Genistein inhibits the release of pro-inflammatory substances from macrophages by suppressing potassium loss- and ROS-mediated caspase-1/gasdermin D pathway activation and pyroptotic cell lysis.

Objectives: The expression of pro-inflammatory substances is closely related to various diseases. Genistein (GEN), a soy isoflavone, has been proven to inhibit the production of pro-inflammatory substances in macrophages. This study aimed to determine whether GEN exerts its inhibitory effect on the expression of pro-inflammatory substances by suppressing the release of these substances via attenuating pyroptotic cell lysis.

Materials And Methods: Mice were treated with lipopolysaccharide (LPS) and GEN. J774A.1 cells were treated with LPS, adenosine triphosphate (ATP), and GEN. The expression of pro-inflammatory cytokines and high mobility group box 1 (HMGB1) was measured by qRT-PCR and ELISA. The activation of caspase-1 (CASP1) and cleavage of gasdermin D (GSDMD) were determined by Western blot assay. Lactic dehydrogenase (LDH) assay and CCK8 assay were performed to determine the integrity of the cell membrane and cell viability. The concentration of intracellular potassium (K) and the production of reactive oxygen species (ROS) were determined by the colorimetric method and flow cytometry, respectively.

Results: GEN inhibited the production of IL-1β and HMGB1 in LPS-challenged mice and LPS+ATP-treated mouse macrophages by inhibiting GSDMD-mediated pyroptotic cell lysis. Mechanistically, GEN could prevent the loss of intracellular K and the production of ROS caused by LPS+ATP treatment, thereby inhibiting the activation of CASP1. The pathological significance of the release of HMGB1 could be partially attributed to its ability to induce cell apoptosis.

Conclusion: GEN inhibits CASP1/GSDMD-mediated pyroptotic cell lysis and the following release of pro-inflammatory substances by suppressing K loss and ROS production of macrophages.