Fig 1: IA suppresses STAT1 signaling through AhR-mediated SOCS5 upregulation(A) Molecular docking analysis of the interaction between IA and AhR.(B) Workflow diagram of the biotin-streptavidin pull-down assay.(C) Immunoblotting detection of AhR protein pulled down by IA.(D) Western blot analysis of p-STAT1 and STAT1 in the ileum of IA-treated NEC mice (n = 6).(E) IF staining of p-STAT1 demonstrating the effect of AhR deficiency on STAT1 activation. The nuclei were counterstained with DAPI (blue). Scale bars: 50 μm.(F) qPCR detection of Socs family mRNA levels in the ileum of IA-treated NEC mice (n = 6).(G) Schematic representation of the Socs5 promoter region.(H) Dual-luciferase reporter assay evaluating the effect of AhR on Socs5 promoter activity (n = 6).(I) IF staining of SOCS5 showing the effect of AhR deficiency on SOCS5 expression. The nuclei were counterstained with DAPI (blue) (n = 6). Scale bars: 50 μm.(J and K) CoIP assay assessing the interaction between STAT1 and SOCS5. Data from at least three independent experiments were expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to the control group; one-way analysis of variance followed by a Tukey’s post hoc test.
Fig 2: Intestinal-epithelial-cell-specific AhR deficiency abolishes the protective effects of IA on necroptosis and NECWT and AhRΔIEC mice were orally administered IA (50 mg/kg) during postnatal days 7–10, followed by TNBS challenge (50 mg/kg) on day 10 for NEC induction.(A) Survival rate of IA-treated mice (n = 12).(B) Body weight dynamics in IA-treated mice (n = 6).(C) DAI of mice recorded during IA treatment in NEC mice, based on body weight change and fecal viscosity (n = 6).(D) Ileal length measurements at 12 h post-TNBS administration in IA-treated mice (n = 6).(E) Representative photomicrographs illustrating H&E-stained ileum sections and histological scores of NEC mice at 12 h post-TNBS administration and treated with IA (n = 6). Scale bars: 50 μm.(F) Western blot analysis of p-RIPK3 and p-MLKL levels in ileum of mice NEC treated with IA. Quantification was preformed using ImageJ software (n = 6). Scale bars: 50 μm.(G) Double IF staining was performed to visualize p-RIPK3 (red) and FABP2 (green) in the ileum of NEC mice treated with IA. The nuclei were counterstained with DAPI (blue) (n = 6). Data were expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to the control group; one-way analysis of variance followed by a Tukey’s post hoc test.
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