The Protective Effect And Mechanisms Of Thymol On Staphylococcus Aureus-induced Inflammatory Response In Bovine Mammary Epithelial Cells

Bovine mastitis, characterized by inflammatory and infectious processes of the mammary gland, is one of the most costly and prevalent diseases in the dairy industry. The damage to mammary tissue decreases the number and activity of epithelial cells and reduces the production and quality of milk. The predominantly contagious pathogen responsible for mastitis in dairy cows is Staphylococcus aureus(S. aureus), which predominately causes subclinical intramammary infections. Currently, conventional methods are unable to prevent the mammary infection. Therefore, it is urgently needed to develop novel drugs to treat with bovine mastitis.Traditional chinese medicine is expected to therapy bovine mastitis, since to its residue is low and relatively safe. Thymol, a monocyclic monoterpene compound isolated from Thymus vulgari,Monarda punctate or Origanum vulgare spp., has been widely used in pharmaceutical industries and veterinary medicine. Previous studies have demonstrated that thymol has anti-bacterial, anti-inflammatory, anti-parasitic, anti-oxidative and anti-tumor properties. However, the effects of thymol on S. aureus-induced inflammatory response in bovine mammary epithelial cells have not been evaluated. Thus, the purpose of this work was to evaluate the effects of thymol on S. aureus-induced primary cultures of bMEC and to further elucidate the possible mechanism of this action.Firstly, the model of S.aureus-infected bMEC was established successfully, and then we observed the protective effects of thymol on S. aureus-induced inflammatory response in primary cultures of bMEC. In this part, the cytotoxicity of thymol on primary cultures of bMEC was determined with MTT. The m RNA expression of TNF-??IL-1? and IL-6 were analysed by q RT-PCR. The levels of p65, p38, JNK and ERK phosphorylation were analysed by Western blotting. The results showed that thymol had no cytotoxicity on primary cultures of bMEC, and thymol decreased the m RNA levels of TNF-??IL-1? and IL-6. Moreover, the mechanisms of thymol protecting S. aureus-induced inflammatory response may be via inhibiting the activation of NF-k B and MAPK signal pathway.Secondly, to further elucidate the protective effects of thymol on S. aureus-induced inflammatory response, we evaluated the effects of thymol on phagocytosis of bMEC. In this part, the expression of tracheal antimicrobial peptide(TAP) and ?-defensin(BNBD5) were analysed by q RT-PCR, and the inhibition of NF-?B activation in bMEC infected with S. aureus was analysed by Western blotting. Our results showed that thymol(16~64 ?g/ml) could reduce the internalization of S. aureus into bMEC and down-regulate the m RNA 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-?B activation in a dose-dependent manner. In conclusion, these results indicated that thymol inhibits S. aureus internalization into bMEC by inhibiting NF-?B activation.Taken together, thymol shows significant protective effects on S. aureus-induced inflammatory response in bovine mammary epithelial cells, and its potential mechanism on one hand is associated with inflammatory signial pathway, and on the other hand is via inhibiting S. aureus internalization into bMEC.