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Difenoconazole (DFZ) is a classical triazole fungicide that causes immunosuppression in non-target organisms. Ferulic acid (FA) is a polyphenolic molecule found in nature that has antioxidant and anti-inflammatory activities. The purpose of this investigation was to see if FA could prevent DFZ-induced immunosuppression and to identify the potential mechanisms. Carp were exposed to 1/10 LC50 of DFZ as well as fed normal feed or feed containing dietary additive FA for 30 d. It was found that DFZ-induced immunosuppression could be improved by FA, as evidenced by upregulation of Hb, C3 and IgM and downregulation of LDH. It was then investigated that FA could ameliorate DFZ-induced splenic injury through p53-mediated apoptosis. At the same time, enhancing the levels of CAT, GSH and T-AOC in spleen and transcription levels Nrf2 signaling pathway related genes indicated that FA reduced oxidative damage caused by DFZ by blocking the Nrf2 signaling pathway. In addition, FA inhibited the inflammatory response triggered by TRAF/TAK1/NF-κB signaling pathway, downregulated the transcript levels of pro-inflammatory factors (il-1β, tnf-α, il-6) and the level of NLRP3 inflammasome (NRLP3, ASC, Caspase 1), and upregulated the transcript levels of anti-inflammatory factors (tgf-β1, il-10). In conclusion, the above results suggested that FA mediated TRAF/TAK1/NF-κB, Nrf2, and p53 pathways to attenuate DFZ-induced inflammation, oxidative stress, and apoptosis thereby enhancing the immune capacity of carp.

Excessive use of hard-to-degrade pesticides threatens the ecological health of aquatic systems. This study aimed to investigate difenoconazole (DFZ) residues in the environment induced neurotoxicity in carp and the underlying mechanisms. A total of thirty-six carps were divided into three groups and exposed to 0, 0.5, and 2.0 mg/L DFZ for 96 h, respectively. The alterations in behavior and blood-brain barrier (BBB) were examined, and potential mechanisms were explored using immunological assays and biochemical methods. The results showed that DFZ exposure caused behavioral freezing, reduced feeding, and neuronal necrosis in carp. Mechanistically, DFZ triggered ROS accumulation and destroyed the balance between oxidation and antioxidation with increased lipid peroxidation product MDA contents and reduced antioxidant enzymes SOD and CAT activities in the carp brain by inhibiting the NF-E2-related factor 2 (Nrf2) pathway. The activation of oxidative stress further reduced tight junction proteins and MMP levels, thereby destroying BBB and leading to DFZ leakage into the brain. Increased BBB permeability additionally led to DFZ activation of nuclear factor kappa-B signaling-mediated inflammatory cytokine storm, exacerbating neuroinflammation. Meanwhile, DFZ exposure activated mitochondria-associated apoptosis in the carp’s brain by up-regulating Bcl-2 associated X protein, cleaved-caspase3, and cytochrome C and decreasing B-cell lymphoma-2 levels. Interestingly, the carp’s brain initiated a protective autophagic response via the PI3K/AKT/TOR pathway intending to counteract the neurotoxicity of DFZ. Overall, we concluded that accumulation of DFZ at high concentrations in the aquatic systems disrupted the BBB and resulted in neurotoxicity in carp through inhibition of Nrf2 pathway-mediated ROS accumulation. This study provides a reference for monitoring DFZ residues in the environment and a new target for the treatment of DFZ-induced neurotoxicity in carp.