Lacticaseibacillus rhamnosus GR-1 Alleviates Escherichia coli-induced Autophagy-dependent Ferroptosis in Bovine Endometrial Epithelial Cells via FOXO1 Signaling.

Endometritis represents a major contributor to infertility in dairy cattle and is characterized histologically by pronounced inflammation and epithelial injury within the endometrium. Here, we investigated the protective efficacy of Lacticaseibacillus rhamnosus GR-1 (L. rhamnosus GR-1) in an Escherichia coli (E. coli)-induced bovine endometrial epithelial (BEND) cell model. We observed that E. coli infection triggered oxidative stress characterized by reactive oxygen species (ROS) accumulation, leading to excessive autophagy (elevated microtubule-associated protein 1 light chain 3 (LC3) II/I, reduced sequestosome 1 (p62)) and subsequent ferroptosis, evidenced by iron overload, nuclear receptor coactivator 4 (NCOA4) upregulation, and ferritin heavy chain 1 (FTH1) downregulation. Mechanistic analysis using the inhibitor 3-methyladenine (3-MA) confirmed this cell death was autophagy-dependent. Furthermore, RNA sequencing (RNA-seq) and molecular validation identified Forkhead box O1 (FOXO1) as the critical upstream regulator; E. coli induced FOXO1 nuclear translocation, while pharmacological inhibition of FOXO1 successfully suppressed the downstream autophagy-ferroptosis cascade. Crucially, pretreatment with L. rhamnosus GR-1 effectively alleviated these pathological changes by blocking FOXO1 hyperactivation, thereby restoring iron homeostasis and preventing cell death. Collectively, these data demonstrate that L. rhamnosus GR-1 protects BEND cells against E. coli-induced injury by inhibiting FOXO1-mediated regulation of autophagy-dependent ferroptosis, providing a theoretical basis for its use in managing bovine endometritis.