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The activity and inhibition of poly(ADP‐ribose) polymerase‐1 in equine peripheral blood mononuclear cells in vitro
Author(s) -
Douglas Hope F.,
Southwood Louise L.,
MeyerFicca Mirella L.,
Hart Samantha K.,
Meyer Ralph G.
Publication year - 2015
Publication title -
journal of veterinary emergency and critical care
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.886
H-Index - 47
eISSN - 1476-4431
pISSN - 1479-3261
DOI - 10.1111/vec.12316
Subject(s) - parp1 , poly adp ribose polymerase , peripheral blood mononuclear cell , medicine , microbiology and biotechnology , in vitro , pharmacology , polymerase , enzyme , immunology , biochemistry , biology
Objectives To evaluate the poly (ADP‐ribose) polymerase‐1 (PARP1) enzyme and its inhibition in horses and explore its potential as a novel therapeutic target for equine intestinal ischemia‐reperfusion injury by (1) identifying poly (ADP‐ribose) (PAR) as an indication of PARP1 activation in equine cells using available immunoblot analytical techniques, (2) inducing PARP1 activation in an in vitro oxidative DNA damage model, (3) and demonstrating the inhibition of PARP1 in equine cells using commercially available PARP1 inhibitors. Design Experimental study. Animals Blood samples were collected from systemically healthy ponies ( n = 3) and horses ( n = 3). Interventions (1) Equine peripheral blood mononuclear cells were exposed to 3 different concentrations of hydrogen peroxide (H 2 O 2 ) and were lysed at specific time points. PARP1 activity was then assessed by using immunoblot analyses to determine PAR levels. (2) Equine peripheral blood mononuclear cells were preincubated with defined concentrations of PARP1 inhibitors prior to H 2 O 2 ‐mediated PARP1 stimulation. PAR levels reflecting PARP1 activity were determined using immunoblot analyses. Measurements and Main Results Commercially available anti‐PAR antibodies were used successfully to identify equine PAR. There was a significant increase in PAR accumulation following treatment with H 2 O 2 . All of the tested PARP inhibitors significantly reduced PAR accumulation to or below basal levels following treatment with H 2 O 2 . Conclusions This proof of principle study demonstrated that PAR, an indicator of PARP1 activity, can be identified in the equine species using immunoblot techniques, that equine PARP1 can be activated by H 2 O 2 ‐induced DNA damage, and that this activation can be inhibited by PARP1 enzyme inhibitors. The data suggest that the PARP1 pathway plays a role in the equine cellular response to oxidative DNA damage and supports its potential as a novel therapeutic target. Further research documenting an increase in PAR levels in vivo and the efficacy of PARP1 inhibitors in an equine intestinal ischemia‐reperfusion model is needed.