反复七氟烷暴露对新生大鼠海马细胞凋亡和远期学习记忆能力的影响

Effects of repeated sevoflurane exposure on hippocampal cell apoptosis and long-term learning and memory ability in neonatal rats

目的:探讨反复七氟烷暴露对新生大鼠海马细胞凋亡和远期学习记忆能力的影响及对PI3K/AKT通路的调控作用。方法:90只SD大鼠按随机数字表法分为对照组(25%的氧气)、1次暴露组(出生后第6日吸入3%的七氟烷、25%氧气)、3次暴露组(出生后第6、7、8日均吸入3%的七氟烷、25%氧气)、5次暴露组(出生后第6、7、8、9、10日均吸入3%的七氟烷、25%氧气)、5次暴露+740Y-P(PI3K激活剂)组(5次吸入七氟烷后腹腔注射0.02 mg/kg 740Y-P)。Morris水迷宫测定大鼠学习记忆能力;HE染色、透射电镜观察大鼠海马组织神经元形态及结构变化;TUNEL检测大鼠海马神经细胞凋亡情况;WB检测大鼠海马组织凋亡相关蛋白(Caspase-3、Bax、Bcl-2)及PI3K/AKT通路相关蛋白的表达。结果:与对照组和1次暴露组相比,3次暴露组和5次暴露组大鼠学习记忆能力严重降低,海马组织神经元形态及结构受到严重损害,海马神经细胞凋亡率增加(P<0.05),海马组织中Capase-3、Bax蛋白表达显著升高(P<0.05),Bcl-2蛋白表达、PI3K/AKT通路蛋白表达显著降低(P<0.05);随着七氟烷暴露次数的增加,大鼠学习记忆能力显著降低,海马神经元细胞受损严重,海马神经细胞凋亡率显著增加(P<0.05),PI3K/AKT通路蛋白表达显著降低(P<0.05);与5次暴露组相比,5次暴露+740Y-P组大鼠学习记忆能力、海马神经元结构得到一定的恢复,海马神经细胞凋亡率、Capase-3、Bax蛋白显著降低(P<0.05),Bcl-2蛋白表达、PI3K/AKT通路蛋白表达显著升高(P<0.05)。结论:反复七氟烷暴露可显著降低新生大鼠的学习记忆能力并加重海马神经细胞凋亡,其机制可能是通过抑制PI3K/AKT通路发挥作用。

Objective:To investigate the effects of repeated sevoflurane exposure on hippocampal cell apoptosis and long-term learning and memory ability in neonatal rats and its regulation on PI3K/AKT pathway.Methods:Ninety SD rats were randomly divided into the control group(25%oxygen),the single exposure group(inhalation of 3%sevoflurane and 25%oxygen on the 6th day after birth),the 3-times exposure group(inhalation of 3%sevoflurane and 25%oxygen on the 6th,7th,and 8th day after birth),the 5-times exposure group(inhalation of 3%sevoflurane and 25%oxygen on the 6th,7th,8th,9th and 10th day after birth),and the 5-times exposure+740Y-P(PI3K activator)group(intraperitoneal injection of 0.02 mg/kg 740Y-P after inhalation of sevoflurane for 5 times)according to the random number table method.Morris water maze was used to measure the learning and memory ability;HE staining and transmission electron microscopy were used to observe the morphological and structural changes of neurons in the hippocampus;TUNEL was used to detect the apoptosis of hippocampal nerve cells;Western blot was used to detect the expressions of apoptosis-related proteins(Caspase-3,Bax,Bcl-2)and PI3K/AKT pathway related proteins in the hippocampus of rats.Results:Compared with the control group and the single exposure group,the learning and memory abilities of rats in the 3-times exposure group and the 5-times exposure group were severely reduced,the morphology and structure of hippocampal neurons were severely damaged,and the apoptosis rate of hippocampal nerve cells was increased(P<0.05),the expressions of Capase-3 and Bax proteins were significantly increased(P<0.05),and the expressions of Bcl-2 protein and PI3K/AKT pathway protein were significantly decreased(P<0.05).With the increase in the number of exposures to sevoflurane,the learning and memory abilities of rats were significantly reduced,the hippocampal neuron cells were severely damaged,the hippocampal neuronal apoptosis rate was significantly increased(P<0.05),and the expressions of PI3K/AKT pathway proteins were significantly reduced(P<0.05).Compared with the 5-times exposure group,the learning and memory abilities and hippocampal neuron structure of rats in the 5-times exposure+740Y-P group were restored to a certain extent,and the hippocampal neuronal apoptosis rate,the levels of capase-3 and Bax protein were significantly reduced(P<0.05),while the expressions of Bcl-2 protein and PI3K/AKT pathway protein were increased significantly(P<0.05).Conclusion:Repeated exposure to sevoflurane can significantly reduce the learning and memory abilities of neonatal rats and exacerbate hippocampal neuronal apoptosis,which may be mediated by inhibiting the PI3K/AKT pathway.