异氟醚对转APP基因小鼠脑内海马神经元蛋白质损伤和蛋白聚集的影响

Influence of isoflurane in neuronal protein damage and aggregation in APP transgenic mouse hippocampus

目的:观察吸入麻醉药异氟醚对转APP基因小鼠脑内海马神经元蛋白质损伤和蛋白聚集的影响,并探讨海藻糖对异氟醚诱导的神经毒性的干预作用。方法:60只12月龄转APP基因小鼠随机分为对照组、异氟醚组(Iso组)和海藻糖组(Tre组),每组20只。对照组小鼠不给予任何药物,将其置于持续通入2L·min-1氧气的麻醉箱中2h;Iso组和Tre组小鼠于麻醉前30min分别经腹腔注射2mL生理盐水或海藻糖(400μg·kg-1)稀释液,然后给予1.4%异氟醚吸入麻醉2h。麻醉后6h取小鼠海马组织制备脑组织匀浆,应用DCFH-DA荧光法检测小鼠海马组织中活性氧(ROS)水平;麻醉后24 h应用免疫组织化学法和Western blotting法检测小鼠海马组织中蛋白羰基化合物、硝基化酪氨酸和β-淀粉样蛋白(Aβ)1-42蛋白表达水平,应用透射电镜检测海马神经元中蛋白聚集物的形成,应用TUNEL染色法观察小鼠海马组织中神经元凋亡率。结果:与对照组比较,Iso组小鼠海马组织中ROS水平、氧化蛋白羰基化合物、硝基化酪氨酸、Aβ1-42蛋白表达水平和神经元凋亡率均明显增加(P<0.05);与Iso组比较,Tre组小鼠海马组织中ROS水平、氧化蛋白羰基化合物、硝基化酪氨酸、Aβ1-42蛋白表达水平和神经元凋亡率均明显降低(P<0.05)。结论:异氟醚能诱导转APP基因小鼠海马神经元蛋白质损伤和蛋白聚集,加剧氧化应激反应,增加脑内海马神经元凋亡,海藻糖能够拮抗异氟醚诱导的神经毒性。

Objective：To observe the influence of inhaled anesthetic isoflurane in the neuronal protein damage and aggregation in the APP transgenic mouse hippocampus,and to investigate the intervention effect of trehalose.Methods：Sixty APP transgenic mice aged 12 months were divided into control group,isoflurane group（Iso group）and trehalose group（Tre group）（n=20）.The rats in control group were not given any drugs and were put into the anesthetic box with continuonsly entering 2L·min^-1 oxygen for 2h;the rats in Iso group and Tre group were respectively injected intraperitoneally with 2mL saline or trehalose（400μg·kg^-1）30min before anesthesia,and then inhalated 1.4%isoflurane for 2h.6hafter anesthesia,the hippocampus tissue of the mice was prepared,and DCFH-DA fluorescence was applied to detect the reactive oxygen species（ROS）level;24hafter anesthesia,immunohistochemical method and Western blotting method were used to detect the contents of carbonyl compounds and nitrotyrosine and the Aβ1-42 protein expression level in hippocampus;TEM was applied to observe the formation of protein aggregates;TUNEL staining was performed to observe the apoptotic rate of hippocampal neurons.Results：Compared with control group,the ROS level,the expression levels of oxidative protein carbonyl compounds and nitrotyrosine,the expression level of Aβ1-42 protein,and the apoptotic rate of hippocampal neurons in Iso group were significantly increased（P〈0.05）;compared with Iso group,the ROS level,the expression levels of oxidative protein carbonyl compounds and nitrotyrosine,the Aβ1-42 protein expression level,and the apoptotic rate of hippocampal neurons in Tre group were significantly decreased（P〈0.05）.Conclusion：Isoflurane can induce the protein damage and aggregation,the apoptotic rate of hippocampal neurons,aggravate oxidative stress reaction,increase the apoptotic rate of brain hippocampal neurons in the APP transgenic mice;trehalose can intervene the neurotoxicity induced by inhaled anesthetics.