Department of Neurology, the First Affiliated Hospital of Kunming Medical University, No.295 Xichang Road, Kunming 650032, Yunnan, China; The Yunnan Province Clinical Research Center for Neurological Diseases, No.295 Xichang Road, Kunming 650032, Yunnan, China.
Department of Neurology, the First Affiliated Hospital of Kunming Medical University, No.295 Xichang Road, Kunming 650032, Yunnan, China.
Brain Res. 2024 Jul 15;1835:148919. doi: 10.1016/j.brainres.2024.148919. Epub 2024 Apr 6.
As a key substance for intercellular communication, exosomes could be a potential strategy for stroke treatment. Activated microglia disrupt the integrity of blood-brain barrier (BBB) to facilitate the stroke process. Hence, this study was designed to investigate the effect of microglia-derived exosomes on BBB cell model injury and to explore the underlying molecular mechanisms.
M1 polarization of BV2 cells was induced with LPS and their derived exosomes were isolated. Astrocytes were cultured in primary culture and constructed with End3 cells as a BBB cell model. After co-culture with exosomes, the BBB cell model was examined for changes in TEER, permeability, and expression of BBB-related proteins (Claudin-1, Occludin, ZO-1 and JAM). Resting and M1-type BV2 cell-derived exosomes perform small RNA sequences and differentially expressed miRNAs (DE-miRNAs) are identified by bioinformatics.
M1-type BV2 cell-derived exosomes decreased End3 cell viability, and increased their apoptotic ratio. Moreover, M1 type BV2 cell-derived exosomes dramatically enhanced the permeability of BBB cell model, and diminished the TEER and BBB-related protein (Claudin-1, Occludin, ZO-1) expression. Notably, resting BV2 cell-derived exosomes had no effect on the integrity of BBB cell model. Sequencing results indicated that 71 DE-miRNAs were present in M1 BV2 cell-derived exosomes, and their targets mediated neurological development and signaling pathways such as MAPK and cAMP. RT-qPCR confirmed the differential expression of mmu-miR-125a-5p, mmu-miR-122b-3p, mmu-miR-139-3p, mmu-miR-330-3p, mmu-miR-3057-5p and mmu-miR-342-3p consistent with the small RNA sequence. Furthermore, Creb1, Jun, Mtor, Frk, Pabpc1 and Sdc1 are the most well-connected proteins in the PPI network.
M1-type microglia-derived exosomes contribute to the injury of BBB cell model, which has the involvement of miRNAs. Our findings provide new perspectives and potential mechanisms for future M1 microglia-derived exosomes as therapeutic targets in stroke.
作为细胞间通讯的关键物质，外泌体可能是治疗中风的一种潜在策略。激活的小胶质细胞破坏血脑屏障（BBB）的完整性，促进中风过程。因此，本研究旨在探讨小胶质细胞衍生的外泌体对 BBB 细胞模型损伤的影响，并探讨其潜在的分子机制。
用 LPS 诱导 BV2 细胞 M1 极化，并分离其衍生的外泌体。原代培养星形胶质细胞，并与 End3 细胞共培养构建 BBB 细胞模型。与外泌体共培养后，检测 BBB 细胞模型的 TEER、通透性和 BBB 相关蛋白（Claudin-1、Occludin、ZO-1 和 JAM）的表达变化。通过生物信息学分析，对静止和 M1 型 BV2 细胞衍生的外泌体进行小 RNA 序列分析，并鉴定差异表达的 miRNAs（DE-miRNAs）。
M1 型 BV2 细胞衍生的外泌体降低了 End3 细胞的活力，并增加了其凋亡比例。此外，M1 型 BV2 细胞衍生的外泌体显著增强了 BBB 细胞模型的通透性，并降低了 TEER 和 BBB 相关蛋白（Claudin-1、Occludin、ZO-1）的表达。值得注意的是，静止的 BV2 细胞衍生的外泌体对 BBB 细胞模型的完整性没有影响。测序结果表明，M1BV2 细胞衍生的外泌体中有 71 个 DE-miRNAs，其靶基因介导神经发育和 MAPK、cAMP 等信号通路。RT-qPCR 证实了 mmu-miR-125a-5p、mmu-miR-122b-3p、mmu-miR-139-3p、mmu-miR-330-3p、mmu-miR-3057-5p 和 mmu-miR-342-3p 的差异表达与小 RNA 序列一致。此外，Creb1、Jun、Mtor、Frk、Pabpc1 和 Sdc1 是 PPI 网络中连接最紧密的蛋白质。
M1 型小胶质细胞衍生的外泌体有助于 BBB 细胞模型的损伤，其中涉及 miRNAs。我们的研究结果为未来中风中 M1 小胶质细胞衍生的外泌体作为治疗靶点提供了新的视角和潜在机制。
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