大鼠全脑缺血再灌注后海马CA1区细胞凋亡及银杏叶提取物的保护作用

Neuronal apoptosis in CA1 region of hippocampus after cerebral ischemia-reperfusion and protective effect of EGb761 in rats

目的:观察大鼠全脑缺血再灌注后神经细胞的凋亡及银杏叶提取物(EGb761)对其的影响,探讨EGb761对脑缺血再灌注损伤的分子保护机制。方法:采用Pulsinelli 4血管闭塞法制备大鼠弥漫性全脑缺血模型。将健康SD大鼠随机分为假手术组(Sham组)、缺血再灌注组(IR组)和EGb761预处理组(EGb组);原位末端标记法检测再灌注48h海马CA1区神经细胞的凋亡;透射电镜观察海马CA1区神经细胞超微结构;实时荧光定量PCR检测Bcl-2和caspase-3mRNA的表达;免疫印迹检测Bcl-2和caspase-3蛋白的表达。结果:IR组海马CA1区有大量凋亡细胞,平均光密度值较Sham组显著升高,EGb组神经细胞凋亡的光密度值较IR组明显减小;IR组可见凋亡的典型形态改变和极少数典型的坏死神经细胞,Sham组仅观察到个别凋亡细胞形态改变,EGb组未见明显的凋亡形态改变;与Sham组相比,IR组Bcl-2mRNA表达显著降低,caspase-3mRNA表达显著升高;EGb组与IR组相比,Bcl-2 mRNA表达明显升高,caspase-3 mRNA表达明显降低;与Sham组相比,IR组Bcl-2蛋白表达显著降低,caspase-3蛋白表达显著升高;EGb组较与IR组相比,Bcl-2蛋白表达明显升高,caspase-3蛋白表达明显降低。结论:细胞凋亡是大鼠全脑缺血再灌注后海马CA1区神经细胞丢失的重要原因;Bcl-2和caspase-3是参与神经细胞凋亡的重要调控基因;EGb761对脑缺血再灌注后的神经细胞具有显著的保护作用,其机制可能与抑制神经细胞凋亡的Bcl-2上调和caspase-3下调有关。

Objective:To observe the effect of EGb761 on the apoptosis of neurons after global cerebral ischemia-reperfusion in rats,and to explore the molecular protective mechanism of EGb761 on cerebral ischemia-reperfusion.Methods:Pulsinelli 4-vessel occlusion was used to prepare the diffuse cerebral ischemia model in rats.Healthy SD rats were randomly divided into sham operated group(Sham group),ischemia-reperfusion group(IR group)and EGb761 pretreatment group(EGb group).DNA fragmentation was identified histochemically by TUNEL and the morphologic features of apoptotic neurons were observed by transmission electron microscopy in the CA1 region of hippocampus at 48 hours.Quantitative real-time PCR(qPCR)was used to analyze the transcription level of Bcl-2 and caspase-3.The protein expression of Bcl-2 and caspase-3 was detected by Western blotting.Results:There was a large number of apoptotic cells in hippocampal CA1 region in IR group,the mean optical density was significantly higher than that in Sham group,and the mean optical density of apoptotic neurons in EGb group was significantly lower than that in IR group.Many apoptotic neurons and a few necrotic neurons appeared in IR group,only a few neuronal apoptosis were seen in Sham group and no obvious apoptotic neuron was observed in the EGb group.Compared with Sham group,the expression of Bcl-2 mRNA was significantly reduced,and the expression of caspase-3 mRNA was significantly increased in IR group.Compared with IR group,the expression of Bcl-2 mRNA was significantly increased,and the expression of caspase-3 mRNA was significantly reduced in EGb group.Compared with sham group,the expression of Bcl-2 protein was significantly reduced and caspase-3 protein expression was significantly increased in IR group.Compared with IR group,the expression of Bcl-2 protein was significantly increased,and the expression of caspase-3 protein was significantly reduced in EGb group.Conclusion:Apoptosis is an important reason for the loss of neurons in hippocampal CA1 region after global cerebral ischemia reperfusion in rats.Bcl-2 and caspase-3 are important regulatory genes involved in the apoptosis of neural cells.EGb761 has highly neuroprotective effects against cerebral ischemia-reperfusion.Its mechanism may be related to the up-regulation of Bcl-2 and down-regulation of caspase-3 which inhibit the apoptosis of nerve cells.