Inhibition of Caspase-1-dependent pyroptosis attenuates copper-induced apoptosis in chicken hepatocytes

The purpose of this study was to investigate the effects of copper (Cu) on hepatocyte pyroptosis and the relationship between pyroptosis and apoptosis in the mechanisms of Cu toxicity. Primary chicken hepatocytes were cultured in different concentrations of Cu sulfate (CuSO₄) (0, 10, 50, and 100 μM), N-acetylcysteine (NAC) (1 mM), and Z-YVAD-fluoromethylketone (Z-YVAD-FMK) (10 μM) for 24 h, and the combination of Cu and NAC or Z-YVAD-FMK for 24 h. Cellular morphology and function, cell viability, mitochondria membrane potential (MMP), apoptosis rate, mRNA expression of pyroptosis-related and apoptosis-related genes, and Caspase-1, Caspase-3 proteins expression were determined. These results indicated that Cu markedly induced the mRNA expression of pyroptosis-related genes (Caspase-1, IL-1β, IL-18, and NLRP3) and Caspase-1 protein expression. Furthermore, contents of Caspase-1, IL-1β, and IL-18 in the supernatant fluid of culture hepatocytes were significantly increased in hepatocytes. NAC relieved excess Cu-caused the changes of above genes and proteins. Additionally, Z-YVAD-FMK, caspase-1 inhibitor, which attenuated Cu-induced the increased lactic dehydrogenase (LDH), aspartate amino transferase (AST), alanine aminotransferase (ALT) activities. Furthermore, treatment with Cu and Z-YVAD-FMK could down-regulate the mRNA levels of Caspase-3, Bak1, Bax, and CytC and Caspase-3 protein expression, up-regulate the mRNA expression of Bcl2, increase the MMP and reduce cell apoptosis compared to treatment with Cu in hepatocytes. Collectively, these finding evidenced that excess Cu induced pyroptosis by generating ROS in hepatocytes, and the inhibition of Caspase-1-dependent pyroptosis might attenuate Cu-induced apoptosis.