Eucalyptol relieves the toxicity of diisobutyl phthalate in Ctenopharyngodon idellus kidney cells through Keap1/Nrf2/HO-1 pathway: Apoptosis-autophagy crosstalk and immunoregulation

Diisobutyl phthalate (DiBP), one of the commonly used plasticizers in industry, is an endocrine disruptor and environmental contaminant that can persist in water and threaten the health of aquatic creatures. Eucalyptol (Euc), a monoterpenoid extracted from plants, has been proved to have anti-inflammatory, antioxidant, and detoxification properties. However, the protective mechanism of Euc against cell injury caused by DiBP exposure and the involvement of apoptosis, autophagy, and immunity remains unknown. In the current investigation, 27.8 μg/mL DiBP or/and 20 μM Euc has been applied to Ctenopharyngodon idellus kidney (CIK) cells for 24 h. The findings showed that exposure to DiBP raised intracellular ROS levels, inducing oxidative stress, and enhanced the rate of apoptosis as well as the expression of the apoptotic markers Bax, Caspase3, Caspase9, and Cytc while decreasing the expression of Bcl-2. Furthermore, DiBP inhibited IL-2, IFN-γ, Hepcidin-1, and β-defensin expression and elevated TNF-α, and IL-1β levels, causing immune dysfunction. DiBP and Euc co-treatment significantly activated the Keap1/Nrf2/HO-1 pathway, restored antioxidant enzyme activity, and elevated autophagy pathway-associated genes ATG5, Beclin1, and LC3B decreased p62 expression, enhanced cell autophagy, reduced apoptosis, and improved immunity. In conclusion, Euc promotes autophagy, alleviates DiBP-induced apoptosis, and improves immunological dysfunction in CIK cells by regulating the Keap1/Nrf2/HO-1 pathway. These results demonstrated the threat of DiBP exposure to fish while providing a theoretical foundation for using Euc in aquaculture.