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 In both models, luteal regression was accompanied by increased ACSL4, decreased GPx4 and GSH, and elevated Fe2⁺ and lipid peroxidation and NOX2 and NOX4 expression. Metabolic analysis revealed 6-phosphogluconate accumulation, reduced pyruvate and pyruvate kinase M activity, and increased G6PD expression and activity, but NADPH depletion and redox imbalance. In vivo, 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.