地龙和水蛭复方制剂干预后大鼠脑缺血耐受和基质金属蛋白酶9的表达

Effect of compound preparation of earth-worm and leech on cerebral ischemic tolerance and expression of matrix metalloproteinase-9 in rats

目的:观察地龙和水蛭复方制剂对缺血预处理诱导的脑缺血耐受的干预作用及对基质金属蛋白酶9表达的影响。方法:于2004-09/2005-02在四川大学华西医院外科实验室完成动物实验部分;于2005-03/05在华西医院移植免疫中心完成图像采集和分析部分。①选用健康雄性SD大鼠48只。随机分为3组:假手术组、预缺血组、预缺血+地龙和水蛭复方制剂组,每组16只。采用二次线栓法制备大鼠脑缺血耐受模型。预缺血+地龙和水蛭复方制剂组:缺血预处理10min,腹腔注射地龙和水蛭复方制剂(主要成分为地龙、水蛭,含生药1g,由牡丹江友搏药业有限责任公司提供,2mL/支,批号:040413-1)10mL/(kg·d),连续3d,3d后给予2h大脑中动脉阻塞,再灌22h后处死大鼠;预缺血组:不给药,余处理同预缺血+地龙和水蛭复方制剂组;假手术组:仅暴露解剖结构,不予10min缺血预处理,余同预缺血组。②各组取8只大鼠于术后清醒2h进行神经功能评分(04分,0分为无神经系统功能缺失;4分为不能自发行走,意识丧失)。③每组随机取5只大鼠,取脑,计算脑含水量[(湿重-干重)/湿重×100%]。④每组随机取5只大鼠,取脑,制切成。计算脑梗死体积(mm3)(各层梗死面积之和×层间隔)。⑤采用免疫组化染色和图像分析比较各组基质金属蛋白酶9蛋白表达。⑥计量资料差异比较采用方差分析。结果:预缺血组和预缺血+地龙和水蛭复方制剂组术后各死亡1只,剔除后随机补充,最终有48只大鼠进入结果分析。①神经功能评分和脑含水量:预缺血组和预缺血+地龙和水蛭复方制剂组大鼠两项指标均明显低于假手术组[神经功能评分:(2.01±0.21),(1.76±0.36),(2.31±0.68)分;脑含水量:(82.01±0.83)%,(78.92±0.76)%,(85.23±1.08)%,P<0.05]。预缺血+地龙和水蛭复方制剂组明显低于预缺血组(P<0.05)。②脑梗死体积和基质金属蛋白酶9蛋白表达:预缺血组和预缺血+地龙和水蛭复方制剂组明显低于假手术组[脑梗死体积:(142.79±17.84),(103.00±12.46),(198.32±19.56)mm3;基质金属蛋白酶9蛋白表达:12473.18±398.47,9185.00±176.32,15687.21±259.49,P<0.05]。预缺血+地龙和水蛭复方制剂组明显低于预缺血组(P<0.05)。结论:缺血预处理可诱导脑缺血耐受作用,基质金属蛋白酶9表达降低是其机制之一;地龙和水蛭复方制剂可进一步下调基质金属蛋白酶9表达,与缺血预处理具有协同作用,减轻再缺血后脑损伤。

AIM： To investigate the effect of compound preparation of earth-worm and leech on cerebral ischemic tolerance in rat induced by focal ischemic preconditioning （1PC） and on expression of matrix metalloproteinase-9 （MMP-9） in rats. METHODS： The animal experiment was completed at the Surgery Laboratory of West China Hospital of Sichuan University from September 2004 to February 2005, and the imaging selection and analysis were completed in the Transplantation Immunity Center of West China Hospital from March to May 2005. ① Totally 48 healthy male SD rats were randomly divided into 3 groups： sham operation group, 1PC group and IPC ＋ compound preparation group with 16 in each group. The model of ischemic tolerance in rats was made by middle cerebral artery occlusion two times. 1PC ＋ compound preparation group： After 10-minute 1PC, rats were injected intravenously with compound preparation of earth-worm and leech （main component： earth-worm, leech and 1 g raw drug; provided by Mudanjiang Youbo Pharmaceutical Limited-liability Company, 2 mL/branch, batch number： 040413-1） 10 mL/（kg·d） for 3 days. Then the animals were subjected to middle cerebral artery occlusion （MCAO） for 2 hours and reperfused for 22 hours. 1PC group： Rats were not treated with drug and other process was as the same as those in 1PC ＋ compound preparation group. Sham operation group： Anatomic structure of rats was exposured, but rats were not treated with 10-minute 1PC, and other process was as the same as those in 1PC group.② Eight rats from each group was waken for 2 hours and tested with neurological score （0-4 points： 0 as without loss of neurological function; 4 as unable to walking independently）. ③ Five rats were selected from each group to obtain their brains to calculate the water content [（wet weight-dry weight）/wet weight×100%]. ④ Five rats were selected from each group to obtain their brains to make cerebral section to calculate infarct volume （mm^3） （total of infarct volume of each layer × interval between layers）. ⑤Expressions of matrix metalloproteinase-9 in each group were compared with immunohistochemical staining and image analysis. ⑥Measurement data were compared with analysis of variance. RESULTS： One rat died in 1PC group and 1PC ＋ compound preparation group respectively. After supplement, 48 rats entered the final analysis.① Neurological scores and water content： Indexes in 1PC group and IPC ＋ compound preparation group were obviously lower than those in sham operation group [neurological scores： （2.01：1：0.21）, （1.76±0.36）, （2.31±0.68） points; water content： （82.01±0.83）%, （78.92±0.76）%, （8513±1.08）%, P 〈 0.05], and those in IPC ＋ compound preparation group was also lower than those in IPC group （P 〈 0.05）. ②Volume of cerebral infarction and expression of MMP-9： Values in IPC group and IPC ＋ compound preparation group were obviously lower than those in sham operation group [volume of cerebral infarction： （142.79±17.84）, （103.00±12.46）, （198.32±19.56） mm3; expression of MMP-9：12 473.18±98.47, 9 185.00±176.32, 15 68711±959.49, P 〈 0.05], and those in IPC ＋ compound preparation group was also lower than those in IPC group （P 〈 0.05）. CONCLUSION： IPC can induce cerebral ischemic tolerance, and decrease of MMP-9 expression is possible one of mechanisms. Compound preparation of earth-worm and leech can down-regulate expression of MMP- 9 further and relieve cerebral injury after ischemia, which is of coincidence with IPC.