Copper Induces Hepatic Pyroptosis In Chicken Liver Through ROS Mediated Caspase-1 Pathway

In order to investigate whether copper can induce hepatocyte pyroptosis and its mechanism,the primary cultured chicken hepatocytes were used to treat cells with different concentrations of copper sulfate（0,10,50,100μmol/L）for 24 h.CCK-8 assay was used to detect the relative vitality of hepatocytes.Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of caspase-1,IL-1β,IL-18,and NLRP3associated with cell death,using the active oxygen scavenger N-acetylcysteine.Nitric acid（NAC）interacts with copper in hepatocytes to observe changes in the above four indicators.Enzyme-linked immunosorbent assay（ELISA）was used to determine the levels of the pyroptosis-associated proteins caspase-1,IL-1β,and IL-18 in the supernatants of the cells,and the caspase-1 inhibitor Z-YVAD-FMK was used together with copper to treat the hepatocytes.After observing the changes of the above three indicators,the biochemical assays for the activity of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,and lactate dehydrogenase（LDH）in culture supernatants can be based on this.Assessment the extent of liver cell damage was examined and the effect of reactive oxygen species scavenger on hepatocyte pyroptosis was explored.Finally,real-time fluorescence quantitative PCR was used to detect apoptosis-related genes Bax,caspase-3,CytC,and P53,and to study the effects of inhibition of pyroptosis on apoptosis.The results show:（1）Copper-induced pyroptosis-related gene caspase-1,IL-1β,IL-18 mRNA expression was significantly upregulated（P<0.01）,and the related protein content was also significantly increased（P<0.01）.（2）The combination of NAC and Cu could significantly downregulate the expression of hepatocyte pyroptosis-associated genes（P<0.01）and reduce the related protein content（P<0.01）.（3）Compared with the cell viability of 100μmol/L Cu²⁺group,the relative activity of copper（100μmol/L）combined with YVAD on hepatocytes was significantly higher（P<0.01）.Compared with normal cells in the control group,the contents of ALT,AST,and LDH in the hepatocyte supernatant of the 100μmol/L Cu²⁺group were significantly increased（P<0.01）.The combination of YVAD and Cu could significantly downregulate the expression of caspase-1 in hepatocyte pyroptosis-associated gene（P<0.01）and reduce the related protein content（P<0.01）.（4）Compared with the control group,the mRNA expression levels of the apoptosis-related genes Bax,caspase-3,CytC,and P53 in the 100μmol/L Cu²⁺group were significantly different（P<0.01）.The mRNA expression of 100μmol/L Cu²⁺plus YVAD group was relative.The amount decreased and the difference was extremely significant（P<0.01）.Conclusion:Copper can induce hepatocyte pyroptosis,and the occurrence of death may be mediated through ROS.YVAD can inhibit the damage of copper to hepatocytes.Inhibition of hepatocyte pyroptosis can reduce the expression level of apoptosis-related gene mRNA.