Copper Exposure Promotes Mitochondrial VDAC Oligomerization and Releases mtDNA to Induce Pyroptosis in Pig Hepatocytes

Copper (Cu) is widely used as a growth-promoting trace element in swine feed, but excessive Cu causes liver toxicity and modulates innate immunity, with unclear molecular mechanisms. In this study, dietary Cu overload in pigs led to Cu accumulation and liver injury, elevated levels of reactive oxygen species (ROS), and decreased mitochondrial membrane potential (MMP). These changes were accompanied by increased VDAC oligomerization in the mitochondrial membrane and mitochondrial DNA (mtDNA) release, which activates the NLRP3 inflammasome and triggers hepatocyte pyroptosis. Mechanistically, the VDAC inhibitor VBIT-4 reduced Cu-induced VDAC oligomerization and mtDNA release. Furthermore, NLRP3 is essential for Cu-mediated pyroptosis. Combined VBIT-4 and the MCC950 (NLRP3 inhibitor) treatment further attenuated VDAC oligomerization, mtDNA release, and pyroptosis. Collectively, our results reveal that Cu exposure promotes VDAC oligomerization and mitochondrial membrane pore formation, leading to mtDNA release, NLRP3 inflammasome activation, and pyroptosis in hepatocytes. These findings provide new insights into Cu-induced hepatotoxicity.