Tangeretin, a bioactive polymethoxyflavone from immature Citrus aurantium L. fruit, ameliorates experimental colitis by targeting the macrophage β2 integrin Mac-1.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhiqiao, the dried immature fruit of Citrus aurantium L., is a traditional Chinese medicine widely used for treating gastrointestinal disorders. Among its varieties, Quzhiqiao, a geo-authentic herb from Quzhou, China, is highly valued for its superior quality, yet the pharmacological basis for its efficacy in inflammatory bowel disease remains unclear.

AIM OF THE STUDY: This study aimed to investigate the therapeutic effects and underlying molecular mechanisms of the ethanol extract of Quzhiqiao (QZCEE) and its key bioactive compound in a mouse model of ulcerative colitis (UC), with a focus on its impact on macrophage-driven inflammation.

MATERIALS AND

METHODS

The efficacy of orally administered QZCEE and its constituent, tangeretin, was evaluated in a dextran sulfate sodium (DSS)-induced colitis mouse model by assessing clinical (DAI score) and pathological (colon length, histology) parameters. Intestinal barrier integrity was examined by Western blot and qPCR. Macrophage infiltration and colonic inflammatory responses were measured by immunohistochemistry, ELISA, and qPCR. The effects of QZCEE and tangeretin on macrophage adhesion, migration, and chemotaxis were assessed in vitro using bone marrow-derived macrophages (BMDMs). The molecular target was identified and validated through molecular docking, pull-down, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and Microscale Thermophoresis (MST) assays. The downstream signaling pathway was investigated by Western blot and Rac1 activation assays.

RESULTS

Oral administration of QZCEE significantly ameliorated the severity of DSS-induced colitis, evidenced by reduced DAI scores, restored colon length, and alleviated histological damage. QZCEE also repaired the intestinal barrier by upregulating the expression of tight junction proteins ZO-1 and Occludin. Mechanistically, QZCEE markedly suppressed the infiltration of F4/80+ macrophages into the inflamed colon and inhibited the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). Intriguingly, QZCEE did not reduce colonic CCL2 levels but directly inhibited macrophage chemotaxis in vitro. Tangeretin, a polymethoxyflavone in QZCEE, was identified as the key bioactive compound responsible for this effect. We provided compelling evidence that tangeretin directly binds to the β2 integrin Mac-1 (αMβ2) with a dissociation constant (Kd) of 3.87 μM, thereby blocking its interaction with ICAM-1 and suppressing the downstream Vav1/Rac1 signaling pathway. Importantly, oral administration of tangeretin alone recapitulated the therapeutic and immunomodulatory effects of the QZCEE extract in vivo.

CONCLUSION

Our findings demonstrate that the geo-authentic herb Quzhiqiao exerts potent anti-colitis effects. This therapeutic action is primarily mediated by its bioactive compound, tangeretin, which functions as a novel, direct antagonist of the β2 integrin Mac-1. By blocking macrophage recruitment, tangeretin represents a promising natural lead compound for the development of targeted therapies for UC. This study provides a robust scientific rationale for the ethnopharmacological use of Quzhiqiao and unveils a precise molecular mechanism for its efficacy.