Electro-acupuncture attenuates cyclophosphamide-induced premature ovarian insufficiency in rats through miRNA regulation and TGFβ/Smad pathway modulation

OBJECTIVE: To investigate the protective effects and molecular mechanisms of electro-acupuncture (EA) in a cyclophosphamide (CTX)-induced rat model of premature ovarian insufficiency (POI), focusing on ovarian microRNA (miRNA) regulation and TGFβ/Smad pathway modulation. METHODS: Twenty-four female Sprague-Dawley rats were classified into four groups: Control, CTX, CTX + EA, and CTX + GnRHa (positive control). POI was induced by CTX injection. EA was applied at six acupoints for 15 min daily (days 10-25). Ovarian function was evaluated through estrous cycle monitoring, histology, follicle counting, and serum hormone assays (FSH, E2, AMH). Serum ALT, AST, BUN, and UA were measured to assess organ toxicity. Ovarian ultrastructure was analyzed by TEM. Differential miRNA expression was profiled by small RNA sequencing and validated by qRT-PCR. Pathway analysis (KEGG, GO) and TGFβ/Smad signaling evaluation (qRT-PCR, western blot) were performed. RESULTS: EA significantly improved body weight and ovarian index (P < 0.01 vs. CTX) and reduced CTX-induced hepatic and renal injury markers. Regular estrous cycles were restored in 83% of EA-treated rats. EA normalized FSH, E2, and AMH levels (P < 0.01 vs. CTX), restored follicle counts and ovarian morphology, and reversed subcellular damage observed under TEM. EA upregulated protective miRNAs (miR-145-3p, miR-25-3p) and inhibited aberrant TGFβ/Smad activation, revealing a miRNA-mediated mechanism underlying ovarian protection. CONCLUSION: EA confers significant protection against CTX-induced POI by preserving ovarian structure, normalizing endocrine function, and regulating miRNA-TGFβ/Smad signaling, supporting its potential as a non-pharmacological therapy for ovarian preservation and fertility maintenance.