Laboratory of Neuro-Trauma and Neurodegenerative Disorders, Tianjin Geriatrics Institute, Tianjin Medical University General Hospital, Tianjin, PR China.
Department of Geriatrics, Tianjin Medical University General Hospital, Tianjin, PR China.
Histol Histopathol. 2022 Feb;37(2):159-168. doi: 10.14670/HH-18-406. Epub 2021 Dec 13.
Traumatic brain injury (TBI) can cause the pathological disruption of the blood-brain barrier (BBB) and associated neurological injury. Reducing the severity of such barrier disruption following TBI can decrease the degree of brain edema, suppress intracranial inflammation, and thereby protect against neurological damage. The BBB is made up of brain microvascular endothelial cells (BMVECs), neurons, pericytes, astrocytes, and extracellular matrix components. In prior analyses, we have demonstrated that miR-124-3p expression is enhanced in microglia-derived exosomes following TBI, with this miRNA being capable of promoting neural repair after such injury. Based upon these results, the present study was formulated to examine the impact of miR-124-3p on BMVEC function and to evaluatethe mechanistic basis for its activity by overexpressing miR-124-3p in these endothelial cells. We utilized a bEnd.3 cell scratch wound in vitro model to simulate TBI-associated brain microvascular endothelial cell injury. Lipofectamine3000 was used to transfect endothelial cells such that they overexpressed miR-124-3p. Fluorescence microscopy was used to observe the effects of miR-124-3p expression on these endothelial cells. TUNEL+CD31 immunofluorescence stainingwas employed to observe endothelial cell apoptosis. Tight junctions were observed via ionconductivity microscopy. Western blotting was used to detect the expression of tight junction proteins (occludin, ZO-1), autophagy-associated proteins (Beclin-1, p62, LC3-II/LC3-I), and mTOR-associated proteins (p-mTOR, PDE4B). Chloroquine was used to treat these injured endothelial cells overexpressing miR-124-3p, and endothelial cell apoptosis was assessed via TUNEL+CD31 immunofluorescence staining. We found that the upregulation of miR-124-3p was sufficient to suppress bEnd.3 cell apoptotic death following in vitro scratch injury while promoting the upregulation of the tight junction proteins ZO-1 and occludin in these cells, thereby reducing the degree of leakage across the cerebral microvascular endothelial barrier. These protective effects may be related to the ability of miR-124-3p to suppress mTOR signaling and to induce autophagic activity within BMVECs. These data support a model wherein miR-124-3p can inhibit mTOR signaling and promote autophagic induction in BMVECs, thereby protecting these cells against TBI-induced damage.
创伤性脑损伤（TBI）可导致血脑屏障（BBB）的病理性破坏和相关的神经损伤。减少 TBI 后这种屏障破坏的严重程度可以降低脑水肿的程度，抑制颅内炎症，从而防止神经损伤。BBB 由脑微血管内皮细胞（BMVEC）、神经元、周细胞、星形胶质细胞和细胞外基质成分组成。在之前的分析中，我们已经证明 miR-124-3p 在 TBI 后微胶质细胞衍生的外泌体中表达增强，这种 miRNA 能够在损伤后促进神经修复。基于这些结果，本研究旨在研究 miR-124-3p 对 BMVEC 功能的影响，并通过在这些内皮细胞中过表达 miR-124-3p 来评估其活性的机制基础。我们利用体外 bEnd.3 细胞划痕伤口模型模拟 TBI 相关的脑微血管内皮细胞损伤。使用 Lipofectamine3000 转染内皮细胞，使其过表达 miR-124-3p。荧光显微镜用于观察 miR-124-3p 表达对这些内皮细胞的影响。TUNEL+CD31 免疫荧光染色用于观察内皮细胞凋亡。通过离子电导率显微镜观察紧密连接。Western blot 用于检测紧密连接蛋白（occludin、ZO-1）、自噬相关蛋白（Beclin-1、p62、LC3-II/LC3-I）和 mTOR 相关蛋白（p-mTOR、PDE4B）的表达。用氯喹处理过表达 miR-124-3p 的受损内皮细胞，并通过 TUNEL+CD31 免疫荧光染色评估内皮细胞凋亡。我们发现，miR-124-3p 的上调足以抑制体外划痕损伤后 bEnd.3 细胞的凋亡死亡，同时促进这些细胞中紧密连接蛋白 ZO-1 和 occludin 的上调，从而降低脑微血管内皮屏障的渗漏程度。这些保护作用可能与 miR-124-3p 抑制 mTOR 信号和诱导 BMVEC 中自噬活性的能力有关。这些数据支持这样一种模型，即 miR-124-3p 可以抑制 mTOR 信号并促进 BMVEC 中的自噬诱导，从而保护这些细胞免受 TBI 诱导的损伤。
