DHA Alleviates Excessive Copper-Induced Hepatocyte Injury by Inhibiting Pyroptosis via the ROS/NLRP3/NF-κB Pathway.

The environmental accumulation of copper (Cu), mainly driven by industrial and agricultural activities, has raised increasing concerns regarding its toxic effects on non-target organisms and its transmission through the food chain. This study explored whether docosahexaenoic acid (DHA, 10 μM), a major ω-3 polyunsaturated fatty acid, could attenuate copper (Cu, 10 μM)-induced hepatocellular injury. The underlying protective mechanisms were systematically investigated using AML12 mouse hepatocytes in vitro. Oxidative damage and cellular injury were assessed through reactive oxygen species (ROS) staining and biochemical assays, including measurements of key antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The activation of inflammasome components, notably NOD-like receptor family pyrin domain containing 3 (NLRP3), and downstream pyroptotic markers such as gasdermin D (GSDMD), were also examined to elucidate the hepatoprotective effects of DHA. These molecular and cellular changes were further validated by quantitative real-time PCR, Western blotting, and immunofluorescence assays, confirming the suppression of copper (Cu)-induced redox imbalance, inflammasome activation, pyroptosis, and inflammatory signaling pathways. Molecular docking analysis revealed strong binding affinities of DHA to both NLRP3 and GSDMD, supporting its dual regulatory potential in modulating inflammasome-mediated inflammation and pyroptotic cell death. This in silico evidence complements the experimental findings, collectively identifying DHA as a promising nutritional intervention to counteract Cu-induced liver toxicity through coordinated inhibition of oxidative stress, pyroptosis, and inflammation.