Impact of varying oxidative stress levels on the secretion characteristics and protein cargo profiling of extracellular vesicles derived from duck intestinal epithelial cells

Oxidative stress is a common challenge in intensive duck production systems, often disrupting intestinal barrier integrity and compromising animal health. This study aimed to explore how oxidative stress alters the secretion and protein cargo of extracellular vesicles (EVs) derived from duck intestinal epithelial cells (IECs), which are crucial mediators of epithelial communication and stress adaptation. Duck IECs were exposed to increasing concentrations of hydrogen peroxide (0-200 μmol/L) to induce oxidative stress. EVs were then isolated and characterized using electron microscopy and nanoparticle tracking, and protein cargo was analyzed via 4D label-free proteomics. The results revealed that oxidative stress caused dose-dependent cytotoxicity and suppressed gene expression patterns linked to barrier function and the oxidative stress response (P < 0.05). Interestingly, low-to-moderate oxidative stress (50-100 μmol/L) increased EV secretion, whereas higher doses (200 μmol/L) reduced it (P < 0.05). Proteomic analysis revealed that the differentially expressed proteins in EVs were associated with RNA metabolism, redox balance, protein folding, and endoplasmic reticulum stress. Notably, EVs from moderately stressed cells were enriched in chaperones (e.g., HSP90AB1 and CALR) and lipid-binding proteins (e.g., FABP5), which may influence epithelial-stem cell signaling. Transcription factor analysis revealed that XBP1, SP1, and BHLHE40 are key regulators of these proteomic changes. In conclusion, oxidative stress modulates both the release and molecular cargo characteristics of EVs in duck IECs in a dose-dependent manner, potentially impairing their signaling roles in intestinal homeostasis. These findings provide mechanistic insights into EV-mediated epithelial communication under stress and suggest potential targets for antioxidant interventions in poultry production systems.