不同剂量右美托咪定对大鼠海马神经元缺氧复氧损伤中线粒体分裂的影响

Effect of different doses of dexmedetomidine on mitochondrial fission in a rat hippocampal neuron model of hypoxia/reoxygenation injury

目的探讨不同剂量的右美托咪定(dexmedetomidine,Dex)在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响。方法 24h内出生的雄性SD大鼠,断头分离海马区神经组织,收集获得神经元细胞进行培养,8d后培养的海马神经元随机分为六组:正常对照组(C组);赋形剂组(V组);缺氧复氧组(HR)组;缺氧复氧+右美托咪定组(D1、D2、D3)组。C组:正常培养;V组:不行缺氧复氧、加入赋形剂二甲基亚砜培养6h,浓度0.01%;HR组:氧糖剥夺法缺氧6h,复氧12h建立缺氧复氧损伤模型;D1、D2、D3组,于缺氧6h后分别加入右美托咪定0.1、1、10μmol/L。采用激光共聚焦显微镜观察各组神经元细胞质Ca2+荧光强度,采用ELISA法检测细胞钙调神经磷酸酶活性,采用透射电镜观察线粒体的超微结构,Western-blot法检测线粒体分裂蛋白Drp1、Fis1的含量。结果与C组比较,HR组、D1组、D2组和D3组线粒体超微结构破坏加重,Ca2+荧光强度、CaN活性明显增强,Fis1、Drp1蛋白含量明显升高(P<0.05);与HR组比较,D1组、D2组和D3组线粒体超微结构破坏减轻,Ca2+荧光强度、CaN活性明显减弱,Fis1、Drp1蛋白含量明显降低(P<0.05);与D1组和D3组比较,D2组线粒体结构更加完整,Ca2+荧光强度、CaN活性明显减弱,Fis1、Drp1蛋白含量明显降低(P<0.05)。C组和V组各指标差异无统计学意义。结论右美托咪定0.1、1、10μmol/L可以减少大鼠海马神经元缺氧复氧损伤中线粒体的分裂,其中1μmol/L是最佳的保护浓度,其机制可能是与其抑制钙超载有关。

Objective To demonstrate the effect of different doses of dexmedetomidine（Dex）on mitochondrial fission in a rat hippocampal neuron model of hypoxia/reoxygenation injury.Methods Sprague-Dawley rats were sacrificed in the hippocampus of the hippocampus.The neurons of the hippocampal neurons were collected and cultured on the 8 th day.After 8 dcultivation,the primary hippocampal neurons were randomly divided into six groups：control group（group C）which was cultured in normal group;vehicle group（groupV）which was not subjected to anoxia and reoxygenation.;groupHR was deprivedoxygen for 6 hours,reoxygenated for 12 hours to establish hypoxiareoxygenation injury model;HR＋Dex treatment groups was further divided into D1,D2 and D3 groups who were respectively given Dex 0.1,1,10μmol/L during oxygen-glucose deprivation and reperfusion period.Fluorescence intensity of Ca2＋（using a laser scanning confocal microscope）,CaN enzymatic activities（by ELISA）,expression of Drp1,Fis1,（by western blot）were measured.Results Compared with group C,mitochondrial ultrastructuredamage in groupHR,group D1,group D2 and group D3 were aggravated,Ca2＋fluorescence intensity and CaN activity were significantly increased,and Fis1 and Drp1 protein content were significantly increased（P〈0.05）;Compared withHR group,the mitochondrial ultrastructural damage of group D1,group D2 and group D3 was lessened,Ca2＋fluorescence intensity and CaN activity were significantly attenuated,and Fis1 and Drp1 protein content were significantly decreased（P〈0.05）.Compared with group D1 and group D3,the mitochondrial ultrastructural of group D2 was more intact,Ca2＋fluorescence intensity,CaN activity were significantly decreased,and Fis1,Drp1protein content was significantly decreased（P〈0.05）.There were no statistically significant differences between the group C and group V.Conclusion Dex 0.1,1 and 10μmol/L can reduce mitochondrial fission during hypoxiareoxygenation injury in rat hippocampal neurons,and 1μmol/L is the best protective concentration.Its mechanism may be related to its inhibition of calcium overload.