Glutaredoxin-1 modulates the NF-κB signaling pathway to activate inducible nitric oxide synthase in experimental necrotizing enterocolitis

Abstract

Inducible nitric oxide synthase (iNOS), regulated by nuclear factor kappa B (NF-κB), is crucial for intestinal inflammation and barrier injury in the progression of necrotizing enterocolitis (NEC). The NF-κB pathway is inhibited by S-glutathionylation of inhibitory κB kinase β (IKKβ), which can be restored by glutaredoxin-1 (Grx1). Thus, we aim to explore the role of Grx1 in experimental NEC. Wild-type (WT) and Grx1-knockout (Grx1^-/-) mice were treated with an NEC-inducing regimen. Primary intestinal epithelial cells (IECs) were subjected to LPS treatment. The production of iNOS, NO, and inflammation injuries were assessed. NF-κB and involved signaling pathways were also explored. The severity of NEC was attenuated in Grx1^-/- mice. Grx1 ablation promoted IKKβ glutathionylation, NF-κB inactivation, and decreased iNOS, NO, and O₂^·- production in NEC mice. Furthermore, Grx1 ablation restrained proinflammatory cytokines and cell apoptosis, ameliorated intestinal barrier damage, and promoted proliferation in NEC mice. Grx1 ablation protected NEC through iNOS and NO inhibition, which related to S-glutathionylation of IKKβ to inhibit NF-κB signaling. Grx1-related signaling pathways provide a new therapeutic target for NEC.

Keywords: NF-κB; S-glutathionylation; glutaredoxin-1; inducible nitric oxide synthase; necrotizing enterocolitis; oxidative stress.

Graphical abstract

Figure 1

Evaluation of iNOS, NO and O₂^·– (A) iNOS expression of intestines were analyzed by western blot. Bottom: densitometry analysis of iNOS normalized by β-actin. ∗∗∗∗p < 0.0001 vs. WT/dwarf (DF); ####p < 0.01 vs. WT/NEC. (B and C) NO (B) and O₂^·– (C) production was evaluated in intestinal tissue between the groups. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. WT/DF mice; ###p < 0.001, ####p < 0.0001 vs. WT/NEC. (D) NO production was assessed in the IECs. (E) Western blot was performed for iNOS expression assessment on IECs, Bottom: densitometry analysis of iNOS normalized by β-actin. ∗∗∗∗p < 0.0001 vs. saline; ##p < 0.01 vs. LPS. (F) Representative fluorescence micrographs with DHE labeling for intracellular ROS detection. Two-sided 1-way ANOVA was used for data comparison with post hoc Tukey test (n = 6–8 mice per group; data are given as means ± SEMs).

Figure 2

Proinflammatory cytokines evaluation in NEC mice (A–C) iNOS, TLR4, TNF-α, and IL-6 mRNA were detected in intestinal tissue (A), IECs (B), and peritoneal macrophages (C) by RT-PCR in different groups. Two-sided 1-way ANOVA was used for data comparison with post hoc Tukey test (n = 6–8 mice per group; data are presented as means ± SEMs). ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. WT/DF mice; ##p < 0.01, ###p < 0.001 vs. WT/NEC; ˆˆp < 0.01,ˆˆˆp < 0.001, ˆˆˆˆp < 0.0001 vs. Grx1^−/−/NEC.

Figure 3

Grx1 regulates IEC proliferation and migration (A) Representative photomicrographs of BrdU immunostaining of the intestine. Scale bar: 100 μm. (B) IEC movement rate (measuring the distance from the bottom of the crypt to the foremost labeled enterocyte [FLE], with the migration rate [lm/h] calculated as FLE/18). (C) IEC movement (characterized as FLE/complete thickness of the mucosa × 100%). (D) IEC multiplication (characterized as BrdU⁺ cells/high-power field). n = 6 animals per group, 6 fields/animal. For data comparison, 2-sided 1-way ANOVA was used with post hoc Tukey test (n = 6–8 mice per group; data are presented as means ± SEMs). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. WT/DF mice; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. WT/NEC; ˆˆp < 0.01 vs. Grx1^−/−/NEC.

Figure 4

Apoptosis and intestinal barrier injury evaluation (A) Immunoblotting analysis of caspase-3 and cleaved caspase-3 proteins in intestine. Right: densitometry analysis of left images. (B) SIgA was estimated from the terminal ileum. (C) The centralization of β-defensin 2 was estimated in the distal ileum. (D) Serum FITC-dextran concentrations in groups were detected. (E) MPO activity was assessed in ileum. (F–H) Bacterial growth was quantified in mesenteric lymph nodes (F), liver (G), and spleen (H) of mice. The data represented 3 independent experiments. For data comparison, 2-sided 1-way ANOVA was used with post hoc Tukey test (n = 6–8 mice per group were adopted; data are presented as means ± SEMs). ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. WT/DF mice; ##p < 0.01, ###p < 0.001 vs. WT/NEC; ˆˆp < 0.01,ˆˆˆp < 0.001, ˆˆˆˆp < 0.0001 vs. Grx1^−/−/NEC.

Figure 5

Grx1 deficiency decreases the severity of experimental NEC (A) Weight changes among different groups. (B) Survival curves in different groups. (C) Severity scores were computed based on morphological changes. (D) Morphology of the ileum was shown. (E) Represent SYTOX Green staining was shown between groups. (F) Groups were compared in terms of necrotic cells. A 2-sided 1-way ANOVA with a Tukey post hoc test (n = 10–15 mice per group; data are presented as means ± SEMs). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 vs. WT/DF mice; #p < 0.05, ##p < 0.01, ####p < 0.0001 vs. WT/NEC mice.

Figure 6

Assessment of NF-κB activation (A–C) Levels of GSH (A), GSSG (B), and the GSSG/GSH ratio (C) in the intestinal homogenates. (D) Western blot analysis of S-glutathionylation of intestinal tissue proteins in different groups. (E) Top: immunoprecipitation (IP) analysis of S-glutathionylation of IKKβ in intestinal tissue lysates; bottom: western blot analysis of whole-cell lysates (WCLs) for total IKKβ, IκBα, and phosphorylated RelA. (F) A western blot assay was used to analyze RelA and p50 proteins in intestines; bottom: densitometry analysis of RelA and p50 normalized by β-actin. (G and H) Evaluation of CCL-20 (G) and GM-CSF (H) concentrations in intestinal tissues using ELISA. For data comparison, we used a post hoc Tukey test with 2-sided 1-way ANOVA (n = 6–8 mice per group; data are presented as means ± SEMs). ∗∗∗∗p < 0.0001 vs. WT/DF mice; ##p < 0.01, ###p < 0.001 vs. WT/NEC mice. ∗p < 0.01, ∗∗p < 0.01, ####p < 0.001 vs. WT/NEC.