The mechanism of exosomes of BMSCs modified with Bu Shen Yi Sui capsule in promoting remyelination via regulating miR-15b/Wnt signaling pathway-mediated differentiation of oligodendrocytes

Ethnopharmacological relevance: The Bu Shen Yi Sui capsule (BSYS), a modified version of the classical Chinese medicine formula Liu Wei Di Huang pill, has demonstrated therapeutic efficacy in the treatment of multiple sclerosis (MS). Nevertheless, the precise mechanism through which BSYS facilitates remyelination remains to be elucidated.

Aim of the study: This research investigates the role and potential mechanisms of BSYS-modified exosomes (exos) derived from bone marrow mesenchymal stem cells (BMSCs) in promoting remyelination in a cuprizone (CPZ)-induced demyelination model in mice.

Materials and methods: C57BL/6J mice were administered a 0.2% CPZ-containing diet for 5 weeks to induce demyelination, followed by treatment with exosomes derived from BMSC (BMSC-exos) and BSYS-modified BMSC exosomes (BSYS-BMSC-exos) twice weekly for 2 weeks. Body weight measurements were recorded, and motor function was evaluated using the rotarod test. Pathological changes in myelin and axons were assessed via Luxol fast blue (LFB) staining, transmission electron microscopy (TEM), and immunofluorescence (IF) staining. Oligodendrocyte proliferation, differentiation, and maturation were analyzed using IF double-staining, Western blot (WB), and real-time quantitative reverse transcription PCR (qRT-PCR). Additionally, microRNA (miRNA) sequencing and a luciferase reporter assay were conducted to verify miRNA binding to its target gene. Key markers of the Wnt/β-catenin signaling pathway were examined using WB and qRT-PCR.

Results: BSYS-BMSC-exos treatment significantly increased both body weight and rotarod performance in CPZ mice. Moreover, BMSC-exos and BSYS-BMSC-exos reversed myelin loss and axonal damage. These treatments enhanced oligodendrocytes proliferation, differentiation, and maturation, with BSYS-BMSC-exos exhibiting a particularly pronounced effect on the expression of adenomatous polyposis coli clone CC1 (CC1), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), and myelin basic protein (MBP). Sequencing and luciferase assays revealed that miR-15b-5p, enriched in BSYS-BMSC-exos, directly targets Wnt3a. Furthermore, BSYS-BMSC-exos elevated axis inhibition protein 2 (Axin2) expression while markedly reducing Wnt family member 3A (Wnt3a), phospho-glycogen synthase kinase-3β (p-GSK3β), β-catenin, and T-cell specific transcription factor 4/transcription factor 7-like 2 (TCF4/TCF7L2) levels.

Conclusions: The findings suggest that BSYS-BMSC-exos alleviate neurological deficits, enhance oligodendrocytes differentiation and maturation, and promote remyelination in CPZ mice. miR-15b-5p, enriched in BSYS-BMSC-exos, targets and downregulates Wnt3a, thereby inhibiting the Wnt/β-catenin signaling pathway.