Table 1_Fresh ginseng-derived vesicle-like nanoparticles protect against intestinal barrier injury in inflammatory bowel disease via the miR-29a-5p/TLR4 axis.docx

Trans-epithelial electrical resistance (TEER) was measured in epithelial cells overexpressing miR-29a-5p, and tight junction protein and TLR4 expression were examined after miR-29a-5p knockdown; direct targeting of TLR4 by miR-29a-5p was confirmed by dual-luciferase reporter assay. Intestinal epithelial cells were also exposed to conditioned medium from fresh GDNP-treated macrophages to assess tight junction protein and miR-29a-5p expression.

In vivo, a dextran sulfate sodium (DSS)-induced murine colitis model was used to evaluate the preventive protective effects of low- or high-dose fresh GDNPs, and body weight, disease activity index (DAI), colon length, histopathology, macrophage polarization, tight junction protein expression, and inflammatory cytokine and miR-29a-5p levels were evaluated.</p>Results<p>Both fresh and dried ginseng extracts alleviated DSS-induced intestinal injury, with fresh ginseng extract showing stronger protective effects in several pathological and inflammatory parameters.

Fresh and dried GDNPs showed comparable spherical morphology and particle-to-protein ratios, but fresh GDNPs were more susceptible to Triton X-100-induced lysis, indicating a higher proportion of intact vesicles. Fresh GDNPs promoted intestinal epithelial cell viability, whereas dried GDNPs at the same concentration had no significant effect, and fresh GDNPs conferred stronger protection against epithelial permeability disruption.

In inflamed epithelial and macrophage models, fresh GDNPs suppressed TLR4/STAT3 signaling and pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) while restoring tight junction proteins (Occludin, Claudin-1) and miR-29a-5p expression. Mechanistically, fresh GDNPs preserved epithelial barrier integrity by upregulating miR-29a-5p, which directly targeted and suppressed TLR4, and by reprogramming the macrophage inflammatory secretome.

In DSS-induced colitis, fresh GDNPs attenuated body weight loss, DAI scores, and colon shortening; reduced M1 macrophage polarization; preserved tight junction protein expression; and restored miR-29a-5p expression in colon tissue.</p>Conclusion<p>Fresh and dried ginseng-derived GDNPs exhibited differential epithelial protective effects in vitro. Fresh ginseng-derived GDNPs protected intestinal barrier integrity through the miR-29a-5p/TLR4 axis and macrophage–epithelial crosstalk.</p>