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

Abstract

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.

Key Findings

• BHLHE40 expression is significantly increased in E. coli-infected endometrial tissues and correlates with tissue damage severity.
• Knockdown of BHLHE40 protects against E. coli-induced ferroptosis by reducing lipid peroxidation, intracellular Fe2+ levels, and restoring glutathione and anti-ferroptosis protein expression.
• BHLHE40 represses CPT1B transcription by recruiting HDAC1 to its promoter, leading to impairment of the NRF2 signaling pathway, while CPT1B overexpression reactivates NRF2 and inhibits ferroptosis.

Clinical Significance

Citation

Yuan Changning, Wang Jue, Wang Zhihaoet al.. BHLHE40 drives ferroptosis in Escherichia coli-induced endometrial injury by recruiting HDAC1 to repress CPT1B and impair NRF2 signaling. Free radical biology & medicine. 2026-Aug-16.