摘要
背景钠钾氯同向转运体与血脑屏障的损伤密切相关,具体调节机制仍有待研究。目的利用体外模型探讨钠钾氯同向转运体对血脑屏障渗透性的调控机制。方法利用bEnd3小鼠脑微血管内皮细胞和BV2小鼠小胶质细胞在Transwell小室中建立血脑屏障模型。通过跨内皮细胞电阻(trans-epithelium electrical resistant,TEER)和荧光素钠渗透实验检测血脑屏障功能,Western blot检测炎症小体NLRP3蛋白含量,ELISA检测白细胞介素-1β(interleukin-1β,IL-1β)和基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)的水平,免疫荧光评估血管内皮细胞间紧密连接蛋白ZO-1的完整性。结果炎症环境下小胶质细胞的激活会破坏血脑屏障模型的完整性,降低TEER值,增加血脑屏障模型的通透性(P<0.05)。在共培养模型中,钠钾氯同向转运体特异性抑制剂显著改善了脂多糖(lipopolysaccharide,LPS)诱导的血脑屏障损伤(P<0.05),而在内皮细胞单培养构建的血脑屏障模型中未观察到此现象。钠钾氯同向转运体特异性抑制剂还降低了小胶质细胞炎症小体NLRP3的表达,抑制了小胶质细胞IL-1β和MMP-9的分泌(P<0.05),在共培养模型中,抑制NLRP3炎症小体改善了LPS诱导的血脑屏障损伤,增加了TEER值,降低了屏障的渗透系数(P<0.05)。结论钠钾氯同向转运体对血脑屏障渗透性的调节作用可能是通过小胶质细胞来源的NLRP3炎症小体介导的。
Background The sodium-potassium-chloride cotransporter is closely related to the damage of the blood-brain barrier(BBB),but the specific regulatory mechanism remains to be studied.Objective To investigate the regulatory mechanism of sodium potassium chloride cotransporter on BBB permeability by in vitro model.Methods The BBB model was established in Transwell chambers using bEnd3 mouse brain microvascular endothelial cells and BV2 mouse microglia.The BBB function was detected by trans-epithelium electrical resistant(TEER)and fluorescein sodium permeation assay,the content of inflammasome NLRP3 protein was detected by Western blot,the levels of IL-1βand MMP-9 were detected by ELISA,and the integrity of the tight junction protein ZO-1 between vascular endothelial cells was assessed by immunofluorescence.Results The activation of microglia in inflammatory environment could destroy the integrity of the blood-brain barrier model,reduce the TEER value,and increase the permeability of the blood-brain barrier model(P<0.05).In the co-culture model,the sodium-potassium-chloride symporter-specific inhibitor significantly ameliorated the LPS-induced BBB damage(P<0.05),while this phenomenon was not observed in the BBB model constructed by endothelial cell monoculture.The specific inhibitor of sodium potassium chloride symporter also reduced the expression level of NLRP3 inflammasome in microglial and inhibited the secretion of IL-1βand MMP-9 in microglial cells(P<0.05),in the co-culture model,NLRP3 inflammasome-specific inhibitor ameliorated LPS-induced BBB damage,increased TEER value,and decreased barrier permeability(P<0.05).Conclusion The regulation of the sodium-potassium-chloride cotransporter on BBB permeability may be mediated by NLRP3 inflammasome derived from microglia.
作者
张泽瀚
陶丙岩
张鼎
张军
ZHANG Zehan;TAO Bingyan;ZHANG Ding;ZHANG Jun(Chinese PLA Medical School,Beijing 100853,China;Department of Neurosurgery,the First Medical Center,Chinese PLA General Hospital,Beijing 100853,China)
出处
《解放军医学院学报》
CAS
北大核心
2022年第5期586-594,共9页
Academic Journal of Chinese PLA Medical School
基金
国家自然科学基金项目(81971167)。
关键词
血脑屏障
钠钾氯同向转运体抑制剂
钠钾氯同向转运体
跨内皮电阻
炎症小体
blood brain barrier
sodium potassium chloride cotransporter inhibitor
sodium-potassium-chloride cotransporter
transendothelial resistance
inflammasome