氧化应激诱导的DNA损伤通过PARP-1信号通路介导七氟醚致发育脑海马神经元死亡的机制研究

Volatile anesthetics are extensively applied in maternal and infant anesthesia, but their potential neurotoxicity in developing brain are drawing more concerns regarding sequelae after anesthesia exposure. Studies have shown that neuron death is the major characteristics of neurotoxicity induced by volatile anesthetics in developing brain, the underlying mechanisms, however, are not well clarified. Researches have found that DNA damage is related to the cellular death of many diseases. Intracellular ROS-induced oxidative stress is of the most crucial factor to DNA damage, and oxidative DNA damage induces PARP-1 overactivation, thus results in caspase independent cellular death, named Parthanatos. Our current research found that sevoflurane, commonly used volatile anesthetics in infants, presented to induce DNA damage and PARP-1 activation. It has also been demonstrated that sevoflurane facilitated intracellular ROS generation. Therefore, we hypothesize that sevoflurane induces DNA damage through ROS generation, leading to activation of PARP-1 signal pathway-mediated Parthanatos, and consequently results in neuronal death of hippocampus in developing brain. In this present study, we aim to clarify the mechanisms of neuronal death induced by sevoflurane in developing brain by using neonatal hippocampal neurons in vitro and neonatal rats in vivo, and expect to provide new strategy to protect from neurodevelopmental toxicity induced by volatile anesthetics.

吸入麻醉药广泛用于孕产妇或婴幼儿麻醉，它对发育脑的神经毒性作用越来越受到关注。研究表明致神经元死亡是吸入麻醉药诱导发育脑神经毒性的主要特征，然而具体机制不清。研究发现DNA损伤与多种疾病的细胞死亡相关，细胞内ROS诱导的氧化应激是最重要的DNA损伤因素，DNA氧化损伤能够引起PARP-1过度活化，导致非caspase依赖性细胞死亡（Parthanatos）。我们最新研究发现，婴幼儿常用的吸入麻醉药七氟醚能够诱导DNA损伤，引起PARP-1活化；研究证实七氟醚还能促进细胞ROS产生。因此我们提出假说：七氟醚能够通过促进ROS生成，诱导DNA损伤，活化PARP-1信号通路介导的Parthanatos死亡途径进而造成发育脑海马神经元死亡。为此，本课题采用体外海马神经元和新生大鼠为研究对象对以上问题进行探讨，以期望阐明七氟醚诱导发育脑神经元死亡的机制，为防治吸入麻醉药的神经发育毒性作用提供新策略。

全世界每年有几千万例的婴幼儿接受麻全身麻醉药物暴露。研究表明，生命早期暴露吸入麻醉药会对婴幼动物及婴幼儿的未成熟大脑产生神经发育毒性，导致大脑神经细胞死亡和神经认知功能障碍，但具体机制尚未阐明。研究发现DNA损伤与多种疾病的细胞死亡相关，细胞内ROS诱导的氧化应激是最重要的DNA损伤因素，DNA氧化损伤能够引起PARP-1过度活化，导致非caspase依赖性细胞死亡（Parthanatos）。本项目应用神经元细胞和新生大鼠观察氧化性DNA损伤和PARP-1信号通路活化在七氟醚诱导神经元Parthanatos死亡中的作用及其机制，并明确Parthanatos在七氟醚致发育脑神经元死亡中的作用。研究发现：七氟醚不仅能够诱导发育脑神经元DNA损伤，还能激活PARP-1依赖性的细胞死亡途径，诱导神经元Parthanatos死亡；细胞内ROS是诱导DNA损伤和PARP-1活化的上游信号分子，七氟醚通过促进ROS积蓄引起氧化应激，诱导DNA损伤，活化PARP-1信号通路，进而介导神经元Parthanatos死亡；七氟醚诱导发育脑神经元Parthanatos死亡不仅与神经元内PARP-1/PAR/AIF/MIF信号通路活化有关，还与PARP-1/NAD+/MPTP/AIF/MIF及PARP-1/NAD+/Bim/Bax/AIF/MIF信号通路激活有关；七氟醚诱导发育脑神经元Parthanatos死亡还与活化神经元JNK信号通路密切相关。研究结果表明，七氟醚能够通过诱导神经元ROS积蓄引起氧化应激，导致广泛的DNA氧化损伤，引起PARP-1过度活化，进而引起NAD+消耗和PAR积蓄，导致线粒体MPTP开放、线粒体内AIF释放和核转位，同时七氟醚还能通过活化JNK信号通路进一步促进AIF释放，最后AIF招募MIF进入细胞核，启动Parthanatos死亡通路，最终导致发育脑海马神经元死亡。