牛膝多肽活性成分对大鼠短暂性脑缺血的保护作用

The effects of an active component isolated from Achyranthes Bidentata polypeptides on rat transient ischemia

目的:研究牛膝多肽(Achyranthes Bidentata polypeptides,ABPP)活性成分(active component k isolated from ABPP,ABPPk)对大鼠短暂性脑缺血的保护作用。方法:体外建立原代胎鼠皮层神经元氧糖剥夺(oxygen and glucose deprivation,OGD)损伤模型,复氧复糖同时分别加入不同质量浓度ABPPk(0.04,0.2,1.0μg/m L)和ABPP(1.0μg/m L,阳性对照)处理皮层神经元,72 h后通过MTT法检测ABPPk对OGD损伤神经元活力的影响;通过Western Blot分析ABPPk对OGD损伤凋亡相关蛋白Bcl-2、Bax、Cleaved caspase-3表达的影响。体内建立大鼠短暂性大脑中动脉栓塞(transient middle cerebral artery occlusion,t-MCAO)模型,缺血2 h后再灌注同时经尾静脉注射给予1.0 mg/kg ABPPk,1次/d,连续3 d。于再灌注72 h行神经行为学评分,评价ABPPk对t-MCAO大鼠神经行为缺陷的影响;再灌注72 h断头取脑,行2,3,5-氯化三苯基四氮唑(2,3,5-Triphenyl tetrazolium chloride,TTC)染色评价ABPPk对t-MCAO大鼠脑梗死体积的影响;再灌注72 h采用末端脱氧核苷酸转移酶(terminal deoxynucleotidyl transferase,TdT)介导的dUTP生物素缺口末端标记法(TdT-mediated dUTP-biotin nick end labeling,TUNEL)试剂盒检测ABPPk对大鼠脑缺血半影区神经元凋亡的影响。结果:ABPPk在体外可有效拮抗OGD引起的皮层神经元凋亡,体内可显著减小脑梗死体积,防止神经元凋亡。结论:ABPPk对大鼠短暂性脑缺血具有神经保护作用,可为开发新型小分子活性肽类神经保护药物提供新的策略。

Objective： To determine the effects of an active component isolated from Achyranthes Bidentata polypeptides （ABPPk） on rat transient ischemia. Methods： In vitro the primary cortex neurons from embryonic rats were subjected to oxygen and glucose deprivation insult. Different concentration（0.04, 0.2 and 1.0 μg/mL） of ABPPk and 1.0 μg/mL ABPP（served as positive control） were added into the medium respectively in the meantime of reperfusion for 72 hours. Cell viability was measured by MTT assay. Western Blot analysis was performed to examine the expression level of apoptosis associated proteins （Bal-2, Bax and Cleaved caspase-3）. A transient middle cerebral artery occlusion（t-MCAO） model was performed to evaluate the neuroprotective effects of ABPPk（1.0 mg/kg） in vivo. Neurological deficit was scored at 72 h after reperfusion. Infarction volume was measured at 72 h after reperfusion by 2, 3, 5-triphenyl tetrazolium chloride（Trc） staining and the apoptosis of neurons in ischemic penumbra was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling（TUNEL） kit. Results： ABPPk could effectively inhibit neuronal apoptosis induced by OGD in vitro and exert neuroprotection on a rat t-MCAO model by improving neurologic deficit, decreasing infarction volume and inhibiting neuronal apoptosis in penumbra area. Conclusion： ABPPk could be considered as a new strategy for developing a novel small molecular active peptide to remedy transient brain ischemia because of its neuroprotective effects.