神经源性丝氨酸蛋白酶抑制剂通过减弱血脑屏障损伤和氧化应激改善组织型纤溶酶原激活物引起的脑缺血再灌注损伤研究  

Research on neurogenic serine protease inhibitor improving cerebral ischemia-reperfusion injury induced by tissue plasminogen activator by reducing blood-brain barrier injury and oxidative stress

作　　者：杨雪莲[1] 江梅[1] 来小音[1] 蔡丽瑛[1] 计海峰[1] 李龙宣[1] YANG Xuelian;JIANG Mei;LAI Xiaoyin;CAI Liying;JI Haifeng;LI Longxuan(Department of Neurology,Shanghai Pudong New Area Gongli Hospotal,Shanghai 200135,China)

机构地区：[1]上海市浦东新区公利医院神经内科,上海200135

出　　处：《世界临床药物》2021年第9期757-765,共9页World Clinical Drug

基　　金：浦东新区卫生和计划生育委员会学科带头人培养计划(PWRd2017-10);上海市卫生和计划生育委员会重点专科(ZK2019A08);上海市新百人计划(2017BR051)。

摘　　要：目的探讨神经源性丝氨酸蛋白酶抑制剂(neuroserpin,NSP)对急性缺血性脑卒中时使用重组人组织型纤溶酶原激活物(tissue plasminogen activator,tPA)治疗的影响和机制。方法构造大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)模型。将大鼠随机分为假手术组、MCAO组、1 h tPA组(再灌注1 h MCAO+tPA)、1h tPA+NSP组(再灌注1 h MCAO+tPA+NSP)、4 h tPA组(再灌注4 h MCAO+tPA)及4 h tPA+NSP组(再灌注4 h MCAO+tPA+NSP)。采用改良神经严重性评分(modified neurologic severity score,mNSS)、Rota-Rod电机测试评估大鼠神经功能改变,采用干湿法和TTC染色评估大鼠脑水肿和脑梗死面积,采用伊文思蓝染色法、苏木精-伊红(Hematoxylin Eosin,HE)染色法检测大鼠血脑屏障完整性,采用酶联免疫吸附试验法检测脑组织丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的表达,采用蛋白质印迹法和甲苯胺蓝染色检测脑组织中半胱门冬氨酸蛋白酶(cysteinyl aspartate specific proteinase,Caspase)-3、B细胞淋巴瘤因子-2(B-cell lymphoma factor 2,Bcl-2)的表达以及脑组织细胞凋亡情况。结果mNSS结果显示,1h tPA+NSP组及4 h tPA+NSP组(联合干预组)分别低于1 h tPA组和4 h tPA组(P<0.05)。Rota-Rod电机测试结果显示,各联合干预组分别长于1 h tPA组和4 htPA组(P<0.05)。各联合干预组脑水肿体积和脑梗死面积均优于相同再灌注时间单纯tPA干预组。各联合干预组MDA含量低于相同再灌注时间单纯tPA干预组(P<0.05),各联合干预组SOD及GSH-Px活性高于相同再灌注时间单纯tPA干预组(P<0.05)。各联合干预组Caspase-3表达低于相同再灌注时间单纯tPA干预组(P<0.05)。各联合干预组Bcl-2表达高于相同再灌注时间单纯tPA干预组(P<0.05)。结论NSP可以改善tPA干预后引起的神经功能缺损,其机制可能是NSP减弱了脑缺血时血脑屏障的损伤和氧化应激�Objective To investigate the effect and mechanism of neuroserpin(NSP)on the treatment of acute ischemic stroke with recombinant human tissue plasminogen activator(tPA).Methods The rats were randomly divided into sham operation group,middle cerebral artery occlusion(MCAO)group,1 h tPA group(reperfusion 1 h MCAO+tPA),1 htPA+NSP group(reperfusion 1 h MCAO+tPA+NSP),4 h tPA group(reperfusion 4 h MCAO+tPA),4 h tPA+NSP group(reperfusion 4 h MCAO+tPA+NSP).Modified neurologic severity score(mNSS)and Rota-Rod motor test were used to evaluate the changes of neurological function.Dry wet method and TTC staining were used to evaluate the brain edema and cerebral infarction.Evans blue staining and Hematoxylin Eosin(HE)staining were used to detect the integrity of blood-brain barrier.Enzyme linked immunosorbent assay was used to detect the expression of malondialdehyde(MDA),Superoxide dismutase(SOD)and Glutathione peroxidase(GSH-Px)in brain tissue.Western blot and toluidine blue staining were used to detect cysteinyl aspartate specific proteinase(Caspase)-3,B-cell lymphoma factor 2(Bcl-2)expression and brain cell apoptosis.Results The results of mNSS showed that 1 h tPA+NSP group and 4 h tPA+NSP group(i.e.combined intervention group)were lower than 1 h tPA group and 4 h tPA group respectively(P<0.05).Rota-Rod motor test showed that combined intervention group were longer than 1 h tPA group and 4 h tPA group respectively(P<0.05).The volume of brain edema and the area of cerebral infarction in combined intervention group were better than those in the simple tPA intervention group with the same reperfusion time.The content of MDA in combined intervention group was lower than that in the tPA intervention group at the same reperfusion time(P<0.05),and the activities of SOD and GSH-Px in combined intervention group were higher than those in the tPA intervention group at the same reperfusion time(P<0.05).The expression of Caspase-3 in combined intervention group was lower than that in the tPA intervention group at the same reperfusion

关 键 词：神经源性丝氨酸蛋白酶抑制剂 组织型纤溶酶原激活剂 脑缺血再灌注损伤 

分 类 号：R743.3[医药卫生—神经病学与精神病学] R-33[医药卫生—临床医学]