七氟烷通过PP2A激活GSK-3α/β引起发育脑认知功能障碍的机制研究

Sevoflurane, the most commonly used inhaled anesthetics in the infants’ surgeries, is reported to induce neurotoxicity in developing brain and cause cognitive dysfunction. However, the mechanisms underlying these deleterious effects of sevoflurane remain unclear. In our previous study, we found that single exposure of sevoflurane could decrease the level of GSK-3α/β Ser21/9 phosphorylation in hippocampal pyramidal neurons. GSK-3α/β specific inhibitor effectively attenuate sevoflurane-induced neuronal apoptosis and cognitive behavioral changes. Secondly, sevoflurane increase the interaction between PP2A and GSK-3β, while PP2A specific inhibitor can reverse sevoflurane-induced GSK-3β Ser9 dephosphorylation and neuronal apoptosis in primary cultured hippocampal pyramidal neurons. Furthermore, sevoflurane exposure promote GSK-3β and BAX translocation to the mitochondria. Accordingly, we hypothesized that sevoflurane increases the interaction between PP2A and GSK-3α/β, inducing GSK-3α/β Ser21/9 dephosphorylation. After that, GSK-3α/β translocate to the mitochondria, activating BAX to mediated hippocampal neuronal apoptosis and cognitive dysfunction. Our project intends to use GSK-3α/β conditional knockout mice and other techniques to confirm that whether GSK-3α/β mediate sevoflurane-induced hippocampus neuronal apoptosis, while the activation of GSK-3α/β is mediated by PP2A. Our purpose is to reveal the mechanisms of sevoflurane’s neurotoxicity to the developing brain, and to provide the scientific evidence of GSK-3 as a target of a protective intervention of the cognitive dysfunction raised by general anesthetics.

七氟烷是临床婴幼儿最常用的吸入麻醉药，已报道其可诱发发育脑神经毒性并引起认知功能障碍，但机制不清。我们发现：①单次暴露七氟烷引起海马神经元促凋亡激酶GSK-3α/β Ser21/9磷酸化下降（活性增加）;GSK-3抑制剂保护性干预神经元凋亡及认知障碍；②七氟烷促使PP2A与GSK-3β相互作用增加；PP2A抑制剂可逆转七氟烷诱导的GSK-3β Ser9去磷酸化；③七氟烷暴露促使GSK-3β和BAX向线粒体转位。据此我们提出假设：七氟烷通过PP2A介导GSK-3激活并转位到线粒体促海马神经元凋亡及认知功能障碍。本项目拟采用GSK-3α/β条件性敲除鼠等工具，证实GSK-3α/β哪一个亚型为主、抑或都介导了七氟烷引起的发育脑认知功能障碍，肯定PP2A是否介导 GSK-3激活，旨在揭示七氟烷对发育脑神经毒性的机制，为确立GSK-3为靶保护性干预七氟烷等全身麻醉药引起的认知功能障碍提供科学依据。

七氟烷是目前临床小儿麻醉中应用最广泛的吸入性麻醉药，但其是对发育期大脑的影响机制仍不明确。本课题运用分子生物学和生物信息学技术研究发现，早期七氟烷暴露后GSK-3β(Ser9)磷酸化下降（活性增加），海马神经元细胞内miRNA-34c表达下调。GSK-3α(Ser21)磷酸化下调在一定程度上由氯化锂所逆转，提示GSK-3在七氟烷引起的发育脑神经元凋亡过程中起到重要作用。通过检测海马神经元p53、Bax、Bcl-2 基因及蛋白表达情况，其表达趋势一致，即P53蛋白上调、Bax/Bcl-2蛋白上调、caspase-3 表达上调。此外，我们另外采用了基因微阵列技术，筛选了海马神经元七氟烷暴露后的长链非编码RNA表达水平，发现与依赖线粒体的促凋亡蛋白Bim正相关的长链非编码RNAENSMUST00000136025在七氟烷处理组表达显著升高，并通过qRT-PCR验证了相关结果。前期研究发现，七氟烷诱导了海马内促凋亡蛋白BIM和Bax的上升，以及抗凋亡蛋白Bcl-2下调。而Bcl-2家族和caspase家族在细胞凋亡转导通路尤其是线粒体途径中发挥了极为重要的调控作用。上述研究提示七氟烷通过作用于GSK-3α/β途径调控发育脑认知功能障碍可能是研究七氟烷神经毒性的新思路。