背景:作为细胞间通讯的关键物质,外泌体可能是卒中治疗的潜在策略.激活的小胶质细胞破坏血脑屏障(BBB)的完整性以促进中风过程。因此,本研究旨在研究小胶质细胞来源的外泌体对BBB细胞模型损伤的影响,并探讨其潜在的分子机制。
方法:用LPS诱导BV2细胞的M1极化,并分离其来源的外泌体。星形胶质细胞在原代培养中培养,并用End3细胞构建作为BBB细胞模型。与外泌体共培养后,检查BBB细胞模型的TEER变化,渗透性,和BBB相关蛋白的表达(Claudin-1,Occludin,ZO-1和JAM)。静息和M1型BV2细胞来源的外泌体执行小RNA序列,并通过生物信息学鉴定差异表达的miRNA(DE-miRNA)。
结果:M1型BV2细胞来源的外泌体降低了End3细胞活力,并增加了它们的凋亡率。此外,M1型BV2细胞来源的外泌体显着增强了BBB细胞模型的通透性,并减少TEER和BBB相关蛋白(Claudin-1,Occludin,ZO-1)表达。值得注意的是,静息BV2细胞来源的外泌体对BBB细胞模型的完整性无影响.测序结果表明,M1BV2细胞来源的外泌体中存在71个DE-miRNA,以及它们的靶标介导神经发育和信号通路如MAPK和cAMP。RT-qPCR证实了mmu-miR-125a-5p的差异表达,mmu-miR-122b-3p,mmu-miR-139-3p,mmu-miR-330-3p,mmu-miR-3057-5p和mmu-miR-342-3p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。
结论:M1型小胶质细胞来源的外泌体有助于BBB细胞模型的损伤,有miRNA的参与。我们的发现为未来M1小胶质细胞来源的外泌体作为卒中治疗靶点提供了新的观点和潜在机制。
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.