Poly(ADP-ribose)polymerases inhibitors prevent early mitochondrial fragmentation and hepatocyte cell death induced by H2O2

Poly(ADP-ribose)polymerases (PARPs) are a family of NAD+ consuming enzymes that play a crucial role in many cellular processes, most clearly in maintaining genome integrity. Here, we present an extensive analysis of the alteration of mitochondrial morphology and the relationship to PARPs activity after oxidative stress using an in vitro model of human hepatic cells. The following outcomes were observed: reactive oxygen species (ROS) induced by oxidative treatment quickly stimulated PARPs activation, promoted changes in mitochondrial morphology associated with early mitochondrial fragmentation and energy dysfunction and finally triggered apoptotic cell death. Pharmacological treatment with specific PARP-1 (the major NAD+ consuming poly(ADP-ribose)polymerases) and PARP-1/PARP-2 inhibitors after the oxidant insult recovered normal mitochondrial morphology and, hence, increased the viability of human hepatic cells. As the PARP-1 and PARP-1/PARP-2 inhibitors achieved similar outcomes, we conclude that most of the PARPs effects were due to PARP-1 activation. NAD+ supplementation had similar effects to those of the PARPs inhibitors. Therefore, PARPs activation and the subsequent NAD+ depletion are crucial events in decreased cell survival (and increased apoptosis) in hepatic cells subjected to oxidative stress. These results suggest that the alterations in mitochondrial morphology and function seem to be related to NAD+ depletion, and show for the first time that PARPs inhibition abrogates mitochondrial fragmentation. In conclusion, the inhibition of PARPs may be a valuable therapeutic approach for treating liver diseases, by reducing the cell death associated with oxidative stress.

聚ADP核糖聚合酶（Poly(ADP-ribose)polymerases, PARPs）是一类消耗烟酰胺腺嘌呤二核苷酸（NAD+）的酶家族，在诸多细胞生理过程中发挥关键作用，其中以维持基因组完整性的功能最为明确。本研究基于人肝细胞体外模型，系统分析了氧化应激后线粒体形态的改变及其与PARPs活性的关联。实验观察到以下结果：氧化处理诱导产生的活性氧（ROS）可快速激活PARPs，促进与早期线粒体碎裂及能量代谢障碍相关的线粒体形态改变，最终触发细胞凋亡。在氧化损伤发生后，采用特异性PARP-1（主要的NAD+消耗型聚ADP核糖聚合酶）及PARP-1/PARP-2抑制剂进行药物干预，可恢复正常的线粒体形态，进而提升人肝细胞的存活率。由于PARP-1与PARP-1/PARP-2抑制剂的干预效果相近，我们推断PARPs介导的多数效应均源于PARP-1的激活。烟酰胺腺嘌呤二核苷酸补充疗法与PARPs抑制剂的作用效果相仿。因此，在遭受氧化应激的肝细胞中，PARPs激活及后续的NAD+耗竭是导致细胞存活率降低（以及细胞凋亡增多）的关键事件。本研究结果表明，线粒体形态与功能的改变似乎与NAD+耗竭密切相关，并首次证实PARPs抑制可消除线粒体碎裂现象。综上，通过减轻氧化应激相关的细胞死亡，抑制PARPs或可成为治疗肝脏疾病的极具价值的治疗策略。