NOX2/NOX4-associated oxidative stress contributes to G6PD-associated metabolic remodeling and ferroptosis during mice luteolysis

OBJECTIVE: Ferroptosis has been shown to participate in luteolysis, yet the upstream signals and metabolic remodeling that trigger it remain unknown. This study aims to clarify these mechanisms. METHODS: Physiological postpartum and PGF2α-induced luteolysis models were established in mice. Targeted energy metabolomics, pharmacological inhibition and adenoviral shRNA-mediated knockdown were employed in vivo and in primary luteal cells to measure ferroptosis markers, G6PD activity, NADP⁺/NADPH ratio and NOX2/NOX4 expression. RESULTS: , combined NOX2/NOX4 knockdown reduced ROS accumulation, suppressed G6PD hyperactivation, alleviated ferroptotic injuryand delayed regression Moreover, G6PD inhibition with 6-AN similarly reduced oxidative stress and ferroptosis and partially restored progesterone secretion. In primary luteal cells, G6PD knockdown or 6-AN treatment attenuated PGF2α-induced ferroptosis and improved cell viability. Simultaneously, NOX2/NOX4 silencing, N-acetylcysteine, or Ferrostatin-1 suppressed PGF2α-induced G6PD activation, and attenuated oxidative stress and ferroptosis. CONCLUSION: NOX2/NOX4-associated oxidative stress contributes to G6PD-associated metabolic remodeling and ferroptosis during mice luteolysis.