Connexin 43在银杏叶提取物抗大鼠脑缺血损伤中的作用

目的通过体内体外实验研究缝隙连接蛋白43(Cx43)与EGb761脑缺血神经保护作用的关系,探讨银杏叶制剂EGb761治疗脑缺血的机制。方法在大鼠原代神经细胞中转染荷载Cx43-shRNA的慢病毒,干扰Cx43蛋白表达,同时给予EGb761 200μg/mL处理观察Cx43的表达以及EGb761对Cx43蛋白表达的影响。建立大鼠脑缺血模型,腹腔给予EGb761 50、100 mg/kg,使用HE染色,TTC染色,免疫组化和westernblot方法检测大鼠脑组织细胞的形态变化和大鼠海马Caspase-3,TUNEL阳性细胞,Cx43,p-Cx43蛋白的表达变化。结果 EGb761给药可以减少脑缺血大鼠脑组织梗死面积,显著性抑制脑缺血大鼠海马Caspase-3表达和TUNEL阳性凋亡细胞数目,显著性抑制大鼠海马神经细胞p-Cx43的表达水平。体外培养的大鼠神经细胞转染Cx43-shRNA慢病毒,给予EGb761处理,处理前后神经细胞Cx43/p-Cx43蛋白表达未见明显变化。结论 EGb761可以减轻脑缺血缺氧引起的细胞损伤,其神经保护作用与抑制缺血引起的缝隙连接蛋白Cx43的表达和激活有关。

Effect of Connexin 43 in Neuroprotective Function of Ginkgo Biloba Extract in Rats with Ischemic Injury

Objective:To explore the roles of Cx43 protein in EGb761-mediated neuroprotection and to investigate the mechanism of ischemic treatment by EGb761.Methods:Rat primary neuronal cells were transfected by Cx43-shRNA and treated with EGb761-200ug/ml.Rats were delivered with EGb761(50 mg/kg and 100 mg/kg) by intraperitoneal injection 30 min before occluding the brain middle artery,then rats brain were obtained and used HE staining,TTC staining,immunohistochemisty and western blot techniques.The level of Cx43 and p-Cx43 were detected and compared in primary neuron cells and brain tissues.Results:EGb761 reduced the brain infarct volume,inhibited significantly the caspase-3 expression and decreased remarkably the neuronal apoptosis in hippocampus area.The p-Cx43 decreased in the hippocampus of ischemic rats after treated with EGb761.In vitro,Cx43 were blocked after transfected by Cx43-shRNA and the level of Cx43 and p-Cx43 in EGb761 treated cells was similar with control group.Conclusion:EGb761 effectively reduce the neuronal apoptosis in ischemic rat brain and EGb761’s neuroprotection effect involved in connexin 43 protein activation.