Guiqi Baizhu prescription attenuates 5-FU-induced intestinal mucositis by targeting IKKβ to inhibit M1 macrophage polarization.

BACKGROUND

Chemotherapy-induced intestinal mucositis (CIM), particularly that induced by agents such as 5-fluorouracil (5-FU), frequently leads to chemotherapy discontinuation. However, effective treatment options remain limited. Guiqi Baizhu prescription (GQBZP), a traditional Chinese medicine formula, has been reported to possess anticancer, analgesic, and anti-inflammatory activities.

PURPOSE: This study aimed to evaluate the therapeutic efficacy of GQBZP against 5-FU-induced intestinal mucositis (IM) and to clarify its underlying molecular mechanisms and material basis.

METHODS

The protective effects and mechanistic actions of GQBZP were investigated using a murine model of 5-FU-induced IM. Potential IKKβ-targeting compounds within GQBZP were screened through virtual docking combined with CCK-8 assays and subsequently evaluated in 5-FU-stimulated human intestinal epithelial cells (HIECs) and lipopolysaccharide/interferon-γ (LPS/IFN-γ)-stimulated THP-1 macrophages. Target specificity and binding characteristics were further validated by molecular dynamics (MD) simulations, surface plasmon resonance (SPR) analysis, and experiments using IKKβ-overexpressing HEK293T cells.

RESULTS

In vivo experiments demonstrated that GQBZP significantly alleviated 5-FU-induced IM by suppressing M1 macrophage polarization within intestinal tissues and restoring intestinal barrier integrity, effects closely associated with modulation of the IKKβ/NF-κB signaling pathway. Virtual screening and CCK-8 assays identified five IKKβ-targeting compounds in GQBZP: Rhamnocitrin, Toralactone, Naringenin, Liquiritigenin, and Carvacrol. These compounds markedly reduced apoptosis and pro-inflammatory cytokine production in 5-FU-treated HIECs. In addition, they inhibited M1 macrophage polarization and cytokine release in LPS/IFN-γ-stimulated THP-1 cells, accompanied by attenuation of IKKβ/NF-κB pathway activation. MD simulations, SPR assays, and functional studies in IKKβ-overexpressing HEK293T cells further confirmed that Toralactone directly binds to and suppresses IKKβ activation, whereas Rhamnocitrin modulates IKKβ activity through an indirect regulatory mechanism.

CONCLUSION

GQBZP alleviates 5-FU-induced IM by promoting recovery of the intestinal epithelial barrier and inhibiting M1 macrophage polarization through an IKKβ/NF-κB-dependent mechanism, thereby exerting synergistic anti-inflammatory effects. Toralactone was identified as a key active constituent responsible for direct inhibition of IKKβ-mediated M1 polarization. These findings suggest that targeting IKKβ to regulate macrophage polarization represents a promising therapeutic strategy for the management of CIM.