BACKGROUND: Breviscapine, extracted from a Chinese herb Erigeron breviscapus (Vant.) Hand-Mazz (Bre), has remarkable activities against platelets and thrombus formation. It can protect against ischemic brain damage through eliminating free radicals and apoptosis.OBJECTIVE: To study the protective effects of Breviscapine on brain damage in rats after ischemia-reperfusion injury, taking MgSO4 as control.DESIGN: Randomized controlled study and analysis of variance.SETTING: College of Life Science and Technology, Xiaogan University.MATERIALS: This experiment was conducted in the College of Life Science and Technology of Xiaogan University between May 2004 and November 2004. Forty male Wistar rats were selected and randomized into five groups: sham-operation group, cerebral ischemia-reperfusion (IR)group, MgSO4 group, 50 mg/kg Breviscapine group and 75 mg/kg Breviscapine group with 8 in each group.METHODS: Rats were subjected to cerebral ischemia-reperfusion induced by inserting a nylon thread into the internal carotid artery to block the origin of middle cerebral artery and removing the thread later. Normal saline of 20 mL/kg was administrated in sham-operation group and IR group, while 50 mg/kg, 75 mg/kg Breviscapine and 30 mg/kg MgSO4 were administrated in other groups respectively 10 minutes after the onset of ischemia. Neurological scoring was performed 1 hour, 2 hours, 5 hours and 23 hours after cerebral ischemia-reperfusion (Five points in total: 0 as unobvious neurological symptom; 1 as inability to completely extend left anterior claw; 2 as rotation toward the left; 3 as inclining to the left side atwalking; 4 as inability to walk by itself. The higher scale, the more severe the behavioral disturbance was). Brain infarcted area was assayed 1 hour and 23 hours after cerebral ischemia-reperfusion, and was expressed as the percentage of stained area to non-stained area. Neuron apoptosis was detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTPfluorescence nick end labeling (TUNEL) method to assess DNA fragments of apoptotic cells 1 hour after cerebral ischemia, and 2 hours, 5 hours and 23 hours after reperfusion. The apoptotic cells were counted under the fluorescence microscope. The percentage of apoptosis cells (%) = (apoptotic neurons ÷ hippocampal neurons) ×100%. The protein expression of caspase-3 was detected by immunohistochemical staining. The positive cells were identified and counted under the light microscope. The percentage of positive cells of caspase-3 (%) = (positive neurons of caspase-3 ÷ hippocampal neurons) ×100%.cells of caspase-3 1 hour after cerebral ischemia, and 2, 5 and 23 hours after reperfusion in each group.scoring: Neurological score of rats in 50 mg/kg, 75 mg/kg Breviscapine groups and MgSO4 group was 1.2±0.4, 0.5±0.4, and 1.3±0.4, respectively,which were all lower than that in IR group [(2.2±0.6)] 23 hours after cerebral ischemia-recirculation (F=6.09, P=0.001). However, it was lower in farcted area in rats of 50 mg/kg and 75 mg/kg Breviscapine groups and MgSO4 group was (0.18±0.03)%, (0.10±0.02)%, and (0.28±0.02)%, respectively, which were all lower than that of IR group [(0.43±0.05) %] 23 hours after cerebral ischemia-reperfusion (F=2.3, P=0.001). It was smaller in hippocampal apoptotic cells: It was (27.2±4.3) %, (20.6±3.6)%, and (35.4±5.5)% in 50 mg/kg and 75 mg/kg Breviscapine groups and MgSO4 group,which were all lower than that of IR group [(60.4±6.2)%] 23 hours after cerebral ischemia-reperfusion (F=6.17, P=0.000 7). It was lower in Brevispositive cells of caspase-3: It was (34.2±5.3)%, (21.6±3.5)%, and (47.4±4.5)% in 50 mg/kgand 75 mg/kg Breviscapine groups and MgSO4 group,which were all lower than that of IR group [(76.3±6.2)%] 23 hours after cerebral ischenia-reperfusion (F=6.88, P=0.000 1). It was lower in Breviscapine group than in MgSO4 group (P ＜ 0.01).CONCLUSION: Breviscapine can protect brain damage induced by cerebral ischemia-reperfusion by markedly decreasing the neurological score,area of cerebral infarction, number of hippocampal apoptotic cells and number of positive cells of caspase-3, and it has better effect than MgSO4.%背景:灯盏花素是从中药灯盏花中提取的,具有显著抗血小板和血栓形成的活性,通过清除自由基和凋亡细胞而保护大脑.目的:观察灯盏花素对大鼠脑缺血再灌注引起脑损伤的保护作用,并以硫酸镁做标准比较.设计:随机对照的实验,方差分析.单位:孝感学院生命科学技术学院.材料:实验于2004-05/11在孝感学院生命科学技术学院完成.实验选用40只雄性Wistar大鼠,随机分成5组:假手术组、脑缺血再灌注组、硫酸镁组、灯盏花素50mg/kg组和灯盏花素75mg/kg组,每组8只.方法:自大鼠颈总动脉插入尼龙线栓塞大脑中动脉,造成大脑缺血,拔出线栓进行再灌注.假手术组、脑缺血再灌注组于脑缺血10 min后给予20mL/kg生理盐水,其余3组分别给予50mg/kg和75 mg/kg灯盏花素及30 mg/kg硫酸镁.各组大鼠分别于脑缺血1 h再灌注2,5,23 h进行神经病学评分(5分制,0分为无明显神经病学症状,1分为不能完全伸展左侧前爪,2分为向左侧旋转,3分为行走时向左侧倾倒,4分为不能自行行走.积分越高,说明动物行为障碍越严重),并于脑缺血1 h再灌注23h时测定脑梗死面积(以染色区与未染色区的百分比表示).脑缺血1 h再灌注2,5,23 h时用脱氧核糖核酸末端转移酶介导的缺口末端标记方法检测脑海马凋亡细胞百分率(%)=(凋亡神经元÷海马神经元)×100%.检测半胱氨酸天冬氨酸蛋白酶3蛋白的表达用免疫组织化学方法[半胱氨酸天冬氨酸蛋白酶3阳性细胞表达百分率(%)=(半胱氨酸天冬氨酸蛋白酶3表达阳性神经元÷海马神经元)×100%].主要观察指标:①各组大鼠脑缺血1 h再灌注2,5,23 h神经病学评分.②各组大鼠脑缺血1 h再灌注23 h时脑梗死面积.③脑缺血1 h再灌注2,5,23 h时脑海马凋亡细胞百分率.④脑缺血1 h再灌注2,5,23 h时脑海马半胱氨酸天冬氨酸蛋白酶3阳性细胞表达百分率.结果:40只大鼠全部进入结果分析.①神经病学评分:脑缺血再灌注23 h时灯盏花素50mg/kg组,灯盏花素75 mg/kg组和硫酸镁组明显低于脑缺血再灌注组[(1.2±0.4)分,(0.5±0.4)分,(1.3±0.4)分,(2.2±0.6)分,F=6.09,P=0.001],但灯盏花素组明显低于硫酸镁组(P＜0.01).②脑梗死面积:脑缺血再灌注23 h时灯盏花素50 mg/kg组,灯盏花素75 mg/kg组和硫酸镁组大鼠大脑梗死区面积明显低于脑缺血再灌注组[(0.18±0.03)%,(0.10±0.02)%,(0.28±0.02)%,(0.43±0.05)%,F=2.3,P=0.001],灯盏花素组明显低于硫酸镁组(P＜0.01).③脑海马凋亡细胞百分率:脑缺血再灌注23 h时灯盏花素50mg/kg组,灯盏花素75mg/kg组和硫酸镁组明显低于脑缺血再灌注组[(27.2±4.3)%,(20.6±3.6)%,(35.4±5.5)%,(60.4±6.2)%,F=6.17,P=0.000 7],灯盏花素组明显低于硫酸镁组(P＜0.01).④半胱氨酸天冬氨酸蛋白酶3阳性细胞表达百分率:脑缺血再灌注23 h时灯盏花素50 mg/kg组,灯盏花素75 mg/kg组和硫酸镁组明显低于脑缺血再灌注组[(34.2±5.3)%,(21.6±3.5)%,(47.4±4.5)%,(76.3±6.2)%,F=6.88,P=0.000 1],灯盏花素组明显低于硫酸镁组(P＜0.01).结论:灯盏花素可显著降低脑缺血再灌注大鼠神经病学评分,缩小脑梗死面积,降低脑海马凋亡细胞数,降低半胱氨酸天冬氨酸蛋白酶3阳性表达细胞数量,起到保护大脑缺血再灌注引起的脑损伤作用,其作用优于硫酸镁.
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