Academic Unit of Mental Health and Clinical Neurosciences, School of Medicine, University of Nottingham, Nottingham, UK.
Academic Unit of Mental Health and Clinical Neurosciences, School of Medicine, University of Nottingham, Nottingham, UK.
Peptides. 2023 Sep;167:171048. doi: 10.1016/j.peptides.2023.171048. Epub 2023 Jun 28.
Substance P (SP) plays a role in vasodilatation and tissue integrity through its receptor, neurokinin 1 (NK1R). However, its specific effect on blood-brain barrier (BBB) remains unknown.
The impact of SP on the integrity/function of human BBB model in vitro, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, was assessed by measurements of transendothelial electrical resistance and paracellular flux of sodium fluorescein (NaF), respectively in the absence/presence of specific inhibitors targeting NK1R (CP96345), Rho-associated protein kinase (ROCK; Y27632) and nitric oxide synthase (NOS; N(G)-nitro-L-arginine methyl ester). Sodium nitroprusside (SNP), a NO donor, was employed as a positive control. The levels of tight junction proteins, zonula occludens-1, occludin and claudin-5 alongside RhoA/ROCK/myosin regulatory light chain-2 (MLC2) and extracellular signal‑regulated protein kinase (Erk1/2) proteins were detected by western analyses. Subcellular localisations of F-actin and tight junction proteins were visualized by immunocytochemistry. Flow cytometry was used to detect transient calcium release.
Exposure to SP increased RhoA, ROCK2 and phosphorylated serine-19 MLC2 protein levels and Erk1/2 phosphorylation in BMECs which were abolished by CP96345. These increases were independent of the changes in intracellular calcium availability. SP perturbed BBB in a time-dependent fashion through induction of stress fibres. Changes in tight junction protein dissolution or relocalisation were not involved in SP-mediated BBB breakdown. Inhibition of NOS, ROCK and NK1R mitigated the effect of SP on BBB characteristics and stress fibre formation.
SP promoted a reversible decline in BBB integrity independent of tight junction proteins expression or localisation.
P 物质（SP）通过其受体神经激肽 1 型（NK1R）在血管舒张和组织完整性中发挥作用。然而，其对血脑屏障（BBB）的具体作用尚不清楚。
通过分别测量钠荧光素（NaF）的跨内皮电阻和旁细胞通透性，评估 SP 对体外由脑微血管内皮细胞（BMEC）、星形胶质细胞和周细胞组成的人 BBB 模型的完整性/功能的影响，在存在/不存在针对 NK1R（CP96345）、Rho 相关蛋白激酶（ROCK；Y27632）和一氧化氮合酶（NOS；N(G)-硝基-L-精氨酸甲酯）的特异性抑制剂的情况下。使用一氧化氮供体硝普钠（SNP）作为阳性对照。通过 Western 分析检测紧密连接蛋白、闭合蛋白-1、occludin 和 Claudin-5 以及 RhoA/ROCK/肌球蛋白调节轻链-2（MLC2）和细胞外信号调节蛋白激酶（Erk1/2）蛋白的水平。通过免疫细胞化学观察 F-肌动蛋白和紧密连接蛋白的亚细胞定位。通过流式细胞术检测瞬时钙释放。
SP 暴露增加了 BMEC 中的 RhoA、ROCK2 和磷酸化丝氨酸-19 MLC2 蛋白水平以及 Erk1/2 磷酸化，这一作用被 CP96345 所阻断。这些变化与细胞内钙可用性的变化无关。SP 通过诱导应激纤维以时间依赖性方式破坏 BBB。紧密连接蛋白溶解或重定位的变化不参与 SP 介导的 BBB 破裂。NOS、ROCK 和 NK1R 的抑制减轻了 SP 对 BBB 特征和应激纤维形成的影响。
SP 促进了 BBB 完整性的可逆下降，与紧密连接蛋白的表达或定位无关。
