缺血预处理对大鼠脑缺血再灌注自噬的影响

Influences of ischemic preconditioning on autophagy in rats with cerebral ischemia reperfusion injury

目的观察缺血预处理后不同间隔时间对大鼠脑缺血再灌注海马自噬的影响。方法参照ZeaLonga线栓法制作大鼠大脑中动脉缺血再灌注模型,将实验动物随机分为3组:假手术组(Sham组,n=10)、缺血再灌注组(I/R组,n=10)、缺血预处理组(IPC组,n=30),缺血预处理组再按缺血预处理后不同间隔时间分为1 d、3 d、5d 3个亚组,每组10只。缺血2 h再灌注24 h后用免疫组织化学法检测自噬相关蛋白BECLIN I、LC3-II的表达情况,透射电镜观察神经细胞中自噬和溶酶体的激活以及细胞超微结构的变化。结果(1)神经功能障碍评分:与Sham组比较,I/R组及IPC组均出现不同程度神经功能障碍(P<0.01),IPC组症状评分低于I/R组(P<0.05)。(2)脑梗死体积:与Sham组比较,I/R组及IPC组均有不同程度梗死灶(P<0.01);IPC组脑梗死灶体积明显低于I/R组(P<0.01),3 d亚组梗死体积少于1 d、5 d亚组(P<0.01)。(3)免疫组织化学染色:与Sham组比较,I/R组及IPC组BECLIN 1及LC3-II阳性表达增多(P<0.01),IPC组阳性表达显著低于I/R组(P<0.01),IPC 3 d亚组阳性表达低于1 d、5 d亚组(P<0.01)。(4)脑组织超微病理形态改变:Sham组细胞正常;I/R组及IPC组可见数量不等、处于不同时期的自噬体和或自噬溶酶体,线粒体肿胀,膜破裂,可见空泡,各级溶酶体显著增多,可见变形次级溶酶体,高尔基体分裂;IPC组自噬体及各级溶酶体数量较I/R组减少,各IPC亚组间自噬体及各级溶酶体数量无可见变化。结论缺血预处理可能通过下调自噬水平减轻缺血再灌注损伤,缺血预处理3 d优于1 d、5 d。

Objective To observe the effect of ischemic preconditioning（IPC） effect on cerebral ischemia reperfusion in rat hippocampus of autophagy.Methods According to method of Zea-Longa filitmene,rats middle cerebral artery ischemia-reperfusion model were established,the experimental animal were randomly divided into 3 groups;sham operation group（Sham group,n =10）,ischemia reperfusion group（I/R group,n =10）,IPC treatment group（IPC group,n =30）,in IPC group,according to ischemic preconditioning time,rats were divided into 1 d,3 d,5 d subgroups,10 rats in each group.After ischemia 2 h and reperfusion 24 h,immunohistochemical method was used to detect the autophagy associated protein BECLIN L,LC3-II expression,transmission electron microscope in nerve cell autophagy and lysosomal activation and cell ultrastructure changes.Results（1） Neurological dysfunction score;compared with Sham group,I/R group and IPC group showed varying degrees of nerve dysfunction（P 〈0.01）,I/R group of symptoms in IPC subgroups（P〈0.05）.（2） The volume of cerebral infarction：compared with Sham group,I/R group and IPC group had different degree of infarction（P 〈0.01）;IPC group was lower than that in I/R group（P〈0.01）,3d subgroup was less than 1 d and 5 d subgroup（P〈 0.01）.（3） Immunohistochemical staining;compared with Sham group,I/R group and IPC group,the number of BECLIN 1and LC3-II positive cells（P 〈0.01）,the expression of IPC positive cells was significantly lower than that of I/R group（P 〈 0.01）,IPC 3 d subgroup was less than 1 d,5 d subgroup（P〈 0.01）.（4） The change of brain tissue ultrastructure morphology of cells;Sham group was normal;I/R group and IPC group showed varying amounts of autophagy,in different period and or autophagic lysosome,mitochondria swelling,rupture of membranes,vesicles,lysosomes increased significantly,visible deformation of secondary lysosomes,Golgi fragmentation;IPC group autophagy and the number of lysosomes was significantly reduced compared with I/R,the number of the IPC between the subgroups of autophagosomes and lysosomes revealed no visible change.Conclosion IPC may inhibit autophagy relieve the ischemic reperfusion injury,3 d IPC is better than 1 d,5 d.