GSK3β-mediated NRF2 degradation drives Escherichia coli-induced ferroptosis in the bovine endometrium

The infection of the bovine endometrium by Escherichia coli often results in endometrial injury with lower fertility in the bovine industry. However, what functions does ferroptosis play during endometritis? This is not completely explained. Therefore, this study aimed to elucidate the potential mechanism of ferroptosis during endometrial injury induced by E. coli. The endometrial tissues from sixteen Holstein cows with E. coli infection were harvested for pathological examination and RNA-seq. Primary bovine endometrial epithelial cells (BEECs) were used to establish an E. coli infection model in vitro. The mechanism by which ferroptosis is involved in endometritis was explored by western blot, flow cytometry, and immunofluorescence methods. The results revealed that the injury of endometrial tissues and BEECs induced by E. coli was associated with ferroptosis via Fe²⁺ accumulation, increased lipid peroxidation, decreased glutathione concentration, and inhibited expression of solute carrier family 7-member 11, glutathione peroxidase 4, and ferritin heavy chain 1. Ferroptosis inhibition alleviated BEEC damage induced by E. coli. Further analysis revealed that the E. coli-induced ferroptosis was associated with impaired nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense, which is characterized by mitigated NRF2 degradation and blunted induction of target antioxidant enzymes, such as NAD(P)H quinone oxidoreductase 1. In contrast, promoting the expression of NRF2 mitigated the cellular damage and ferroptosis induced by E. coli. In particular, glycogen synthase kinase (GSK)-3β exhibited sustained expression and hyperactivity in endometrial tissue and BEECs infected with E. coli, which confirmed that the suppression of GSK-3β could significantly inhibit NRF2 degradation and attenuate ferroptosis. This study demonstrated that E. coli can trigger ferroptosis in endometrial tissues and BEECs through GSK-3β-mediated NRF2 degradation. Modulating GSK-3β/NRF2 holds promise as a potential therapeutic strategy for alleviating endometritis induced by E. coli.