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Bovine mastitis is one of the most costly and prevalent diseases in the dairy industry and is characterised by inflammatory and infectious processes.Staphylococcus aureus (S.aureus), a Gram-positive organism, is a frequent cause of subclinical ,chronic mastitis.Thymol, a monocyclic monoterpene compound isolated from Thymus vulgaris,has been reported to have antibacterial properties.However, the effect of thymol on S.aureus internalization into bovine mammary epithelial cells (bMEC) has not been investigated.In this study, the growth of S.aureus supplemented with thymol (16-64 μg/ml) was monitored turbidimetrically at 600 nm for 24 h and cell viability was measured by the Cell Counting Kit-8 (CCK-8).Gentamicin protection assay was carried out to determine the effect of thymol on S.aureus internalization into bMEC.To determine the potential mechanism, tracheal antimicrobial peptide (TAP) and β-defensin (BNBD5) expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR).The activation of nuclear factor-kappa B (NF-κB) was determined by Western blotting.Our results showed that thymol (16-64 μg/ml) could reduce the internalization of S.aureus into bMEC and down-regulate the mRNA expression of TAP and BNBD5 in bMEC infected with S.aureus.In addition, thymol was found to inhibit S.aureus-induced nitric oxide (NO) production in bMEC and suppress S.aureus-induced NF-κB activation in a dose-dependent manner.In conclusion, these results indicated that thymol inhibits S.aureus internalization into bMEC by inhibiting NF-κB activation.