Douglas Hope F, Southwood Louise L, Meyer-Ficca Mirella L, Hart Samantha K, Meyer Ralph G
School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, 19104.
Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA, 19348.
J Vet Emerg Crit Care (San Antonio). 2015 Jul-Aug;25(4):528-37. doi: 10.1111/vec.12316. Epub 2015 Jun 3.
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.
Experimental study.
Blood samples were collected from systemically healthy ponies (n = 3) and horses (n = 3).
(1) Equine peripheral blood mononuclear cells were exposed to 3 different concentrations of hydrogen peroxide (H2 O2 ) 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 H2 O2 -mediated PARP1 stimulation. PAR levels reflecting PARP1 activity were determined using immunoblot analyses.
Commercially available anti-PAR antibodies were used successfully to identify equine PAR. There was a significant increase in PAR accumulation following treatment with H2 O2 . All of the tested PARP inhibitors significantly reduced PAR accumulation to or below basal levels following treatment with H2 O2 .
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 H2 O2 -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.
评估聚（ADP - 核糖）聚合酶 -1（PARP1）酶及其在马体内的抑制作用，并探索其作为马肠道缺血再灌注损伤新治疗靶点的潜力，具体方法包括：（1）使用现有的免疫印迹分析技术，将聚（ADP - 核糖）（PAR）鉴定为马细胞中PARP1激活的指标；（2）在体外氧化DNA损伤模型中诱导PARP1激活；（3）使用市售的PARP1抑制剂证明其对马细胞中PARP1的抑制作用。
实验研究。
从全身健康的小马（n = 3）和马（n = 3）采集血样。
（1）将马外周血单个核细胞暴露于3种不同浓度的过氧化氢（H2O2），并在特定时间点进行裂解。然后通过免疫印迹分析评估PARP1活性，以确定PAR水平。（2）在H2O2介导的PARP1刺激之前，将马外周血单个核细胞与特定浓度的PARP1抑制剂进行预孵育。使用免疫印迹分析确定反映PARP1活性的PAR水平。
成功使用市售的抗PAR抗体鉴定出马的PAR。用H2O2处理后，PAR积累显著增加。所有测试的PARP抑制剂在用H2O2处理后均显著降低PAR积累至基础水平或以下。
这项原理验证研究表明，使用免疫印迹技术可在马体内鉴定出作为PARP1活性指标的PAR，马的PARP1可被H2O2诱导的DNA损伤激活，且这种激活可被PARP1酶抑制剂抑制。数据表明，PARP1途径在马细胞对氧化DNA损伤的反应中起作用，并支持其作为新治疗靶点的潜力。需要进一步研究记录体内PAR水平的升高以及PARP1抑制剂在马肠道缺血再灌注模型中的疗效。
