Helenine blocks NLRP3 activation by disrupting the NEK7-NLRP3 interaction and ameliorates inflammatory diseases

The involvement of NLRP3 inflammasome is associated with the progress of numerous inflammatory conditions. However, there is currently no single compound used in the clinic. Search for the inhibitor of NLRP3 inflammasome from natural products is an attractive direction. The compound Helenin (Hel), which is obtained from Inula helenium L., is reported to have anti-inflammatory activities. However, the underlying molecular mechanisms and specific inflammatory signal pathway remains not well understood. This research aims to determine the impacts of Hel on NLRP3 inflammasome and the underlying mechanism involved, meanwhile also assessing its potential as a therapeutic intervention for inflammatory diseases mediated by NLRP3 overactivation. Pretreated with Hel in BMDMs (bone marrow-derived macrophages), then stimulated with NLRP3 triggers and measured the expression of active caspase-1 and interleukin 1β (IL-1β). Determination of intracellular K+ and Ca2+, ASC oligomerization and mitochondrial reactive oxygen species (mtROS) production were employed to explore the preliminary mechanism of Hel on NLRP3 activation. Subsequently, Co-immunoprecipitation was used to investigate protein-protein interaction and reduction of covalent bonds of Hel was to explore the binding mode between drugs and proteins. Finally, in vivo experiments, we utilized mouse lethal sepsis and monosodium urate(MSU)-induced peritonitis models to evaluate the effectiveness of Hel in inhibiting inflammatory diseases. The findings revealed that Hel exhibited a specific blocking effect on NLRP3, with no impact on the assembly of NLRC4 and AIM2 inflammasome. Through the analysis of mechanisms targeting key upstream factors in NLRP3 activation, Hel inhibited NLRP3-dependent ASC oligomerization but did not regulating inflammasome priming, K+ efflux, Ca2+ influx, or mitochondrial damage and mtROS. Moreover, Hel effectively interrupted the binding of NEK7-NLRP3, which was dependent on the active double C=C of the α,β-unsaturated carbonyl units in Hel. In mouse models, Hel showed promising therapeutic effects in the treatment of NLRP3 overactivation-associated diseases, including the lethal sepsis and acute systemic inflammation induced by lipopolysaccharide (LPS) and peritonitis induced by MSU. Our results indicate that Hel dependent α,β-unsaturated carbonyl units interrupt the formation of the NLRP3-NEK7 interaction, thereby blocks the inflammasome assemblage and activation. These fundings would suggest that Hel is a promising inhibitor for treating diseases driven by NLRP3 overactivation.