氧化／抗氧化失衡与大鼠前脑缺血后海马CA1区神经元损伤的关系

Relationship of oxidation / antioxidation imbalance and hippocampal CA1 neuronal injury in rats at early stage of vascular dementia

目的探索大鼠永久性结扎双侧颈总动脉(bilateral common carotid arteries occlusion,BCCAO)后1 h至21 d海马CA1区神经元氧化/抗氧化平衡的变化,揭示血管性痴呆早期神经元损伤的分子机制。方法雄性SD大鼠按完全随机分组法分为假手术组(sham)和BCCAO(1 h,1、3、7、21 d)组,采用激光共聚焦和Western blot技术检测海马CA1区氧化应激损伤的标志蛋白和抗氧化应激损伤蛋白表达的变化,采用透射电镜观察BCCAO对海马CA1区神经元、血管及星形胶质细胞超微结构的影响。结果 1与sham组比较,BCCAO后1 h,3、7、21 d氧自由基探针HEt荧光强度呈时间依赖性增强;免疫荧光双染色及Western blot检测结果显示:过氧亚硝酸自由基(ONOO-)标志蛋白3NT、膜脂质损伤标志蛋白4HNE的表达于BCCAO后7、21 d较sham组升高(P<0.05);2抗氧化损伤的转录因子E2相关因子2(Nrf2)及其下游抗氧化产物SOD2、HO-1的表达于BCCAO后7、21 d较sham组降低(P<0.05);3透射电镜观察结果显示:BCCAO后1 h,3、21 d,海马CA1区神经元、血管及星形胶质细胞均有不同程度损伤,并且于21 d损伤最严重,出现染色质边集、核膜溶解,线粒体空泡样变,内质网严重扩张,血管周围严重水肿等病理改变。结论血管性痴呆早期海马CA1区神经元损伤呈现进行性加重,其机制可能与BCCAO后细胞内氧化/抗氧化失衡密切相关。

Objective To explore the change of oxidation/antioxidation balance from 1 h to 21 d after bilateral common carotid artery occlusion （BCCAO） in hippocampal CA1 region, and further to reveal the molecular mechanism of neuron damage in the early stage of vascular dementia. Methods Adult male Sprague-Dawley rats were randomly assigned into 2 groups： sham group and BCCAO groups （1 h, and 1, 3, 7 and 21 d）. The markers of oxidative stress damage and anti-oxidant level in hippocampal CA1 region were detected by laser scanning confocal microscopy （LSCM） and Western blot analysis. The neuron, vascular and astrocyte ultrastructures were observed after BCCAO in hippocampal CA1 region by transmission electron microscopy （TEM）. Results Compared with the sham group, the fluorescence intensity of HEt （oxygen free radical probe） were significantly increased in time-dependent manner （in 1 h and 3, 7 and 21 d after BCCAO）. Double immunofluorescent staining and Western blot results indicated that levels of 3NT （peroxynitrite radical marker） and 4HNE （membrane lipid damage marker） were significantly increased in the BCCAO 7 d and 21 d groups than the sham group （P〈0.05）. Compared with the sham group, the levels of antioxidant transcription factor Nrf2 and its downstream antioxidation products SOD2 and HO-1 were significantly decreased in the BCCAO 7 d and 21 d groups （P〈0.05）. Ultrastructure analysis revealed that different degrees of damage were found in the neuron, vascular and astrocyte ultrastructures at 1 h, 3 d and 21 d after BCCAO in hippocampal CA1 region. And the damage was the most serious in the BCCAO 21 d group, showing chromatin edge accumulation, dissolved nuclear membrane, swollen mitochondria, dilatation of endoplasmic reticulum, as well as severe edema around the blood vessels. Conclusion The damage of hippocampal CA1 neuron is increased in time-dependent manner at the early stage of vascular dementia, and the mechanism may be closely related with the intracellular oxidation/antioxidation imbalance after BCCAO.