The primers used in this study.

Pyroptosis is linked to the development of acute lung injury (ALI), and circular RNAs (circRNAs) play a role in ALI-related inflammation. However, the mechanisms by which circRNAs contribute to macrophage pyroptosis in ALI remain unclear. This study constructed an in vitro ALI model by inducing THP-1 cells with phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). The expression and potential mechanism of circ-CARD8 in macrophage pyroptosis were then investigated. The interaction between circ-CARD8, hsa-miR-580-3p, and caspase recruitment domain family member 8 (CARD8) was confirmed through luciferase reporter assays and RNA-binding protein immunoprecipitation. Our data showed that circ-CARD8 was expressed at low levels. Meanwhile, the pyroptotic proteins caspase-1 and GSDMD, along with the secretion of chemokine (C-C motif) ligand 18 and interleukin 1 beta, were upregulated in the ALI cell model. Overexpression of circ-CARD8 reversed macrophage pyroptosis, whereas inhibition of circ-CARD8 promoted it. Furthermore, the expression of miR-580-3p, a downstream microRNA that binds to circ-CARD8, was reduced upon circ-CARD8 overexpression and increased following its inhibition. Additionally, overexpression of miR-580-3p suppressed the expression of CARD8, a downstream target of miR-580-3p, thereby promoting macrophage pyroptosis. The inhibition of miR-580-3p reversed the effect of circ-CARD8 silencing on macrophage pyroptosis and CARD8 expression. Therefore, our study confirms that the low expression of circ-CARD8 reduces the sponge adsorption of miR-580-3p, increasing its expression, which in turn targets and inhibits CARD8, ultimately promoting macrophage pyroptosis induced by LPS in THP-1 cells.