KAJF alleviated colorectal cancer liver metastasis by reshaping the gut microbiota and inhibiting M2-like macrophage polarization

Abstract

Background

Colorectal cancer liver metastasis (CRLM) represents one of the most severe complications of colorectal cancer (CRC), often associated with unfavorable prognosis. The herbal formulation Kang-Ai-Jing-Fang (KAJF) has been employed clinically against CRC for several decades, although its precise mechanism of action remains elusive.

Purpose

This study investigates the anti-metastatic potential of KAJF in CRLM, focusing on its modulatory effects on gut microbiota and inhibition of M2-like macrophage activation.

Methods

KAJF was administered orally to CRLM model mice established through intrasplenic injection of murine CRC cells. To elucidate the role of gut microbiota reshaped by KAJF, fecal microbiota transplantation (FMT) was utilized to assess its impact on CRLM inhibition. Core targets and active compounds were identified through network pharmacology and molecular docking. To characterize microbiota composition, metabolite profiles, and gene expression variations, 16S rRNA sequencing, untargeted liquid chromatography-mass spectrometry (LC-MS/MS) metabolomics, and transcriptomics analyses were performed.

Results

KAJF demonstrated significant inhibitory effects on CRLM and ameliorated gut microbiota dysbiosis by enhancing the abundance of butyrate-producing bacteria (Lactobacillus, Bacteroides, Bifidobacterium). The therapeutic efficacy of KAJF-induced bacterial alterations in delaying CRLM and promoting butyrate-producing microbiota enrichment was further substantiated by FMT. Network pharmacology identified active ingredients in KAJF, including asperulosidic acid, polyphyllin II, ganoderenic acid B, calycosin, and ganoderic acid C, which exhibit substantial interactions with TLR4, PPARγ, SIRT1, PTGS2, and TNF. Molecular dynamics simulations and surface plasmon resonance (SPR) analysis demonstrated that ganoderic acid C2 exhibits a strong binding affinity for PPARγ. Moreover, KAJF administration led to a marked reduction in F4/80⁺ CD206⁺ macrophages and their associated cytokines (CCL17, CCL22, IL10, IL4, TGF), accompanied by a decrease in CD4⁺ T cells and myeloid-derived suppressor cells (MDSCs), while increasing CD8⁺ T cell populations.

Conclusions

This study demonstrates that KAJF mitigates CRLM, primarily through the regulation of gut microbiota and microbial metabolites, alongside inhibition of M2-like macrophage polarization. By integrating metabolomics, transcriptomics, and network pharmacology, this research elucidates the molecular mechanisms underpinning KAJF's therapeutic effects against CRLM, offering a promising approach for clinical intervention.