Structural integrity facilitates multi-layer modulation of the brain-metabolome-microbiota network by Lilium lancifolium-derived vesicles in depression

Abstract

Plant-derived vesicles (PDVs) have emerged as promising natural nanotherapeutics with multi-component cargo profiles and potential biological activity. However, their delivery behavior and mechanisms of action, particularly in complex neuro-immune-metabolic disorders such as depression, remain incompletely understood. This study investigates the therapeutic efficacy, biodistribution, and multi-layer modulatory network of Lilium lancifolium -derived vesicles (LLEs) in chronic unpredictable mild stress (CUMS)-induced depression models, with a focus on vesicular structural integrity and brain-metabolome-microbiota associations. LLEs were isolated from fresh Lilium lancifolium Thunb and comprehensively characterized in terms of morphology, particle size, zeta potential, particle-to-protein ratio, proteomic cargo and simulated gastrointestinal stability. Therapeutic efficacy was evaluated in CUMS mice and LPS-challenged neuronal cell models using behavioral tests, histopathology, immunofluorescence, and multi-omics approaches (hippocampal transcriptomics, serum metabolomics, and gut metagenomics). Cellular internalization was assessed using pharmacological inhibitors and fluorescent tracking. Brain-associated delivery was evaluated using an in vitro BBB Transwell model, ex vivo brain imaging, and LC–MS detection of LLEs-associated small molecules in perfused brain tissues. The contribution of intact vesicular architecture was assessed by comparing native LLEs with disrupted LLEs (D-LLEs) and single bioactive components. Integrative multi-omics analysis was used to construct an associative brain-metabolome-microbiota modulatory network. LLEs exhibited nanoscale vesicle-like morphology, a negatively charged surface, and partial environmental resilience, with stability in simulated salivary and gastric fluids but vulnerability to aggregation under simulated intestinal conditions. Proteomic profiling and western blot validation supported the presence of vesicle-associated protein components, including HSP70. Intact LLEs showed stronger neuroprotective, anti-neuroinflammatory, and antidepressant-like effects than D-LLEs, supporting the functional contribution of intact vesicular architecture. Cellular uptake assays suggested that macropinocytosis and clathrin-mediated endocytosis may contribute to LLE internalization. In vitro BBB Transwell model, ex vivo brain imaging, and LC–MS tissue quantification supported the brain-associated delivery potential of LLEs. Multi-omics integration revealed LLEs-associated changes in brain inflammatory and lipid metabolic pathways, circulating metabolites, and gut microbial composition, suggesting an associative brain-metabolome-microbiota modulatory network. This study identifies LLEs as a class of plant-derived nanovesicles that integrate structural stability with intrinsic neuroprotective potential. The present study supports the importance of intact vesicular architecture and provides an integrated, evidence-calibrated framework linking LLEs treatment to central neuroimmune modulation, systemic metabolic shifts, and gut microbiota remodeling. These findings provide a basis for the future development of plant-derived vesicles as natural nanovesicle platforms for neuroimmune and metabolic modulation, while highlighting the need for further causal validation of the proposed multi-layer regulatory network. Native Lilium lancifolium -derived vesicles (LLEs) contain multiple classes of vesicle-associated cargos, including proteins, regulatory RNAs, and small molecules, and show a pH-responsive stability profile. Multi-omics annotation suggested that these cargos are associated with inflammatory signaling, lipid metabolism, cellular stress responses, and neurological regulation.In vitro BBB model transport assays,ex vivo brain imaging, and LC-MS detection in perfused brain tissues support the brain-associated delivery potential of LLEs. At the systems level,LLEs treatment was associated with coordinated modulation of hippocampal transcriptomic signatures, circulating metabolites, inflammatory cytokines,and gut microbial composition. Representative changes include the reprogramming of hippocampal transcriptomic signatures (e.g., C1qa, Cr2, Itgal,Grin3b), the suppression of peripheral inflammatory cytokines (TNF-α, IL-1β, and IL-6), shifts in circulating metabolic mediators (ALA, PUFAs, and succinic acid), and the reshaping of the gut microbiota (enriching Adlercreutzia and Roseburia while suppressing Helicobacter and Alistipes ). Together, this schematic conceptualizes an associative brain-metabolome-microbiota network through which LLEs may facilitate neuroimmune and metabolic homeostasis in CUMS-induced depression-like mice.