黄角颗粒对大脑中动脉栓塞老年大鼠脑神经细胞凋亡形态学的影响

背景:黄角颗粒为纯中药制剂,具有通腑泄热、开窍熄风、活血化淤等功效。目的:观察黄角颗粒对大鼠大脑中动脉栓塞模型神经细胞凋亡的影响,探讨黄角颗粒治疗急性期脑梗死的可能机制。设计:完全随机分组设计,对照动物实验。单位:华中科技大学同济医学院附属中西医结合医院神经内科。材料:实验于2003-09/2003-12在湖北中医学院中心实验室完成。采用鼠龄为22个月的老年Wistar大鼠60只。大鼠适应性喂养3d后,按体质量随机分为6组:正常对照组、模型组、尼莫地平组、黄角颗粒10g/kg组、黄角颗粒5g/kg组、黄角颗粒2.5g/kg组,每组10只。方法:①采用大脑中动脉栓塞法将大鼠造成脑缺血模型。黄角颗粒(主要成分为生大黄、水牛角等,武汉市第一医院制剂室提供,批号:021120)10,5,2.5g/kg组于造模前30min分别给予含生药1,0.5,0.25kg/L的黄角颗粒水溶液按10,5,2.5g/kg灌胃,每只灌胃1次;尼莫地平组于造模前30min给予含生药1g/L的尼莫地平水溶液按10mg/kg灌胃,每只灌胃1次;模型组和正常对照组(只结扎颈外动脉)于造模前30min给予生理盐水按10mL/kg灌胃,每只灌胃1次。②采用光学显微镜观察原位细胞凋亡染色切片,在高倍镜(20×10)下观察5个视野,观察每个高倍视野平均阳性细胞数,并计算细胞凋亡率。普通光镜下观察皮质缺血局部及周围组织学变化。电镜下观察大鼠大脑皮质细胞超微结构变化。③计量资料差异比较采用F检验及Q检验。主要观察指标:黄角颗粒对大脑中动脉栓塞老年大鼠脑组织神经细胞凋亡率及大脑皮质细胞形态学的影响。结果:Wistar大鼠60只均进入结果分析。①模型组大鼠脑组织凋亡阳性细胞数及细胞凋亡率明显高于正常对照组(P<0.01);黄角颗粒各剂量组及尼莫地平组大鼠脑组织凋亡阳性细胞数及细胞凋亡率明显低于模型组(P<0.01);黄角颗粒10g/kg组大鼠脑组织凋亡阳性细胞数及细胞凋亡率与尼莫地平组比较,差异不明显(P>0.05)。黄角颗粒10g/kg组大鼠脑组织凋亡阳性细胞数及细胞凋亡率明显低于黄角颗粒5和2.5g/kg组(P<0.05,0.01)。②光镜及电镜观察结果显示模型组大鼠局部脑组织细胞有明显的凋亡现象,而黄角颗粒有明显的改善作用。结论:黄角颗粒对急性缺血性神经细胞凋亡有一定的拮抗作用,该种作用可能是黄角颗粒治疗急性期脑梗死的可能机制之一。

Morphologic effect of huangjiao granule on apoptosis of cerebral neurocytes in elderly rats with middle cerebral arterial occlusion

BACKGROUND: Huangiao granule is pure Chinese drugs pharmaceu-tics, which can eliminate heat by purgatingfu-organs, clear unconscious-ness and stop endogenous wind as well as promote blood flow and removeblood stasis etc.OBJECTIVE: To observe the effect of huangjiao granule on apoptosis ofneurocytes in rat models with middle cerebral arterial occlusion, and probeinto the possible mechanism of huangjiao granule in the treatment of acutecerebral infarction.DESIGN: Complete randomized grouping design, control animal experi-ment.SETTING: Department of Neurology, Affiliated Hospital of TraditionalChinese Medicine Combined with Western Medicine, Tongji Medical Col-lege of Huazhong University of Science and Technology.MATERIALS: The experiment was conducted in the Central Laboratory,Hubei College of Traditional Chinese Medicine. Sixty old Wistar rats aged22 months were selected. After 3-day-feeding for adaptability, rats wererandomized into 6 groups according to body mass: Normal control group,model group, nimodipine gorup, 10 g/kg huangjia ogranule group and 5 g/kghuangjiao granule group with 10 rats in each group.METHODS: ①Rats were made into cerebral ischemic models with mid-dle cerebral arterial occlusion method. Thirty minutes before modeling, ratsin the 10, 5, 2.5 g/kg huangjiao granule (main gredients were radix et rhi-zoma rhei, cornu bubali etc. provided by Manufacturing Laboratory ofWuhan First Hospital with the batch number of 021120) groups respec-tively received gastric perfusion of 1, 0.5, 0.25 g/mL huangjiao water so-lution containing crude drug at the dose of 10, 5, 2.5 g/kg with each rats perfused once; Rats in the nimodipine group received gastric perfusion of1 mg/mL nimodipine water solution, containing crude drug at the dose of 10 mg/kg with each rats perfused once; Rats in the model group and normal control group received gastric perfusion of normal saline at the dose of 10 mL/kg with each rats perfused once. ② Apoptotic stainning sections in situ were observed under light microscope, 5 fields were observed under high power (20×10). Observed the number of mean positive cells of each highpower field, and then caculated the percentage of apoptosis. Histologic changes of local and peripheral ischemic cortexes were observed under light microscope. Changes of ultramicrostructure of cells in cerebral cortexes of rats were observed under electron microscope. ③Differences of measurement data were compared with F test and Q test.MAIN OUTCOME MEASURES: Morphologic effects of huangjiao granule on apoptotic rate of neurocytes in brain tissues of old rats with middle cerebral arterial occlusion (MACO) and cells in cerebral cortexes.RESULTS: A total of 60 Wistar rats were involved in the analysis of results. ①Number of apoptotic positive cells in brain tissues and apoptotic rate in rats of model group were significantly higher than those in the normal control group (P ＜ 0.01); Those in each huangjiao granule groups and nimodipine group were obviously lower than those in the model group (P ＜ 0.01); Differences in numbers of apoptotic positive cells in brain tissues and apoptotic rates in rats of 10 g/kg huangiao group and nimodipine groupwere not obvious (P ＜ 0.05). Number of apoptotic positive cells in brain tissues and apoptotic rate in rats of 10 g/kg huangjiao granule group were markedly lower than those in the 5, 2.5 g/kg huangjiao granule groups (P ＜ 0.05, 0.01).②Observation with light microscope and electron microscope showed that there were obvious apoptosis in local cells of brain tissues in rats of model group, which can significantly ameliorate by haungjiao granule.CONCLUSION: Huangjiao granule has some antagonistic effects on acute ischemic apoptosis of neurocytes, which may be one of the possible mechanisms of huangjiao granule in the treatment of acute cerebral infarction.