七氟醚促进孕早期胚胎干细胞m6A RNA甲基化并抑制hnRNPA2/B1表达致子代认知功能损伤的机制

The long-term effects of anesthetic exposure on fetal development have been a focus of the patients and the anesthesia community. Our previous studies suggested that sevoflurane is associated with cognitive dysfunction of offspring mice if the mother mice were exposed to sevoflurane in early pregnancy. We further found that sevoflurane repressed stemness genes expression, stem cells proliferation, and promoted stem cells differentiation via increasing the m6A RNA methylation of Nanog, Sox2, Myc and Lin28. So we speculated that sevoflurane could improve stemness genes m6A RNA methylation levels, repress proliferation and promote differentiation of stem cells, and finally cause cognitive dysfunction in offspring mice by modulating the m6A RNA methylation related proteins. We also found that sevoflurane inhibited hnRNP A2/B1 expression and hnRNP A2/B1 participated in the alternative splicing of mRNA, so we speculated that sevoflurane inhibited stemness genes expression, promoted the stem cells differentiation, caused cognitive dysfunction of offspring mice via repressing hnRNP A2/B1. We will observe the effect of sevoflurane on neural cells differentiation in embryonic stem cells and early pregnant mice and illustrate its mechanism in this study, which will provide new theoretical evidence for prenatal exposure to anesthesia causing cognitive dysfunction.

麻醉药物暴露对胎儿发育的长期影响一直是麻醉学界和患者关注的焦点。我们前期研究证实孕早期母鼠暴露于吸入性麻醉药七氟醚可导致子代小鼠认知功能受损。我们进一步研究发现，七氟醚通过提高胚胎干细胞干性基因Nanog、Sox2、Myc和Lin28 m6A RNA甲基化水平抑制干性基因表达，抑制干细胞增殖，促进干细胞分化。因此我们推测七氟醚通过影响m6A RNA甲基化相关蛋白提高干性基因m6A RNA甲基化水平，抑制干细胞增殖，促进干细胞分化，导致子代小鼠认知功能受损。我们还发现七氟醚抑制hnRNP A2/B1表达下调，而hnRNP A2/B1参与mRNA的选择性剪接，因此我们推测七氟醚通过下调hnRNP A2/B1表达抑制干性基因表达，促进干细胞分化，导致子代小鼠认知功能受损。本研究将在胚胎干细胞和孕早期母鼠中观察七氟醚对神经细胞分化的影响并研究其具体机制。

七氟醚在胚胎中枢神经系统发育敏期内，对于子代中枢神经系统各个重要功能发育的具体影响及其表观遗传学机制一直是研究的热点问题。通过建立孕早期以及孕中期的七氟醚反复暴露模型，从行为学、脑组织细胞脆性、m6A甲基化调节以及干细胞分化角度进行了细致考察后发现：孕早期、孕中期七氟醚的暴露可导致后代的学习记忆障碍；海马作为大脑学习和记忆的关键组成部分，是七氟醚的反复暴露损伤的关键脑区，海马轴突发育发生障碍。孕中期反复七氟醚暴露导致子代小鼠海马区m6A甲基化转移酶Mettl3表达下降，而在4周时出现反跳性升高，可能是导致子代神经性为发育异常的关键分子。而在孕早期七氟醚反复暴露所致子代小鼠学习认知功能障碍与胚胎干细胞向神经前体定向分化的细胞比例下降则是关键因素。上述研究成果对于明确七氟醚的胚胎毒性发病机制，从而寻找到有效的治疗措施提供可能。.此外，七氟醚所致的意识转换的过程也是麻醉学重要的科学问题之一。我们的研究发现，七氟醚通过作用于伏隔核（nucleus accumbens, NAc）多巴胺1型受体（dopamine 1 receptor，D1R）中型多棘神经元（medium spiny neurons, MSNs）对意识起到调控作用。NAc D1R-MSNs活性动态变化与七氟醚麻醉诱导期和苏醒期意识状态转换的具有相关性；NAc D1R-MSNs激活后减弱七氟醚敏感性，七氟醚麻醉诱导时间延长、苏醒时间缩短；NAc D1R-MSNs抑制后七氟醚麻醉敏感性增强，七氟醚麻醉诱导时间延长、苏醒时间缩短；在麻醉维持期激活NAc D1R-MSNs可同时促进大脑皮层活化和行为苏醒，并且NAc到下游腹侧苍白球（ventral pallidum，VP）的投射参与了NAc D1R-MSNs对意识状态的调控作用。这一发现对于理解七氟醚的全麻作用机制具有一定的启示作用。