异氟烷神经毒性：线粒体通透性转换孔mPTP分子机制研究

Inhalation 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 mechanism(s) by which isoflurane induces apoptosis remains largely unknown. Our preliminary data showed that isoflurane can induce the opening of mitochondrial permeability transition pore (mPTP), cytochrome C release, caspase-3 activation and apoptosis, leading to cognitive impairment in mice. Moreover, neural progenitor cells (NPCs) of mice have been found to be resistant to the isoflurane-induced caspase-3 activation, whereas mice neurons have not been resistant. .We therefore will use the established system in both NPCs and neurons to test a hypothesis that isoflurane regulates mPTP components voltage-dependent anion channel (VDAC), ADP/ATP translocator (ANT) and Cyclophilin D (Cyp D), leading to caspase activation and apoptosis. In Aim 1, we will compare the levels of VDAC, ANT and Cyp D, as well as reactive oxygen species (ROS), mPTP opening, mitochondrial membrane potential (MMP) between neurons and NPCs of mice. We expect that isoflurane will still increase ROS levels. However, it will cause lesser effects on the levels of VDAC, ANT, Cyp D, mPTP, MMP, caspase-3 activation and apoptosis in NPCs as compared to the effects on mice neurons. In Aim 2, we will use RNA interference to knock down VDAC, ANT and Cyp D in mice neurons. We expect that RNAi of VDAC, ANT, Cyp D will affect the isoflurane-induced mPTP opening, MMP and caspase-3 activation. In Aim 3, we will assess whether isoflurane will have a lesser neurotoxicity and neurobehavioral deficits in Cyp D knockout mice. .The proposed studies will test a novel hypothesis using innovative system and new technology. The outcomes from the studies could .demonstrate the mitochondria-associated molecular basis of the isoflurane-induced caspase-3 activation, apoptosis, as well as neurotoxicity and neurobehavioral deficits. These findings would likely lead to the development of targeted interventions of the anesthesia neurotoxicity.

吸入麻醉药异氟烷可诱发神经细胞凋亡，从而引起术后认知功能障碍并促进阿尔茨海默病的神经病理发生，但其机制不明。我们前期研究证实：异氟烷可通过线粒体通透性转换孔(mPTP)过渡开放，释放细胞色素C，激活caspase，导致神经细胞凋亡，最终影响小鼠的认知功能。由此假设：异氟烷通过诱导mPTP过渡开放导致神经细胞凋亡。.然而，异氟烷诱导的凋亡在神经元和神经前体细胞中的反应却截然不同；异氟烷仅诱发神经元的凋亡，而神经前体细胞对其诱发的凋亡却具有天然的“免疫作用”。因此，本课题首次采用异氟烷对神经元和神经前体细胞凋亡的不同诱导作用这一创新模型，应用基因敲除、小干扰RNA、免疫印迹、流式细胞等技术，研究异氟烷对mPTP相关构成蛋白(CypD, VDAC, ANT)的调控作用；并探讨上述蛋白在其诱导神经元凋亡中的作用机制。本结果将揭示异氟烷诱导神经细胞凋亡的分子机制，为防治麻醉药神经毒性提供理论依据。

吸入麻醉药异氟烷可诱发神经细胞凋亡，从而引起术后认知功能障碍并促进阿尔茨海默病的神经病理发生，但其机制不明。本课题首次采用异氟烷对神经元和神经前体细胞凋亡的不同诱导作用这一创新模型，研究异氟烷对线粒体/mPTP相关构成蛋白(CypD, VDAC, ANT)的调控作用；并探讨上述蛋白在其诱导神经元凋亡中的作用机制。. 我们在进展报告中提到过在进行WT的神经元在异氟烷处理后凋亡信号因子检测的过程中,我们意外的发现 ,mild hypothermia(温度在35-35.5°C的情况下)也出现了保护异氟烷诱导的凋亡的过 程。结果和在CYPD knock out的神经元中的情况十分相似。并将这一特别的发现作为我们工作的新的方向。我们发现异氟烷可以诱导DNA的损失以及产生细胞毒性，在此过程中，线粒体ATP的降低起到重要重要。我们通过降低温度达到Mild hypothermia的状态，可以通过减缓ATP的减低，从而保护线粒体的功能，从而保护细胞不受损伤。在此实验中，我们用启用了human neuroglioma cells作为实验model。我们通过 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) 和lactate dehydrogenase (LDH) 的检测，以及phosphorylation of the histone protein H2A variant X 在 Ser139 (gamma H2A.X)位点上的变化来评估上述实验结果。. 同时,我们完成了在原代神经前体细胞上进行细胞线粒体通透性转换孔mPTP开放的检测。并且发现细胞线粒体通透性转换孔上的调节蛋白CYPD与mPTP的开放直接相关。具体来说：CYPD蛋白的升高，可以直接调控MPTP的开放，我们在WT的神经前体细胞看到，当CYPD蛋白水平提高后，MPTP会被持续打开，而在CYPD deficeincy的神经前体细胞上，MPTP不会被开放。