The Study Of NEFAs And BHBA Inducing Oxidative Stress On NF-κB Signaling Pathway In Calf Hepatocytes

Higher plasma levels of nonesterified fatty acids (NEFAs) and β-hydroxybutyrate(BHBA) exist in the dairy cows with ketosisi that is related to oxidative stress. Higherconcentrations of NEFAs and BHBA are closely related with inflammatory diseasessuch as mastitis and metritis in dairy cows. The NF-κB signaling pathway plays acentrol role in the inflammatory responses and is avtivated by oxidative stress.However, it is unclear whether high concentrations of NEFAs and BHBA can activatethe NF-κB signaling pathways to induce hepatocytes inflammatory damage in dairycows. Therefore, in this study, primary cultured calf hepatocytes were treated withdifferent concentrations of NEFAs, BHBA, PDTC (an NF-κB pathway inhibitor) andNAC (antioxidant) to research the oxidative stress, NF-κB pathway and the release ofpro-inflammatory factors in cultured calf hepatocytes. And we measured the contentof plasma TNFα, IL-6and IL-1β in healthy, subclinical ketosis and clinical ketosiscows, respectively, in order to understand the mechanism that high plasma NEFAs andBHBA induce inflammatory injury in calf hepatocytes.Detecting the concentration of plasma TNFα, IL-6and IL-1β in subclinicalketosis and clinical ketosis cows, the results showed that the release ofpro-inflammatory factors in ketosis cows was significantly higher than in healthycows, it demonstrated that there was potentially low intensity inflammatory reactionin ketosis cows.This study is to observe the effect of different concentrations of NEFAs andBHBA on the oxidative stress, NF-κB pathway and the release of pro-inflammatoryfactors in cultured calf hepatocytes. Firstly, calf hepatocytes were incubated with1.2mM NEFAs and1.8mM BHBA respectively, and treated with multiple time points.The results showed that the the phosphorylation level of IκBα was highest at9h intreatment with NEFAs, and was highest at24h in treatment with BHBA. Then, calfhepatocytes were incubated with different concentrations of NEFAs (0,0.6,1.2and2.4mM),10μM PDTC,10μM PDTC+1.2mM NEFAs,10mM NAC,10mM NAC+1.2mM NEFAs for9h, and different concentrations of BHBA (0,0.6,1.2,1.8and2.4mM),10μM PDTC,10μM PDTC+1.8mM BHBA,10mM NAC,10mMNAC+1.8mM BHBA for24h. The result showed that treatment with NEFAs andBHBA significantly increased the IKKβ activity and the phosphorylation level ofIκBα. These increases were accompanied by increasing protein expression andtranscription activity of NF-κB p65. Activated NF-κB p65makerly upregulated theexpression and release of pro-inflammatory cytokines TNF-α, IL-6and IL-1β, itshowed that high concentration of NEFAs and BHBA can induce inflammatoryresponse in bovine hepatocytes. When PDTC was added, the phosphorylation level ofIκBα as well as the expression and avtivity of NF-κB p65and its target genes weremarkerly decreased. NEFAs treatment results in the overproduction of the markers ofoxidative stress reactive oxygen (ROS), and BHBA treatment results in theoverproduction of the oxidative stress markers NO and iNOS. Additionally, addedwith the NAC could significantly decrease the activity of ROS and the expression andrelease of pro-inflammatory cytokines TNF-α, IL-6and IL-1β in calf hepatocytes.In conclusion, the present data indicate that higher concentrations of NEFAs, andBHBA can activate the NF-κB signaling pathway, which may be activated byoxidative stress, thereby to regulate the release of pro-inflammatory factors TNF-α,IL-6and IL-1β, then inducing cattle hepatocyte inflammatory response.