七氟醚激活海马线粒体自噬诱导老年小鼠认知功能损伤

目的探讨七氟醚对老年小鼠海马线粒体自噬和认知功能的影响。方法雄性C57BL/6J小鼠48只,18~20月龄,体重35~40 g。采用随机数字表法将小鼠为三组:对照组(C组)、吸入3%七氟醚组(S组)和吸入3%七氟醚+自噬抑制剂三甲基腺嘌呤组(3-MA组),每组16只。C组小鼠吸入空氧混合气体6 h。S组和3-MA组吸入3%七氟醚6 h。3-MA组吸入七氟醚前1 h腹腔注射三甲基腺嘌呤30 mg/kg。七氟醚吸毕24 h后采用Morris水迷宫实验观察小鼠行为学变化,第1~4天训练,第5天进行空间探索实验。行为学测试结束后立即处死小鼠。采用HE染色法观察海马组织病理学改变,免疫荧光法观察海马组织中自噬情况,Western blot法检测海马组织中PINK1、Parkin、LC3和Beclin1蛋白含量。结果与C组比较,S组、3-MA组水迷宫实验第2~4天逃避潜伏期明显延长(P<0.05),第5天目标象限停留时间明显缩短(P<0.05),穿越次数明显减少(P<0.05),海马组织出现病理形态学损伤,海马组织中LC3阳性细胞数明显增多(P<0.05),海马组织PINK1、Parkin、Beclin1蛋白含量和LC3Ⅱ/LC3Ⅰ比值明显升高(P<0.05)。与S组比较,3-MA组水迷宫实验第2~4天逃避潜伏期明显缩短(P<0.05),第5天目标象限停留的时间明显延长(P<0.05),穿越次数明显增多(P<0.05),海马组织病理形态学损伤程度减轻,海马组织中LC3阳性细胞数明显减少(P<0.05),海马组织PINK1、Parkin、Beclin1蛋白含量和LC3Ⅱ/LC3Ⅰ比值明显降低(P<0.05)。结论吸入3%七氟醚6 h的老年小鼠,海马病理形态学发生改变,伴随学习和记忆能力下降,其机制可能与七氟醚激活线粒体自噬有关。

Sevoflurane activates hippocampal mitophagy and thereby induces cognitive decline in aged mice

Objective To investigate the effects of sevoflurane on hippocampal mitophagy and cognitive function in aged mice.Methods Forty-eight male C57BL/6J mice,aged 18-20 months,weighing 35-40 g,were randomly divided into three groups(n=16 in each group):control group(group C),3%sevoflurane group(group S)and 3%sevoflurane plus 3-MA group(group 3-MA).Mice in group C inhaled air for 6 hours.Mice in group S and group 3-MA inhaled 3%sevoflurane for 6 hours.Mice in group 3-MA received an intraperitoneal injection of 3-methyladenine(30 mg/kg)1 hour before sevoflurane inhalation.The Morris water maze(MWM)test was performed 24 hours after sevoflurane inhalation in all groups.Training was performed on the 1 st to 4 th day and testing was performed on the 5 th day.Mice were sacrificed immediately after the behavior test and the brains were taken out for hematoxylin-eosin staining and immunofluorescence.Hippocampal expressions of PINK1,Parkin,LC3 and Beclin1 were detected by Western blot.Results Compared with group C,the escape latency prolonged significantly on the 2 nd to 4 th day of MWM test,and the residence time and number of crossings in the target quadrant on the 5 th day decreased significantly in groups S and 3-MA(P<0.05);the pathomorphology of the hippocampus was obviously damaged in groups S and 3-MA;the hippocampal number of LC3 positive cells,the ratio of LC3Ⅱ/LC3Ⅰand the expressions of PINK1,Parkin and Beclin1in mice in groups S and 3-MA were higher(P<0.05).Compared with group S,the escape latency shortened significantly on the 2 nd to 4 th day of MWM test,and the residence time and number of crossings in the target quadrant on the 5 th day increased significantly in group 3-MA;the pathomorphological damage of the hippocampus was attenuated in group 3-MA;the hippocampal number of LC3 positive cells,the ratio of LC3Ⅱ/LC3Ⅰand the expressions of PINK1,Parkin and Beclin1 were decreased in group 3-MA(P<0.05).Conclusion Aged mice inhaled 3%sevoflurane for 6 hours display hippocampal morphology damage and decline in the ability of learning and memory.The mechanism may be related to the activation of mitophagy.