Integrity of the blood-brain barrier structure is essential for maintaining the internal environment of the brain.Development of cerebral infarction and brain edema is strongly associated with blood-brain barrier leak...Integrity of the blood-brain barrier structure is essential for maintaining the internal environment of the brain.Development of cerebral infarction and brain edema is strongly associated with blood-brain barrier leakage.Therefore,studies have suggested that protecting the blood-brain barrier may be an effective method for treating acute stroke.To examine this possibility,stroke model rats were established by middle cerebral artery occlusion and reperfusion.Remote ischemic postconditioning was immediately induced by three cycles of 10-minute ischemia/10-minute reperfusion of bilateral hind limbs at the beginning of middle cerebral artery occlusion reperfusion.Neurological function of rat models was evaluated using Zea Longa’s method.Permeability of the blood-brain barrier was assessed by Evans blue leakage.Infarct volume and brain edema were evaluated using 2,3,5-triphenyltetrazolium chloride staining.Expression of matrix metalloproteinase-9 and claudin-5 m RNA was determined by real-time quantitative reverse transcription-polymerase chain reaction.Expression of matrix metalloproteinase-9 and claudin-5 protein was measured by western blot assay.The number of matrix metalloproteinase-9-and claudin-5-positive cells was analyzed using immunohistochemistry.Our results showed that remote ischemic postconditioning alleviated disruption of the blood-brain barrier,reduced infarct volume and edema,decreased expression of matrix metalloproteinase-9 m RNA and protein and the number of positive cells,increased expression of claudin-5 m RNA and protein and the number of positive cells,and remarkably improved neurological function.These findings confirm that by suppressing expression of matrix metalloproteinase-9 and claudin-5 induced by acute ischemia/reperfusion,remote ischemic postconditioning reduces blood-brain barrier injury,mitigates ischemic injury,and exerts protective effects on the brain.展开更多
Background:The increased permeability of the blood-brain barrier (BBB) induced by ischemia/hypoxia is generally correlated with alteration of tight junctions (TJs). DL-3-n-butylphthalide (NBP) has been shown to exert ...Background:The increased permeability of the blood-brain barrier (BBB) induced by ischemia/hypoxia is generally correlated with alteration of tight junctions (TJs). DL-3-n-butylphthalide (NBP) has been shown to exert neuroprotective effects after ischemic injury. However, few studies have assessed the correlation between NBP and TJs. This study aimed to investigate the potential effect of NBP on the TJ proteins claudin-5, zonula occludens-1 (ZO-1), and occludin during brain ischemia. Methods: A chronic cerebral hypoperfusion (CCH) Sprague-Dawley rat model was established, and NBP (20, 40, or 80 mg/kg, gavage, once a day) treatment was performed for 14 days. NBP (0.1 or 1.0μmol/L) pre-treatment was applied to an in vitro hypoxia microvascular endothelial cell model (1%〇2, 24 h). BBB permeability was assessed by performing the Evans blue assay. The expressions and localization of claudin-5, ZO-1, occludin, phosphorylated/total protein kinase B (p-Akt/Akt), phosphorylated/total glycogen synthase kinase 3p (GSK-3(3)/GSK-3p, and (3-catenin/p-actin were evaluated by Western blotting or immunofluorescence. Reactive oxygen species (ROS) generation was measured by flow cytometry analysis. TJ ultrastructure was observed by transmission electron microscopy. Results: In CCH rats, treatment with 40 and 80 mg/kg NBP decreased the Evans blue content in brain tissue (9.0 ± 0.9 (μg/g vs. 12.3 ± 1.9 (μg/g, P = 0.005;6.7 ± 0.6 μg/g vs. 12.3 ± 1.9μg/g, P < 0.01), increased the expression of claudin-5 (0.79 ± 0.08 mvs. 0.41 ± 0.06, P < 0.01;0.07 ± 0 .0 7 vs. 0.41 ± 0 .0 6 , P < 0 .61 ), and elevated the ZO-1 protein level (P < 0.05) in brain microvascular segments in a dose-dependent manner in comparison with the corresponding values in the model group. There was no significant difference in occludin expression (P > 0.05). In the hypoxia cell model, NBP pre-treatment improved TJ ultrastructure, decreased intracellular ROS level, and increased the expression of claudin-5 (P < 0.01) and ZO-1 (P < 0.01) in comparison with展开更多
Objective To study the effect of electromagnetic pulse （EMP） exposure on permeability of in vitro blood-brain-barrier （BBB） model. Methods An in vitro BBB model, established by co-culturing brain microvascular end...Objective To study the effect of electromagnetic pulse （EMP） exposure on permeability of in vitro blood-brain-barrier （BBB） model. Methods An in vitro BBB model, established by co-culturing brain microvascular endothelial cells （BMVEC） and astroglial cells （AC） isolated from rat brain, was exposed to EMP at 100 kV/m and 400 kV/m, respectively. Permeability of the model was assayed by measuring the transendothelial electrical resistance （TEER） and the horseradish peroxidase （HRP） transmission at different time points. Levels of BBB tight junction-related proteins were measured at O, 1, 2, 4, 8, 12, 16, 20, 24 h after EMP exposure by Western blotting. Results The TEER level was lower in BBB model group than in control group at 12 h after EMP, exposure which returned to its normal level at 24 h. The 24 h recovery process was triphasic and biphasic respectively after EMP exposure at 100 kV/m and 400 kV/m. Following exposure to 400 kV/m EMP, the HRP permeability increased at 1-12 h and returned to its normal level at 24 h. Western blotting showed that the claudin-5 and ZO-1 protein levels were changed after EMP exposure. Conclusion EMP exposure at 100 kV/m and 400 kV/m can increase the permeability of in vitro BBB model and BBB tight junction-related proteins such as ZO-1 and claudin-5 may change EMP-induced BBB permeability.展开更多
基金国家自然科学基金(81571283)%山东省自然科学基金(ZR2016HL21,ZR2017LH068)%山东省中医药科技发展计划项目(2015-420)National Natural Science Foundation of China(81571283)%Natural Science Foundation of Shandong Province(ZR2016HL21,ZR2017LH068)%Traditional Chinese Medicine Science and Technology Development Project of Shandong Province(2015-420)
文摘目的 探讨丁苯酞(NBP)对急性一氧化碳(CO)中毒大鼠血脑屏障(BBB)超微结构损伤的干预作用及其对紧密连接相关蛋白ZO-1、claudin-5表达的影响.方法 按随机数字表法将144只健康雄性SD大鼠分为正常对照组、CO中毒组和NBP干预组,每组48只.采用高压氧舱吸入法建立急性CO中毒动物模型并给予高压氧舱治疗,正常对照组自由呼吸新鲜空气.NBP干预组大鼠于中毒2 h后灌胃NBP 60 mg/kg、每日2次,直至处死;正常对照组和CO中毒组灌胃等量纯橄榄油.各组分别于制模后1、3、7、14 d取4只大鼠,透射电镜下观察BBB超微结构改变,用免疫荧光标记及蛋白质免疫印迹试验(Western Blot)检测ZO-1和claudin-5的蛋白表达,用免疫荧光双标染色观察两种蛋白在脑组织中的定位关系,用线性回归分析两种蛋白的相关性.结果 正常对照组大鼠BBB超微结构无明显异常,可见部分ZO-1和大量claudin-5阳性细胞.CO中毒组BBB超微结构严重损坏;脑组织ZO-1和claudin-5阳性细胞明显减少,中毒后1 d蛋白表达即较正常对照组明显降低(ZO-1蛋白:3.38±0.30比24.50±5.62,claudin-5蛋白:11.38±0.93比46.35±6.88,均P<0.05), 14 d时仍维持在较低水平,与正常对照组比较差异有统计学意义(ZO-1蛋白:10.35±0.80比24.63±3.57, claudin-5蛋白:32.35±3.11比46.43±7.20,均P<0.05).NBP干预可明显改善急性CO中毒导致的大鼠BBB超微结构改变;脑组织ZO-1和claudin-5阳性细胞明显增多,蛋白表达亦明显升高,分别于1 d、3 d起明显高于CO中毒组(1 d ZO-1蛋白:7.57±0.69比3.38±0.30,3 d claudin-5蛋白:20.46±1.42比11.43±0.86,均P<0.05),并随时间延长呈升高趋势.免疫荧光双标染色结果提示,ZO-1和claudin-5蛋白不仅可以在同一细胞中共存,也可以在不同细胞中单独表达.线性回归分析显示,急性CO中毒大鼠脑组织ZO-1与claudin-5表达呈线性正相关(R2=0.917,P=0.022).结论 NBP可能通过上调ZO-1和claudin-5蛋白表达维持急性CO中毒
基金supported by the National Natural Science Foundation of China,No.30960107the Natural Science Foundation of the Education Department of Sichuan Province of China,No.14ZA0223
文摘Integrity of the blood-brain barrier structure is essential for maintaining the internal environment of the brain.Development of cerebral infarction and brain edema is strongly associated with blood-brain barrier leakage.Therefore,studies have suggested that protecting the blood-brain barrier may be an effective method for treating acute stroke.To examine this possibility,stroke model rats were established by middle cerebral artery occlusion and reperfusion.Remote ischemic postconditioning was immediately induced by three cycles of 10-minute ischemia/10-minute reperfusion of bilateral hind limbs at the beginning of middle cerebral artery occlusion reperfusion.Neurological function of rat models was evaluated using Zea Longa’s method.Permeability of the blood-brain barrier was assessed by Evans blue leakage.Infarct volume and brain edema were evaluated using 2,3,5-triphenyltetrazolium chloride staining.Expression of matrix metalloproteinase-9 and claudin-5 m RNA was determined by real-time quantitative reverse transcription-polymerase chain reaction.Expression of matrix metalloproteinase-9 and claudin-5 protein was measured by western blot assay.The number of matrix metalloproteinase-9-and claudin-5-positive cells was analyzed using immunohistochemistry.Our results showed that remote ischemic postconditioning alleviated disruption of the blood-brain barrier,reduced infarct volume and edema,decreased expression of matrix metalloproteinase-9 m RNA and protein and the number of positive cells,increased expression of claudin-5 m RNA and protein and the number of positive cells,and remarkably improved neurological function.These findings confirm that by suppressing expression of matrix metalloproteinase-9 and claudin-5 induced by acute ischemia/reperfusion,remote ischemic postconditioning reduces blood-brain barrier injury,mitigates ischemic injury,and exerts protective effects on the brain.
文摘Background:The increased permeability of the blood-brain barrier (BBB) induced by ischemia/hypoxia is generally correlated with alteration of tight junctions (TJs). DL-3-n-butylphthalide (NBP) has been shown to exert neuroprotective effects after ischemic injury. However, few studies have assessed the correlation between NBP and TJs. This study aimed to investigate the potential effect of NBP on the TJ proteins claudin-5, zonula occludens-1 (ZO-1), and occludin during brain ischemia. Methods: A chronic cerebral hypoperfusion (CCH) Sprague-Dawley rat model was established, and NBP (20, 40, or 80 mg/kg, gavage, once a day) treatment was performed for 14 days. NBP (0.1 or 1.0μmol/L) pre-treatment was applied to an in vitro hypoxia microvascular endothelial cell model (1%〇2, 24 h). BBB permeability was assessed by performing the Evans blue assay. The expressions and localization of claudin-5, ZO-1, occludin, phosphorylated/total protein kinase B (p-Akt/Akt), phosphorylated/total glycogen synthase kinase 3p (GSK-3(3)/GSK-3p, and (3-catenin/p-actin were evaluated by Western blotting or immunofluorescence. Reactive oxygen species (ROS) generation was measured by flow cytometry analysis. TJ ultrastructure was observed by transmission electron microscopy. Results: In CCH rats, treatment with 40 and 80 mg/kg NBP decreased the Evans blue content in brain tissue (9.0 ± 0.9 (μg/g vs. 12.3 ± 1.9 (μg/g, P = 0.005;6.7 ± 0.6 μg/g vs. 12.3 ± 1.9μg/g, P < 0.01), increased the expression of claudin-5 (0.79 ± 0.08 mvs. 0.41 ± 0.06, P < 0.01;0.07 ± 0 .0 7 vs. 0.41 ± 0 .0 6 , P < 0 .61 ), and elevated the ZO-1 protein level (P < 0.05) in brain microvascular segments in a dose-dependent manner in comparison with the corresponding values in the model group. There was no significant difference in occludin expression (P > 0.05). In the hypoxia cell model, NBP pre-treatment improved TJ ultrastructure, decreased intracellular ROS level, and increased the expression of claudin-5 (P < 0.01) and ZO-1 (P < 0.01) in comparison with
基金supported by the National Basic Research Program of China(2011CB503704,2011CB503705)National Natural Science Foundation of China (No. 30970670, 60871068)
文摘Objective To study the effect of electromagnetic pulse （EMP） exposure on permeability of in vitro blood-brain-barrier （BBB） model. Methods An in vitro BBB model, established by co-culturing brain microvascular endothelial cells （BMVEC） and astroglial cells （AC） isolated from rat brain, was exposed to EMP at 100 kV/m and 400 kV/m, respectively. Permeability of the model was assayed by measuring the transendothelial electrical resistance （TEER） and the horseradish peroxidase （HRP） transmission at different time points. Levels of BBB tight junction-related proteins were measured at O, 1, 2, 4, 8, 12, 16, 20, 24 h after EMP exposure by Western blotting. Results The TEER level was lower in BBB model group than in control group at 12 h after EMP, exposure which returned to its normal level at 24 h. The 24 h recovery process was triphasic and biphasic respectively after EMP exposure at 100 kV/m and 400 kV/m. Following exposure to 400 kV/m EMP, the HRP permeability increased at 1-12 h and returned to its normal level at 24 h. Western blotting showed that the claudin-5 and ZO-1 protein levels were changed after EMP exposure. Conclusion EMP exposure at 100 kV/m and 400 kV/m can increase the permeability of in vitro BBB model and BBB tight junction-related proteins such as ZO-1 and claudin-5 may change EMP-induced BBB permeability.
