格列苯脲调控HSP70/p-Akt/MMP-9/COX-2炎性信号通路保护脑缺血再灌注损伤大鼠的神经血管单元  

Glibenclamide protects neurovascular units in rats with cerebral ischemia/reperfusion injury by regulating inflammatory signaling pathway HSP70/p-Akt/MMP-9/COX-2

作　　者：祝淑贞[1] 潘速跃[2] Zhu Shuzhen;Pan Suyue(Department of Neurology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China;Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China)

机构地区：[1]南方医科大学珠江医院神经内科,广州510282 [2]南方医科大学南方医院神经内科,广州510515

出　　处：《中华神经医学杂志》2019年第8期767-778,共12页Chinese Journal of Neuromedicine

基　　金：广东省自然科学基金(2017A030313672).

摘　　要：目的探讨磺酰脲类受体1(SUR1)-转化受体电位阳离子通道亚家族M成员4(TRPM4)特异性抑制剂格列苯脲对脑缺血再灌注损伤大鼠神经血管单元的保护作用及其机制。方法采用随机数字表法将120只雄性SD大鼠分为假手术组、模型组及格列苯脲组,每组40只。后2组采用线栓法制备成急性局灶性大脑中动脉闭塞缺血2 h再灌注模型,其中模型组于缺血2 h后给予单次腹腔注射含0.05%二甲基亚砜的生理盐水,格列苯脲组给予单次腹腔注射10 μg/kg格列苯脲(5 μg/mL)。再灌注后8 h时,采用免疫荧光双标染色及Western blotting检测各组大鼠脑组织中SUR1、TRPM4的表达,采用酶联免疫吸附法(ELISA)检测血浆中基质金属蛋白酶-9(MMP-9)含量。再灌注后24 h时,采用Zea Longa评分法评估各组大鼠的神经功能缺损程度,采用干湿重法检测脑组织含水量,采用伊文思蓝(EB)染色检测血脑屏障通透性,采用尼氏染色检测脑组织中存活神经元数量,采用免疫组化染色检测IgG渗出量及离子钙接头蛋白-1(Iba-1)、环氧化酶-2(COX-2)阳性细胞的表达,采用Western blotting检测热休克蛋白70(HSP70)、磷酸化蛋白激酶B(p-Akt)、磷酸化c-Jun氨基末端激酶(p-JNK)、磷脂酰肌醇-3激酶(PI3K)蛋白的表达。结果(1)再灌注后8 h时,与假手术组比较,模型组大鼠脑组织中SUR1、TRPM4蛋白表达均明显升高,差异均有统计学意义(P<0.05),且该两种蛋白存在共定位;与模型组比较,格列苯脲组的SUR1、TRPM4蛋白表达降低,但差异无统计学意义(P>0.05)。与假手术组比较,模型组大鼠的MMP-9含量明显升高,差异有统计学意义(P<0.05);与模型组比较,格列苯脲组的MMP-9含量明显降低,差异有统计学意义(P<0.05)。(2)再灌注后24 h时,与假手术组比较,模型组大鼠的神经功能缺损评分明显升高,脑组织含水量明显升高,EB渗出量明显升高,IgG渗出量明显升高,尼氏染色阳性神经元数量明显降低,Iba-ObjectiveTo investigate the protective effect of glibenclamide on neurovascular units (NVUs) and its possible mechanism in cerebral ischemia/reperfusion injury models. MethodsOne hundred and twenty healthy male SD rats were randomly divided into sham-operated group, model group, and glibenclamide (GBC) treatment group (n=40). Two h reperfusion models of acute focal middle cerebral artery occlusion were prepared by thread occlusion in rats of the latter two groups;rats in the model group were treated with 0.05% DMSO saline solution two h after ischemia, and rats in the GBC treatment group were given intraperitoneal injection of 10 μg/kg GBC with single dose. Immunofluorescence and Western blotting were used to detect the protein levels of sulfonylurea receptor 1 (SUR1) and transient receptor potential cation channel subfamily M member 4 (TRPM4) 8 h after reperfusion, and ELISA was used to detect the plasma level of matrix metalloproteinase 9 (MMP-9). At 24 h after reperfusion, Zea Longa scale was used to determine the neurological deficits;water content in the brain tissues was detected by dry and wet weight method, and blood-brain barrier (BBB) permeability was detected by Evans blue (EB) staining;Nissl staining was employed to detect the survival neurons;ionized calcium bindingadaptor molecule-1 (Iba-1) and cyclooxygenase-2 (COX-2) positive cells and IgG seepage quantity were detected by immumohistochemical staining to assess the neuro-vascular inflammation;the expressions of heat shock protein 70 (HSP70), phosphorylated protein kinase B (p-Akt), phosphorylated c-jun amino-terminal kinase (p-JNK), and phosphatidylinositol-3 kinase (PI3K) were detected by Western blotting. Results(1) At 8 h after reperfusion, the protein expressions of SUR1 and TRPM4 in the brain tissues of the model group were significantly increased as compared with those of the sham-operated group (P<0.05), and the two proteins were co-located;as compared with those in the model group, the protein expressions of SUR1 and TRPM4 in GBC treatmen

关 键 词：磺酰服类受体1 转化受体电位阳离子通道亚家族M成员4 格列苯腺 脑缺血再灌注损伤 热休克蛋白70 蛋白激酶B 基质金属蛋白酶-9 环氧化酶-2 神经血管单元 

分 类 号：R743.34[医药卫生—神经病学与精神病学]