星形胶质细胞CB1R-TREK1通路在七氟烷麻醉致神经发育毒性中的作用

There are many controversies about neurodevelopmental toxicity associated with general anesthesia. TWIK-related potassium channel-1 (TREK-1) is an important target of inhaled anesthetics and can be activated by CB1R to mediate glutamate release. Our previous studies have confirmed that astrocytic TREK1 participate in apoptosis by regulating BDNF and EAAT2 in hippocampal and play an important role in anesthetic-related short-term memory injury in adult mice. Preliminary experiments found that repeated exposure to sevoflurane after birth could cause TREK1 up-regulation in hippocampal astrocytes, which was negatively correlated with cognitive level in adult mice. Meanwhile, sevoflurane exposure could increase the release of glutamate in primary cultured astrocytes, and up-regulate the expression of NR2A but not NR2B in the hippocampus. Accordingly, we speculated that sevoflurane anesthesia enhanced the release of astrocytic glutamate by activating CB1R-TREK1 pathway and further leaded to neurodevelopmental toxicity by regulating the generation of postsynaptic LTD and the balance of NR2A/NR2B. This project intends to use transgenic animals and electrophysiological techniques to clarify the mechanism of the astrocytic CB1R-TREK1 pathway in neurodevelopmental toxicity induced by sevoflurane anesthesia and lay the foundation for finding potential intervention targets.

全麻药物对幼儿神经发育的影响尚存诸多争议。TWIK相关钾离子通道1（TREK1）是吸入麻醉药作用的重要靶点，可被大麻素受体1（CB1R）激活介导星形胶质细胞谷氨酸快速释放。申请者前期证实海马星形胶质细胞TREK1可通过调控BDNF及EAAT2参与细胞凋亡的发生，在成年小鼠麻醉相关短期记忆损伤中发挥重要作用。最新预实验发现发育早期七氟烷暴露可引起海马星形胶质细胞中TREK1表达上调，并与成年后认知水平呈负相关。同时七氟烷暴露可提高星型胶质细胞谷氨酸释放水平，上调突触后NR2A的表达。据此我们推测七氟烷麻醉激活海马星形胶质细胞CB1R-TREK1通路进而介导谷氨酸释放，作用于突触后膜，通过调节突触后兴奋性传递及NR2A/NR2B的平衡最终导致长期认知损伤。本课题拟利用转基因动物和电生理技术，阐明星形胶质细胞CB1R-TREK1通路在麻醉相关神经发育毒性中的作用，为寻找潜在干预靶点奠定基础。

“全麻药物和围手术期应激对发育脑功能的影响及其远期效应”已被列为麻醉学亟待解决的十大科学问题之一。TWIK相关钾离子通道1（TREK1）是吸入麻醉药作用的重要靶点，可被大麻素受体1（CB1R）激活介导星形胶质细胞谷氨酸快速释放。课题组前期研究证实海马星形胶质细胞TREK1可通过调控BDNF及EAAT2参与细胞凋亡的发生，在成年小鼠麻醉相关短期记忆损伤中发挥重要作用，但其在发育脑中的作用尚未可知。进一步研究发现发育早期七氟烷暴露可引起海马星形胶质细胞中TREK1表达上调，并与成年后认知水平呈负相关。此外，发现星形胶质细胞特异性TREK1基因敲除小鼠较同等暴露条件下的Wild-type小鼠成年后认知能力有明显改善，提示星形胶质细胞TREK1参与了麻醉诱导的神经发育毒性损伤。本课题利用在体、离体模型，证实七氟烷麻醉激活海马星形胶质细胞CB1R-TREK1通路，介导谷氨酸释放，作用于突触后膜调节突触后兴奋性传递及NR2A/NR2B平衡，引起长期认知损伤。同时发现七氟烷长时程暴露可上调海马神经干细胞中TREK1表达，抑制PKA、AKT、GSK3β磷酸化，进而抑制cyclinD1、cyclinE/CDK2，导致G1期停滞，同时TREK1通过抑制PKA磷酸化抑制了CREB，引起细胞增殖抑制，两者共同导致海马神经发生异常，造成老鼠成年后认知功能受损。使用TREK1特异性阻断剂Spadin可逆转上述现象，改善七氟烷长时程暴露所致的认知功能损伤。上述结果表明TREK1是麻醉相关神经发育毒性的关键分子，且在不同细胞中介导不同的下游信号通路，为开发靶向性防治策略奠定了坚实的理论基础。