Notch1对局灶性脑缺血再灌注损伤后神经细胞凋亡的影响

Effect of Notch1 on neuronal apoptosis after focal cerebral ischemia reperfusion injury

目的研究Notch1对脑局灶性缺血再灌注损伤后神经细胞凋亡的影响,为缺血性脑血管病临床治疗寻找新的靶点。方法 72只SD雄性成年大鼠随机分为等量4组:假手术组(Sham组),缺血再灌组(I/R组),Notch1抑制剂组(DAPT组),溶剂对照组(Vehicle组)。采用线栓法大脑中动脉阻塞(MCAO)模型制作大鼠局灶性脑缺血再灌注模型,缺血90 min为标准。Sham组仅分离血管,不插入线栓,Notch1抑制剂组或Vehicle组在模型建立再灌注恢复后立即向缺血对侧侧脑室分别注射DAPT或单纯溶剂(PBS+DMSO),每天1次,分别于术后1、3、7 d断头取脑,病理学观察缺血再灌注侧皮层损伤情况,TUNEL法检测神经细胞凋亡指数(AI),Weatern blot检测PARP裂解片段含量、Notch1活性片段NICD、Akt和Bad磷酸化水平。结果与Sham组相比,I/R组神经细胞凋亡指数及PARP裂解片段含量增加,NICD、磷酸化Akt及Bad水平升高,差异有明显统计学意义(P<0.05);与I/R组相比,给予Notch1抑制剂DAPT后,神经细胞凋亡指数及PARP裂解片段含量明显增加,NICD、磷酸化Akt及Bad水平明显下降,差异有显著性(P<0.05),而溶剂对照Vehicle组的各项指标无明显差异。结论 Notch1在脑缺血再灌注损伤中具有抑制神经细胞凋亡作用,其作用机制可能与增强Akt磷酸化水平,促进Bad失活有关。

Objective To study effect of nerve cell apoptosis after injury of Notch1 on focal cerebral ischemia reperfusion,and find new targets for the treatment of ischemic cerebrovascular disease. Methods 72 male adult SD rats were randomly divided into four equal groups： sham operation group（ Sham group）,ischemia reperfusion group（ I/R group）,Notch1 inhibitor group（ DAPT group）,solvent control group（ Vehicle group）. By the suture method of middle cerebral artery occlusion（ MCAO） for rat focal cerebral ischemia reperfusion model,the rats ischemic for 90 minutes as the standard. The Sham group only isolated vascular,suture was not inserted. After the recovery of ischemia-reperfusion,the rats in inhibitor of Notch1 group or Vehicle group were immediately injected with DAPT or pure solvent（ PBS ＋ DMSO） to the contralateral ventricle,once daily,respectively. After 1 d,3 d,7 d,rats were cut off the head to take out the brain tissue,pathological observation of ischemia perfusion of cortex damage detection index,neural cell apoptosis by TUNEL（ AI）,Western blot detection PARP cleavage fragment content,activity of Notch1 fragment of NICD,Akt and Bad phosphorylation. Results Compared with Sham group,the apoptotic index,PARP cleavage fragment content,NICD,phosphorylation of Akt,Bad level increased in I / R group,and there were significant difference（ P〈 0. 05）; compared with I / R group,apoptotic index and PARP fragments were markedly increased,NICD,phosphorylation Akt and Bad levels decreased significantly in DAPT group,and the differences were significant（ P〈 0. 05）,while among the solvent control indexes of the Vehicle group. Conclusion Notch1 can inhibit nerve cell apoptosis in cerebral ischemia reperfusion injury,the mechanism may be related to enhance Akt phosphorylation,promote Bad inactivation.