The Effect Of NEFAs And BHBAs On TLR2/4Mediated NF-κB Signaling Pathway In Cow Neutrophil

It’s clinical pathology characteristiced of high concerntration no-esterified fattyacids(NEFAs) and-hydroxybutyric acid(BHBA) in blood for ketosis dairy cows.High NEFAs and BHBA can involve in regulate neutrophil(PMN) immunefunction,espescially in inflammatory diseases(such as mastitis and endomertritis).But, It is unclear whether high concentration of NEFAs and BHBA can activateTLR2/4-NF-κB signaling pathway in bovine neutrophils.Therefore, the firstexperiment in vivo is to understand that whether ketosis cow exists inflammatory.The results shows that the mRNA expression level of TLR2,TLR4,NF-κBp65,IL-1β,IL-6and TNF-α in blood of ketosis cow is significantly improved as well asthe release of pro-inflammatory(IL-1β,IL-6,TNF-α), at the same time, activity ofIKKβ strengthens significantly.These results prove that ketosis cow existinflammatory response at some extent.In order to determine the mechanism of inflammatory damage to PMN， thesecond experiment in vitro was designed. High concentration of NEFAs,BHBAand PDTC(inhibitor of NF-κB) was added into PMN media to detect theTLR2/4-NF-κB signaling pathway, then observe the release of pro-inflammatoryfactors (IL-1β,IL-6,TNF-α).At first, the PMN were isolated and identified,subsequent to ascertain the culture time point of primary cow PMN by the mean ofproliferation activity of MTT and CCK8. Add different concerntrationNEFAs(0,0.125,0.25,0.5,1,2mM,10μM PDTC(inhibitor of NF-κB),10μM PDTC+0.5mM) and BHBA(0,0.5,1,2,4,6mM、10μM PDTC、10μM PDTC+2mM）to themedia，and cultured for4h and5h，respectively.The experiment results indicatethat compared with the control group, activity of IKKβ and the protein expressionlevel of p-IκB in PMN media were significantly enhanced with the high NEFAs(≥0.5 mM) and BHBA(≥1.2mM), also the same to the protein expression level and thetranscriptional acticity of NF-κBp65. The synthesis and release ofpro-inflammatory factors (IL-1β,IL-6,TNF-α) were significantly strengthened.However, both the protein expression level of p-IκB and p-NF-κBp65weredescended after added PDTC.Above all, high NEFAs(≥0.5mM) and BHBA(≥1.2mM) can upregulate TLR2and TLR4,activated downsream signaling pathway NF-κB, leading to excessiverelease of pro-inflammatory factors (IL-1β,IL-6,TNF-α) of PMN, which induce theinflammatory damage of PMN. It is a reason why immunosuppression occures inketosis cows.