In vitro effects of poly(adp-ribose) polymerase inhibitors in an equine model of inflammation.

Introduction: Endotoxemia and ischemia/reperfusion (I/R) injury following strangulating gastrointestinal tract diseases are leading causes of morbidity and mortality in horses. Poly(ADP-ribose) polymerase (PARP) may play a role when activated in response to DNA injury induced by these types of lesions, and synthetizes PAR polymers using NADþ as donor of ADP-ribose units. Hyperactivation of this process leads to rapid depletion of the intracellular NADþ andATP stores culminating in energy crisis-induced cell necrosis. Cell death and increased inflammatory response is also due toPAR-dependent apoptosis-inducing factor translocation to the nucleus and release of the pro-inflammatory mediator high mobility group box 1. Because of these functions, PARP pharmacologic inhibitionmay be protective and therefore beneficial if used in conjunction with the current therapy for I/R injury and endotoxemia. The aim of the present study was to utilize an equine in-vitro model in order to determine the TNF-a and NO production after lipopolysaccharide (LPS)/interferon-g (IFN-g) stimulation of mononuclear equine cells, and evaluate the protective effect of PARP inhibition on TNF-a and NO production. Methods: All procedures were IACUCapproved. Following a pilot study (2 horses) to determine the TNFa and NO production after LPS/IFN-g stimulation of mononuclear cells, peripheral venous blood was collected from 6 healthy horses; white blood cells were separated and mononuclear cells were cultured. Various concentrations of IFN-g and LPS (none, low and high stimulation) were added to cultured cells to mimic endotoxemia, and PARPinhibitors (ABT888,AZD2281, PJ34) were added at 1mM final concentration to wells before stimulation to determine their protective effects. Triplicates were performed for each combination of stimulation and inhibition. After 24 h of incubation a NO assay and TNF-a ELISA were performed. ANOVA for repeated measures was used for statistical analysis (P<0.05). Results: Results of the pilot study revealed no increase in NO production. Therefore, only TNF-a concentration was measured in samples from the 6 horses. There was no difference in TNF-a production between control (untreated) and treatment with ABT888 or AZD228. When data from experiments using PJ34 under high stimulation condition were analyzed separately, a significant difference between control group and PJ34 inhibited samples was detected (P¼0.0457). Discussion and conclusion: According to our findings, inhibition of PARP with PJ34 decreases TFN-a production in LPS/IFN-g stimulated equine mononuclear cells, suggesting that this inhibitor may become a promising novel therapy helping prevent inflammation and thereby reducing endotoxin-induced organ damage in horses.