Heat Stress-Derived Plasma Extracellular Vesicles Protect Hepatocytes in Chickens by Suppressing MYD88/NF-κB/MAPK Signaling

Heat stress is a major systemic challenge in poultry, but the role of circulating extracellular vesicles (EVs) in liver-directed adaptation remains unclear. This study investigated whether plasma-derived EVs from heat-stressed chickens (HS_EV) mediate hepatoprotective responses under thermal stress. EVs were isolated from the plasma of control and heat-stressed chickens and characterized by morphology, size distribution, and marker expression. Their biodistribution in vivo and uptake by primary hepatocytes in vitro were also evaluated. Hepatocyte injury was induced by heat exposure, and the effects of HS_EV on proliferation, apoptosis, inflammatory cytokine production, transcriptomic reprogramming, and MYD88/NF-κB/MAPK signaling were assessed. Heat stress induced systemic injury in chickens, increased the release of plasma-derived EVs, and promoted their preferential accumulation in the liver. Whole-transcriptome analysis further showed that HS_EV enhanced glutathione metabolism and related metabolic pathways while suppressing apoptosis- and inflammation-related signaling. In primary hepatocytes, HS_EV, but not control EVs, restored proliferative capacity, reduced apoptosis, suppressed the expression and secretion of IL-1β, IL-6, and TNF-α under heat stress, and was associated with attenuation of the MYD88/NF-κB/MAPK axis. These findings suggest that circulating EVs participate in adaptive intercellular communication during heat stress and identify HS_EV as a potential endogenous mediator of hepatoprotection in chickens.