吸入麻醉药通过影响CRMP蛋白干扰新生大鼠脑突触发育的机制研究

Inhaled anesthetics can induce neurotoxicity in developing brain by disturbing synaptic development, the mechanism is underdetermined. Our proteomic research suggested sevoflurane decrease the protein expression of collapsing response mediator protein (CRMP) 1 and CRMP4 in neonatal rat cortice which closely related with synaptic development. Recently, our primary experimental results showed that isoflurane also decreased the protein expression of CRMP1 and CRMP4, activated GSK-3β and increased phosphorylation of CRMP2, while dexmedetomidine reduced isoflurane-induce neuroapotosis and increased expression of CRMP1 and CRMP4 and inhibited phosphorylation of CRMP2. Therefore, we presumed inhaled anesthetics may disturb synaptic development and induce neurotoxicity by interfering with the function of CRMP proteins. In this project, we will study 1) the effects of inhaled anesthetics on morphology changes of dendrites and axons development in hippocampal neurons of neonatal rats and their relationship with distribution and expression of CRMP1/2/4 protein by using immunostaining methods; 2) the signal pathway of inhaled anesthetics influencing CRMP1/2/4 proteins by using pharmacological approaches; 3) whether CRMP1/2/4 proteins will be involved in inhaled anesthetics-induced cognitive dysfunction by using patch clamp technique to measure LTP in hippocampus slices and behavior methods to measure learning and memory of rats. These results will better explain how inhaled anesthetics disturbing synaptic development and also provide the new clues about interventions against the anesthetics-induced neurotoxicity in developing brain.

吸入麻醉药可以干扰突触发育而诱导发育神经细胞毒性，但机制未明。我们近期蛋白质组学研究证明，七氟醚麻醉可以降低幼鼠皮质与突触发育相关的坍塌反应调节蛋白(CRMP)1和4的表达。预实验结果发现异氟醚也降低CRMP1/4表达，激活GSK-3β，增加CRMP2的磷酸化。而右美托咪啶可以抑制异氟醚诱导的发育幼鼠神经毒性并恢复CRMP1/4的表达，降低CRMP2的磷酸化。因此我们推论吸入麻醉药可能通过影响CRMP蛋白的功能，干扰突触发育而诱导神经毒性。本课题通过免疫染色观察吸入麻醉药对发育海马神经元树突和轴突的形态学影响及其与CRMP1/2/4表达和分布的关系，通过药理学方法研究吸入麻醉药影响CRMP1/2/4的信号通路，通过膜片钳检测脑片LTP和行为学研究探讨CRMP1/2/4变化是否介导吸入麻醉药诱导的认知功能障碍，以明确吸入麻醉药干扰突触发育的机制，为麻醉药毒性作用的防治提供新靶点。

本研究通过建立2.8%七氟醚处理P7新生大鼠4h模型，首先发现Cdk5，GSK-3β或RoA抑制剂（roscovitine，SB415286，Y27632）均能减轻幼鼠海马cleaved caspase-3表达，并确定了最佳的保护作用的剂量。进一步研究发现七氟醚激活了cdk5和GSK-3β的活性，增加了其下游CRMP2 Ser522、Thr514位点磷酸化表达，降低了CRMP1，CRMP2与 CRMP4总蛋白表达，而Cdk5和GSK-3β抑制剂逆转了七氟醚诱导的上述变化；七氟醚对RoA以及其下游CRMP2 Thr555位点磷酸化无影响。此外，cdk5抑制剂可逆转七氟醚诱导的p38及其下游NF-κB磷酸化表达增加。使用Golgi 染色方法研究发现，七氟醚处理后的幼鼠在P33天前额叶皮层以及海马CA1，CA3，DG各区神经元树突总长度，第二、三、四级树突长度和树突分支均显著减少，而Cdk5，GSK-3β或RoA抑制剂均减轻了七氟醚对树突的形态学影响。幼鼠在P25-P27进行Open-field Test，结果发现各组幼鼠运动的总距离和穿过中心区的次数无差异，提示七氟醚不会影响幼鼠的运动能力以及情感状况；幼鼠在P31-P32进行Fear conditioning test，结果发现七氟醚减少了电刺激后幼鼠的“木僵”时间，提示七氟醚损害了与海马相关的恐惧记忆；Cdk5或GSK-3β抑制剂，而非RhoA抑制剂，可以逆转七氟醚引起的幼鼠 “木僵”时间减少。对P33幼鼠海马突触相关蛋白表达的研究发现，七氟醚降低了PSD-95、Synaptophysin和drebrin蛋白表达，预处理Cdk5或GSK-3β抑制剂，可以逆转七氟醚引起的上述蛋白变化。P33幼鼠取脑片检测LTP，发现七氟醚可导致LTP的抑制，Cdk5或GSK-3β抑制剂，而非RhoA抑制剂，逆转了七氟醚对LTP的抑制。综上所述，七氟醚主要通过激活Cdk5/GSK-3β-CRMPs通路抑制神经元树突发育，降低PSD-95，突触素等蛋白的表达，从而导致幼鼠学习记忆损害。此外，我们还证明了异氟醚可通过抑制Akt/Bad通路并激活JNK和p38 MAPK通路诱导发育幼鼠神经细胞凋亡，而右美托嘧啶预处理通过激活Akt/Bad通路并抑制JNK和p38 MAPK通路减轻异氟醚诱导的神经毒性作用。