孕中期七氟醚麻醉通过自噬调节Nrf2干扰子代神经干细胞分化的机制研究

Emerging evidence has demonstrated that exposure to anesthetics early in life caused neurohistopathologic changes and persistent behavioral impairments. The second trimester is a critical period of fetal neurogenesis, during which sevoflurane exposure may interfere with brain development in the offspring. Our previous research has found that sevoflurane exposure promoted the differentiation of neural stem cells (NSCs), reduced the number of NSCs, and caused the learning and memory dysfunction. Sevoflurane enhanced autophagy of NSCs and increased expression of nuclear transcription factor Nrf2. Autophagy-lysosomal pathway can activate Nrf2, which triggers cell differentiation. Accordingly, we hypothesized that mid-gestational sevoflurane anesthesia induces up-regulation of Nrf2 by activating autophagy, and Nrf2-mediated downstream signaling promotes NSCs differentiation in the offspring. First, we aim to investigate whether sevoflurane anesthesia during the second trimester can promote NSCs differentiation by activating autophagy. Then, we explore whether Nrf2 promotes NSCs differentiation as an effector of autophagy activation. Finally, we focus on whether Nrf2 up-regulates the expression of RNA methyltransferases, Mettl3, in order to inhibit Nanog, the pluripotency regulator of stem cells, promoting NSCs differentiation. Our research explores the effect and underlying mechanism of sevoflurane anesthesia during second trimester on neurogenesis and learning and memory ability, to provide a theoretical basis for safer and more rational application of anesthetics in second trimester.

麻醉药物对发育期脑神经毒性研究备受关注。孕中期是胎儿神经发生的关键时期，七氟醚麻醉可能干扰子代神经发育。我们前期发现，七氟醚促进子代神经干细胞(NSCs)分化，NSCs数量减少，学习记忆能力受损；七氟醚致NSCs自噬激活，核转录因子Nrf2表达升高。自噬是一种细胞自我降解过程，参与调控NSCs的分化潜能。自噬溶酶体途径可以激活Nrf2；Nrf2能激活细胞分化程序。据此我们推测，七氟醚通过激活自噬上调Nrf2，启动下游信号促进NSCs分化。本项目首先研究七氟醚是否通过激活自噬促进NSCs分化；进一步研究Nrf2是否作为自噬的效应因子促进NSCs分化；最后研究自噬调控下Nrf2是否通过上调RNA甲基转移酶Mettl3，抑制神经干细胞多潜能调节蛋白Nanog进而促进NSCs分化。本课题探究孕中期七氟醚麻醉对子代神经发生和学习记忆的影响及分子机制，旨在为孕中期麻醉药物的安全合理应用提供理论依据。

麻醉药物对发育期脑影响越来越受到关注，是麻醉领域的研究热点。大量动物实验发现全麻药物可影响发育期脑的突触形态和认知功能，临床研究也提示全身麻醉可能导致儿童学习及行为异常发生。作为胎儿神经发育的重要时期，孕中期麻醉药物暴露是否干扰子代神经发育逐渐受到关注。孕中期是胎儿神经系统发育的重要时期，此时脑内广泛存在神经发生，即神经干细胞(NSCs)经历增殖和不对称分裂产生功能性的有丝分裂后神经元，并整合入神经网络的生物学过程。本研究在明确孕中期大鼠暴露七氟醚过度激活自噬，引起 NSCs 数目减少，损伤学习记忆能力的基础上，首先明确了孕中期单次或多次七氟醚麻醉促进子代 NSCs 分化，干扰神经发生，影响子代远期学习记忆功能。结果发现，多次七氟醚麻醉导致子代大鼠神经干细胞及原代海马神经干细胞过早分化为神经元和星形胶质细胞，并在远期导致神经元减少和星形胶质细胞增加；单次七氟醚麻醉对子代大鼠神经干细胞及原代海马神经干细胞的分化并无影响。其次探讨了孕中期七氟醚麻醉是否通过自噬激活促进子代 NSCs分化。结果发现，与对照组相比，七氟醚暴露后，神经元和星形胶质细胞的标记物和FOXO3的表达增加。抑制自噬减轻七氟醚致NSCs分化异常。氯化锂抑制FOXO3表达，抑制自噬激活，调节NSCs分化和Nestin蛋白表达下降。结果表明，孕中期母体暴露于七氟醚通过激活 FOXO3 诱导胎鼠大脑中的NSCs分化。自噬抑制剂或氯化锂逆转了NSCs 分化异常。最后阐明了孕中期七氟醚麻醉是否通过激活Nrf2促进子代 NSCs 分化及造成远期学习记忆功能受损的分子机制。结果发现，七氟醚孕中期麻醉会使子代大鼠NRF2表达激活；NRF2作为调控氧化应激关键转录因子，可通过SHH/GLI1促进神经干细胞分化，造成子代大鼠神经干细胞过早分化为神经元和星形胶质细胞；最终造成子代远期学习记忆损伤；抑制NRF2能够减轻七氟醚对子代大鼠海马神经干细胞早期和远期分化水平的影响。本项目探究孕中期七氟醚暴露对子代神经发生及学习记忆的影响及分子机制，旨在为孕中期麻醉药物的合理选择提供理论依据。