PARP-1依赖性细胞死亡（Parthanatos）在异氟烷诱导发育期脑神经毒性中的作用及机制研究

Inhaled anesthetics are widely used in maternal and pediatric anesthesia, but their potential neurotoxicity in immature brain are raising significant concerns regarding sequelae after anesthesia. Studies have demonstrated that the damage and death of neuron are the major characteristics of neurotoxicity induced by inhaled anesthetics in developing brain, the mechanisms, however, are not well understood. Our current research found that the mechanisms of neuron death in developing brain caused by inhaled anesthetics isoflurane was not only involved with apoptosis, but also associated closely with PARP-1-dependent cell death, that is Parthanatos. However, it is still unclear the mechanism underlying the upstream signal of PARP-1 activation, the activation of PARP-1-mediated Parthanatos and its subsequent biological effects after isoflurane exposure. In this present study, we aim to clarify the role of PARP-1-dependent Parthanatos in isoflurane-induced neurodevelopment neurotoxicity and its mechanisms by using neonatal hippocampal neurons in vitro and neonatal rats in vivo, and expect to prove whether Parthanatos inhibitor could be a new therapeutic strategy to protect from neurotoxicity induced by inhaled anesthetics in developing brain.

吸入麻醉药广泛用于孕产妇或婴幼儿麻醉，它对未成熟脑的神经发育毒性作用越来越受到关注。研究表明致神经元损伤及死亡是吸入麻醉药诱导发育期神经毒性的主要特征，然而具体机制不清。我们最新研究发现，吸入麻醉药异氟烷造成发育期神经元死亡的机制不仅与促进神经元凋亡有关，还与其它死亡途径即PARP-1依赖性细胞死亡（Parthanatos）密切相关。但在异氟烷作用下，神经元内调节PARP-1活化的上游信号分子，PARP-1介导的Parthanatos激活的机制以及PARP-1激活后的生物学效应尚不清楚。为阐明该问题，本项目采用体外神经细胞和新生大鼠为研究对象，采用RNA干扰、蛋白印迹等方法对以上问题进行探讨。我们期望通过该研究，明确PARP-1依赖性的Parthanatos在异氟烷诱导发育期脑神经毒性中的作用及其机制，并探讨阻断Parthanatos能否成为预防和治疗吸入麻醉药致发育期神经毒性的新措施。

全世界每年有上千万例的婴幼儿、孕妇和胎儿接受麻醉药物暴露和手术治疗。研究表明临床常用的吸入麻醉药能够诱导发育期脑神经毒性作用，导致婴幼动物未成熟大脑广泛的神经元变性和死亡，但具体机制尚未阐明。Parthanatos为一种独特的非caspase依赖的细胞死亡方式，是基于DNA损伤诱发，依赖于PARP-1激活而介导的一种新的程序性细胞死亡，与多种神经疾病的细胞死亡密切相关。本研究应用体外神经元细胞和体内新生大鼠观察吸入麻醉药异氟烷对发育期神经元PARP-1依赖性Parthanatos死亡的影响及其分子机制，并明确Parthanatos在异氟烷的神经发育毒性中的作用。研究发现：异氟烷能够通过诱导神经元内ROS生成，引起DNA广泛损伤，进而引起PARP-1过度活化，介导发育期神经元Parthanatos死亡；异氟烷诱导发育期神经元Parthanatos死亡不仅与神经元PARP-1-PAR-AIF信号通路活化有关、还与PARP-1-NAD+-MPTP-AIF及PARP-1-NAD+-Bim-Bax-AIF信号通路激活有关；同时异氟烷还能够通过诱导PARP-1活化，抑制神经元内糖酵解限速酶（己糖激酶HKII和丙酮酸激酶PKM2）的活性，引起能量代谢抑制，减少NAD+和ATP生成，进而促进Parthanatos死亡发生；异氟烷诱导发育期神经元Parthanatos死亡还与活化神经元JNK信号通路及HMGB1炎性信号通路密切相关。研究结果表明，异氟烷能够诱导神经元ROS生成，引起氧化应激进而造成DNA广泛损伤，导致PARP-1过度激活，引起NAD+消耗和PAR积累，导致线粒体MPTP开放、AIF释放和核易位，同时激活的PARP-1还能通过抑制糖酵解和能量代谢、活化JNK信号通路及HMGB1炎性信号通路进一步引起AIF释放，启动Parthanatos死亡通路，最终导致发育期神经元死亡。