Effect of firocoxib on cyclooxygenase 2, microsomal prostaglandin E2 synthase 1, and cytosolic phospholipase A2 gene expression in equine mononuclear cells.
Abstract: To validate primer sets for use in reverse transcription quantitative PCR assays to measure gene expression of cytosolic phospholipase A2 (cPLA2) and microsomal prostaglandin E2 synthase 1 (mPGES1) in equine mononuclear cells and determine the effects of firocoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, on COX-2, cPLA2, and mPGES1 gene expression following incubation of mononuclear cells with lipopolysaccharide (LPS). Methods: 8 healthy adult horses. Methods: Peripheral blood mononuclear cells were isolated by density gradient centrifugation and incubated at 37°C with medium alone, firocoxib (100 ng/mL), LPS (1 ng/mL or 1 μg/mL), or combinations of firocoxib and both LPS concentrations. After 4 hours, supernatants were collected and tested for prostaglandin E2 (PGE2) concentration with an enzyme inhibition assay, and gene expression in cell lysates was measured with PCR assays. Results: Primer pairs for cPLA2 and mPGES1 yielded single products on dissociation curve analyses, with mean assay efficiencies of 102% and 100%, respectively. Incubation with firocoxib and LPS significantly decreased PGE2 supernatant concentrations and significantly reduced COX-2 and mPGES1 gene expression, compared with values following incubation with LPS alone. Conclusions: Primer sets for mPGES1 and cPLA2 gene expression in equine mononuclear cells were successfully validated. Firocoxib significantly decreased LPS-induced COX-2 and mPGES1 expression, suggesting that it may be useful in the control of diseases in which expression of these genes is upregulated.
Publication Date: 2015-12-01 PubMed ID: 26618729PubMed Central: PMC7048238DOI: 10.2460/ajvr.76.12.1051Google Scholar: Lookup
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- Journal Article
- Research Support
- N.I.H.
- Extramural
- Research Support
- Non-U.S. Gov't
Summary
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This research study aimed to evaluate the impact of firocoxib, a specific inhibitor of the cyclooxygenase 2 (COX-2) enzyme, on the expression of COX-2, cytosolic phospholipase A2 (cPLA2), and microsomal prostaglandin E2 synthase 1 (mPGES1) genes in equine mononuclear cells. The researchers also validated custom primer sets for reverse transcription quantitative PCR assays used to measure the expression of those genes in equine mononuclear cells.
Methods
- The author of the paper initiated the research using healthy adult horses. The mononuclear cells from the blood of these horses were isolated using a process known as density gradient centrifugation.
- These isolated cells were then treated with either firocoxib alone, lipopolysaccharide (LPS) alone, combinations of firocoxib and two different LPS concentrations, or simply medium with no added treatments. The cells were kept in an incubator at 37°C for this step of the experiment. LPS was used to stimulate an immune response in the cells.
- After 4 hours of incubation, supernatants (the fluid above settled cells in a test tube) were collected from the samples. These supernatants were then tested for their prostaglandin E2 (PGE2) concentration with an enzyme inhibition assay.
- The genomic data from the cell debris was then analyzed by PCR assays to observe any changes in the expression of COX-2, cPLA2, and mPGES1 genes.
Results
- The primer pairs used for assessing the expressions of cPLA2 and mPGES1 genes were validated by producing single products during the dissociation curve analyses. They yielded efficient results with mean assay efficiencies being 102% and 100%, respectively.
- The study found that the combination treatment of firocoxib and LPS significantly decreased the concentrations of PGE2 in the supernatants. It also significantly reduced the expression of COX-2 and mPGES1 genes when compared to the treatment using LPS alone. This means that the use of firocoxib had a regulatory effect on the expressions of COX-2 and mPGES1 genes.
Conclusion
- The primer sets used for the expression of mPGES1 and cPLA2 in equine mononuclear cells were successfully validated. Hence, they can be effectively used in future gene expression studies.
- The study concluded that firocoxib could significantly decrease the expressions of COX-2 and mPGES1 genes that were induced by LPS. Thus, it can be inferred that firocoxib may hold potential for controlling diseases where expressions of these genes are upregulated.
Cite This Article
APA
Barton MH, Darden JE, Clifton S, Vandenplas M.
(2015).
Effect of firocoxib on cyclooxygenase 2, microsomal prostaglandin E2 synthase 1, and cytosolic phospholipase A2 gene expression in equine mononuclear cells.
Am J Vet Res, 76(12), 1051-1057.
https://doi.org/10.2460/ajvr.76.12.1051 Publication
Researcher Affiliations
MeSH Terms
- 4-Butyrolactone / administration & dosage
- 4-Butyrolactone / analogs & derivatives
- Animals
- Centrifugation, Density Gradient
- Cyclooxygenase 2 / metabolism
- Cyclooxygenase 2 Inhibitors
- Cytosol / enzymology
- Dinoprostone / metabolism
- Gene Expression Regulation / drug effects
- Horses
- Intramolecular Oxidoreductases / metabolism
- Leukocytes, Mononuclear / metabolism
- Lipopolysaccharides / pharmacology
- Membrane Proteins / metabolism
- Microsomes / enzymology
- Phospholipases A2 / metabolism
- Polymerase Chain Reaction
- Prostaglandin-E Synthases
- Sulfones / administration & dosage
Grant Funding
- T35 OD010433 / NIH HHS
- 5T35 OD01043307 / NIH HHS
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Citations
This article has been cited 1 times.- Han C, Fu R, Lei W. Beneficial effects of dexmedetomidine on early postoperative cognitive dysfunction in pediatric patients with tonsillectomy. Exp Ther Med 2018 Jul;16(1):420-426.
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