Kaempferitrin attenuates DSS-induced colitis by promoting ubiquitination-mediated degradation of the nuclear factor kappa B p65

Background: Inflammatory bowel disease (IBD), including colitis, is commonly associated with dysfunction of the intestinal barrier and inflammatory responses. Current medications used to treat IBD may cause severe side effects with long-term use. Previous studies have confirmed that Bupleuri Radix extract exhibits significant anti-inflammatory effects. However, the therapeutic effects of its active component, kaempferitrin (KPN), on dextran sulfate sodium (DSS)-induced colitis in mice and the underlying mechanisms remain largely unexplored.

Purpose: The aim of this study was to investigate the therapeutic effects of KPN on colitis in mice and explore its potential mechanisms.

Methods: This study employed a DSS-induced colitis mouse model to evaluate KPN's therapeutic effects. Mice were divided into control, model, 5-Aminosalicylic Acid (5-ASA), and KPN treatment groups, followed by DAI, histopathology, inflammatory cytokines, gut barrier proteins, and gene expression.

Results: This investigation revealed that Kaempferitrin (KPN), an active constituent within Bupleuri Radix extract, attenuates pathological manifestations, inflammatory cascades, and barrier dysfunction in dextran sulfate sodium (DSS)-induced murine colitis. Integrative network pharmacology and transcriptomic analyses identify NDRG2 as a potential target of KPN in the treatment of colitis. Co-immunoprecipitation (Co-IP) and mass spectrometry showed that KPN enhanced the interaction between NDRG2 and NF-κB p65, whereas mechanistic studies in bone marrow-derived macrophages (BMDMs), supported by public single-cell RNA-seq analysis and NDRG2/F4/80 co-localization in inflamed colon tissues, indicated that macrophages represent a relevant cellular context for the KPNNDRG2-p65 axis. Through the utilization of a biotin-conjugated KPN probe, direct molecular interaction between KPN and NDRG2 was established, with site-directed mutagenesis revealing E164, P280, and M314 as critical amino acid residues mediating this interaction. Mechanistically, KPN promoted NDRG2-associated recruitment of FBXO11, facilitating K48-linked ubiquitination and proteasomal degradation of p65 in BMDMs, thereby suppressing NF-κB activation and inflammatory cytokine expression. Additionally, in vivo experimental evidence indicated that NDRG2 ablation (NDRG2^-/-) substantially diminished the protective efficacy of KPN against DSS-induced colitis and compromised its capacity to inhibit p65.

Conclusion: KPN directly engages NDRG2 and modulates a BMDMs-associated NDRG2-FBXO11-p65 axis to restrain NF-κB signaling, providing mechanistic insight into the anti-inflammatory effects of KPN in experimental colitis.