Paeoniflorin alleviates inflammation in bovine mammary epithelial cells induced by Staphylococcus haemolyticus through TLR2/NF-?B signaling pathways

Staphylococcus haemolyticus (S. haemolyticus) is one of the most common coagulase-negative staphylococci (CoNS) isolates from bovine mastitis. Paeoniflorin (PF) shows anti-inflammatory effects on different inflammatory dis-eases in vitro studies and in vivo animal experiments. In this study, the viability of bovine mammary epithelial cells (bMECs) was detected by the cell counting kit-8 experiment. Subsequently, bMECs were induced with S. haemolyticus, and the induction dosage was determined. The expression of pro-inflammatory cytokines and toll-like receptor (TLR2) and nuclear factor kappa-B (NF-kappa B) signaling pathway-related genes were investigated by quantitative real-time PCR. The critical pathway proteins were detected by western blot. The results showed that the multiplicity of infection (MOI; the ratio of bacteria to bMECs) 5:1 of S. haemolyticus for 12 h could cause cellular inflammation, which was selected to establish the inflammatory model. Incubation with 50 mu g/ml PF for 12 h was the best intervention condition for cells stimulated by S. hemolyticus. Quantitative real-time PCR and western blot analysis showed that PF inhibited the activation of TLR2 and NF-kappa B pathway-related genes and the expression of related proteins. Western blot results showed that PF suppressed the expression of NF-kappa B unit p65, NF-kappa B unit p50, and MyD88 in bMECs stimulated by S. haemolyticus. The inflammatory response pathway and molecular mechanism caused by S. haemolyticus on bMECs are related to TLR2-mediated NF-kappa B signaling pathways. The anti-inflammatory mechanism of PF may also be through this pathway. Therefore, PF is expected to develop potential drugs against CoNS-induced bovine mastitis.