Gengnianchun Against H₂O₂‐Induced Oxidative Damage in KGN Cells via miR‐548m/FOXO3 Signaling

Gengnianchun (GNC) is a traditional remedy used for diminished ovarian reserve, but its underlying mechanisms remain unclear. This study aimed to explore these mechanisms in human granulosa-like cancer (KGN) cells pretreated with medicated rat serum (MRS) before H₂O₂ exposure. MRS pretreatment significantly alleviated H₂O₂-induced cell damage, including improvements in cell viability, superoxide dismutase and GSH-Px activities, and Bcl-2 expression. Conversely, H₂O₂ treatment increased apoptosis, autophagosomes, IL-1β, TNF-α, reactive oxygen species, malondialdehyde levels, and the expression of LC-II/LC3-I, Bax, and Beclin-1. GEO database analysis revealed significant differential expression of several miRNAs, including miR-548m. qPCR confirmed that MRS upregulated miR-548m expression, which was downregulated by H₂O₂ in a dose-dependent manner. Preincubation with MRS prevented the decline in miR-548m expression and mitigated H₂O₂-induced damage, including improvements in cell viability, apoptosis, autophagy, and oxidative stress. miR-548m suppressed FOXO3 3'-UTR luciferase activity, and anti-miR-548m enhanced it. Transfection with miR-548m reduced FOXO3 mRNA and protein levels, while anti-miR-548m increased them. These findings suggest that GNC protects against H₂O₂-induced ovarian damage by modulating the miR-548m/FOXO3 axis, triggering autophagy and apoptosis.