Plumbagin ameliorates ferroptosis of ovarian granulosa cells in polycystic ovary syndrome by down-regulating SLC7A5 m6A methylation modification through inhibition of YTHDF1.

BACKGROUND AND

objectivePolycystic ovary syndrome (PCOS) is a common endocrine-metabolic disease in women of reproductive age. One of its core pathologies is ovarian granulosa cell (GC) dysfunction, and ferroptosis, as a novel cell death mode dependent on iron ions and lipid peroxidation, may be involved in the PCOS process, but the exact mechanism is unknown. Plumbagin (PLB) shows potential in PCOS treatment due to its antioxidant properties. The present study aimed to elucidate the molecular mechanisms by which PLB ameliorates mitochondrial dysfunction and ferroptosis in PCOS GCs through the YTH N6-methyladenosine RNA binding protein 1/L-type amino acid transporter 1 (YTHDF1/SLC7A5) axis.

methodsAn in vitro model of PCOS was constructed by treating KGN cells with dihydrotestosterone (DHT), and PLB treatment, YTHDF1 knockdown (si-YTHDF1), and SLC7A5 overexpression (pcDNA 3.1-SLC7A5) were intervened respectively. Cell viability was measured by cell counting kit-8. Lactate dehydrogenase (LDH) release, adenosine triphosphate (ATP) level, iron ion, and lipid peroxidation (LPO) content were detected by commercial kits. Mitochondrial membrane potential (MMP) was analyzed by JC-1 staining combined with flow cytometry. Reactive oxygen species (ROS) levels were assessed by C11-BODIPY probe, oxidative stress indicators including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase were measured by kits, and Cytochrome C, Ferritin, mitochondrial transcription factor A (TFAM), glutathione peroxidase 4 (GPX4) and SLC7A5 expression were detected by Western blot. Fluorescence in situ hybridization, RNA immunoprecipitation, and m6A quantitative real-time polymerase chain reaction verified the interaction and translational regulation of YTHDF1 and SLC7A5.

resultsDHT treatment significantly decreased KGN cell viability, MMP and ATP levels, increased LDH release, ROS, MDA, iron ions and LPO content, up-regulated Cytochrome C expression, and down-regulated Ferritin, TFAM, and GPX4 expression. Both PLB treatment and YTHDF1 knockdown significantly reversed the above changes, but YTHDF1 overexpression reversed the protective effect of PLB. YTHDF1 co-localized with SLC7A5 mRNA and enhanced its translation through m6A modification. YTHDF1 knockdown reduced SLC7A5 protein levels without affecting mRNA expression. SLC7A5 overexpression weakened the protective effect of YTHDF1 knockdown, resulting in decreased cell viability, deterioration of mitochondrial function, and increased ferroptosis.

conclusionPLB ameliorates DHT-induced mitochondrial dysfunction and ferroptosis in KGN cells by inhibiting YTHDF1 expression, and its action is dependent on the mechanism by which YTHDF1 regulates SLC7A5 translation through m6A modification. Downregulating YTHDF1 or SLC7A5 significantly enhances GC survival and function.