ATPIF1/ATP合酶在异氟烷致秀丽线虫全身损伤中的机制研究

Currently, the study of inhalational anesthetic-induced neurotoxicity and subsequent cognitive impairment is of great research interest in anesthesiology. The inhalational anesthetic isoflurane has been reported to induce caspase activation and apoptosis, potentially leading to postoperative cognitive dysfunction and promotion of Alzheimer’s disease neuropathogenesis. However, the underlying mechanisms by which isoflurane induce apoptosis remains largely unknown. Our preliminary data has shown that: firstly, isoflurane can activate mitochondrial apoptotic pathway, leading systemic injury in Caenorhabditis elegans; secondly, isoflurane can induce ATPase inhibitory factor 1(ATPIF1) increasing in neuron, the inhibitor of ATP synthase can improve isoflurane-induced neuronal apoptosis, which doesn’t affect the increasing of ATPIF1.It is newly reported that the increasing expression of ATPIF1 can inhibit the activity of ATP synthase in hydrolysis of ATP and induce the decrease of mitochondrial membrane potential. Based on the results of our preliminary studies and the aforementioned findings, we hypothesize that isoflurane induces systemic injury in Caenorhabditis elegans by increasing expression of ATPIF1, which could inhibit the activity of ATP synthase in hydrolysis of ATP, lead to declining mitochondrial membrane potential and activate mitochondrial apoptotic pathway, which could be prevented by administration of the inhibitor of ATP synthase. . Both apoptotic neurons model and systemic injury Caenorhabditis elegans model induced by isoflurane are used to test the hypothesis that isoflurane induces systemic injury in Caenorhabditis elegans by increasing expression of ATPIF1, which could inhibit the activity of ATP synthase in hydrolysis of ATP, lead to declining mitochondrial membrane potential and activate mitochondrial apoptotic pathway, the potential protective role of the inhibitor of ATP synthase on this process. To test this hypothesis, we will apply gene overexpression, small interfering RNA, RNA interfering, co-Immunoprecipitation, western blot, immunofluorescence confocal microscopy, electron microscopy, and behavioral tests to study the novel mechanism underlying isoflurane-induced cellular apoptosis at multiple aspects of molecular signaling, mitochondrial morphology, mitochondrial function, cell function, and animal behavior. The results will reveal the molecular mechanism of isoflurane-induced neuronal apoptosis and systemic injury, provide a theoretical basis for the prevention of inhalational anesthetic neurotoxicity.

吸入麻醉药异氟烷导致术后认知功能障碍（POCD），与神经元线粒体凋亡密切相关，但其上游机制未明。申请者前期研究证实：①异氟烷可激活线粒体凋亡，造成秀丽线虫损伤；②异氟烷诱导神经细胞ATP酶抑制因子1（ATPIF1）表达上调，ATP合酶抑制剂可改善异氟烷的神经细胞凋亡，且不影响ATPIF1表达。最新研究报道：ATPIF1可抑制ATP合酶水解ATP的活性，加重线粒体膜电位降低。我们推断：异氟烷通过诱导ATPIF1表达上调，降低ATP合酶水解ATP的活性，导致线粒体膜电位持续下降，激活线粒体凋亡通路，最终造成秀丽线虫损伤。本研究采用异氟烷诱导秀丽线虫全身损伤创新模型和神经细胞凋亡模型，通过调控ATPIF1表达或应用ATP合酶抑制剂干预，研究ATPIF1与ATP合酶结合能力、线粒体形态和功能、细胞凋亡以及线虫行为学的变化，从而揭示异氟烷导致POCD和线虫损伤的机制，为防治吸入麻醉药毒性提供新靶点。

术后认知功能障碍是麻醉领域的研究热点。吸入麻醉药物具有神经毒性作用，会导致学习记忆等认知功能减退，但确切机制未明。本研究的主要目的是明确ATPIF1是否介导吸入麻醉药异氟烷引起的神经细胞毒性，进而导致动物认知功能损伤。（1）首先建立异氟烷引起H4-APP细胞凋亡细胞模型，检测细胞内活性氧簇（ROS）试剂盒、线粒体膜电位（MMP）、ATP等线粒体功能指标；通过线粒体肿胀实验检测线粒体通透性转化孔（mPTP）的开放状态；通过western blotting实验和RT-PCR检测ATPIF1、线粒体凋亡效应蛋白Caspase-3蛋白及mRNA基因表达；研究ATP合酶抑制剂—二环己基碳二亚胺（DCCD）预处理，对异氟烷引起H4-APP细胞凋亡的保护作用。（2）通过构建低表达或过表达ATPIF1 H4-APP细胞，研究异氟烷对上述神经细胞的影响。（3）在体动物研究，验证ATP合酶抑制剂在异氟烷引起小鼠认知功能损伤中的保护作用。本研究结果表明：（1）异氟烷会使H4-APP细胞ATPIF1表达升高，mPTP持续过度开放，引起ROS蓄积、MMP降低、ATP生成减少； Caspase-3 mRNA基因表达增加和蛋白激活。DCCD可以阻断上述变化。（2）异氟烷不会引起低表达ATPIF1 H4-APP细胞mPTP持续过度开放，不会引起细胞内ROS、MMP和ATP的变化以及 Caspase-3 mRNA基因表达和蛋白活化。异氟烷会使过表达ATPIF1 H4-APP细胞mPTP大量过度开放，引起细胞内ROS蓄积、MMP降低、ATP生成减少；导致 Caspase-3 mRNA表达增加和蛋白活化。DCCD可以阻断异氟烷引起的过表达ATPIF1 H4-APP细胞变化。（3）ATP合酶抑制剂DCCD预处理，可以明显改善异氟烷引起的小鼠海马依赖性和非海马依赖性学习记忆功能损伤。本研究证实，异氟烷可以使ATPIF1表达升高，与ATP合酶结合，导致ATP生成减少，引起细胞内活性氧簇蓄积、线粒体损伤和神经细胞凋亡，进而损伤动物的认知功能。ATP合酶抑制剂可以阻断ATPIF1与ATP合酶的结合，对异氟烷的神经毒性产生保护作用。说明，ATPIF1是异氟烷引起神经细胞凋亡和动物认知功能损伤的作用靶点。本研究为术后认知功能障碍的防治提供可能的靶点和理论支持。

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