Convallatoxin inhibits IL-1 beta production by suppressing zinc finger protein 91 (ZFP91)-mediated pro-IL-1 beta ubiquitination and caspase-8 inflammasome activity

Background and Purpose: ZFP91 positively regulates IL-1 beta production in macrophages and may be a potential therapeutic target to treat inflammatory-related diseases. We investigated whether this process is modulated by convallatoxin, which is a cardiac glycoside isolated from the traditional Chinese medicinal plant Adonis amurensis Regel et Radde. Experimental Approach: In vitro, the mechanisms by which convallatoxin inhibits ZFP91-regulated IL-1 beta expression were investigated using molecular docking, western blotting, RT-PCR, ELISA, immunofluorescence and immunoprecipitation assays.In vivo, mice liver injury was induced by an intraperitoneal injection of D-GalN and LPS, colitis was induced by oral administration of dextran sulfate sodium (DSS) in drinking water and peritonitis was induced by an intraperitoneal injection of alum. Key Results: We confirmed that convallatoxin inhibited the release of IL-1 beta by down-regulating ZFP91. Importantly, we found that convallatoxin significantly reduced K63-linked polyubiquitination of pro-IL-1 beta regulated by ZFP91 and decreased the efficacy of pro-IL-1 beta cleavage. Moreover, convallatoxin suppressed ZFP91-mediated activation of the non-canonical cysteine-requiring aspartate protease-8 (caspase-8) inflammasome and MAPK signalling pathways in macrophages. Furthermore, we showed that ZFP91 promoted the assembly of the caspase-8 inflammasome complex, whereas convallatoxin treatment reversed this result. Mice in vivo studies further demonstrated that convallatoxin ameliorated D-GalN/LPS-induced liver injury, DSS-induced colitis and alum-induced peritonitis by down-regulating ZFP91. Conclusion and Implications: We show for the first time that convallatoxin-mediated inhibition of ZFP91 is an important regulatory event that prevents inappropriate inflammatory responses to maintain immune homeostasis. This mechanism provides new insight for the development of convallatoxin as a novel anti-inflammatory drug targeting ZFP91.