found in Supplementary Table 1.

Western blotting

Protease and phosphatase inhibitors were employed in conjunction with RIPA lysis buffer (Cwbio, China) to lyse exosomes, cells, and half of each rat ovary in order to extract proteins. The BCA protein assay kit (Beyotime Biotechnology, China) was utilized to ascertain the protein concentration. Following protein denaturation, electrophoresis was carried out using SDS-PAGE, and the gel was subsequently transferred to a PVDF membrane (Millipore, USA). After transfer, a 5% BSA solution (MCR, China) was used to block the membrane. It was then incubated with particular primary antibodies for an entire night. These antibodies included anti-CD9 (HuaBio, China), anti-TSG101 (HuaBio, China), anti-CD81 (Abmart, China), anti-NLRP3 (Abmart, China), anti-CASP1 (Abmart, China), anti-GSDMD (Abmart, China), anti-pP65 (Abmart, China), anti-pIKK (Abmart, China), anti-pIKB (Abmart, China), (Proteintech, China). Following a 1-h exposure to a secondary antibody from Cwbio (China), the membrane was cleaned using Tris-buffered saline–Tween (TBS-T; Boster, China). The detection technique was carried out using an electrochemiluminescence (ECL) apparatus manufactured by Beyotime Biotechnology in China. The reference standard was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using ImageJ software, protein levels were examined. The target protein's relative content in the samples was calculated by dividing its grayscale value by GAPDH's grayscale value.

Dual luciferase reporter gene analysis

The putative mRNA predicted for miR-21 miRNA were identified and compared using the online target prediction algorithms Tarbase (miRNA target gene database, https://dianalab.e-ce.uth.gr/tarbasev9, predicted 1588 miR-21 target genes), miRDB (miRNA target gene predictions, https://mirdb.org/, predicted 469 miR-21 target genes), DIANA tools (Various miRNA analysis tools, https://dianalab.e-ce.uth.gr/html/mirpathv3/index.php?r=mirpath, predicted 515 miR-21 target genes), miRTarBase (miRNA target gene database, primarily based on experimental validation, https://mirtarbase.cuhk.edu.cn/, predicted 625 miR-21 target genes), and miRWalk (miRNA target gene predictions and database, http://mirwalk.umm.uni-heidelberg.de/, predicted 2214 miR-21 target genes). The 27 genes closely associated with pyroptosis are derived from rectome (https://reactome.org/). The 55 genes that are closely associated with NF-κB signaling are sourced from GeneCards (https://www.genecards.org/).The miRDB website was used to estimate the binding sites of MALT1 and miR-21. The psiCHECK-2 reporter vector containing the wild-type (MALT1-wt) and mutant (MALT1-MUT) MALT1 constructs was used to build the reporter vector for the dual-luciferase reporter gene experiment. The MALT1-WT and MALT1-MUT reporter plasmids were simultaneously introduced into HEK293T cells together with either a miR-21 mimic or a control mimic (IGE Biotechnology, China) using Lipofectamine 3000 (Invitrogen, USA). The luciferase activity was assessed using the Dual-Luciferase Reporter Assay System provided by Beyotime Biotechnology (China) following a 48-h transfection period.

LDH Detection

The supernatant of the cell culture was collected and the concentration of lactate dehydrogenase (LDH) was measured using an LDH test kit from Nanjing Jiancheng Bioengineering Institute, China. The results were obtained using a spectrophotometer with a light path of 1 cm at a wavelength of 440 nm.

TEM examination of cell pyroptosis

Following the digestion of the cell samples with trypsin and subsequent centrifugation, they were allowed to remain at room temperature for 1 h. Subsequently, they were stored at a temperature of 4 °C until 12 h before to their submission for evaluation. After replacing the fixative with a PBS solution, the samples were sent for testing. The Center Laboratory of Southern Medical University processed these samples and viewed and photographed them using a Hitachi H-7500 transmission electron microscope.

Cryopreservation and Cryosectioning

Tissue samples are quickly frozen and immersed in cryoprotectant to solidify, then sliced into thin sections with a cryostat. The sections are placed on slides for DAPI staining, and fluorescence is observed using a confocal microscope.

In vivo toxicity assessment: hematological analysis and histology

For the purpose of assessing toxicity in living organisms, evaluations were performed on day 28 following the last injection. AST (aspartate aminotransferase), ALT (alanine aminotransferase), BUN (blood urea nitrogen), and creatinine levels were determined using commercially available assay kits obtained from Nanjing Jiancheng Bioengineering Institute, China. Tissue samples from many organs (including the heart, liver, spleen, lung, kidney, and uterine) were obtained 28 days after the final injection. The samples were conserved in a solution of containing 4% paraformaldehyde, thereafter encased in paraffin, sectioned into 5 μm slices, and stained with H&E for histological analysis.

Statistical Analysis

Data analysis was performed using SPSS Statistics 20 and GraphPad Prism 9.0.0. Average values and standard deviations (SD) were reported. The Student's t-test was used to compare two groups, while one-way analysis of variance (ANOVA) was applied for multiple groups. For normally distributed data, the least significant difference (LSD) method was used; for non-normally distributed data, Dunnett's T3 test was utilized. Statistical significance was defined as a P-value of less than 0.05. Each experiment was independently replicated at least three times.