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- Title
The In Vitro Effects of Carprofen on Lipopolysaccharide-Induced Neutrophil Extracellular Trap Formation in Dairy Cows.
- Authors
Zhi, Jianbo; Qiao, Kaixi; Xie, Lei; Pascottini, Osvaldo Bogado; Opsomer, Geert; Dong, Qiang
- Abstract
Simple Summary: In the present study, white blood cells (neutrophils) were isolated from the blood of postpartum dairy cows and subjected in vitro to conditions designed to mimic an inflammatory reaction induced by the bacterial toxin named lipopolysaccharide (LPS). The production of neutrophil extracellular traps (NETs), reactive oxygen species (ROS), gene expression of NETs-related proteins and inflammatory factors were quantified. The results indicate that LPS stimulation resulted in a concentration-dependent release of NETs, while ROS exhibited rather minor changes. Nevertheless, the presence of the anti-inflammatory drug Carprofen (CA) resulted in a decrease in NETs formation, a downregulation of gene expression of related proteins as well as pro-inflammatory genes. These results indicate that CA has the potential to serve as a therapeutic for alleviating an excessive inflammatory reaction characterized by an overabundance of NETs. Furthermore, this study illuminates the potential of CA in regulating the immune response and its potential benefits in the treatment of inflammatory conditions in (transition) dairy cows. The objective of this study was to develop an in vitro model that mimics inflammatory reactions and neutrophil extracellular traps (NETs) formation by polymorphonuclear leukocytes (PMNs) in dairy cows. This model was used to examine the effect of carprofen (CA) on lipopolysaccharide (LPS)-induced NETs formation and expression of inflammatory factors. Peripheral blood samples were collected from 24 Holstein cows (3–11 days postpartum) and PMNs were isolated. In three replicates, PMNs were exposed to various treatments to establish an appropriate in vitro model, including 80 μg/mL of LPS for 2 h, followed by co-incubation for 1 h with 60 μmol/L CA and 80 μg/mL LPS. The effects of these treatments were evaluated by assessing NETs formation by extracellular DNA release, gene expression of pro-inflammatory cytokines, reactive oxygen species (ROS) production, and the expression of NETs-related proteins, including histone3 (H3), citrullinated histone (Cit-H3), cathepsin G (CG), and peptidyl arginine deiminase 4 (PAD4). The assessment of these parameters would elucidate the specific mechanism by which CA inhibits the formation of NETs through the PAD4 pathway instead of modulating the Nox2 pathway. This highlights CA's effect on chromatin decondensation during NETs formation. Statistical analyses were performed utilizing one-way ANOVA with Bonferroni correction. The results demonstrated that LPS led to an elevated formation of NETs, while CA mitigated most of these effects, concurrent the PAD4 protein level increased with LPS stimulating and decreased after CA administration. Nevertheless, the intracellular levels of ROS did not change under the presence of LPS. LPS supplementation resulted in an upregulation of H3 and Cit-H3 protein expression levels. Conversely, the CA administration inhibited their expression. Additionally, there was no change in the expression of CG with either LPS or LPS + CA co-stimulation. The gene expression of pro-inflammatory cytokines (tumor necrosis factor -α, interleukin (IL)-18, IL-1β, and IL-6) upregulated with LPS stimulation, while the treatment with CA inhibited this phenomenon. In conclusion, CA demonstrated a pronounced inhibitory effect on both LPS-induced NETs formation as well as the associated inflammatory response.
- Subjects
DAIRY cattle; LEUCOCYTES; NEUTROPHILS; GENE expression; TUMOR necrosis factors; BACTERIAL toxins
- Publication
Animals (2076-2615), 2024, Vol 14, Issue 6, p985
- ISSN
2076-2615
- Publication type
Article
- DOI
10.3390/ani14060985