Study On ACAC-induced Neutrophil Immune Dysfunction Via TLR2/4-NF-κB Signalling Pathway In Dairy Cows

In the current experiment,we aim to investigate the effects of acetoacetic acid（ACAC）on the expression of related proteins and genes in TLR2/4-NF-κB signalling pathway during the induction of inflammatory lesions of dairy cow neutrophils（PMN）which will reveal the signal transduction mechanism of high ACAC on PMN,and the mechanism of immune dysfunction in ketotic dairy cows.Eight nonketotic and eight ketotic Holstein dairy cows were selected respectively,blood samples were collected from jugular vein and serum was separated by centrifugating for future analysis.The changes of serum ketotic indexes,oxidative and antioxidant indexes and inflammatory factors were detected by enzyme-linked immunosorbent assay（ELISA）.PMN were isolated from fresh blood of the nonketotic dairy cows,then cultured and stained with giemsa for the morphological observation.PMN proliferation and activity were measured by Cell Counting Kit-8（CCK-8）method after PMN were treated with various concentrations of ACAC.The optimal concentration and time of ACAC were determined after being treated at different time points.Subsequently,PMN were stimulated with different concentrations of ACAC（0,0.6,1.2,1.8 mM）,10μM ammonium pyrrolidinedithiocarbamate（PDTC）,1.2 mM ACAC+10μM PDTC,10 mM N-acetylcysteine（NAC）,1.2 mM ACAC+10 mM NAC.The concentrations of interleukin-6（IL-6）,interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）and the activity of IKKβkinase were measured by ELISA.The protein expression level of TLR2,TLR4,IκBα,p-IκBα,NF-κB p65,p-NF-κB p65,IL-6,IL-1βand TNF-αwere detected by western blot.The mRNA relative expression of toll like receptor 2（TLR2）,toll like receptor 4（TLR4）,nucleus factor-κB（NF-κB）p65,IL-6,IL-1βand TNF-αwere detected by real time fluorescence quantitative PCR（qRT-PCR）.The effect of ACAC on NF-κB p65expression in the nucleus was measured by electrophoretic mobility shift assay（EMSA）.The distribution of NF-κB p65 in nucleus and cytoplasm was detected by immunofluorescence（IF）.The level of reactive oxygen species（ROS）was detected by flow cytometer.The results obtained were as follows:1.Compared with the nonketotic dairy cows,the concentrations ofβ-hydroxybutyric acid（BHBA）,mon-esterified fatty acid（NEFA）,ACAC and malondialdehyde（MDA）in the serum of ketotic dairy cows were highly significantly increased（P<0.01）.The concentrations of glucose（GLU）,glutathione-peroxidase（GSH-Px）and catalase（CAT）were highly significantly decreased（P<0.01）.The concentration of hydrogen peroxide（H₂O₂）was significantly increased（P<0.05）.The concentration of TNF-αwas highly significantly increased（P<0.01）.2.1.2 mM and 4 h were determined as the optimal concentration and time point of ACAC.Compared with the control group,the activity of IKKβkinase was highly significantly increased in 1.2 mM ACAC and 1.8 mM ACAC treated groups（P<0.01）.Compared with 1.2 mM ACAC treated group,the activity of IKKβkinase was highly significantly decreased in 1.2 mM ACAC+10 mM NAC treated group（P<0.01）.3.Compared with the control group,the protein expression of TLR2,TLR4,IκBα,p-IκBα,NF-κB p65,p-NF-κB p65,IL-6,IL-1βand TNF-αwas highly significantly increased after different concentrations of ACAC was added（P<0.01）.The protein expression of p-IκBα,NF-κB p65 and p-NF-κB p65 and the mRNA relative expression of NF-κB p65 in 10μM PDTC treated group were decreased.The protein expression of TLR2,TLR4,IκBα,NF-κB p65,p-NF-κB p65 and IL-1βand the mRNA relative expression of TLR2,TLR4,NF-κB p65,IL-1βand TNF-αin 10 mM NAC treated group were decreased.The concentrations of IL-6,IL-1βand TNF-αin the supernatant of the cells increased with the increase of ACAC concentrations and decreased in 10μM PDTC treated group.Compared with the 1.2 mM ACAC treated group,the protein expression of IκBα,p-IκBα,p-NF-κB p65,IL-1βand the mRNA relative expression of IL-6,TNF-αin 1.2 mM ACAC+10μM PDTC treated group was highly significantly decreased（P<0.01）.The protein expression of TLR2,IκBα,p-NF-κB p65,IL-6,IL-1βand the mRNA relative expression of TLR4 in 1.2 mM ACAC+10 mM NAC treated group were highly significantly decreased（P<0.01）.4.The results of EMSA showed that the expression of NF-κB p65 in nucleus increased with the increasing of ACAC concentrations（P<0.05）,but decreased in 10μM PDTC treated group.The results of IF were consistent with EMSA results,and decreased in 10 mM NAC treated group.5.Compared with the control group,the level of ROS were significantly decreased in0.6 mM ACAC treated group（P<0.05）,and highly significantly decreased in 1.8 mM ACAC and 10 mM NAC treated groups（P<0.01）.In conclusion,inflammation existed in the ketotic dairy cows,ACAC causes inflammatory damage and immune dysfunction in PMN via activating the TLR2/4-NF-κB inflammatory signalling transduction pathway.Moreover,oxidative stress existed in the ketotic dairy cows,ACAC causes oxidative stress of PMN and can be alleviated by NAC.