背景:载有脂质液滴(LD)的小胶质细胞是多发性硬化症的关键病理标志。最近发现的这种新的小胶质细胞亚型,脂滴积聚小胶质细胞(LDAM),值得注意的是炎症因子分泌增加和吞噬能力减弱。Lipopagy,自噬介导的LDs选择性降解,在这方面起着至关重要的作用。这项研究调查了在脱髓鞘疾病期间microRNAs(miRNAs)参与脂质吞噬,评估了他们调节LDAM亚型的能力,并阐明了潜在的潜在机制。
方法:C57BL/6小鼠用于体内实验。宫颈4级脱髓鞘诱导后两周(C4),进行组织学评估和共聚焦成像以检查病变部位小胶质细胞中LD的积累。使用透射电子显微镜观察自噬变化。miRNA和mRNA多组学分析鉴定了在脱髓鞘条件下差异表达的miRNA和mRNA以及相关的自噬靶基因。具体探讨了miR-223在这些条件下的脂质吞噬中的作用。体外研究,包括miR-223在BV2细胞中通过慢病毒感染上调,验证了生物信息学的发现。免疫荧光染色用于测量LD积累,自噬水平,靶基因表达,和炎症介质水平来阐明miR-223在LDAM中的作用机制。
结果:油红O染色和共聚焦成像显示脱髓鞘脊髓中大量LD积累。透射电子显微镜显示损伤部位的自噬液泡数量增加。多组学分析显示miR-223是脱髓鞘过程中噬脂症的关键调控基因。已确定组织蛋白酶B(CTSB)靶向自噬中的miR-223整合miRNA,mRNA和自噬基因数据库。体外,miR-223上调抑制BV2细胞CTSB表达,增强自噬,减少LD积累,并降低炎症介质IL-1β的表达。
结论:这些研究结果表明,miR-223在脱髓鞘条件下的吸脂症中起关键作用。通过抑制CTSB,miR-223促进选择性LD降解,从而降低LDAM中的脂质负荷和炎症表型。这项研究扩大了对吸脂性的分子机制的理解,并提出了吸脂性诱导作为减轻脱髓鞘疾病炎症反应的潜在治疗方法。
BACKGROUND: Lipid droplet (LD)-laden microglia is a key pathological hallmark of multiple sclerosis. The recent discovery of this novel microglial subtype, lipid-droplet-accumulating microglia (LDAM), is notable for increased inflammatory factor secretion and diminished phagocytic capability.
Lipophagy, the autophagy-mediated selective degradation of LDs, plays a critical role in this context. This study investigated the involvement of microRNAs (miRNAs) in
lipophagy during demyelinating diseases, assessed their capacity to modulate LDAM subtypes, and elucidated the potential underlying mechanisms involved.
METHODS: C57BL/6 mice were used for in vivo experiments. Two weeks post demyelination induction at cervical level 4 (C4), histological assessments and confocal imaging were performed to examine LD accumulation in microglia within the lesion site. Autophagic changes were observed using transmission electron microscopy. miRNA and mRNA multi-omics analyses identified differentially expressed miRNAs and mRNAs under demyelinating conditions and the related autophagy target genes. The role of miR-223 in
lipophagy under these conditions was specifically explored. In vitro studies, including miR-223 upregulation in BV2 cells via lentiviral infection, validated the bioinformatics findings. Immunofluorescence staining was used to measure LD accumulation, autophagy levels, target gene expression, and inflammatory mediator levels to elucidate the mechanisms of action of miR-223 in LDAM.
RESULTS: Oil Red O staining and confocal imaging revealed substantial LD accumulation in the demyelinated spinal cord. Transmission electron microscopy revealed increased numbers of autophagic vacuoles at the injury site. Multi-omics analysis revealed miR-223 as a crucial regulatory gene in
lipophagy during demyelination. It was identified that cathepsin B (CTSB) targets miR-223 in autophagy to integrate miRNA, mRNA, and autophagy gene databases. In vitro, miR-223 upregulation suppressed CTSB expression in BV2 cells, augmented autophagy, alleviated LD accumulation, and decreased the expression of the inflammatory mediator IL-1β.
CONCLUSIONS: These findings indicate that miR-223 plays a pivotal role in lipophagy under demyelinating conditions. By inhibiting CTSB, miR-223 promotes selective LD degradation, thereby reducing the lipid burden and inflammatory phenotype in LDAM. This study broadens the understanding of the molecular mechanisms of
lipophagy and proposes
lipophagy induction as a potential therapeutic approach to mitigate inflammatory responses in demyelinating diseases.