title: Effect of firocoxib on cyclooxygenase 2, microsomal prostaglandin E2 synthase 1, and cytosolic phospholipase A2 gene expression in equine mononuclear cells.
abstract: OBJECTIVE
  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.
authors.forename: Michelle H
authors.initials: MH
authors.lastname: Barton
authors.forename: Joshua E
authors.initials: JE
authors.lastname: Darden
authors.forename: Sarah
authors.initials: S
authors.lastname: Clifton
authors.forename: Michel
authors.initials: M
authors.lastname: Vandenplas
mesh_terms: D015107:4-Butyrolactone / Q000008:administration & dosage / Q000031:analogs & derivatives*; D000818:Animals; D002499:Centrifugation, Density Gradient; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D052246:Cyclooxygenase 2 Inhibitors; D003600:Cytosol / Q000201:enzymology; D015232:Dinoprostone / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006736:Horses; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D054467:Phospholipases A2 / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D000071876:Prostaglandin-E Synthases; D013450:Sulfones / Q000008:administration & dosage*
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D013450:Sulfones; D051546:Cyclooxygenase 2; D054467:Phospholipases A2; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; D015232:Dinoprostone; D015107:4-Butyrolactone; C487384:firocoxib