水蛭酶解前后对缺血性脑卒中大鼠治疗作用比较

Comparative Study of the Therapeutic Effects of Hirudo Before and After Enzymolysis on Ischemic Stroke Rats

目的:利用酶解法制备水蛭肽,与水蛭原粉平行作用于脑卒中模型大鼠,比较酶解前后的药效。方法:对大鼠实施脑中动脉电凝术,80只大鼠腹腔注射水合氯醛0.35 g·kg^(-1)(10%,3.5 m L·kg^(-1))麻醉,20只大鼠作为假手术组,仅切开皮肤,然后缝合饲养。于脑缺血后24 h进行行为评分。取造模后≥1分的大鼠,随机分为3组:模型组(无菌蒸馏水),水蛭组(水蛭0.8 g·kg^(-1)),水蛭肽组(水蛭肽0.64 g·kg^(-1))。每组20只大鼠。灌胃给药,每天1次,连续7 d,给药容积均为1.0 ml/100 g体质量。末次给药后24 h进行各项指标的检测。各组大鼠腹腔注射水合氯醛0.35 g·kg^(-1)(10%,3.5 ml·kg^(-1)),股动脉取血2 m L,3 500转·min-1,离心10 min,取血清,测定SOD和MDA。取大脑(去掉嗅球、小脑和低位脑干),-20℃速冻10 min后切成厚约2 mm的冠状脑片(5~6片),立刻置于1%TTC溶液中,37℃孵育40 min。缺血区呈现白色,非缺血区呈现红色。根据染色分离缺血区和非缺血区,分别称重,并计算缺血区重量占整个脑组织的百分比(%)。结果:给药7天后,与假手术组比较,模型对照组动物出现明显的向外侧转圈、行走时向对侧倾倒等神经功能损伤症状,行为评分显著高于假手术组(P<0.01)。与模型对照组比较,水蛭肽组和水蛭组神经行为评分明显降低(P<0.01,P<0.05)。结论:水蛭酶解后所得的水蛭肽,对缺血性脑卒中大鼠的治疗作用优于水蛭。

Objective： To prepare hirudin by using enzyme hydrolysis method which will act on rats with stroke in parallel, and compare the efficacies before and after enzymatic hydrolysis. Methods ： All the rats were implanted with electrocoagulation of middle cerebral artery; 80 rats were anesthetized with chloral hydrate 0.35 g ·kg^-1（ 10%, 3.5 mL·kg^-1） ; 20 rats, chosen into the sham operated group, were only cut in the skin and then sutured and fed. The rats＇behaviors were scored after cerebral ischemia for 24 hours. The ones who ≥ 1 score after modeling were chosen and randomly divided into 3 groups ： the model group （ sterile distilled water）, the leech group 0.8 g ·kg^-1, and the hirudin group 0.64 g·kg^-1, with 20 rats in each group. They received intragastric administration, once a day, for 7 days continuously, and the volume of administration was 1.0 mL/100 g body mass. And 24 hours after the last administration, various indexes were checked. The rats in each group were injected with chloral hydrate 0.35 g·kg^-1（ 10% . 3.5 mL ·kg^-1） ; femoral artery blood 2ml was taken, 3 500 circles each minute. 10 minutes after being centrifuged, serum was taken, and SOD and MDA were measured. The brain was taken （ removing the olfactory bulb, cerebellum and lower brainstem）, frozen for 10 minutes with -20 degrees, and then cut into the coronal slices with a thickness of about 2mm（ 5 or 6 slices）, immediately placed in 1% TFC solution, incubated for 40 minutes with 37 ℃. The ischemic areas were white and the non ischemic areas were red. The ischemic areas and non ischemic areas were separated according to coloration. and weighed respectively, and the percentage of brain weight in the ischemic zone （ % ） was calculated. Results ： After 7 days＇ treatment, compared with the sham operation group, the rats in the model control were circling to the right, toppling to the opposite side when walking and other symptoms of neurological damage, and their behavior scores were significantly higher than those of the sham operation group （ P 〈 0. 01 ）. Compared with the model control group, the neurobehavioral score of the leech peptide group and the leech group were significantly reduced （ P 〈 0.01, P 〈 0.05 ）. Conclusion ： The hirudin peptide obtained from hirudo by the enzymatic hydrolysis is superior to leech in the treatment of ischemic stroke rats.