Abstract
Background: Premature ovarian insufficiency (POI) is a challenging condition with limited effective treatments. Adipose-derived stem cells (ADSCs) have demonstrated potential in tissue repair, and their extracellular vesicles (ADSC-EVs) show more safety in clinical translation. However, the role and mechanism of ADSC-EVs in the treatment of POI are not yet fully understood. This study aims to investigate the protective effects of ADSC-EVs on rat POI models induced by 4-vinylcyclohexene diepoxide (VCD) and to explore the potential therapeutic mechanisms. Methods: Rat ADSCs and ADSC-EVs were isolated and characterized. The POI rat model was established via intraperitoneal injection of VCD for 15 consecutive days. ADSCs and ADSC-EVs were injected into the ovaries for treatment. Ovary function was assessed by monitoring estrous cycles, follicle counts, sexual hormone levels, and ovulation. Molecular mechanisms were investigated using TUNEL staining, immunohistochemistry, quantitative polymerase chain reaction, and western blotting. In vitro, primary rat granulosa cells were treated with VCD in the presence or absence of ADSC-EVs. Cell proliferative ability, hormone secretion, apoptosis rate, and relative molecular expression were measured. Whole-transcriptome sequencing and DIA proteomics of ADSC-EVs were performed to identify bioactive molecules. Results: ADSC-EVs protected granulosa cells from VCD-induced apoptosis, promoted cell proliferation, enhanced hormone secretion, and upregulated KITL expression. Treatment with ADSCs and ADSC-EVs in POI rats significantly improved estrous cycles, follicle counts, and serum levels of estradiol (E2), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH). These treatments activated the KITL/KIT/PI3K/AKT signaling pathway, downregulated pro-apoptotic genes (Bax, Caspase3), and upregulated anti-apoptosis genes (Bcl2). ADSC-EVs are highly enriched in mRNA of Kit and PI3K, and both transcriptomic and proteomics enrichment analysis predominantly focused on PI3K/AKT pathway. Conclusions: ADSCs and ADSC-EVs effectively protect and restore ovarian function in VCD-induced POI rats. The mechanism involves inhibiting apoptosis in granulosa cells and activating the KITL/KIT/PI3K/AKT pathway in ovary. ADSC-EVs, with advantages in clinical translation, hold significant potential for POI treatment.