The University of Georgia College of Veterinary Medicine

Georgia Veterinary Scholar Program

Georgia Veterinary Scholar	Faculty Mentor

Jenn Winnick University of Georgia Class of 2007	Dr. Jim Moore

Effects of non-steroidal anti-inflammatory drugs on activation of NF- kB in equine cells

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used for the treatment of pain, inflammation and endotoxemia in horses. Of the vast array of NSAIDs available to veterinarians, a few have been shown to be far more effective than the others in treating inflammatory processes such as endotoxemia. It is well known that NSAIDS exert their effects by inhibiting cyclo-oxygenase. However, some NSAIDs also exert anti-inflammatory effects via an alternate mechanism that prevents activation of the pro-inflammatory transcription factor nuclear factor kappa B (NF- k B). We hypothesize that some NSAID inhibit activation of NF- k B to a greater extent than the others, thereby accounting for their enhanced efficacy in treating complex inflammatory processes. In this study cells are incubated with phenylbutazone, ketoprofen or flunixin meglumine and subsequently stimulated with lipopolysaccaride. The nuclear proteins are extracted, incubated with a radioactively labeled oligonucleotide containing the binding site for NF- k B. Complexes formed were separated using an electrophoretic mobility shift assay. Using optical densities, comparisons are made based on the amount of NF- k B that is attached to the radioactively labeled oligonucleotide. From this information, concentrations of individual NSAIDs that inhibit lipopolysaccharride-dependent nuclear translocation of NF- k B by can be identified. In order to carry out this study, we first had to establish the conditions for performing the electrophoretic mobility shift assay using the nuclei from equine cells. At this point, the assay now works reliably with equine cells and data have been obtained using phenylbutazone at concentrations of 0.1-100 µg/ml. A decrease in the amount of NF- k B bound to the radioactively labeled oligonucleotide was observed at a concentration of 10 µg/ml. Continuation of this study will allow for data to be collected for ketoprofen and flunixin meglumine.