Apis mellifera Royal Jelly-derived Extracellular Vesicles (RJ-EVs) as Novel Anti-inflammatory Nanotherapeutics for Equine Metabolic Syndrome-Affected Adipose Stem Cells 

Equine metabolic syndrome (EMS) is a multifactorial endocrine disorder characterized by insulin resistance, regional adiposity, and chronic low-grade inflammation, collectively driving functional impairment of adipose tissue-derived stem cells (EqASCs). Royal Jelly-derived extracellular vesicles (RJ-EVs) are emerging as bioactive nanoconstructs endowed with immunomodulatory and pro-regenerative properties; however, their potential in metabolic disease contexts remains largely unexplored. This study aimed to isolate and characterize RJ-EVs and to evaluate their cytocompatibility and immunomodulatory capacity in EMS-affected EqASCs in vitro.

RJ-EVs were isolated from raw Royal Jelly using the miRCURY Exosome Isolation Kit and characterized by scanning electron microscopy (SEM) and flow cytometry-based immunophenotyping targeting the surface markers CD9 and CD63, and, following bead permeabilization, the intraluminal marker Syntenin-1 and the cargo protein MRJP1. EqASCs isolated from healthy (EqASC_HE) and EMS-affected (EqASC_EMS) horses were treated with RJ-EVs at different doses for 24, 48, and 72 hours under serum free conditions. Cytocompatibility was assessed by MTS assay and Annexin V flow cytometry, and inflammatory profiling was performed by RT-qPCR and Western blot.

SEM confirmed a heterogeneous nanoscale vesicle population with spherical to ovoid morphology. Immunophenotyping validated CD63 and CD9 as EV surface markers, confirmed Syntenin-1 as an intraluminal EV marker, and identified MRJP1 as a candidate RJ-EV-associated cargo protein. RJ-EV treatment enhanced EqASC_EMS viability in a dose and time dependent manner with no cytotoxic effects, and Annexin V analysis indicated a reduced apoptotic fraction, accompanied by downregulation of p53 and BAX and upregulation of BCL-2. At the inflammatory level, RJ-EVs suppressed the pro-inflammatory mediators IL-1β and MCP-1, with concurrent changes in IL-4 and IL-6 expression at both transcriptional and protein levels.

RJ-EVs exert cytoprotective and immunomodulatory effects on EqASC_EMS in vitro, supporting a rationale for their further investigation as a naturally-derived nanotherapeutic candidate in metabolic disease.