Allosteric regulation of Keap1 by 8 β-hydroxy- α-cyclocostunolide for the treatment of acute lung injury

Abstract

Image 1.

Keywords: 8β-Hydroxy-α-cyclocostunolide; Acute lung injury; Keap1; Macrophage overactivation; Nrf2.

Graphical abstract

Figure 1

Keap1 served as a cellular direct target of HCC. (A) The synthesis of the biotinylated probe Bio-HCC and the schematic diagram of the biotin-streptavidin strategy and silver staining plot; (B) Pull down result detected by Western blot; (C) CETSA plot; (D) DARTS plot; (E) MST result of HCC and Keap1; (F) Immunofluorescence co-localization of HCC with Keap1; (G) IAA abolished the interaction of HCC with Keap1; (H) LC–MS/MS plot after the incubation of HCC with Keap1; (I) Molecular docking result; (J) Cys257Ala and Cys288Ala mutations weakened the binding of HCC with Keap1; (K) HCC led to the conformational change of Keap1 through the fluorescence titration experiment; (L) Co-IP result of Keap1 with Nrf2; (M) HCC inhibited Keap1-mediated Nrf2 ubiquitination; (N) HCC promoted the nuclear translocation of Nrf2.

Figure 2

Keap1 genetic KO abolished the pulmonary protective effects of HCC in vivo. (A) The construction of Keap1 KO mice; (B, C) Genotyping and confirmation of Keap1 KO mice; (D) Schematic diagram of LPS-mediated ALI in Keap1^+/+ and Keap1^+/− mice; (E) Representative H&E plot; (F) Representative CD68 staining plot; (G) Keap1 genetic KO abolished effects of HCC on MPO, TNF-α, IL-6, SOD, and GSH (mean ± SEM, n = 5).