Rosmarinic acid and dihydrorosmarinic acid enhance antioxidant defense and barrier function in chicken intestinal epithelial cells via the AHR and Nrf2 pathways.

Rosmarinic acid (RA) is a phenolic compound with antioxidant and intestinal barrier-protective effects, yet the underlying molecular mechanisms remain incompletely understood. This study aimed to investigate the antioxidant mechanisms of RA and dihydrorosmarinic acid (DHRA) in chickens. In vivo experiments revealed that administration of 100 mg/kg RA for 21 days activated the AHR and Nrf2 pathways, and upregulated antioxidant enzymes and tight junction proteins in the jejunum of chickens (P < 0.05). For in vitro experiments, chicken intestinal epithelial cells (cIECs) were cultured with 100 μM RA or DHRA, with or without Nrf2 and AHR inhibitors. RA and DHRA significantly activated AHR and Nrf2 pathways (P < 0.05), while the Nrf2 inhibitor blocked Nrf2 activation (P < 0.05), and the AHR inhibitor abrogated the activation of both AHR and Nrf2 pathways (P < 0.05). RA and DHRA significantly upregulated the expression of intestinal tight junction proteins (P < 0.05), an effect that was reversed by both AHR and Nrf2 inhibitors (P < 0.05). The activation of AHR and Nrf2 by RA and DHRA was associated with their oxidative products, such as RA-quinone, DHRA-quinone, and H₂O₂ in the culture medium. Both GSH and catalase decreased the concentration of H₂O₂ and quinones in cell culture medium and attenuated the activation of Nrf2 and AHR pathways in cIECs. In summary, the oxidation products of RA and DHRA act as dual activators of the AHR and Nrf2 pathways, enhancing antioxidant defenses and barrier function in cIECs.