Curcumin attenuates combined DON+AFB1 induced apoptosis via Nrf2-associated signaling in porcine alveolar macrophages 3D4/21 cell line: an in vitro study

Deoxynivalenol (DON) and Aflatoxin B1 (AFB1) are among the most prevalent mycotoxin contaminants of grain based feed and frequently co-occur, yet most evidence on their combined toxicity comes from hepatic, intestinal, or lymphoid models. Alveolar macrophages are among the first immune cells to encounter mycotoxins carried on inhaled feed dust in swine housing, this respiratory exposure route is toxicologically distinct from oral uptake and has not been characterized DON+AFB1 combination. In the present in vitro study, the porcine alveolar macrophages (PAMs) cell line 3D4/21 was chosen as a model and treated by various combined concentrations of DON+AFB1. Co-exposure induced cytotoxicity in a dose-dependent manner, as evidenced by reduced cell viability, elevated LDH release, a suppressed Bcl-2/Bax ratio, and increased intracellular ROS accumulation. These changes are consistent with activation of the intrinsic apoptotic pathway. Curcumin, a natural product widely used in traditional Chinese medicine and known for its antioxidant properties, increased cell viability, reduced LDH release and ROS accumulation, and reversed the apoptotic shift in Bcl-2/Bax/Cytochrome c and Caspase-3 expression. DON+AFB1 co-exposure also suppressed the Nrf2/HO-1/NQO1 antioxidant axis, whereas curcumin restored Nrf2, HO-1, and NQO1 expression at both mRNA and protein levels. Pharmacological inhibition of Nrf2 with ML385 reduced the protective effects of curcumin by preventing restoration of the antioxidant gene expression and elevating apoptotic markers. These findings indicate that Nrf2/HO-1 signaling is associated with the protective effects of curcumin against DON+AFB1 induced oxidative stress and apoptotic changes in PAMs and support further evaluation of curcumin as a candidate intervention against inhalation mycotoxin co-exposure in swine production environments.