MiR-223 Enhances Lipophagy by Suppressing CTSB in Microglia Following Lysolecithin-Induced Demyelination in Mice

Abstract

Background Lipid droplet (LD)-laden microglia are key pathological hallmarks of multiple sclerosis. The recent discovery of a 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 investigates microRNA (miRNA) involvement in lipophagy during demyelinating diseases, assesses their capacity to modulate LDAM subtypes, and elucidates potential underlying mechanisms. Methods We conducted in vivo experiments using C57BL/6 mice. Two weeks post-demyelination induction at the cervical level 4 (C4), we performed histological assessments and confocal imaging 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. We specifically explored the role of miR-223 in lipophagy in these conditions. In vitro studies, including miR-223 upregulation in BV2 cells via lentiviral transfection, validated bioinformatics findings. Immunofluorescence staining measured LD accumulation, autophagy levels, target gene expression, and inflammatory mediators, elucidating miR-223's mechanisms in LDAM. Results Oil Red O staining and confocal imaging revealed substantial LD accumulation in the demyelinated spinal cord. Transmission electron microscopy showed increased autophagic vacuoles at the injury site. Multi-omics analysis pinpointed miR-223 as a crucial regulatory gene in lipophagy during demyelination. Integrating miRNA, mRNA, and autophagy gene databases identified Cathepsin B (CTSB) as a miR-223 target in autophagy. In vitro, miR-223 upregulation suppressed CTSB expression in BV2 cells, augmented autophagy, alleviated LD accumulation, and decreased IL-1β, an inflammatory mediator. Conclusion These findings indicate miR-223's pivotal role in lipophagy during demyelinating conditions. By inhibiting CTSB, miR-223 promotes selective LD degradation, thereby reducing the lipid burden and inflammatory phenotype in LDAM. This study broadens our understanding of lipophagy's molecular mechanisms and proposes lipophagy induction as a potential therapeutic approach to mitigate inflammatory responses in demyelinating diseases.