谷红注射液抗脑缺血的药效学与作用机制研究
摘要
背景：脑卒中是世界范围内及中国最主要的死亡原因之一,也是世界范围内最主要的疾病负担之一。脑卒中约80%为缺血性脑卒中。谷红注射液是国内用于治疗急性缺血性脑卒中的药物之一,虽然疗效得到一些临床试验证实,但目前尚缺乏抗脑缺血的药效学和作用机制相关基础研究。
目的：评价谷红注射液抗脑缺血的有效性,在一定程度上探讨其抗脑缺血的作用机制。
方法：采用大鼠双侧颈总动脉结扎模型和大脑中动脉栓塞模型分别研究谷红注射液在慢性脑缺血和急性局灶性脑缺血再灌注损伤中的作用。对于大鼠双侧颈总动脉结扎模型,造模后分别给予生理盐水、乙酰谷酰胺、红花提取物和谷红注射液等药物干预；术后30天进行自发运动实验、Morris水迷宫实验和Y迷宫实验等行为学实验；行为学评估后留取样本,通过NeuN染色、Ibal染色和BrdU染色分别显示海马CA1区神经元、小胶质细胞及海马齿状回神经再生情况,通过实时定量PCR显示突触相关基因表达情况。对于大鼠大脑中动脉栓塞模型,造模后分别给予生理盐水、乙酰谷酰胺、红花提取物和谷红注射液(两种剂量)等药物干预；术后1天、7天、14天进行神经缺损症状评分和贴纸去除实验,术后14天进行平衡杆实验和穿梭箱实验；行为学评估后留取样本,通过TTC染色显示梗死情况。
结果：对于大鼠双侧颈总动脉结扎模型,双侧颈总动脉结扎显著增加Morris水迷宫实验寻台潜伏期(P=0.021),显著减少海马CA1区神经元(P<0.001),对自发运动距离(P=0.577)、Y迷宫实验连续入臂百分比(P=0.547)、海马齿状回BrdU阳性细胞数(P=0.052)、海马CA1区小胶质细胞数(P=0.750)和所选突触相关基因表达量无显著影响。乙酰谷酰胺组、红花组和谷红组大鼠海马CA1区神经元数量显著多于模型组(三者均有P<0.001),而三种药物对Morris水迷宫实验寻台潜伏期无显著影响(P=0.282)。对于大鼠大脑中动脉栓塞模型,大脑中动脉栓塞显著增加神经缺损症状评分(P<0.001)、贴纸去除实验不对称分(P=0.010)、平衡杆实验过杆总时间(P=0.003)和相对梗死体积(P=0.037),对穿梭箱实验入暗箱潜伏期(P=0.952)和次数(P=0.905)无显著影响。谷红注射液可显著减少平衡杆实验过杆总时间(P=0.010),乙酰谷酰胺(P=0.139)和红花(P=0.093)均对过杆总时间无显著影响。乙酰谷酰胺、红花和谷红对神经缺损症状评分(P=0.568)、贴纸去除实验不对称分(P=0.688)和相对梗死体积(P=0.350)均无显著影响。
结论：谷红注射液在慢性脑缺血中具有保护海马CA1区神经元的作用。谷红注射液在急性局灶性脑缺血再灌注损伤中具有改善运动与共济运动的作用,且优于单用乙酰谷酰胺或红花注射液。
Background:Stroke is one of the most common causes of mortality and disease burden worldwide.67.3～80.5%of strokes are ischemic stroke. The effectiveness of Guhong injection for acute ischemic stroke is proved by several clinical trials. The effectiveness and mechanism of Guhong injection for cerebral ischemia in animal experiment are not clear.
Objective:To evaluate the effectiveness and mechanism of Guhong injection for cerebral ischemia.
Methods:Two models of cerebral ischemia, permanent bilateral carotid artery occlusion (2VO) and middle cerebral artery occlusion (MCAO) were used in our study. Aceglutamine, safflower and Guhong injection were evaluated in each model. For2VO, behavior changes were evaluated by open field test, Morris water maze test and Y maze test. The changes of neurons and microglia in hippocampus region CA1were evaluated by NeuN and Ibal staining separately. The initial evaluation of the neurogenesis in dentate gyrus was done by BrdU staining. Several synapsis-related genes were evaluated by real time PCR. For MCAO, behavior changes were evaluated by neurologic deficit scores, adhesive removal test, balance beam test and shuttle box test. TTC staining was used to demonstrate the cerebral infarction.
Result:For2VO, the2VO surgery increased the escape latency in Morris water maze test (P<0.001), and decreased the number of neurons in hippocampus region CA1(P<0.001) significantly. Open field test (P=0.577), Y maze test (P=0.547), BrdU positive cells in DG (P=0.052), microglia in region CA1(P=0.750) and the synapsis-related gene tested were not significantly affected by the surgery. Aceglutamine, safflower and Guhong injection seemed to protect the neurons in region CA1(P<0.001, respectively), although their protective effect was not showed by Morris water maze test (P=0.282). For MCAO, the neurologic deficit scores (P<0.001), the asymmetric index in adhesive removal test (P=0.010), the beam walking latency (P=0.003) and the relative infarction volume (P=0.037) were significantly increased by MCAO surgery. The result of shuttle box was not significantly affected by the surgery (latency:P=0.952, counts:P=0.905). The beam walking latency was significantly decreased by Guhong injection (P=0.010), and was not significantly affected by aceglutamine (P=0.139) or safflower (P=0.093). The neurologic deficit scores (P=0.568), the asymmetric index in adhesive removal test (P=0.688), and relative infarction volume (P=0.350) were unaffected by Guhong injection, aceglutamine or safflower.
Conclusion:Guhong injection, as well as aceglutamine and safflower injection, showed protective effect on neurons in region CA1for chronic cerebral ischemia. Guhong injection seemed to improve the behavior results for acute cerebral ischemia. Aceglutamine and safflower, on the other hand, didn't show protective effect for acute cerebral ischemia.
目的：评价谷红注射液抗脑缺血的有效性,在一定程度上探讨其抗脑缺血的作用机制。
方法：采用大鼠双侧颈总动脉结扎模型和大脑中动脉栓塞模型分别研究谷红注射液在慢性脑缺血和急性局灶性脑缺血再灌注损伤中的作用。对于大鼠双侧颈总动脉结扎模型,造模后分别给予生理盐水、乙酰谷酰胺、红花提取物和谷红注射液等药物干预；术后30天进行自发运动实验、Morris水迷宫实验和Y迷宫实验等行为学实验；行为学评估后留取样本,通过NeuN染色、Ibal染色和BrdU染色分别显示海马CA1区神经元、小胶质细胞及海马齿状回神经再生情况,通过实时定量PCR显示突触相关基因表达情况。对于大鼠大脑中动脉栓塞模型,造模后分别给予生理盐水、乙酰谷酰胺、红花提取物和谷红注射液(两种剂量)等药物干预；术后1天、7天、14天进行神经缺损症状评分和贴纸去除实验,术后14天进行平衡杆实验和穿梭箱实验；行为学评估后留取样本,通过TTC染色显示梗死情况。
结果：对于大鼠双侧颈总动脉结扎模型,双侧颈总动脉结扎显著增加Morris水迷宫实验寻台潜伏期(P=0.021),显著减少海马CA1区神经元(P<0.001),对自发运动距离(P=0.577)、Y迷宫实验连续入臂百分比(P=0.547)、海马齿状回BrdU阳性细胞数(P=0.052)、海马CA1区小胶质细胞数(P=0.750)和所选突触相关基因表达量无显著影响。乙酰谷酰胺组、红花组和谷红组大鼠海马CA1区神经元数量显著多于模型组(三者均有P<0.001),而三种药物对Morris水迷宫实验寻台潜伏期无显著影响(P=0.282)。对于大鼠大脑中动脉栓塞模型,大脑中动脉栓塞显著增加神经缺损症状评分(P<0.001)、贴纸去除实验不对称分(P=0.010)、平衡杆实验过杆总时间(P=0.003)和相对梗死体积(P=0.037),对穿梭箱实验入暗箱潜伏期(P=0.952)和次数(P=0.905)无显著影响。谷红注射液可显著减少平衡杆实验过杆总时间(P=0.010),乙酰谷酰胺(P=0.139)和红花(P=0.093)均对过杆总时间无显著影响。乙酰谷酰胺、红花和谷红对神经缺损症状评分(P=0.568)、贴纸去除实验不对称分(P=0.688)和相对梗死体积(P=0.350)均无显著影响。
结论：谷红注射液在慢性脑缺血中具有保护海马CA1区神经元的作用。谷红注射液在急性局灶性脑缺血再灌注损伤中具有改善运动与共济运动的作用,且优于单用乙酰谷酰胺或红花注射液。
Background:Stroke is one of the most common causes of mortality and disease burden worldwide.67.3～80.5%of strokes are ischemic stroke. The effectiveness of Guhong injection for acute ischemic stroke is proved by several clinical trials. The effectiveness and mechanism of Guhong injection for cerebral ischemia in animal experiment are not clear.
Objective:To evaluate the effectiveness and mechanism of Guhong injection for cerebral ischemia.
Methods:Two models of cerebral ischemia, permanent bilateral carotid artery occlusion (2VO) and middle cerebral artery occlusion (MCAO) were used in our study. Aceglutamine, safflower and Guhong injection were evaluated in each model. For2VO, behavior changes were evaluated by open field test, Morris water maze test and Y maze test. The changes of neurons and microglia in hippocampus region CA1were evaluated by NeuN and Ibal staining separately. The initial evaluation of the neurogenesis in dentate gyrus was done by BrdU staining. Several synapsis-related genes were evaluated by real time PCR. For MCAO, behavior changes were evaluated by neurologic deficit scores, adhesive removal test, balance beam test and shuttle box test. TTC staining was used to demonstrate the cerebral infarction.
Result:For2VO, the2VO surgery increased the escape latency in Morris water maze test (P<0.001), and decreased the number of neurons in hippocampus region CA1(P<0.001) significantly. Open field test (P=0.577), Y maze test (P=0.547), BrdU positive cells in DG (P=0.052), microglia in region CA1(P=0.750) and the synapsis-related gene tested were not significantly affected by the surgery. Aceglutamine, safflower and Guhong injection seemed to protect the neurons in region CA1(P<0.001, respectively), although their protective effect was not showed by Morris water maze test (P=0.282). For MCAO, the neurologic deficit scores (P<0.001), the asymmetric index in adhesive removal test (P=0.010), the beam walking latency (P=0.003) and the relative infarction volume (P=0.037) were significantly increased by MCAO surgery. The result of shuttle box was not significantly affected by the surgery (latency:P=0.952, counts:P=0.905). The beam walking latency was significantly decreased by Guhong injection (P=0.010), and was not significantly affected by aceglutamine (P=0.139) or safflower (P=0.093). The neurologic deficit scores (P=0.568), the asymmetric index in adhesive removal test (P=0.688), and relative infarction volume (P=0.350) were unaffected by Guhong injection, aceglutamine or safflower.
Conclusion:Guhong injection, as well as aceglutamine and safflower injection, showed protective effect on neurons in region CA1for chronic cerebral ischemia. Guhong injection seemed to improve the behavior results for acute cerebral ischemia. Aceglutamine and safflower, on the other hand, didn't show protective effect for acute cerebral ischemia.
引文
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