HDAC6调控Tau蛋白磷酸化在七氟醚发育期神经毒性中的作用及机制

Developmental neurotoxicity of general anesthetics has caused widespread concerns for anesthesiologists, even the society in recent years. It has been demonstrated that postnatal sevoflurane anesthesia could induce cognitive dysfunction in adulthood. Tau phosphorylation is proved to be an initial factor. Blocking tau phosphorylation may be a promising way to prevent the impairment above. However, no specific targets could be used so far. Previous studies indicated that histone deacetylase 6 (HDAC6) promoted degradation of phosphorylated tau. Our preliminary experiment has found that HDAC6 expression increased after sevoflurane anesthesia in postnatal mice. Therefore, we speculate that HDAC6 participates in the cognitive dysfunction in adulthood caused by postnatal sevoflurane anesthesia through regulating tau phosphorylation. On the basis of pilot studies, the present research subject will used models of HDAC6 knockout mice and HDAC6 overexpressed or silent hippocampal neurons to test this speculation from molecular, cellular and animal levels. First, we will investigate expressions of HDAC6 and phosphorylated tau at different time points and cognitive performance in adulthood after sevoflurane anesthesia in postnatal mice. Then the role of HDAC6 in cognitive dysfunction induced by postnatal sevoflurane anesthesia will be confirmed. Finally the involved mechanism of HDAC6 in regulation of Tau phosphorylation will be explored. The current research subject will provide new thoughts and targets to prevent developmental neurotoxicity of sevoflurane.

全身麻醉药发育期神经毒性近年来受到麻醉学界乃至社会的广泛关注。发育期七氟醚麻醉可导致成年后认知功能障碍，Tau蛋白磷酸化是始动因素之一。阻断Tau蛋白磷酸化是拮抗上述损害的潜在有效方法，但目前仍缺乏特异性干预靶点。研究表明，组蛋白去乙酰化酶6(HDAC6)可促进磷酸化Tau蛋白降解。我们预实验发现，新生鼠七氟醚麻醉后HDAC6表达显著上调。因此，我们推测，HDAC6调控Tau蛋白磷酸化参与发育期七氟醚麻醉致成年后认知功能障碍的过程。本课题拟在前期研究基础上，利用HDAC6基因敲除小鼠和HDAC6过表达/沉默海马神经元麻醉模型，从分子、细胞和动物水平等多层次观察新生鼠七氟醚麻醉后不同时程HDAC6和磷酸化Tau蛋白表达及成年后认知功能表现；明确HDAC6在七氟醚麻醉致成年后认知功能障碍中的作用；探索HDAC6调控Tau蛋白磷酸化的机制。本课题将为预防七氟醚发育期神经毒性提供新思路、新靶点。

项目的背景：全球每年数以千万的小儿因手术接受全身麻醉，我国小儿麻醉数量也达到500至600万例/年。接受全身麻醉的小孩是否会出现认知功能障碍的问题，引起人们极大关注。七氟醚起效快，可控性好，是小儿麻醉中最常用的全身麻醉药之一。基础研究证实发育期七氟醚麻醉可导致以学习记忆能力下降为主的成年后认知功能障碍。但是，其具体机制目前仍不明确。主要研究内容：观察新生鼠七氟醚多次麻醉后HDAC6表达和活性变化；明确HDAC6在七氟醚多次麻醉致成年后认知功能障碍中的作用；从Tau蛋白磷酸化方面探索HDAC6的作用机制。重要结果和关键数据：我们发现，新生鼠在出生后第7、8、9天连续3天接受七氟醚麻醉后海马组织HDAC6蛋白质、mRNA和活性显著上升。每次七氟醚暴露前给与HDAC6特异性抑制剂Tubastatin A，将新生鼠喂养至49天采用Morris水迷宫实验检测学习记忆能力，发现抑制HDCA6活性可显著改善学习记忆水平。进一步运用HDAC6敲除小鼠验证，发现敲除HDAC6后七氟醚麻醉引起的学习记忆损害得到缓解。机制研究显示，抑制HDAC6可提升海马组织synaptophysin和PSD95蛋白表达量，突触显微结构明显改善。采用原代培养神经元和基因敲除小鼠为模型，发现抑制HDAC6或敲除HDAC6可降低p-Tau(AT8)和p-Tau(Ser396)蛋白质表达水平。科学意义：本研究结果为临床预防和治疗七氟醚麻醉所致发育期神经毒性提供干预靶点，具有重要的理论和临床意义。