Extracellular vesicles from Li-Qi-Yang-Yin formula attenuate LPS-stimulated inflammatory injury in human colonic epithelial cells in vitro by delivering bioactive metabolites and suppressing the oxidative stress–inflammation axis

Background The Li-Qi-Yang-Yin (LQYY) formula, a traditional Chinese medicine (TCMs) compound, holds potential in managing gastrointestinal disorders. Extracellular vesicles (EVs) derived from medicinal plants are emerging as novel carriers of bioactive compounds, yet their role in multi-herb formulations like LQYY remains largely unexplored. This study aimed to isolate LEVs from the LQYY formula and investigate their protective effects against inflammation and oxidative stress in intestinal epithelial cells. Methods Li-Qi-Yang-Yin formula contains EVs were isolated from the LQYY decoction via ultracentrifugation. Their physicochemical characteristics were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The metabolite profile was analyzed by untargeted metabolomics. Using a lipopolysaccharide (LPS)-stimulated inflammatory model in human NCM460 colon epithelial cells, we assessed nitric oxide (NO) production, inflammatory cytokine levels, and the expression of proteins in the Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway. Results We successfully isolated LEVs with a typical cup-shaped morphology and an average size of 201.1 nm. Metabolomic analysis identified 862 metabolites, with significant enrichment in fatty acids, phenylpropanoids, and flavonoids. These LEVs were efficiently internalized by NCM460 cells. Pretreatment with LEVs, particularly at a high dose (50 μg/mL), significantly attenuated LPS-stimulated cytotoxicity and intracellular NO overproduction. Furthermore, LEVs dose-dependently suppressed the secretion of pro-inflammatory cytokines (IL-6, TNF-α), promoted the anti-inflammatory cytokine IL-10, and inhibited the activation of the TLR4/NF-κB signaling pathway. Conclusion Our findings demonstrate that LEVs from the LQYY formula are natural nanoparticles enriched with anti-inflammatory and antioxidant metabolites and can alleviate LPS-stimulated inflammatory injury in human colonic epithelial cells in vitro . These results provide proof-of-concept evidence and warrant further validation in animal models to determine disease relevance.