Influence of endometritis and LPS stimulation on the expression of bta-miR-200b and PI3K/AKT and NF-κB pathways in in vivo or cultured bovine endometrial epithelial cells.

Endometritis in dairy cows cause severe economic losses in the dairy farming industry. Although bta-miR-200b expression is decreased in cows with endometritis, role of bta-miR-200b in moderation of endometritis in dairy cows is unknown. In the present study, bovine endometrial epithelial (BEND) cells stimulated with lipopolysaccharide (LPS, 0-100 μg/mL) for 24 h significantly decreased cell viability and the expression of bta-miR-200b consistent with the expression pattern of bta-miR-200b in endometritis. Subsequently, western blotting revealed that overexpression of bta-miR-200b significantly suppressed the LPS-induced increase in phosphorylated p65 protein levels in BEND cells, thereby decreasing the release of proinflammatory factors and inhibiting apoptosis. As predicted by KEGG analysis, LPS induced overexpression of bta-miR-200b significantly increased the protein abundance of phosphorylated AKT and the Bcl-2/Bax ratio. These findings suggested that the inhibition of inflammatory injury by bta-miR-200b is mediated by the PI3K/AKT pathway. The results of western blotting further revealed that the overexpression of bta-miR-200b decreased the protein abundance of PTEN, indicating that PTEN is a target of bta-miR-200b. Knocking down both bta-miR-200b and PTEN in BEND cells resulted in decreasing level of phosphorylated AKT and the Bcl-2/Bax ratio while promoting p65 nuclear transcription. Furthermore, immunofluorescence staining and flow cytometry verified that PTEN knockdown inhibited the effect of bta-miR-200b inhibition on LPS-induced apoptosis. Collectively, these findings suggest that bta-miR-200b attenuates LPS-induced inflammatory injury by targeting the PTEN/AKT/NF-κB axis. These findings provide a theoretical basis for further studies on the in vivo application of miRNA-based therapy to mitigate endometritis.