PI3K/Akt/GSK-3β信号通路在右美托咪定逆转异丙酚神经毒性中的作用

Dexmedetomidine is a highly selective a2-adrenoceptor agonist which has the effects including sedation and analgesia. It is used as an adjuvant drug for general anesthesia and local anesthesia. A number of studies indicated that dexmedetomidine has neuroprotective properties and it could attenuate hypoxic-ischemic and drug-related brain injury. Our preliminary experiments showed that dexmedetomidine reversed propofol-induced neurotoxicity, upregulated pAkt and downregulated GSK-3β in hippocampal tissues. Because PI3K/Akt/GSK-3β signaling pathway may have important roles on dexmedetomidine reversing propofol-induced neurotoxicity, the objectives of our project are: ① to investigate the effects of PI3K/Akt/GSK-3β inhibitors or dexmedetomidine on metabolites of brain, the expression of PI3K/Akt/GSK-3β pathway and apoptosis-related moleculars, growth and apoptosis of neurons, and adult cognitive function in neonatal rats of propofol anesthesia; ② to investigate metabolites of hippocampus,the expression of PI3K/Akt/GSK-3β pathway and apoptosis-related moleculars, and the functions of neurons after the hippocampal slices and hippocampal neuron cells of rats were incubated with PI3K inhibitor, GSK-3β inhibitor, dexmedetomidine or propofol; ③ to test interacting of PI3K, Akt and GSK-3β by GST pull-down method and to elucidate the roles of PI3K/Akt/GSK-3β signaling pathway on dexmedetomidine reversing propofol-induced neurotoxicity.

右美托咪定是高选择性的α2肾上腺素受体激动剂，具有镇静镇痛作用，可作为全身麻醉、局部麻醉的辅助用药。研究表明，右美托咪定有神经保护功能，可减轻缺血缺氧及药物对大脑的损害。我们前期的实验发现右美托咪定逆转异丙酚的神经毒性，上调pAkt而下调GSK-3β。针对PI3K/Akt/GSK-3β通路在右美托咪定逆转异丙酚神经毒性中具有重要作用的特点，本项目：①观察PI3K/Akt/GSK-3β抑制剂、右美托咪定对异丙酚麻醉大鼠大脑代谢产物、PI3K/Akt/GSK-3β通路、凋亡相关分子及神经元存活、凋亡以及成年后认知功能的影响；②以上述药物孵育大鼠海马脑片及海马神经元，观察海马代谢物质、PI3K/Akt/GSK-3β通路、凋亡相关分子和细胞功能的变化；③GST pull-down验证PI3K、Akt、GSK-3β的相互作用，阐明PI3K/Akt/GSK-3β在右美托咪定逆转异丙酚神经毒性中的作用。

右美托咪定是高选择性的α2肾上腺素受体激动剂，具有镇静镇痛作用。近年来，右美托咪定在临床中应用较为广泛，很多研究提示其具有神经保护作用。本项目中，我们以7天的新生大鼠作为实验对象，分别注射右美托咪定、异丙酚、PI3K/Akt抑制剂或GSK-3β抑制剂等，并将部分大鼠养至9周，应用透射电镜、水迷宫、TUNEL、免疫组化、RT-PCR、Western blot以及免疫荧光等技术观察到右美托咪定可以逆转异丙酚麻醉导致的7天和9周大鼠海马神经元和突触结构的损害，减少海马神经元的凋亡，而且右美托咪定减轻了异丙酚对PSD95、pAkt(ser473)和 pGSK-3β(ser9)等蛋白表达的抑制作用，并使9周大鼠空间学习记忆功能得以恢复；体外实验中，通过分离胎鼠海马神经元细胞，应用CCK-8、流式细胞仪、RT-PCR以及Western blot等方法进一步发现右美托咪定可以剂量依赖性的减轻异丙酚对体外培养的海马神经元细胞的毒性作用，上调pCREB、BDNF、Bcl-2、pAkt(ser473)和 pGSK-3β(ser9)等的表达。无论是体内实验还是体外实验，本研究均发现PI3k/Akt抑制剂LY294002可减弱右美托咪定对海马神经元的保护作用，而GSK-3β抑制剂 TDZD-8可增强右美托咪定的神经保护作用，这说明右美托咪定可能是通过激活 PI3K/Akt/GSK-3β信号通路从而逆转异丙酚的神经毒性作用。