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Endometritis is a major infectious disease affecting dairy development. MicroRNAs are recognized as critical regulators of the innate immune response. However, the role and mechanism of Bta-miR-24-3p in the development of endometritis are still unclear. This study aimed to investigate the effect of Bta-miR-24-3p on the inflammatory response triggered by lipopolysaccharide (LPS) and to clarify the possible mechanism. LPS-treated bovine endometrial epithelial cells (BEECs) were cultured to investigate the role of Bta-miR-24-3p. The expression levels of Bta-miR-24-3p were downregulated, and galectin-9 (LGALS9) were measured by quantitative real-time polymerase chain reaction. The LPS-induced inflammatory response was assessed by the elevated secretion of inflammatory cytokines measured by using enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction. Activation of nuclear factor-κB (NF-κB) and TLR4 pathway was assessed by Western blot. The interaction between Bta-miR-24-3p and LGALS9 was validated by bioinformatics analysis and a luciferase reporter assay. LPS-induction in BEECs with Bta-miR-24-3p was overexpressed leads inhibition of pro-inflammatory cytokines, LGALS9 expression, and TLR4/NF-ĸB pathway deactivation. Knockdown of LGALS9 inhibited the LPS-induced inflammatory response in BEECs. LGALS9 was validated as a target of Bta-miR-24-3p. Cloned overexpression of LGALS9 failed to alter the effect of Bta-miR-24-3p on the inflammatory response in BEECs. Overall, Bta-miR-24-3p attenuated the LPS-induced inflammatory response via targeting LGALS9. The immunotherapeutic stabilisation of Bta-miR-24-3p could give a therapeutic option for endometritis and other disorders commonly associated with endometritis, suggesting a novel avenue for endometritis treatment.

Neutrophils are involved in the development of endometritis, but it remains unknown how neutrophils induce inflammation and tissue damage. Neutrophil extracellular traps (NETs) clear invading pathogens during infection but induce pyroptosis, leading to inflammation and tissue damage. Thus, our objective was to investigate whether NETs participate in bovine endometrial epithelial cell (BEEC) pyroptosis during endometritis. To confirm this, NETs and caspase-1/4; apoptosis-associated speck-like protein containing a caspase-recruitment domain(ASC); nod-like receptor protein-3 (NLRP3); and gasdermin D N-terminal (GSDMD-N), TNF-a, IL-1β, IL-6, and IL-18 in endometrial tissue were detected. Pathological section and vaginal discharge smears were performed to visually determine endometritis in the uterus. BEECs were stimulated with NETs to induce pyroptosis, which was treated with DNase I against pyroptosis. Caspase-1/4, ASC, NLRP3, GSDMD-N, TNF-a, IL-1β, IL-6, and IL-18 in BEECs were analyzed in endometrial tissue. The results showed that NET formation, as well as pyroptosis-related proteins and proinflammatory, cytokines were elevated in the endometrial tissue of cows with endometritis. Pathological sections and vaginal discharge smears showed increased neutrophils and plasma cells in the uterus, as well as tissue congestion. In BEECs, NETs increased the level of pyroptosis-related proteins and proinflammatory cytokines and were diminished by DNase I. In summary NETs participate BEEC pyroptosis during endometritis in dairy cows. [ABSTRACT FROM AUTHOR]