背景:脉络丛(ChP)血-脑脊液(CSF)屏障功能障碍和淋巴细胞侵袭在中风的神经炎症反应中的潜在机制尚不清楚。在这项研究中,我们调查了中风是否由于主要的ChP离子转运系统的失调而损害了血-CSF屏障的完整性,Na+-K+-Cl-共转运蛋白1(NKCC1),和调节性Ste20相关的富含脯氨酸-丙氨酸激酶(SPAK)。
方法:在C57Bl/6J小鼠中诱导假或缺血性中风。通过免疫荧光染色和免疫印迹定量ChP中SPAK-NKCC1复合物和紧密连接蛋白(TJs)的变化。通过流式细胞术和免疫染色评估ChP中的免疫细胞浸润。使用培养的ChP上皮细胞(CPEC)和皮质神经元来评估H2O2介导的氧化应激刺激SPAK-NKCC1复合物和细胞损伤。检查了SPAK抑制剂ZT-1a或NKCC1抑制剂布美他尼对ChPSPAK-NKCC1级联的体内或体外药理阻断。
结果:缺血性卒中刺激了CPECs顶端膜SPAK-NKCC1复合物的激活,NF-κB,和MMP9,这与血液-CSF屏障完整性的丧失和增加的免疫细胞浸润到ChP有关。氧化应激直接激活SPAK-NKCC1通路,导致细胞凋亡,神经变性,和NKCC1介导的离子流入。SPAK-NKCC1通路的药理学阻断保护了ChP屏障的完整性,减弱的ChP免疫细胞浸润或神经元死亡。
结论:卒中诱导的SPAK-NKCC1级联的病理刺激导致CPECs损伤和TJs在血-CSF屏障的破坏。ChPSPAK-NKCC1复合物可作为减轻卒中后ChP功能障碍和淋巴细胞侵袭的治疗靶点。
BACKGROUND: The mechanisms underlying dysfunction of choroid plexus (ChP) blood-cerebrospinal fluid (CSF) barrier and lymphocyte invasion in neuroinflammatory responses to stroke are not well understood. In this study, we investigated whether stroke damaged the blood-CSF barrier integrity due to dysregulation of major ChP ion transport system, Na+-K+-Cl- cotransporter 1 (NKCC1), and regulatory Ste20-related proline-alanine-rich kinase (
SPAK).
METHODS: Sham or ischemic stroke was induced in C57Bl/6J mice. Changes on the SPAK-NKCC1 complex and tight junction proteins (TJs) in the ChP were quantified by immunofluorescence staining and immunoblotting. Immune cell infiltration in the ChP was assessed by flow cytometry and immunostaining. Cultured ChP epithelium cells (CPECs) and cortical neurons were used to evaluate H2O2-mediated oxidative stress in stimulating the SPAK-NKCC1 complex and cellular damage. In vivo or in vitro pharmacological blockade of the ChP
SPAK-NKCC1 cascade with
SPAK inhibitor ZT-1a or NKCC1 inhibitor bumetanide were examined.
RESULTS: Ischemic stroke stimulated activation of the CPECs apical membrane SPAK-NKCC1 complex, NF-κB, and MMP9, which was associated with loss of the blood-CSF barrier integrity and increased immune cell infiltration into the ChP. Oxidative stress directly activated the
SPAK-NKCC1 pathway and resulted in apoptosis, neurodegeneration, and NKCC1-mediated ion influx. Pharmacological blockade of the
SPAK-NKCC1 pathway protected the ChP barrier integrity, attenuated ChP immune cell infiltration or neuronal death.
CONCLUSIONS: Stroke-induced pathological stimulation of the
SPAK-NKCC1 cascade caused CPECs damage and disruption of TJs at the blood-CSF barrier. The ChP SPAK-NKCC1 complex emerged as a therapeutic target for attenuating ChP dysfunction and lymphocyte invasion after stroke.