GABA(A)R激活NCX上调PI3K/Akt/mTOR信号通路在七氟烷致神经发育毒性中的作用及机制研究

临床研究显示3岁前多次七氟烷麻醉下手术的患儿神经系统发育受到影响，FDA警示患儿3岁前接受2次以上麻醉可能影响小儿脑发育。我们之前的研究发现七氟烷通过GABA(A)R引起发育期神经元去极化，导致兴奋性损伤。预实验中我们用单细胞测序证明七氟烷引起少突胶质细胞前体细胞(OPC)集落数减少。据此提出研究假设：七氟烷通过作用于突触后膜GABA(A)R，引起OPC去极化，激活Na+/Ca2+交换器(NCX)导致细胞内Ca2+聚集，上调PI3K/Akt/mTOR通路，ULK1自噬启动复合物形成障碍，髓鞘少突胶质细胞生成减少，影响髓鞘发育，最终引起认知功能下降。本项目将应用基因敲除、siRNA基因沉默、CRISPR等技术建立离体及在体模型，通过检测自噬相关指标、突触后电位、髓鞘发育、细胞内Ca2+等，来验证本假说。本课题从髓鞘发育角度阐明七氟烷引起神经发育毒性的机制，为临床上预防提供新的靶点。

Clinical studies have shown that the neurological development of children undergoing sevoflurane anesthesia for several times is affected before the age of 3. The FDA warns that more than 2 times of anesthesia before the age of 3 may affect the brain development of children. Our previous study found that sevoflurane causes depolarization of developmental neurons through GABA(A)R, leading to excitatory injury. In preliminary experiments, we demonstrated that sevoflurane caused a decrease in the colony number of oligodendrocytes (OPC) by single cell sequencing. Therefore, the research hypothesis was put forward: sevoflurane caused OPC depolarization by acting on GABA(A)R in the postsynaptic membrane, activated Na+/Ca2+ exchange (NCX), led to intracellular Ca2+ accumulation, up-regulated PI3K/Akt/mTOR pathway, impaired formation of ULK1 autophagy initiation complex, decreased myelin oligodendrocytes, and eventually caused the decline of cognitive function. In this project, gene knockout, siRNA gene silencing, CRISPR and other technologies were used to establish in vitro and in vivo models, and the hypothesis was tested by detecting autophagy related indicators, postsynaptic potential, myelin development, intracellular Ca2+, etc. This study will elucidate the mechanism of neurodevelopmental toxicity induced by sevoflurane from the perspective of myelin development, and provide a new target for clinical prevention.