Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai 200040, China.
School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China.
Aging Dis. 2024 May 7;15(3):1344-1356. doi: 10.14336/AD.2023.0714.
Protecting the integrity of the blood-brain barrier (BBB) is crucial for maintaining brain homeostasis after ischemic stroke. Previous studies showed that M2 microglial extracellular vesicles (EVs) played a neuroprotective role in cerebral ischemia. However, the role of M2 microglial EVs in maintaining BBB integrity is unclear. Therefore, we explored the mechanisms of M2 microglial EVs in regulating BBB integrity. To identify microglial EVs, we used nanoparticle tracking analysis, transmission electron microscopy, and western blot analysis. Adult male ICR mice were subjected to 90-min middle cerebral artery occlusion (MCAO), followed by the injection of PKH26-labeled M2 microglial EVs via the tail vein. After MCAO, we assessed brain infarct and edema volume, as well as modified neurological severity score. BBB integrity was measured by assessing IgG leakage. The effects of M2 microglial EVs on astrocytes and endothelial cells were also examined. To investigate the molecular mechanisms, we performed RNA sequencing, miR-23a-5p knockdown, and luciferase reporter assays. Our results showed that PKH26-labeled microglial EVs were mainly taken up by neurons and glial cells. M2 microglial EVs treatment decreased brain infarct and edema volume, modified neurological severity score, and IgG leakage, while increasing the ZO-1, occludin, and claudin-5 expression after MCAO. Knockdown of miR-23a-5p reversed these effects. RNA sequencing revealed that the TNF, MMP3 and NFκB signaling pathway involved in regulating BBB integrity. Luciferase reporter assay showed that miR-23a-5p could bind to the 3' UTR of TNF. M2 microglial EVs-derived miR-23a-5p decreased TNF, MMP3 and NFκB p65 expression in astrocytes after oxygen-glucose deprivation, thereby increasing ZO-1 and Claudin-5 expression in bEnd.3 cells. In conclusion, our findings demonstrated that M2 microglial EVs attenuated BBB disruption after cerebral ischemia by delivering miR-23a-5p, which targeted TNF and regulated MMP3 and NFκB p65 expression.
保护血脑屏障（BBB）的完整性对于维持缺血性中风后的脑内稳态至关重要。先前的研究表明，M2 小胶质细胞细胞外囊泡（EVs）在脑缺血中发挥神经保护作用。然而，M2 小胶质细胞 EVs 维持 BBB 完整性的作用尚不清楚。因此，我们探讨了 M2 小胶质细胞 EVs 调节 BBB 完整性的机制。为了鉴定小胶质细胞 EVs，我们使用纳米颗粒跟踪分析、透射电子显微镜和 Western blot 分析。成年雄性 ICR 小鼠接受 90 分钟大脑中动脉闭塞（MCAO），随后通过尾静脉注射 PKH26 标记的 M2 小胶质细胞 EVs。MCAO 后，我们评估脑梗死和水肿体积以及改良神经功能严重程度评分。通过评估 IgG 渗漏来测量 BBB 完整性。还检查了 M2 小胶质细胞 EVs 对星形胶质细胞和内皮细胞的影响。为了研究分子机制，我们进行了 RNA 测序、miR-23a-5p 敲低和荧光素酶报告基因测定。我们的结果表明，PKH26 标记的小胶质细胞 EVs 主要被神经元和神经胶质细胞摄取。M2 小胶质细胞 EVs 治疗可降低 MCAO 后脑梗死和水肿体积、改良神经功能严重程度评分和 IgG 渗漏，同时增加 ZO-1、occludin 和 claudin-5 的表达。miR-23a-5p 的敲低逆转了这些作用。RNA 测序显示，TNF、MMP3 和 NFκB 信号通路参与调节 BBB 完整性。荧光素酶报告基因测定显示，miR-23a-5p 可以与 TNF 的 3'UTR 结合。M2 小胶质细胞 EVs 衍生的 miR-23a-5p 可降低氧葡萄糖剥夺后星形胶质细胞中 TNF、MMP3 和 NFκB p65 的表达，从而增加 bEnd.3 细胞中 ZO-1 和 Claudin-5 的表达。总之，我们的研究结果表明，M2 小胶质细胞 EVs 通过递送 miR-23a-5p 减轻脑缺血后 BBB 破坏，miR-23a-5p 靶向 TNF 并调节 MMP3 和 NFκB p65 的表达。
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