Goat bone marrow mesenchymal stem cell angiotensin-converting enzyme 2 attenuates Streptococcus uberis-induced ferroptosis in goat mammary epithelial cells by downregulating the reactive oxygen species-chaperone-mediated autophagy pathway.

Streptococcus uberis is a leading cause of environmental mastitis globally. Ferroptosis, a cell death pathway driven by Fe2+-mediated lipid peroxide accumulation, with reactive oxygen species (ROS) participating in this process, is related to the mechanisms of infections caused by other pathogens inducing mastitis, such as Escherichia coli and Staphylococcus aureus. This study aims to investigate whether ferroptosis occurs in S. uberis-induced mastitis and to develop corresponding therapeutic interventions. Goat bone marrow mesenchymal stem cells (GBMSC) were isolated from a 3-mo-old healthy goat. After verification via surface marker analysis and trilineage differentiation, angiotensin-converting enzyme 2 (ACE2)-overexpressing GBMSC (GBMSC-ACE2) were generated using lentiviral vectors. In vitro, a coculture model of GBMSC, GBMSC-GFP, or GBMSC-ACE2 with goat mammary epithelial cells (GMEC) infected by S. uberis was established. Results showed that GBMSC-ACE2 effectively reduced ROS accumulation and inflammatory cytokine secretion, inhibited excessive activation of chaperone-mediated autophagy (CMA), and maintained iron metabolism homeostasis, thus alleviating S. uberis-induced ferroptosis in GMEC better than GBMSC or GBMSC-GFP. The main manifestations were: 1) inhibiting ADAM metallopeptidase domain 17 (ADAM17) and promoting ACE2 expression to suppress the Angiotensin II (Ang II)/Ang II Type 1 Receptor (AT1R)/NADPH oxidase 2 (NOX2) pathway; 2) inhibiting lysosomal associated membrane protein 2A (LAMP2A), heat shock cognate protein 70 (Hsc70), heat shock protein 90 (Hsp90) while promoting glutathione peroxidase 4 (GPX4) expression; 3) reducing intracellular Fe2+ accumulation; 4) delaying lipid peroxidation. In vivo, 9 lactating goats were randomly divided into 3 groups (n = 3 each): control, S. uberis-infected, and GBMSC-ACE2-treated. The experiment lasted 7 d. The GBMSC-ACE2 significantly decreased SCC, improved milk yield and quality, inhibited bacterial proliferation, enhanced ACE2 expression in mammary tissue, and alleviated ferroptosis by suppressing CMA-mediated GPX4 degradation, thus reducing damage to the blood-milk barrier and mammary tissue. In conclusion, this study suggests that GBMSC-ACE2 is a novel treatment for S. uberis mastitis, capable of inhibiting ferroptosis in mammary epithelial cells and promoting tissue regeneration.