脑出血大鼠脑组织膜型基质金属蛋白酶、基质金属蛋白酶2和基质金属蛋白酶9表达及益气活血中药的干预效应

Effects of yiqi huoxue herb on the expressions of membrane type 1-matrix metalloproteinase, matrix metalloproteinase-2 and matrix metalloproteinase-9 in intracerebral hemorrhagic rat brains

目的:观察益气活血中药对脑出血大鼠脑组织中膜型基质金属蛋白酶、基质金属蛋白酶2和基质金属蛋白酶9表达量的影响,从脑出血损伤区微血管系统重建的角度,探讨益气活血中药治疗脑出血的作用机制。方法:实验于2006-03/10在中南大学湘雅医院中西医结合研究所实验室完成。实验材料:补阳还五汤全方(黄芪,当归,赤芍,红花,川芎,地龙,桃仁按20∶3∶3∶3∶2∶3∶3的比例);补阳还五汤益气成分(黄芪按上述比例);补阳还五汤活血成分(当归,赤芍,地龙,川芎,桃仁,红花按3∶3∶3∶2∶3∶3的比例)。用蒸馏水两次水煎,分别浓缩为1.54,0.81和0.73g/mL。实验分组:155只SD大鼠随机分为正常对照组、假手术组、模型组、益气活血组、益气组、活血组。正常对照组5只大鼠,其余每组30只,再随机分为术后灌胃2,4,7,14,21,28d6个观察时间点,各个时间点5只大鼠。实验干预:造模:采用立体定位技术将胶原酶Ⅶ注入大鼠大脑右苍白球制成脑出血大鼠模型。假手术组大鼠仅注入2μL生理盐水,其余手术过程相同。给药:正常对照组:普通饲养,自由饮水;假手术组和模型组术后予蒸馏水灌胃2次/d,2mL/次;益气活血组、益气组、活血组分别给予补阳还五汤全方、补阳还五汤益气成分、补阳还五汤活血成分30.80,16.20,14.60g/(kg·d)(按体表面积计算为临床70kg成人剂量的3倍)灌胃,2次/d,2mL/次。各组大鼠分别于灌胃2,4,7,14,21,28d麻醉下取脑,制备切片;正常组动物于28d处死。实验评估:免疫组织化学染色方法检测各组灌胃不同时间脑组织基质金属蛋白酶2、基质金属蛋白酶9和膜型基质金属蛋白酶的阳性微血管数。结果:155只大鼠均进入结果分析。①正常组、假手术组皮质偶见膜型基质金属蛋白酶、基质金属蛋白酶2和基质金属蛋白酶9表达。②模型组膜型基质金属蛋白酶、基质金属蛋白酶2呈双峰表达,4d为最高峰,至14～21d再有小高峰出现。③益气活血组给药4d时,膜型基质金属蛋白酶、基质金属蛋白酶2为表达低谷,低于模型组(P<0.01)、益气组和活血组(P<0.05);在中后期,益气活血组膜型基质金属蛋白酶表达高峰为7～14d,较模型组提前出现,21d后与模型组比,各治疗组膜型基质金属蛋白酶已表达极少(P<0.01);益气活血组基质金属蛋白酶2在7d后一直呈高水平维持,高于其他各组(P<0.05),28d后开始逐渐下降。④模型组基质金属蛋白酶9在造模后4d达最高峰(P<0.01),两周后几乎无表达。益气活血组基质金属蛋白酶9高峰推迟至7d出现(P<0.05),之后逐渐下降。结论:益气活血中药可通过调节脑出血后膜型基质金属蛋白酶、基质金属蛋白酶2和基质金属蛋白酶9表达,改善出血后脑组织的微环境,有利于微血管系统重建,促进组织修复。

AIM： To investigate the action mechanism of yiqi huoxue （YQHX） therapy by observing the expressions of membrane type 1-matrix metalloproteinase （MT1-MMP）, MMP-2 And MMP-9 in intracerebral hemorrhagic （ICH） rat brains from the microvessel network reconstruction aspect. METHODS： The experiment was done in the laboratory of Institute of Integrated Traditional Chinese and Western Medicine, Xiangya Hospital, Central South University from March to October in 2006. Experiment materials： Buyang Huanwu Decoction （milkvetch root, Chinese Angelica, red peony root, Carthamus tinctorius, Szechwan Lovage Rhizome, Pheretima asiatica, peach seed at the ratio of 20：3：3：3：2：3：3）, Yi-qi composition of Buyang Huanwu Decoction （milkvetch root at the same ratio）, Huo-xue composition of Buyang Huanwu Decoction （Chinese Angelica, red peony root, Carthamus tinctorius, Szechwan Lovage Rhizome, Pheretima asiatica, peach seed at the ratio of 3：3：3：2：3：3）. All the herbs were decocted twice with distilled water into 1.54,0.81 and 0.73 g/mE Experiment groups： 155 SD rats were randomly divided into six groups： normal control group （n =5）, sham group （n =30）, ICH group （n =30）, YQHX-treated group （n =30）, YQ-treated group （n =30）, HX-treated group （n =30）. Animals were observed at 2, 4, 7, 14, 21, 28 days postoperation, 5 rats at each time point. Experiment intervention： ICH model was induced by injecting collagenase type stereotaxically into right globus pallidus, while sham group was injected with 2 μL saline. Normal group was routinely fed. The sham group and ICH group were intragastricaUy administrated with distilled water, 2 μL once, twice daily. The animals of YQHX-treated group, YQ-treated group, and HX-treated group were respectively intragastrically administrated with Buyang Huanwu Decoction, the Yi-qi composition of Buyang Huanwu Decoction and the Huo-xue composition of Buyang Huanwu Decoction at the dosage of three times of the 70 kg adult according to body surface area calculation （30.80,16.20,14.60 g/kg daily, 2 mL once, twice daily）. Then, the animals of every group were anesthetized through intragastric injection at 2, 4, 7, 14, 21, 28 days postoperation and the brain tissue of the animals were used to prepare frozen sections. Normal group was anesthetized to death at 28 days postoperation. Experiment evaluation： The expressions of MT1-MMP, MMP-2 and MMP-9 were evaluated by immunohistochemistry. RESULTS： All the 155 rats were involved in the result analysis. （1）Expressions of MT1-MMP, MMP-2 and MMP-9 were observed in the cortex of normal group and sham group occasionally.（2）In ICH group, the expressions of MT1-MMP and MMP-2 peaked at 4 days, then gradually decreased but peaked at 14-21 days again. （3）In YQHX-treated group, the expressions of MT1-MMP and MMP-2 were lower than those in ICH group （P 〈 0.01） and other therapy-treated groups at 4 days （P 〈 0.05）. Later the expression of MT1-MMP in YQHX-treated group peaked at 7-14 days, which was earlier than that in ICH group. In all therapy-treated groups, the expression of MT1-MMP was obviously lower compared with ICH group after 21 days （P 〈 0.01 ）. In YQHX-treated group, the expression of MMP-2 was higher than those in the other groups after 7 days （P 〈 0.05）, then began to depress at 28 days.（4）In ICH group, the expression of MMP-9 peaked at 4 days （P 〈 0.01） and decreased gradually, then could not be observed after two weeks, but in YQHX-treated group, the expression of MMP-9 peaked at 7 days （P 〈 0.05）, which delayed compared with in ICH group, then decreased gradually. CONCLUSION： YQHX therapy can regulate the expressions of MT1-MMP, MMP-2 and MMP-9 in ICH rat brains, which may improve the microenvironment of the damaged brain tissue, promote the reconstruction of microvessel network, and facilitate the tissue repair after ICH.