右美托咪定对七氟醚麻醉下老年大鼠海马神经元自噬的影响

Influence of dexmedetomidine on autophagy of hippocampal neurons in senile rats under sevoflurane anesthesia

目的探讨右美托咪定(dexmedetomidine,DEX)对七氟醚麻醉下老年大鼠海马神经元自噬的影响。方法60只雄性SD大鼠随机分为对照组、七氟醚组、DEX预处理组,每组20只,对照组大鼠吸入体积分数60%O 26h,七氟醚组大鼠吸入体积分数3.4%~3.6%七氟醚6h,DEX预处理组大鼠在吸入七氟醚前1h腹腔注射15μg/kg DEX。分别于麻醉前(T 1)、麻醉后12h(T 2)、24h(T 3)、48h(T 4)每组各处死大鼠5只并取出其海马,HE染色进行组织病理学观察,采用荧光双标法检测LC-3与NeuN、mTOR共定位表达。采用免疫组织化学法检测海马组织自噬蛋白Beclin-1相对表达量,采用Western blot法检测海马组织自噬蛋白LC3-Ⅰ、LC3-Ⅱ及p-Akt、p-mTOR通路蛋白相对表达量。于麻醉后48h处死大鼠前进行神经功能评分,记录各组Morris水迷宫实验结果。结果七氟醚组海马组织神经细胞胞质染色变浅、细胞核染色增深,DEX预处理组神经细胞形态改善和胞核染色较七氟醚组明显减轻;七氟醚组、DEX预处理组大鼠神经运动功能评分[(7.69±2.79)、(13.38±2.36)分]低于对照组[(22.15±1.47)分](P<0.05),七氟醚组低于DEX预处理组(P<0.05);七氟醚组、DEX预处理组、对照组大鼠逃避潜伏期依次缩短(P<0.05),平台象限停留时间、穿越平台象限次数依次增加(P<0.05);T 1~T 4时,对照组大鼠海马组织LC3-Ⅰ、LC3-Ⅱ、Beclin-1及p-Akt、p-mTOR蛋白相对表达量比较差异均无统计学意义(P>0.05);T 2~T 4时,七氟醚组、DEX预处理组海马组织LC3-Ⅰ、LC3-Ⅱ及Beclin-1蛋白相对表达量较T 1时增高(P<0.05),p-Akt、p-mTOR蛋白相对表达量较T 1时降低(P<0.05),DEX预处理组LC3-Ⅰ、LC3-Ⅱ、Beclin蛋白相对表达量低于七氟醚组(P<0.05),p-Akt、p-mTOR蛋白相对表达量高于七氟醚组(P<0.05)。结论右美托咪定可明显改善七氟醚麻醉下老年大鼠的神经运动和认知功能,可能与激活PI3K/Akt/mTOR通路、抑制海马神经元自噬,从而减轻脑损伤有关。

Objective To investigate the influence of dexmedetomidine(DEX)on autophagy in hippocampal neurons of senile rats under sevoflurane anesthesia.Methods Sixty male rats were randomly divided into control group,sevoflurane group and DEX pretreatment group,with 20rats in each group.Control group inhaled 60%O 2 for 6h,sevoflurane group inhaled 3.4%to 3.6%sevoflurane for 6h,and DEX pretreatment group was intraperitoneally injected with 15μg/kg DEX 1hbefore inhalation of sevoflurane.Five rats in each group were sacrificed and their hippocampus tissues were removed before anesthesia(T 1),and 12h(T 2),24h(T 3)and 48h(T 4)after anesthesia,respectively.HE staining was used for pathological examination.Fluorescent double-labeled LC-3was co-localized with NeuN and mTOR.The relative expression of autophagy protein Beclin-1in the hippocampus was detected by immunohistochemistry.The contents of autophagy proteins LC3-Ⅰ,LC3-Ⅱ,p-Akt and p-mTOR pathway proteins in the hippocampus were detected by Western blot.The neurological function scores were performed before the rats were sacrificed 48hafter anesthesia,and the Morris water maze test results of each group were recorded.Results In sevoflurane group,the cytoplasmic staining was shallow and the nucleus staining was deepened.The neuronal morphology was better and nucleus staining was shallower in DEX pretreatment group compared with sevoflurane group.The neuromotor function score was significantly lower in sevoflurane group(7.69±2.79)and DEX pretreatment group(13.38±2.36)than that in control group(22.15±1.47)(P<0.05),and lower in sevoflurane group than that in DEX pretreatment group(P<0.05).The escape latency was shortened gradually in turn in sevoflurane group,DEX pretreatment group and control group(P<0.05),the platform quadrant residence time and the number of crossing the platform quadrants increased sequentially(P<0.05).At T 1-T 4,the relative expressions of LC3-Ⅰ,LC3-Ⅱ,Beclin-1,p-Akt and p-mTOR protein showed no significant differences in control group(P>0.05).The relative expressions of LC3-Ⅰ,LC3-Ⅱand Beclin-1were higher and the relative expressions of p-Akt and p-mTOR were lower in sevoflurane group,DEX pretreatment group and control group At T 2-T 4 than those at T 1(P<0.05).The relative expressions of LC3-Ⅰ,LC3-Ⅱand Beclin prefeins were lower and the relative expressions of p-Akt and p-mTOR proteins were higher in DEX pretreatment group than those in sevoflurane group(P<0.05).Conclusion DEX can significantly improve the neuromotor and cognitive functions of senile rats under sevoflurane anesthesia,which may be correlated with the activation of PI3K/Akt/mTOR pathway,inhibition of hippocampal neuronal autophagy,and reduction of brain injury.