Lumefantrine ameliorates DSS-induced colitis by targeting FLI-1 to suppress NF-κB signaling

Abstract

Background: Current therapeutic options for inflammatory bowel disease (IBD) remain suboptimal due to limited efficacy, significant side effects, and high relapse rates, necessitating novel treatment strategies. Lumefantrine, a clinically established antimalarial drug, emerges as a compelling repurposing candidate based on its putative anti-inflammatory activity, though its efficacy and mechanism in IBD remain unexplored.

Methods: A murine IBD model was induced by 3% dextran sulfate sodium (DSS). Mice received oral Lumefantrine (20 mg/kg/day) for 7 days. Disease progression was monitored via disease activity index (DAI) scoring and histological analysis. Serum cytokines (IL-1β, IL-6, TNF-α) and colonic inflammatory mediators (Cox-2, iNos) were quantified by ELISA and qPCR. Tight junction proteins (Claudin-1, ZO-1) were assessed by immunohistochemistry and Western blot. Molecular targets were identified through computational docking and pull-down assays. Additionally, NF-κB signaling modulation was assessed in lipopolysaccharide (LPS)-stimulated intestinal epithelial cells (IEC-6 and NCM460) via Western blot analysis.

Results: Oral administration of Lumefantrine significantly attenuated disease activity index (DAI) scores and restored intestinal barrier integrity through upregulation of epithelial tight junction proteins Claudin-1 and ZO-1. Treated mice exhibited reduced serum levels of IL-1β, IL-6 and TNF-α, along with decreased colonic expression of inflammatory mediators cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNos). Computational and experimental approaches identified FLI-1 a transcription factor upregulated in IBD colon tissues as Lumefantrine's direct binding target. This interaction mediated suppression of NF-κB signaling, specifically downregulating phosphorylation of IκBα and p65 in LPS-stimulated intestinal epithelial cells.

Conclusion: Lumefantrine ameliorates experimental colitis through FLI-1-dependent inhibition of the NF-κB pathway, demonstrating high repurposing potential as an IBD therapeutic.

Keywords: Fli-1; Lumefantrine; NF-κB; colitis; inflammatory bowel disease (IBD).

FIGURE 1

Lumefantrine ameliorates DSS-induced colitis in mice. (A) Body weight changes in mice. (B) Disease Activity Index (DAI) scores. (C) Colon length measurement. (D) Hematoxylin and eosin, (H&E)-stained colon sections (scale bar: 200 μm above and 100 μm below), showing the colon pathological score on the right side. (E) Immunohistochemical and (F) Western blot analysis of tight junction proteins Claudin-1 and ZO-1 expression in colonic tissues. (scale bar: 100 μm).

FIGURE 2

Anti-inflammatory effects of Lumefantrine. (A–E) mRNA expression levels of pro-inflammatory cytokines (IL-1β, IL-6, Tnf-α) and mediators (Cox-2, iNos) in murine model colon tissues. (F–H) Serum protein concentrations of IL-1β, IL-6, and TNF-α measured by ELISA.

FIGURE 3

Molecular docking and dynamics simulations of Lumefantrine-FLI-1 interaction. (A) Predicted tertiary structure of FLI-1 (UniProt database). (B) Chemical structure of Lumefantrine. (C) 2D interaction diagram showing intermolecular force between FLI-1 and Lumefantrine. (D–F) Predicted ligand-binding pockets on FLI-1. (G) Binding conformation of the Lumefantrine-FLI-1 complex during molecular dynamics simulations. (H–K) Molecular dynamics parameters: Root mean square deviation [RMSD, (H)], root mean square fluctuation [RMSF, (I)], radius of gyration [Rg, (J)], and solvent-accessible surface area [SASA, (K)], confirming stable complex formation.

FIGURE 4

Binding of Lumefantrine to FLI-1 protein. Pull-down analysis of Lumefantrine conjugated magnetic beads and cell lysates from (A) IEC-6 and (B) NCM460, followed by WB detection using FLI-1 antibody. (C) Western blot was used to detect the expression of FLI-1 protein in IEC-6 after LPS modeling and treatment with Lumefantrine or Camptothecin (CPT).

FIGURE 5

Lumefantrine modulates the NF-κB pathway. (A,B) CCK-8 assay showing cytotoxicity of Lumefantrine in NCM460 and IEC-6 cells after 24/48 h exposure. (C) Western blot analysis demonstrating Lumefantrine’s suppression of LPS-induced phosphorylation of p65 and IκB. FLI-1 inhibitors YK-4-279 and CPT used as positive controls. (D) Western blot assessment of Lumefantrine’s effect on NF-κB pathway proteins under CPT-mediated FLI-1 suppression.

FIGURE 6

FLI-1 overexpression in IBD colonic tissues and its suppression by Lumefantrine. (A) Differential expression of FLI-1 in intestinal tissues between healthy individuals and IBD patients. Data sourced from the GSE255720 dataset (RNA-Seq libraries constructed from single-cell suspensions of colon biopsy tissues from 5 non-IBD healthy controls and 5 IBD patients), p = 0.011. (B) Western blot analysis of FLI-1 protein expression in normal controls, DSS-induced colitis mice and DSS mice treated with Lumefantrine.