BHLHE40 drives ferroptosis in Escherichia coli-induced endometrial injury by recruiting HDAC1 to repress CPT1B and impair NRF2 signaling.

Endometritis is a common disease of the reproductive system that leads to decreased fertility and potential miscarriage, resulting in substantial economic losses. Ferroptosis is an iron-dependent mode of cell death driven by lipid peroxidation. Basic helix-loop-helix family member E40 (BHLHE40) has been developed as a molecular marker and a therapeutic target for bacterial infectious diseases. However, the underlying role of BHLHE40 needs to be further studied. This study investigated whether BHLHE40 drives Escherichia coli (E. coli) -induced endometrial ferroptosis by transcriptionally repressing carnitine palmitoyltransferase 1B (CPT1B) and attenuating the NRF2 signaling pathway, with the aim of elucidating the regulatory mechanism of the BHLHE40-CPT1B-NRF2 axis. In endometrial tissues from E. coli-infected cows (n = 8 per group, in vivo), the expression of BHLHE40 was increased by 3.6 times (p < 0.05), and its protein level positively correlated with the severity of tissue damage (Pearson r = 0.6722, p = 0.0034). Functionally, knockdown of BHLHE40 conferred significant protection against E. coli-induced cell death (decreased from 36% to 18%, p < 0.01), concurrently reversing the features of ferroptosis (p < 0.05), including reduced lipid peroxidation, decreased intracellular Fe2+ concentration, and restored glutathione levels and the expression of anti-ferroptosis proteins. Integrated analysis of CUT&Tag and RNA-sequencing data revealed that BHLHE40 binds to the promoter region of CPT1B (enrichment increased by 5 times, p < 0.01) and transcriptionally represses its expression. Further investigation demonstrated that BHLHE40 mediates CPT1B repression by recruiting HDAC1 to the CPT1B promoter, a process reversible upon HDAC1 inhibition. Crucially, BHLHE40-mediated suppression of CPT1B led to functional impairment of the NRF2 signaling pathway. Overexpression of CPT1B inhibited ferroptosis by reactivating NRF2 and its downstream targets (p < 0.05), whereas inhibition of NRF2 abolished the protective effect (cell death increased from 16% to 27%, p < 0.01), confirming NRF2 as the critical downstream effector. This study elucidates the BHLHE40-CPT1B-NRF2-ferroptosis axis as a novel pathway and potential target for E. coli to cause endometrial epithelial injury.