依达拉奉联合灯盏花素对局灶性脑缺血大鼠皮层神经元凋亡的影响

The effects of edaravone combined with breviscapine treatment on cortical neuronal apoptosis following focal cerebral ischemia in rats

目的:探讨依达拉奉联合灯盏花素(简称灯盏花素)治疗对大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型大鼠局灶性脑缺血后大脑皮层神经细胞凋亡的影响。方法:将25只SD大鼠随机分为5组:假手术组、MCAO组、MCAO+依达拉奉组、MCAO+灯盏花素组、MCAO+依达拉奉+灯盏花素(n=5),应用微创开颅法制作大鼠MCAO模型,药物组于术前2 h,术后12、24、36、48、60 h经腹腔注射依达拉奉5 mg/kg,灯盏花素100 mg/kg,依达拉奉+灯盏花素(5 mg/kg+100 mg/kg)。假手术组和MCAO组均给予生理盐水。各组分别于术后1 d、3 d和7 d断头取脑,制备常规石蜡组织切片。应用神经功能缺损评分法评估大脑缺血后的神经功能。利用尼氏染色观察不同时间点脑缺血大鼠大脑皮层神经元的形态和数量变化。采用TUNEL方法显示脑缺血后细胞凋亡。应用免疫组织化学法检测凋亡相关蛋白Bcl-2和Bax在脑缺血大脑皮层中的表达变化。结果:(1)大鼠脑缺血后神经功能缺损评分结果显示:假手术组大鼠神经功能正常;MCAO组大鼠神经功能评分在不同点显著增高于假手术组(P<0.05);MCAO+依达拉奉+灯盏花素组大鼠神经功能评分在10 d、14 d较依达拉奉组、灯盏花素组明显降低,与单药组相比差异有统计学意义(P<0.05),但各时间点均高于假手术组(P<0.05)。(2)假手术组大鼠脑缺血大脑皮层尼氏染色和TUNEI检测显示依达拉奉+灯盏花素组各时间点大脑皮层核固缩神经元及凋亡细胞与两种药物单独使用相比数量明显减少(P<0.01)。(3)免疫组化结果显示:依达拉奉+灯盏花素组缺血侧大脑皮层Bcl-2阳性细胞表达显著增强,Bax阳性细胞表达明显减少、减弱,与单独用药相比差异有统计学意义(P<0.01)。结论:依达拉奉联合灯盏花素治疗大鼠脑缺血可能通过增强/抑制神经元凋亡相关蛋白Bcl-2/Bax的表达,提高Bcl-2/Bax的比值,抑制神经细胞凋亡和增强神经元的抗凋亡作用,对缺血脑组织发挥神经保护作用。

Objective;To probe the impact of cerebral cortex neurons apoptosis after focal cerebral ischemia by using adaravone combined with breviscapine as a treatment to the modal of middle cerebral artery occlusion（MCAO） in rats.Methods：A total of 25 Sprague-Dawley adult male rats were randomly divided into five groups including（n = 5）：Sham operation group,MCAO group,MCAO ＋ edaravonne group,MCAO ＋ breviscapine group and MCAO ＋ edaravonne ＋ breviscapine group.The focal cerebral ischemia model of MCAO in rats was duplicated following a minimal invasive craniotomy.The rats in the drug group were injected by intraperitonealedaravone（5 mg/kg）,breviscapine（100 mg/kg）,and edaravone ＋ breviscapine（5 mg/kg ＋ 100 mg/kg） at 2 h before operation andat 12,24,48,60 h postoperation.The sham group and MCAO group were given normalsaline.The brains were removed at 1 d,3 d and 1 week after operation,and the routine paraffin sections were prepared.Application neurological deficit score assessed neurological function after focal cerebral ischemia in rats.The morphological and number changes of neurons of the cerebral cortex were observed using Nissl＇s staining.The apoptotic cells of the cerebral cortex of rats were detected via TUNEL method and the expression changes of apoptosis related protein Bcl-2 and Bax were examined using immunohistochemical method.Results：（1）The results of neuronlogical degicits scores after cerebral ischemia showed that the neurological function of rats in sham group was normal,the neurological degicits scores of MCAO group were all significantly increased compared with the sham group at different time points（P0.05）.The neuronlogical degicits scores of MCAO ＋ edaravone ＋ breviscapine group was significantly decreased at the 10 d and 14 d compared with the treatment using edaravonne or breviscapine group respectively（P〈0.05）.But the neuronlogical degicits scores of MCAO ＋ edaravone ＋ breviscapine group was higher than sham guoup all the time（P0.05）.（2） Nissl staining and TUNEL assay indicated that the combination of edaravone and breviscapine decreased the number of degenerated pyknotic neurons in cerebral cortexafter MCAO in rats（P〈0.01）.（3） Immunohistochemical staining results showed that the expression of Bcl-2 positive cells in the ischemic side of the MCAO ＋ edaravone ＋ breviscapine group increased,and the expression of Bax positive cells decreasedcompared with the treatment using edaravonne or breviscapine respectively（P〈0.01）.Conclusion;Edaravone combined with breviscapine may partly effect by increasing/inhibiting the expression of Bcl-2/Bax,increase the ratio of Bcl-2/Bax,inhibiting the apoptosis of neurons to play a neuroprotective function.