七氟醚介导的神经元自噬抑制对幼年大鼠神经发生和学习记忆功能的影响

Effects of sevoflurane-mediated neuronal autophagy inhibition on neurogenesis,learning and memory function in newborn rats

目的探讨七氟醚对幼龄大鼠学习记忆功能的影响及机制。方法48只幼鼠随机分为对照组、七氟醚-1 h组、七氟醚-3 h组与七氟醚-3 h+雷帕霉素(20 mg/kg,于麻醉前连续ip 3 d)组,每组12只。麻醉当天,对照组幼鼠吸入氧气混合氮气3 h,七氟醚组以含3%七氟醚的混合气体麻醉1 h或3 h。4周后,应用Morris水迷宫检测幼鼠学习与记忆功能;使用电子透射显微镜观察神经元自噬;采用BrdU免疫荧光法观察幼鼠海马组织中神经元新生;Western blotting法检测幼鼠海马组织中NeuN、DCX、PI3K、mTOR、LC3-II/LC3-I和Beclin-1蛋白表达。结果与对照组比较,七氟醚-3 h组幼鼠逃避潜伏期显著增加(P<0.05),穿越平台次数显著减少(P<0.05),靶象限停留时间显著减少(P<0.05);与七氟醚-3 h组比较,雷帕霉素显著缩短幼鼠逃避潜伏期(P<0.05),增加测试中穿越平台次数(P<0.05)与靶象限停留时间(P<0.05)。与对照组比较,七氟醚-3 h组幼鼠齿状回中神经元新生显著减少(P<0.05),神经元新生相关蛋白NeuN与DCX表达显著降低(P<0.05);海马组织中PI3K表达显著降低(P<0.05),且mTOR蛋白表达显著增加(P<0.05),而LC3-II/LC3-I与Beclin-1表达显著下调(P<0.05),造成自噬小体显著减少(P<0.05)。与七氟醚-3 h组比较,雷帕霉素显著增加NeuN与DCX蛋白表达(P<0.05),显著提高PI3K蛋白表达(P<0.05),显著下调mTOR表达(P<0.05),显著上调LC3-II/LC3-I与Beclin-1表达(P<0.05),显著增加自噬小体的形成。结论七氟醚通过下调PI3K,增加mTOR表达,抑制LC3-II/LC3-I与Beclin-1表达,减少自噬小体形成并抑制神经元新生,最终诱导幼鼠学习和记忆功能障碍。

Objective To investigate the effect and mechanism of sevoflurane on the learning and memory function in newborn rats.Methods 48 young rats were randomly divided into control group,sevoflurane-1 h group,sevoflurane-3 h group and sevoflurane-3 h+rapamycin group,with 12 rats in each group.Before anesthesia,rats in each group were intraperitoneally injected with saline or rapamycin(20 mg/kg)for three days.On the day of anesthesia,the newborn rats in control group were inhaled with oxygen and nitrogen for three hours,and the newborn rat in sevoflurane group were anesthetized with 3%sevoflurane for one or three h.Morris water maze was used to detect the learning and memory function of young mice after four weeks.Transmission electron microscope was applied for observation of autophagosome.In addition,Immunofluorescence and Western blotting were used to explore the expression of neuron regeneration and autophagy-related proteins in the hippocampus of newborn mice.Results Compared with control group,sevoflurane increased the escape latency of newborn rats(P<0.05),decreased the number of crossing platforms(P<0.05),and reduced the residence time in the target quadrant(P<0.05).However,the intervention of rapamycin significantly reduced the escape latency of young mice(P<0.05),increased the number of platform crossings(P<0.05)and the target quadrant stay time(P<0.05).In addition,sevoflurane caused decreased neurogenesis in the dentate gyrus of newborn rats,and inhibited the expression of neuron regeneration-related proteins NeuN and DCX.At the same time,sevoflurane mediated the decrease of PI3 K expression in the hippocampus of newborn rats(P<0.05),and caused an increase in mTOR protein expression(P<0.05),while the expression of LC3-II/LC3-I and Beclin-1 was significantly down-regulated(P<0.05),resulting in a decrease in autophagosome(P<0.05).However,the administration of autophagy inducer rapamycin significantly increased the expression of PI3 K(P<0.05),downregulated the expression of mTOR(P<0.05),and up-regulated the expression of LC3-II/LC3-I and Beclin-1(P<0.05)which increased the formation of autophagosome.Conclusion Sevoflurane mediates down-regulation of PI3 K,up-regulation of mTOR,inhibition of LC3-II/LC3-I and Beclin-1,to reduce formation of autophagosomes and inhibit neurogenesis,which ultimately induces learning and memory dysfunction in newborn rats.