HDAC2调控NR2B/HGN表达在孕早期母体接受氯胺酮麻醉致大鼠子代学习记忆损害中的作用

The critical role of the expression balance between possitive and negative regulating factors in the formation and maintenance of learning and memory function has been well recognized.N-methyl-D-Aspartate receptor 2B unit (NR2B) and hippyragranin (HGN) are the established positive and negative regulating factors of learning and memory function in the hippocampus.Our preliminary study of national natural science fund project (81060093) showed that maternal ketamine anesthesia during early gestation could impair the learning and memory function of offspring rats' through down-regulating the expression of NR2B while up-regulating the expression of HGN.But role of the change of the expression balance of NR2B and HGN in the pathogenesis and it's upstream mechanism is still not known yet.It is known that histone deacetylase 2 (HDAC2) upstream regulating factors of learning and memory function by the pathway "HDAC2-cyclic adenosine monophosphate response element binding protein (CREB)-NR2B",while it's role in the pathogenesis of learning and memory function impairment of the offspring rats' induce by ketamine anesthesia during the early gestation is not known yet.In present study,both of the changes of the expression levels and activity of HDAC2 in the hippocampus and their relationship to the expression levels of CREB,NR2B,HGN and learning and memory function of the offspring rats' with maternal ketamine exposure during early gestation will be researched.Thus to clarify whether ketamine anesthesia during early gestation impair the learning and memory function by disturbing the expression balance between NR2B and HGN through the pathway "HDAC2-NR2B-CREB".Then,the inhibitors of NR2B and HDAC2,HGN antisense and HDAC2 gene knockout rats will be used to prove it further.It is critical to the clinical anesthesia selection.

正负调控平衡在学习记忆中的作用已得到重视。NR2B和HGN是海马内重要的正、负调控因子。课题组2013年结题的国家自然科学基金课题研究发现，孕早期母体接受氯胺酮麻醉通过抑制NR2B表达、促进HGN表达损害子代学习记忆，但其上游机制尚不清楚。HDAC2是调控学习记忆的上游因子，通过"HDAC2-CREB-NR2B"通路影响学习记忆，但其在氯胺酮对子代学习记忆损害中的作用不清楚。本项目拟分析孕早期母体接受氯胺酮麻醉后，SD大鼠子代海马HDAC2表达及活性变化与磷酸化CREB表达水平、NR2B/HGN表达平衡的关联及其与学习记忆的关系，以证实"孕早期母体接受氯胺酮麻醉是否通过HDAC2-CREB-NR2B通路破坏子代大鼠海马区NR2B/HGN表达平衡，从而损害子代学习记忆功能"，并用NR2B、HDAC2抑制剂、HGN反义核苷酸及HDAC2基因敲除大鼠模型进一步证实。对临床麻醉选择有重要意义。

课题组前期发现，孕早期母体接受氯胺酮麻醉对子代大鼠学习记忆的损害与其抑制NR2B、促进HGN表达有关，但NR2B/HGN表达平衡改变在其中的作用及上游机制需深入研究。本实验研究了HDAC2调控NR2B/HGN表达及其下游机制在孕早期母体接受氯胺酮麻醉、孕中期母体接受氯胺酮麻醉手术、不同孕期（早、中、晚期）母体接受丙泊酚麻醉及孕晚期母体接受异氟醚致子代认知功能损害中的作用。结果发现：（1）孕早期母体接受氯胺酮麻醉可引起子代大鼠学习记忆功能损害，机制与其促进子代海马区HGN表达、抑制NR2B表达，从而破坏子代海马区NR2B/HGN表达平衡有关，与HDAC2表达关系不密切。（2）孕中期母体单纯接受氯胺酮麻醉对子代认知功能影响不明显，孕中期氯胺酮麻醉手术则对子代空间感知功能及空间学习记忆功能有明显损害，机制与其上调子代海马区HDACs活性、引起组蛋白乙酰化/去乙酰化失衡，下调NEDD9、BDNF、p-TrkB、突触小泡蛋白和PSD-95蛋白表达水平，从而使海马CA1、 CA3、DG区树突棘密度下降有关。出生后使用SEN可通过上述机制改善子代认知功能，SAHA则无明显改善作用。（2）孕早期母体接受丙泊酚麻醉对子代学习记忆的损害，与其促进HDAC2表达、抑制子代海马区组蛋白乙酰化（H3、H4位点）、下调CREB磷酸化水平、导致NR2B/HGN表达失衡、下调突触小泡蛋白表达有关。SAHA、SEN和HGC反义核苷酸对受损的学习记忆有治疗作用。（3）孕中母体接受期丙泊酚对子代学习记忆的损害与其抑制KIF17表达，从而抑制NR2B蛋白向细胞膜转移有关，与总NR2B蛋白表达水平关系不明显。（4）孕晚期母体接受丙泊酚麻醉对子代空间学习记忆的损害与其干扰子代海马区BDNF-TrkB信号通路有关，TrkB拮抗剂7,8-DHF对其有治疗作用。（5）孕晚期母体接受异氟醚麻醉对子代学习记忆的损害，与其改变子代海马区 HDAC2-CREB -NR2B信号通路、损害子代海马神经元超微结构有关，SAHA对其有治疗效应。本研究结果可为孕期非产科手术麻醉选择及胎儿出生后认知功能障碍的诊治提供实验依据。