Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, Jiangsu Province, China; Department of neurosurgery, The Affiliated Hospital of Yangzhou University, Yangzhou, Jiangsu Province, China.
Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, Jiangsu Province, China; Department of Neurosurgery, Yancheng City No.1 People's Hospital, Yancheng First Hospital, Affiliated Hospital of Nanjing University Medical School, Yancheng 224006, Jiangsu Province, China.
Exp Neurol. 2023 Oct;368:114508. doi: 10.1016/j.expneurol.2023.114508. Epub 2023 Aug 19.
Intracerebral hemorrhage (ICH) is one of the stroke subtypes with the highest mortality. Secondary brain injury is associated with neurological dysfunction and poor prognosis after ICH. Caveolin-1 (CAV1) is the key protein of Caveolae. Previous studies have shown that CAV1 plays an important role in central nervous system diseases, and pointed out that in a collagenase-induced ICH model in vivo, CAV1 is associated with neuroinflammatory activation and poor neurological prognosis. In this study, we explore the role and the molecular mechanism of CAV1 in brain injury via a rat autologous whole blood injection model and an in vitro model of ICH.
Adult male Sprague-Dawley rats ICH model was induced through autologous whole blood injecting into the right basal ganglia. The changes in protein levels of CAV1 in brain tissues of ICH rats were detected by western blot analysis. The immunofluorescent staining was used to explore the changes of CAV1 in microglia/macrophages (Iba1+ cells). Lentivirus vectors were administered by intracerebroventricular injection to induce CAV1 overexpression and knockdown respectively. The western blot analysis, immunofluorescence staining, enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase dUTP nick end labeling and Nissl staining were performed to explore the role of CAV1 in secondary brain injury after ICH. Meanwhile, the rotarod test, foot fault test, adhesive-removal test, and Modified Garcia Test, as well as Morris Water Maze test, were performed to evaluate the behavioral cognitive impairment of ICH rats after genetic intervention. Additionally, BV-2 cells treated with oxygen hemoglobin for 24 h, were used as an in vitro model of ICH in this study to explore the molecular mechanism of CAV1 in brain injury; we performed western blot analysis after precise regulation of CAV1 in BV2 cells to observe changes in protein levels and phosphorylated levels of C-Src, IKK-β, and NF-κB.
The expression of CAV1 in microglia/macrophages (Iba1+ cells) was elevated and reached the peak at 24 h after ICH. CAV1 knockdown ameliorated ICH-induced neurological deficits, while CAV1 overexpression significantly worsened neurological dysfunction of ICH rats. CAV1 knockdown attenuated cellular apoptosis and promoted neuronal survival in brain tissues of ICH rats, while the ICH rats with CAV1 overexpression presented more cellular apoptosis and neuronal loss. Meanwhile, CAV1 knockdown inhibited the microglia activation and neuroinflammatory response, while CAV1 overexpression abolished these effects and aggravated neuroinflammation in brain tissues of ICH rats. Additionally, by inducing to CAV1 knockdown in BV2 cells in an in vitro model of ICH, the levels of p-C-Src, CAV-1, p-CAV-1, and p-IKK-β in cytoplasm and the level of NF-κB p65 in nucleus of BV2 cells were significantly decreased, while they were increased by inducing to CAV1 overexpression.
Our research revealed CAV1 aggravated neurological dysfunction in a rat ICH model. CAV1 knockdown exerted neuroprotective effect by suppressing microglia activation and neuroinflammation after ICH might via the C-Src/CAV1/IKK-β/NF-κB signaling pathway.
脑出血（ICH）是死亡率最高的中风亚型之一。继发性脑损伤与 ICH 后的神经功能障碍和不良预后有关。小窝蛋白-1（CAV1）是小窝的关键蛋白。先前的研究表明，CAV1 在中枢神经系统疾病中发挥重要作用，并指出在体内胶原酶诱导的 ICH 模型中，CAV1 与神经炎症激活和不良神经预后有关。在这项研究中，我们通过大鼠自体全血注射模型和 ICH 的体外模型探索了 CAV1 在脑损伤中的作用和分子机制。
通过向右侧基底节注射自体全血诱导成年雄性 Sprague-Dawley 大鼠 ICH 模型。通过 Western blot 分析检测 ICH 大鼠脑组织中 CAV1 蛋白水平的变化。免疫荧光染色用于探索 CAV1 在小胶质细胞/巨噬细胞（Iba1+细胞）中的变化。通过脑室内注射慢病毒载体分别诱导 CAV1 过表达和敲低。通过 Western blot 分析、免疫荧光染色、酶联免疫吸附试验、末端脱氧核苷酸转移酶 dUTP 缺口末端标记和尼氏染色，探讨 CAV1 在 ICH 后继发性脑损伤中的作用。同时，通过对 ICH 大鼠进行基因干预，进行旋转棒试验、足误试验、粘扣移除试验、改良 Garcia 试验以及 Morris 水迷宫试验，评估 ICH 大鼠的行为认知障碍。此外，在本研究中，氧合血红蛋白处理 24 小时的 BV-2 细胞被用作 ICH 的体外模型，以探索 CAV1 在脑损伤中的分子机制；我们通过精确调节 BV2 细胞中的 CAV1，观察 CAV1 蛋白水平和磷酸化 C-Src、IKK-β 和 NF-κB 的变化。
ICH 后 24 小时，小胶质细胞/巨噬细胞（Iba1+细胞）中 CAV1 的表达升高并达到峰值。CAV1 敲低改善了 ICH 引起的神经功能缺损，而 CAV1 过表达显著加重了 ICH 大鼠的神经功能障碍。CAV1 敲低减轻了 ICH 大鼠脑组织中的细胞凋亡并促进了神经元存活，而 CAV1 过表达导致更多的细胞凋亡和神经元丢失。同时，CAV1 敲低抑制了小胶质细胞活化和神经炎症反应，而 CAV1 过表达消除了这些作用，并加重了 ICH 大鼠脑组织中的神经炎症。此外，通过在 ICH 的体外模型中诱导 CAV1 敲低，BV2 细胞中的细胞质中 p-C-Src、CAV-1、p-CAV-1 和 p-IKK-β 水平以及核中的 NF-κB p65 水平显著降低，而通过诱导 CAV1 过表达，这些水平增加。
我们的研究表明 CAV1 加重了大鼠 ICH 模型的神经功能障碍。CAV1 敲低通过抑制 ICH 后小胶质细胞活化和神经炎症发挥神经保护作用，可能通过 C-Src/CAV1/IKK-β/NF-κB 信号通路。
