Objective:To evaluate the role of Z-DNA-binding protein 1 (ZBP1)/receptor-interacting protein kinase 1 (RIPK1) signaling pathway in lipopolysaccharide (LPS)-adenosine triphosphate (ATP)-induced pyroptosis in macrophages of mice.Methods:The RAW264.7 macrophages from mice were routinely cultured and divided into 6 groups ( n=9 each) using a random number table method: control group (group C), LPS-ATP group, LPS-ATP+ transfection negative control scRNA group (group LPS-ATP+ scRNA), LPS-ATP+ ZBP1 small interference RNA group (group LPS-ATP+ siRNA), LPS-ATP+ dimethyl sulfoxide group (group LPS-ATP+ DSMO), and LPS-ATP+ RIPK1 inhibitor nec-1 group (group LPS-ATP+ nec-1). The siRNA technique was used to inhibit the expression of ZBP1 in group LPS-ATP+ siRNA. The RIPK1 inhibitor nec-1 was given to inhibit the expression of RIPK1 protein in group LPS-ATP+ nec-1. Group C was routinely cultured. Cells were incubated with 10 μg/ml LPS for 24 h, then 5 mmol/L ATP was added, and the cells were incubated for 30 min to develop the cell pyroptosis model in the remaining 5 groups. The cell survival was detected by the CCK-8 assay. The concentrations of interleukin-1beta (IL-1β), IL-6, IL-18 and tumor necrosis factor-alpha (TNF-α) in cell supernatant were determined by enzyme-linked immunosorbent assay. The pyroptosis was determined by propidium iodide fluorescence staining. Western blot was used to detect the expression of ZBP1, RIPK1, caspase-1 and GSDMD. Results:Compared with group C, the cell survival rate was significantly decreased, the cell pyroptosis rate and concentrations of IL-1β, IL-6, IL-18 and TNF-α in the supernatant were increased, and the expression of ZBP1, RIPK1, caspase-1 and GSDMD was up-regulated in group LPS-ATP ( P<0.05). Compared with group LPS-ATP, no significant change was found in the parameters mentioned above in group LPS-ATP+ scRNA and group LPS-ATP+ DSMO ( P>0.05). Compared with group LPS-ATP+ scRNA, the cell survival rate was significantly increased, the cell pyroptosis rate and concentrations of IL-1β, IL-6, IL-18 and TNF-α in the supernatant were decreased, and the expression of ZBP1, RIPK1, caspase-1 and GSDMD was down-regulated in group LPS-ATP+ siRNA ( P<0.05). Compared with group LPS-ATP+ DMSO, the cell survival rate was significantly increased, the cell pyroptosis rate and concentrations of IL-1β, IL-6, IL-18 and TNF-α in the supernatant were decreased, the expression of ZBP1, caspase-1 and GSDMD was down-regulated ( P<0.05), and no significant change was found in the expression of ZBP1 in group LPS-ATP+ nec-1 ( P>0.05). Conclusions:Activation of ZBP1/RIPK1 signaling pathway is involved in LPS-ATP-induced pyroptosis in macrophages of mice.