大鼠脑缺血再灌注后大脑皮层神经细胞NOS表达与细胞凋亡及复方丹参的保护作用

Expression of NOS and neuron apoptosis in the cerebral cortex after brain ischemia reperfusion injury in rat and protective effects of salvia miltiorrhiza

目的:探讨大鼠局灶性脑缺血再灌注后大脑皮层神经细胞一氧化氮合酶(NOS)的表达与神经细胞凋亡的关系及中药复方丹参的保护作用。方法:采用大脑中动脉内栓线法造模,应用原位细胞凋亡检测方法观察神经细胞凋亡,用免疫组织化学方法检测大鼠大脑皮层神经细胞nNOS、iNOS的表达并做图像分析。结果:与假手术组比较,脑缺血再灌注2h后缺血侧大脑皮层缺血边缘区神经细胞nNOS、iNOS表达升高,并出现神经细胞凋亡,随着再灌注时间的延长,缺血侧大脑皮层缺血边缘区神经细胞iNOS表达明显增强,凋亡神经细胞数逐渐增多,至24h达高峰,但神经细胞nNOS的表达增强不如iNOS表达明显。复方丹参保护组神经细胞nNOS、iNOS的表达和凋亡神经细胞数明显低于缺血再灌组。结论:脑缺血再灌注后缺血侧大脑皮层缺血边缘区神经细胞nNOS的表达增强,尤其是iNOS的表达显著升高,使NO产生增加可能是介导脑缺血再灌注后神经细胞凋亡的机制之一,复方丹参具有下调神经细胞nNOS、iNOS表达,减少NO生成,抑制细胞凋亡,减轻缺血再灌注对大鼠大脑皮层神经细胞损伤的作用。

Objective： To explore the relationship between expression of NOS with neuron apoptosis in the cerebral cortex after focal brain isehemic reperfusion injury in rats and the protective effect of salvia miltiorrhiza （SM）. Methods： The method of reversible middle cerebral artery occlusion （MCAO） without craniectomy was used to establish model of focal brain ischemic reperfusion injury in rats. Immunohistehemistry was used to detect the expression of neuronal nitric oxide synthase （nNOS） and inducible NOS （iNOS） in the cerebral cortex of rats and image analysis system were used. The apoptosis of neurons was observed with an terminal deoxynucleotidyl tranferase mediated dUTP-fluorescein nik end-labeling （TUNEL） assay. Results ： The expression of nNOS and iNOS in ischemie penumbra of the cerebral cortex in the ischemic side increased at 2 h after brain ischemic reperfusion and TUNEL-positive cell appeared in isehemic penumbra of the cerebral cortex in the ischemic side compared with the sham control group. The expression of iNOS and TUNEL-positive cell was progressively increasing as time went by and peaked at 24 h after reperfusion, whereas there was no significant change in the expression of nNOS. The expressions of nNOS and iNOS and neuron apoptosis in the salvia miltiorrhiza group were significantly decreased compared with those the IR group. Conclusion： An increase in the expression of NOS and NO might be one of the mechanisms of apoptosis. Salvia miltiorrhiza could significantly inhibit the apoptosis by means of inhibiting the expression of NOS and reducing the production of NO.