Dehydroevodiamine targeting IKKβ to alleviate acute gastric injury via inhibiting the p65/NLRP3 axis

Abstract

BackgroundAcute gastric injury, a common and recurring global digestive disorder, significantly impairs patient quality of life and overall health. Dehydroevodiamine (DHE), a bioactive natural product derived from Tetradium ruticarpum (A. Juss.) Hartley, shows potential therapeutic effects on acute gastric injury. This study investigates the underlying mechanisms of DHE's alleviating effects on acute gastric injury. MethodsThe gastric mucosal protective effect of DHE was confirmed through in vivo and in vitro acute gastric injury models. Biotin pulldown MS and molecular dynamics simulations identified DHE's target. CETSA and SPR assays validated DHE's affinity for IKKβ. Protein site mutation validation and MST pinpointed the direct binding sites of DHE on IKKβ. Additionally, the potential mechanism by which DHE ameliorates acute gastric injury was elucidated using WB, IHC, and IF methods, and further confirmed by rescue experiments. ResultsDHE effectively ameliorated IDO-induced gastric injury in GES-1 cells and rat gastric mucosa, both in vitro and in vivo. Biotin pulldown MS identified IKKβ as the target of DHE in alleviating gastric injury. CETSA and SPR assays confirmed DHE's direct binding to IKKβ. Molecular dynamics simulations, protein mutation experiments, and MST results pinpointed GLU-149, GLU-49, and ASP-103 in the ATP-binding pocket as the binding sites of DHE on IKKβ. Notably, DHE was found to competitively bind to IKKβ with ATP. Mechanistically, DHE attenuated IDO-induced gastric injury by inhibiting the IKKβ-p65/NLRP3 signaling pathway. Importantly, exogenous activation of IKKβ reversed the therapeutic effect of DHE, indicating that DHE's efficacy depends on IKKβ. ConclusionDHE attenuated IDO-induced gastric injury by inhibiting the IKKβ-p65/NLRP3 signaling pathway. Notably, DHE is a novel ATP-competitive IKKβ inhibitor that prevents phosphorylation by targeting GLU-149, GLU-49, and ASP-103 in the ATP-binding pocket. This study reveals new targets of action for DHE, providing a new molecular basis for using DHE in treating inflammation-related diseases.