摘要
目的探究高糖条件下,腺苷酸活化蛋白激酶(adenine monophosphate activated protein kinase, AMPK)调控的氧化应激对血脑屏障通透性的影响。方法采用人脑微血管内皮细胞(human brain microvascular endothelial cells, HBMEC)作为体外血脑屏障细胞模型,采取如下两种方式处理:①不同浓度(5.5mmol/L或27.5mmol/L)葡萄糖处理HBMEC 24h;②AMPK激动剂氨基咪唑-4-甲酰胺核苷酸(4-amino-1H-imida-zole-5-carboxamide, AICAR)预处理2h后,再给予27.5mmol/L葡萄糖处理24h。Western blot及免疫荧光染色检测细胞间血脑屏障相关的闭锁连接蛋白(zonula occludens protein 1, ZO-1)水平,活性氧检测试剂盒检测细胞内活性氧(reactive oxygen species, ROS)水平,透射电镜观察细胞内线粒体超微结构改变。结果高糖处理后HBMEC细胞ZO-1水平显著降低,ROS水平升高,线粒体结构损伤;AICAR预处理可显著逆转高糖处理引起的HBMEC细胞中ZO-1水平降低、ROS水平升高和线粒体结构损伤。结论 AMPK激活可通过抑制ROS的产生,修复高糖所致HBMEC损伤。
Objective To investigate the effect of AMPK-regulated ROS on the permeability of blood-brain barrier under high glucose conditions. Methods Human brain microvascular endothelial cells(HBMEC) were used as the in vitro blood-brain barrier cell model, which were treated in the following two ways: ① HBMEC was treated with different glucose concentrations(5.5 mmol/L or 27.5 mmol/L) for 24 hours;② HBMEC was pre-treated with or without AICAR for 2 hours and then incubated in the culture medium with 27.5 mmol/L glucose for 24 hours. The level of ZO-1 was detected by Western blot and immunofluorescence staining. The ROS level was measured by ROS detection kit. The ultrastructural changes of mitochondria in cells were observed by transmission electron microscopy. Results Compared with the normal group, the expression of ZO-1 decreased significantly in the high glucose group, while the level of ROS increased and the mitochondrial ultrastructure damaged. The expression of ZO-1 was significantly increased in the AICAR pretreated group, while the level of ROS was significantly decreased and mitochondrial ultrastructure damage was improved. Conclusion AMPK activation repairs the damage of HBMEC induced by high-glucose by inhibiting the production of ROS.
作者
杨梦珍
汪琳
Yang Mengzhen;Wang Lin(Department of Histology and Embryology,School of Basic Medical Sciences,Wuhan University,Wuhan 430071,China)
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2020年第1期1-7,共7页
Chinese Journal of Histochemistry and Cytochemistry
基金
国家自然科学基金面上项目(81671534,81070514)。
关键词
糖尿病
人脑微血管内皮细胞
血脑屏障
氧化应激
腺苷酸活化蛋白激酶
Diabetes
human brain microvascular endothelial cells
blood-brain barrier
reactive oxygen species
adenine monophosphate activated protein kinase