艾司氯胺酮对小鼠脑缺血再灌注损伤的影响及其与线粒体应激的关系
Effect of esketamine on cerebral ischemia-reperfusion injury and association with mitochondrial stress in mice
王霞 1李培龙 2黄亚茹 3迟文英 3王公明 4孟凡军3
作者信息
- 1. 山东第一医科大学(山东省医学科学院)研究生部,济南 250117
- 2. 山东第一医科大学附属中心医院烧伤整复外科,济南 250013
- 3. 山东第一医科大学附属中心医院麻醉科,济南 250013
- 4. 山东第一医科大学附属省立医院麻醉科,济南 250021
- 折叠
摘要
目的 评价艾司氯胺酮对小鼠脑缺血再灌注损伤的影响及其与线粒体应激的关系.方法 实验Ⅰ SPF级雄性C57BL/6小鼠18只,8～12周龄,体质量28～30 g,采用随机数字表法分为3组(n=6):假手术组(S组)、脑缺血再灌注组(IR组)和艾司氯胺酮+脑缺血再灌注组(E+IR组).采用大脑中动脉栓塞1 h,再灌注24 h的方法制备脑缺血再灌注损伤模型;E组造模前20 min腹腔注射艾司氯胺酮10 mg/kg.采用Zea Longa评分及平衡木实验(Feeney评分)评估小鼠神经功能;TTC染色法测定脑梗死体积.实验Ⅱ将原代皮层神经元采用随机数字表法分为3组(n=42):对照组(C组)、氧糖剥夺/复氧复糖组(OGD/R组)和艾司氯胺酮+OGD/R组(E+OGD/R组).采用氧糖剥夺1 h,复氧复糖24 h的方法制备OGD/R模型.E+OGD/R组加入25 μmol/L艾司氯胺酮处理40 min后制备模型.采用CCK-8法检测神经元活力,透射电镜下观察神经元超微结构,检测ROS、谷胱甘肽过氧化物酶(GSH-px)和MDA的水平,JC-1试剂盒检测线粒体膜电位,TUNEL染色法测定神经元凋亡率,Western blot法测定 Bax、细胞色素 C(Cyt C)、cleaved-caspase-9、caspase-3 和 cleaved-caspase-3 的表达.结果 实验 Ⅰ与S组相比,IR组Zea Longa评分、Feeney评分和脑梗死体积升高(P＜0.01);与IR组相比,E+IR组Zea Longa评分、Feeney评分和脑梗死体积降低(P＜0.01).实验Ⅱ与C组相比,OGD/R组神经元活力和GSH-px活性降低,神经元凋亡率、ROS、MDA水平、线粒体膜电位和cleaved-caspase-3/caspase-3比值升高,Bax、Cyt C和cleaved-caspase-9表达上调(P＜0.01);与OGD/R组相比,E+OGD/R组神经元活力和GSH-px活性升高,神经元凋亡率、ROS、MDA水平、线粒体膜电位和cleaved-caspase-3/caspase-3比值降低,Bax、Cyt C和cleaved-caspase-9表达下调(P＜0.01).结论 艾司氯胺酮可减轻小鼠脑缺血再灌注损伤,其机制可能与抑制神经元线粒体应激,改善线粒体功能,抑制线粒体途径的神经元凋亡有关.
Abstract
Objective To evaluate the effect of esketamine on cerebral ischemia-reperfusion(I/R)injury and the association with mitochondrial stress in mice.Methods The experiment was performed in two parts.Part Ⅰ Eighteen SPF male C57BL/6 mice,aged 8-12 weeks,with body mass index of 28-30 g,were divided into 3 groups(n=6 each)by a random number table method:sham operation group(S group),cerebral I/R group(IR group),and esketamine plus cerebral I/R group(E+IR group).Cerebral I/R was produced by occlusion of middle cerebral artery for 1 h followed by 24-h reperfusion in anesthetized mice.Esketamine 10 mg/kg was intraperitoneally injected at 20 min before developing the model in E group.Neurological function was evaluated using the Zea Longa score and balance beam test(Feeney score).The cerebral infarct size was determined by TTC staining.Part Ⅱ Primary cortical neurons were isolated and cultured and then divided into 3 groups(n=42 each)using a random number table method:control group(group C),oxygen-glucose deprivation-reoxygenation(OGD/R)group,and esketamine plus OGD/R group(group E+OGD/R).Cells were subjected to 02-glucose deprivation for 1 h followed by restoration of O2-glu-cose supply for 24 h.The cells were treated with 25 μmol/L esketamine for 40 min before preparing the mod-el in E+OGD/R group.The neuronal viability was measured by the CCK-8 assay.The ultrastructure of neu-rons was observed with a transmission electron microscope.The levels of reactive oxygen species(ROS),glutathione peroxidase(GSH-px)and malondialdehyde(MDA)were determined,and the mitochondrial membrane potential was determined by JC-1 kit.The neuronal apoptosis was detected by TUNEL staining,and the apoptosis rate of neurons was calculated.The expression of Bax,cytochrome C(CytC),cleaved-caspase-9,caspase-3 and cleaved-caspase-3 was detected by Western blot.Results Part Ⅰ Compared with S group,the Zea Longa score,Feeney score and cerebral infarct size were significantly increased in IR group(P＜0.01).Compared with IR group,the Zea Longa score,Feeney score and cerebral infarct size were significantly decreased in E+IR group(P＜0.01).Part Ⅱ Compared with C group,the cell viability and activity of GSH-px were significantly decreased,the apoptosis rate of neurons,levels of ROS and MDA,mitochondrial membrane potential,and cleaved-caspase-3/caspase-3 ratio were increased,and the expres-sion of Bax,Cyt C and cleaved-caspase-9 was up-regulated in OGD/R group(P＜0.01).Compared with OGD/R group,the cell viability and activity of GSH-px were significantly increased,the apoptosis rate of neurons,levels of ROS and MDA,mitochondrial membrane potential,and cleaved-caspase-3/caspase-3 rati-o were decreased,and the expression of Bax,Cyt C and cleaved-caspase-9 was down-regulated in E+OGD/R group(P＜0.01).Conclusions Esketamine can alleviate cerebral I/R injury in mice,and the mecha-nism may be related to inhibition of mitochondrial stress in neurons,improvement in mitochondrial function,and inhibition of mitochondria-dependent apoptosis in neurons.
关键词
艾司氯胺酮/再灌注损伤/脑/线粒体/应激,生理学Key words
Esketamine/Reperfusion injury/Brain/Mitochondria/Stress,physiological引用本文复制引用
基金项目
山东省自然科学基金(ZR2020MH125)
山东省医药卫生科技发展计划(202104110344)
山东第一医科大学青年科学基金培育计划(202201-128)
出版年
2024
NETL
NSTL
万方数据