健脑安对大鼠海马神经元糖皮质激素受体的影响

背景:先前实验中已经证实补肾活血化痰中药健脑安可抑制脑缺血再灌注后2～24h内糖皮质激素含量增高,降低高糖皮质激素的促神经细胞凋亡毒性作用。这一作用是否在受体环节也有影响尚不清楚。目的:观察中药健脑安对糖皮质激素受体的干预作用,进一步明确健脑安保护脑缺血再灌注后海马神经细胞的作用靶向;并与复方阿米三嗪进行阳性对照。设计:随机对照动物实验。单位:北京中医药大学中心实验室。材料:实验于2002-07/2003-03在北京中医药大学中心实验室进行。取SD雄性大鼠80只,随机分为假手术组、模型组、治疗组、阳性对照组和拮抗剂组5组,每组16只,每组又分为再灌注2,6,12和24h4个时点,每个时点4只。方法:①给药:除模型组外,其他4组大鼠于造模前7d开始灌胃给药,1次/d。假手术组给予7μL/(g·d)蒸馏水;阳性对照组予复方阿米三嗪7.39mg/(kg·d);治疗组予健脑安(肉苁蓉、石菖蒲、大黄等组成)生药6.7g/(kg·d);拮抗剂组给予糖皮质激素受体拮抗剂米非司酮10g/(kg·d),7μL/g。②给药7d后用大脑中动脉阻塞线栓法制备大鼠脑缺血再灌注模型,假手术组麻醉后,剥离颈总动脉后缝合,不予进栓线。主要观察指标:各组在再灌注相应时间点处死大鼠取脑,利用免疫组化方法观察大鼠糖皮质激素受体蛋白表达的变化,计算CA2区3个200倍视野内阳性细胞数。结果:80只大鼠全部进入结果分析。假手术组糖皮质激素受体表达损伤侧与健侧双侧对称,糖皮质激素受体表达无明显差别。模型组、治疗组、阳性对照组、拮抗剂组损伤侧较健侧糖皮质激素受体表达明显减少,亦明显少于假手术组(P<0.05)。治疗组、阳性对照组、拮抗剂组在再灌注各时间点的糖皮质激素受体表达量无明显变化,各组与模型组相比差异不显著(P>0.05)。结论:健脑安对糖皮质激素受体和糖皮质激素含量的调节有选择性,不能调节糖皮质激素受体的表达,与复方阿米三嗪相似;但健脑安可通过降低血浆和脑组织中糖皮质激素含量,发挥对脑缺血再灌注后的神经细胞的保护作用。

Effects of Jiannaoan on glucocorticoid receptor in hippocampus neurons of rats

BACKGROUND: The previous experiments have conformed the traditional Chinese medicine (TCM) Jiannaaan, with the effects of tonifying kidney, promoting blood circulation and resolving phlegm, can inhibit the increased content of glucocorticoid (GC) in 2-24 hours after cerebral ischemic reperfusion (CIR), and reduce toxic effects of promoting nervous cell apoptosis induced by high GC. However, it is unclear whether this effect exists in GC receptor (GR).OBJECTIVE: To observe the intervention of TCM Jiannaoan on GR,further study protective mechanism of Jiannaoan power to hippocampal neurons after CIR, and perform positive control with compound almitrine.DESIGN: A randomized and controlled trial taken animals as subjects.SETTING: Center Laboratory of Beijing University of Chinese Medicine.MATERIALS: The experiment was conducted at the Center Laboratory of Beijing University of Traditional Chinese Medicine between July 2002 and March 2003. Eighty male SD rats were randomized into 5 groups with 16 in each: Sham group, model group, treatment group, positive control group and antagonist group. And each group was divided into 4 subgroups: 2, 6,12 and 24 hours after CIR, with 4 rats at every time point.METHODS:①Administration: Except model group, rats in other 4 groups were administrated by intragastric infusion since 7 days before model establishment, once per day, with dose of 7 μL/g per day distilled water in sham group, 7.39 mg/kg per day compound almitrine in positive control group, 6.7 g/kg per day Jiannaoan crude drug (consisted of desertliving cistanche herb, tatarinowii sweetflag rhizome and rhubarb, etc) in treatment group and 10 g/kg per day GR antagonist mifepristone in antagonist group.② After 7-day administration, the CIR models were prepared on the experimental rats with middle cerebral artery occlusion (MCAO) filament method, while the rats in sham group were sutured after common carotid artery detachment at anesthesia, without filament.MAIN OUTCOME MEASURES: All the rats were executed to take out brains at different time points of reperfusion, and the change of GR protein expression was observed with immunohistochemical method then the amount of positive cells were calculated in 3×200 sight of CA2 region.RESULTS: Totally 80 rats were entered into the result analysis. Compared with uninjured side, the protein expression of GR in model group,treatment group, positive control group and antagonist group were significantly lower than that of sham group (P ＜ 0.05), in which GR expression of injured side was equal to that of uninjured side without significant difference. No obvious change was found in the protein expression of GR among treatment group, positive group and antagonist group at different time points of reperfusion, and no significant difference was found between above groups and model group (P ＞ 0.05).CONCLUSION: Jiannaoan power is selective for adjusting GR and content of GC: Jiannaoan can not adjust expression of GR, identical as compound almitrine; But Jiannaoan can protect the neurons through decreasing the content of GC in plasm and brain tissues after CIR.