Electroacupuncture Ameliorates Premature Ovarian Insufficiency by Inhibiting METTL3-Mediated m6A Methylation of Beclin 1 mRNA in Granulosa Cells.

Abstract

Premature ovarian insufficiency (POI) is a major cause of infertility in young women. In this study, we investigated the therapeutic effects of electroacupuncture (EA) on ovarian function and granulosa cell autophagy in a high-fat, high-sugar (HFHS) diet-induced POI mouse model. POI was established by HFHS feeding, and mice received EA stimulation at CV4, ST36, and SP6. Ovarian morphology was evaluated using hematoxylin and eosin (HE), Masson's trichrome, and Sirius Red staining. Estrous cycles were monitored by crystal violet staining, and serum sex hormones and inflammatory cytokines were measured by ELISA. Cholesterol accumulation, apoptosis, and FSHR expression were assessed using filipin staining, TUNEL assays, and immunofluorescence, respectively. Single-cell RNA sequencing was performed to characterize ovarian cell subpopulations and autophagy-related transcriptional signatures. Autophagosome formation and the expression of autophagy-related markers (Beclin1, LC3, and P62), as well as METTL3, were analyzed by transmission electron microscopy (TEM), RT-qPCR, Western blotting, and immunohistochemistry. m6A modification levels on Beclin1 mRNA were determined using LC-MS, colorimetric assays, and RIP-qPCR. The results demonstrated that EA significantly improved follicular development, normalized estrous cycles, reduced granulosa cell apoptosis, and decreased inflammatory cytokine levels in POI mice. Single-cell analysis revealed that granulosa cells exhibited the highest autophagy activity and were strongly associated with senescence-related pathways. EA markedly reduced autophagosome formation, downregulated Beclin1 and LC3 expression, and upregulated P62, indicating suppression of excessive autophagy. Furthermore, EA decreased METTL3 expression and reduced m6A modification of Beclin1 mRNA, thereby limiting Beclin1 translation. Collectively, these findings suggest that EA ameliorates HFHS-induced POI by inhibiting METTL3-mediated m6A methylation of Beclin1 mRNA, suppressing excessive granulosa cell autophagy, and restoring ovarian function, thereby revealing a potential epigenetic mechanism underlying the therapeutic effects of EA in diet-related ovarian dysfunction.