细胞自噬在七氟醚致老年小鼠海马区神经元细胞凋亡中的保护作用

Protective effect of autophagy on sevoflurane induced apoptosis of hippocampal neurons in aged mice

目的探讨细胞自噬在七氟醚致老年小鼠海马区神经元细胞凋亡中的作用机制。方法18月龄BL/C57小鼠16只,按随机数字表法分为两组(每组8只):七氟醚组和对照组。七氟醚组吸入47.5%空气+2.5%七氟醚+50%氧气3 h,对照组吸入50%空气+50%氧气3 h。建模后24 h进行新物体识别实验,随后断头取海马组织,尼氏染色检测海马神经元细胞凋亡情况,Western blot法检测海马神经元细胞自噬相关蛋白[微管关联蛋白(microtubule⁃associated protein,LC)3Ⅱ/LC3Ⅰ、Becline⁃1、p62蛋白]水平。另取18月龄BL/C57小鼠18只,按随机数字表法分为3组(每组6只):七氟醚+3⁃甲基腺嘌呤组(M组,吸入麻醉前2 h经腹腔注射3⁃甲基腺嘌呤)、七氟醚+雷帕霉素组(R组,吸入麻醉前24 h和2 h经腹腔注射雷帕霉素)、七氟醚+生理盐水组(N组,吸入麻醉前2 h经腹腔注射生理盐水100μl)。经腹腔注药及七氟醚处理后,尼氏染色检测海马神经元细胞凋亡情况,Western blot法检测海马神经元细胞自噬相关蛋白(LC3Ⅱ/LC3Ⅰ、Becline⁃1、p62蛋白)水平。结果与对照组比较,七氟醚组小鼠新物体识别率和辨别系数降低(P<0.05),海马神经元细胞凋亡增加(P<0.05),LC3Ⅱ/LC3Ⅰ、Becline⁃1水平升高(P<0.05),p62蛋白水平降低(P<0.05)。与N组比较:M组LC3Ⅱ/LC3Ⅰ、Becline⁃1水平降低(P<0.05)、p62蛋白水平升高(P<0.05),海马神经元细胞凋亡增加(P<0.05);R组LC3Ⅱ/LC3Ⅰ、Becline⁃1水平升高(P<0.05),p62蛋白水平降低(P<0.05),海马神经元细胞凋亡降低(P<0.05)。结论老年小鼠经七氟醚吸入麻醉后对新物体的识别率和辨别系数均降低,海马神经元细胞凋亡增加,同时存在细胞自噬的激活,并负反馈抑制神经元细胞凋亡,发挥神经保护机制。

Objective To explore the mechanism of autophagy in sevoflurane⁃induced neuronal apoptosis in the hippocampus of aged mice.Methods According to the random number table method,sixteen 18⁃month⁃old BL/C57 mice were divided into two groups(n=8):a sevoflurane group and a control group.The sevoflurane group inhaled 47.5%air+2.5%sevoflurane+50%oxygen for 3 h,while the control group inhaled 50%air+50%oxygen for 3 h.Then,24 h after modeling,the novel object recognition experiment was performed,and the brains were removed to collect the hippocampus.Nissl staining was performed to detect the apoptosis of neurons in the hippocampus,and Western blot was used to detect the levels of autophagy⁃related proteins in neurons[microtubule⁃associated pro⁃tein(LC3)Ⅱ/LC3Ⅰ,Becline⁃1,and p62].Furthermore,according to the random number table method,eighteen 18⁃month⁃old BL/C57 mice were divided into three groups(n=6):a sevoflurane+3⁃methyladenie group(group M,intraperitoneal injection of 3⁃methyladenine 2 h before inhaling anesthesia),a sevoflurane+rapamycin group(group R,intraperitoneal injection of rapamycin 24 h and 2 h before in⁃haling anesthesia),and a sevoflurane+normal saline group(group N,intraperitoneal injection of 100μl normal saline 2 h before inhal⁃ing anesthesia).After intraperitoneal injection and sevoflurane treatment,Nissl staining was used to detect the apoptosis of hippocam⁃pal neurons,and Western blot was used to detect the level of autophagy related proteins(LC3Ⅱ/LC3Ⅰ,Becline⁃1,p62)in hippocam⁃pal neurons.Results Compared with the control group,the sevoflurane group showed significant decreases in the recognition rate and resolution coefficient of novel objects(P<0.05),increases in hippocampal neuronal apoptosis(P<0.05),increases in the levels of LC3Ⅱ/LC3Ⅰand Becline⁃1(P<0.05),and decreases in the amounts of p62 protein(P<0.05).Compared with group N,group M pre⁃sented decreases in the levels of LC3Ⅱ/LC3Ⅰand Becline⁃1(P<0.05),increases in the amounts of p62(P<0.05),and increases in neu⁃ronal apoptosis in the hippocampus(P<0.05);group R presented increases in the levels of LC3Ⅱ/LC3Ⅰand Becline⁃1(P<0.05),de⁃creases in the amounts of p62(P<0.05),and decreases in neuronal apoptosis in the hippocampus(P<0.05).Conclusions After sevo⁃flurane inhalation,aged mice show decreases in the recognition rate and resolution coefficient of new objects,and increases in neuronal apoptosis in the hippocampus,with activation of autophagy at the same time,which negatively inhibits neuronal apoptosis and exerts a neuroprotective effect.