Department of Neurology, The First Affiliated Hospital, Guangxi Medical University, Nanning, Guangxi, 530021, PR China; Department of Neurology, Changsha Central Hospital, Yuhua District, Changsha, Hunan, 410000, China.
Department of Neurology, The First Affiliated Hospital, Guangxi Medical University, Nanning, Guangxi, 530021, PR China.
Neurochem Int. 2022 Mar;154:105278. doi: 10.1016/j.neuint.2022.105278. Epub 2022 Jan 10.
Glutamate can activate the N-methyl-D-aspartatic acid (NMDA) receptor (NMDAR), damage brain microvascular endothelial cells, and disturb the intercellular tight junctions (TJs). These result in changes in the permeability of the blood brain barrier (BBB). In neurons, the activation of Rho/ROCK signaling pathway is related to the activation of NMDAR，however, whether human brain vascular endothelial cells NMDAR mediates the Rho/ROCK pathway is not fully understood. The present study evaluates the effects of excessive NMDAR activation induced by NMDA (a glutamate analog) on the Rho/ROCK signaling pathway and the permeability of BBB by using a primary human brain microvascular endothelial cell (HBMEC) model. NMDAR subunit GluN1 was expressed in HBMECs and promoted by NMDA detected by Western blot and qRT-PCR. Furthermore, NMDA exposure decreased HBMEC viability, promoted HBMEC apoptosis, increased intracellular reactive oxygen species (ROS) levels, and destroyed the endothelial cytoskeleton. Additionally, NMDA exposure suppressed transendothelial electrical resistance (TEER) values and the expression of TJ proteins occludin and claudin5; it also promoted ROCK activated substrate myosin phosphatase target subunit-1 (MYPT)-1 phosphorylation and the transmittance of sodium fluorescein. In contrast, these effects were attenuated by ROCK inhibitor hydroxyfasudil (HF) and NMDAR antagonist MK801, respectively. Therefore, these results indicate that excessive endothelial NMDAR activation induced by NMDA may induce TJs and cytoskeleton damage, while HF attenuated NMDA-induced cytotoxicity in HBMECs by inhibiting the Rho/ROCK signaling pathway.
谷氨酸可以激活 N-甲基-D-天冬氨酸（NMDA）受体（NMDAR），损伤脑微血管内皮细胞，并破坏细胞间紧密连接（TJ）。这会导致血脑屏障（BBB）通透性发生变化。在神经元中，Rho/ROCK 信号通路的激活与 NMDAR 的激活有关，然而，人脑血管内皮细胞 NMDAR 是否介导 Rho/ROCK 通路尚不完全清楚。本研究通过原代人脑微血管内皮细胞（HBMEC）模型，评估了 NMDA（谷氨酸类似物）诱导的 NMDAR 过度激活对 Rho/ROCK 信号通路和 BBB 通透性的影响。Western blot 和 qRT-PCR 检测到 HBMEC 中表达 NMDAR 亚基 GluN1，并受 NMDA 促进。此外，NMDA 暴露降低了 HBMEC 的活力，促进了 HBMEC 的凋亡，增加了细胞内活性氧（ROS）水平，并破坏了内皮细胞骨架。此外，NMDA 暴露抑制了跨内皮电阻（TEER）值和 TJ 蛋白紧密连接蛋白（occludin）和 Claudin5 的表达；它还促进了 ROCK 激活的底物肌球蛋白磷酸酶靶亚基-1（MYPT-1）磷酸化和荧光素钠的通透性。相比之下，这些作用分别被 ROCK 抑制剂羟基法舒地尔（HF）和 NMDAR 拮抗剂 MK801 减弱。因此，这些结果表明，NMDA 诱导的内皮 NMDAR 过度激活可能导致 TJ 和细胞骨架损伤，而 HF 通过抑制 Rho/ROCK 信号通路减弱了 NMDA 诱导的 HBMEC 细胞毒性。
