他罗利姆对癫痫大鼠海马氧化应激、凋亡诱导因子及神经元凋亡的影响

Effect of tacrolimus ( FK506) on oxidative stress,apoptosis induce factor and apoptosis of neurons in the hippocampus of rats with status epilepticus

目的研究免疫抑制剂他罗利姆(FK506)对癫痫持续状态(SE)大鼠海马组织一氧化氮(NO)、一氧化氮合酶(NOS)、丙二醛(MDA)及线粒体膜电位、凋亡诱导因子(AIF)的影响。方法 (1)将126只Wistar大鼠随机分为癫痫组、FK506干预组、对照组各42只。建立氯化锂-匹鲁卡品癫痫模型,FK506干预组在注射匹鲁卡品之前24、1 h 2次腹腔注射FK506,采用比色法和羟胺法分别检测海马组织中NO、MDA含量、NOS活性;采用免疫组化法检测海马组织神经元型一氧化氮合酶(nNOS)、诱导型一氧化氮合酶(iNOS)的表达;尼氏染色观察神经元形态变化。(2)24只Wistar大鼠随机分为癫痫组、FK506干预组、生理盐水干预组、对照组,每组6只。FK506干预组在注射匹鲁卡品之前24 h、1 h 2次腹腔注射FK506,生理盐水干预组注射等体积生理盐水;于癫痫发作24 h后处死,应用流式细胞仪测定线粒体内罗丹明123的荧光强度和线粒体的大小;Western印迹检测大鼠海马线粒体和细胞核AIF的水平。结果与癫痫组相比,FK506干预组NO、MDA含量、NOS活性明显降低(P均<0.01);海马nNOS、iNOS的表达降低(P<0.01);线粒体膜电位升高(P<0.05);海马线粒体AIF水平显著增高,而细胞核AIF水平显著降低(P均<0.05);海马存活神经元增加。结论 FK506可能通过抑制NO引起的氧化应激损伤,稳定线粒体膜电位,抑制AIF的易位,发挥脑保护作用。

Objective To investigate the effect of tacrolimus on expressions of nitric oxide （NO）,nitric oxide synthase （NOS）,malondialdehyde （MDA） and changes of apoptosis induce factor （AIF） and the effect of mitochondrial membrane potential （MMP） in the hippocampus of rats with status epilepticus （SE）.Methods 126 male Wistar rats were randomly divided into epilepsy,tacrolimus and the control groups.Rats in epilepsy and the tacrolimus groups were injected with pilocarpine to establish SE models.Rats in tacrolimus group were pretreated with tacrolimus at 24 h and 1 h before pilocarpine injection.The contents of NO,NOS,MDA of the hippocampus were assayed and expression of neuronal nitric oxide synthase （nNOS） and inducible nitric oxide synthase （iNOS） in the hippocampus was determined by immunohistochemical techniques.24 Wistar rats were randomly divided into epilepsy,tacrolimus,saline and the control groups.Rats in tacrolimus group were pretreated with tacrolimus at 24 h and 1 h before pilocarpine injection.Rats in saline group only received normal saline.Then fluorescence intensity of rhodamine 123 and size of mitochondria were detected by flow cytometry.Western blot analysis was used to detect AIF levels in mitochondria and nucleus in hippocampus.Results Compared with epilepsy group,the contents of NO,NOS and MDA and expressions of nNOS,iNOS in tacrolimus group were obviously decreased （P ＜ 0.01） and the mitochondrial membrane potential was increased （P ＜ 0.05）,and AIF levels were obviously increased in the mitochondrial fraction while decreased in the nuclear （P ＜0.05）.And the brain injury was less severe in tacrolimus group.Conclusions FK506 plays an important role by suppressing oxidative stress and the changes of MMP and AIF,which may underlie one of the potential mechanisms of neuroprotection.