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Neutrophil immune dysfunction induced by acetoacetic acid via TLR2/4-NF-κB signaling pathway in dairy cows.

H. Ding



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Neutrophil immune dysfunction induced by acetoacetic acid via TLR2/4-NF-κB signaling pathway in dairy cows.
H. Ding*1, Y. Li1, S. Feng1, J. Li1, X. Wang1, J. Wu1, Y. Liang2, J. J. Loor2. 1Anhui Agricultural University Hefei, Anhui, China, 2University of Illinois at Urbana-Champaign Urbana, IL.

The objective of the current study was to investigate the effects of acetoacetic acid (ACAC) on mRNA and protein abundance related to the toll-like receptor 2/4-nuclear factor-κB (TLR2/4-NF-κB) signaling pathway in neutrophils (PMN) during the induction of inflammatory response in dairy cows. Nonketotic (n = 8) and ketotic (n = 8) Holstein cows were selected based on serum concentrations of β-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA), ACAC and glucose. PMN were treated with 0.6, 1.2 and 1.8 mM ACAC using 0 mM ACAC as control group, with or without ammonium pyrrolidinedithiocarbamate (PDTC, 10 μM, 30 min), and N-acetylcysteine (NAC, 10 mM, 30 min). Total RNA and protein were used for quantitative real-time polymerase chain reaction and Western blotting, respectively. One-way ANOVA was performed by Statistical Package for the Social Science (SPSS) 17.0 and multiple comparisons of data were done using the Duncan test. Compared with nonketotic cows, serum concentrations of BHBA, NEFA, ACAC, hydrogen peroxide, malondialdehyde (MDA), glutathione peroxidase and tumor necrosis factor α (TNF-α) increased (P < 0.05) while glucose and catalase decreased (P < 0.01). Compared with control group, IKKβ activity, the protein abundance of TLR2 and phosphorylated-NF-κB p65 (p-NF-κB p65) in PMN increased in response to ACAC (P < 0.01). Additionally, ACAC increased transcription activity of p65. Moreover, ACAC treatment led to greater protein abundance of TNF-α (P < 0.01), while decrease after NAC treatment. ACAC could increase MDA (P < 0.01), where decrease the concentrations of CAT and Cu/Zn SOD (P < 0.05) in PMN. However, abundance of p-NF-κB p65, transcription activity of p65 and target TNF-α were decreased in PDTC + ACAC group compared with ACAC (1.2 mM) group. In conclusion, inflammation and oxidative stress existed in the ketotic dairy cows and ACAC caused inflammatory damage and immune dysfunction in PMN by activating the TLR2/4-NF-κB inflammatory signaling transduction pathway, moreover, which could be alleviated by NAC.

Keywords: dairy cow, immune dysfunction, TLR2/4-NF-κB.