The miR-6538 in extracellular vesicles derived from lycorine-pretreated mesenchymal stem cells enhances therapeutic efficacy in rheumatoid arthritis.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) were engineered as a targeted platform for Lycorine (LYC), a natural isoquinoline alkaloid with anti-inflammatory and anti-neoplastic properties that exhibits therapeutic potential in inflammatory diseases but is limited by systemic toxicity in clinical applications. We thus established a novel LYC-MSC-EV therapeutic system for the treatment of rheumatoid arthritis (RA). EVs isolated from LYC-pretreated MSCs were characterized and evaluated in vitro and in a collagen-induced arthritis (CIA) murine model. LYC-MSC-EVs demonstrated superior anti-inflammatory efficacy compared to naïve MSC-EVs, significantly suppressing pro-inflammatory cytokine release (TNF-α, IL-6) and macrophage hyperactivation. In CIA mice, LYC-MSC-EVs attenuated joint inflammation, reduced bone erosion, and modulated immune microenvironment polarization. High-throughput analysis revealed that LYC-MSC-EVs possess a reshaped miRNA cargo profile due to the specific enrichment of miR-6538, and thereby exert potent anti-inflammatory bioactivity. Mechanistic studies revealed miR-6538 enrichment in LYC-MSC-EVs as the primary mediator, targeting the NF-κB-PD-L1 signaling axis through direct regulation of Cd274 (PD-L1) expression. RNA sequencing confirmed pathway modulation, while miR-6538 inhibition abolished therapeutic effects. This study establishes that MSC-EV-mediated delivery enhances LYC's anti-arthritic activity while mitigating toxicity, with miR-6538 identified as a critical functional component. The NF-κB-PD-L1 axis targeting mechanism provides a foundation for developing EV-based cell-free therapies for RA and related inflammatory disorders.