Modulation of gut microbiota and its metabolite Equol by Huaier granule suppresses hepatocellular carcinoma via the gut-liver axis.

HEG suppressed tumor development in an orthotopic HCC mouse model in a gut microbiota-dependent manner.

• Anti-tumor effects of HEG were demonstrated using an orthotopic HCC mouse model.
• The gut microbiota-dependent nature of HEG's efficacy was established, suggesting that disruption of gut microbiota would attenuate HEG's anti-HCC effects.
• HEG modified the gut microbiota composition in treated animals.

Adlercreutzia was identified as the primary differential bacterial genus between the Model group and the HEG group.

• 16S rRNA sequencing was used to characterize gut microbiota composition differences between groups.
• Adlercreutzia was enriched in the HEG-treated group compared to the Model group.
• Adlercreutzia is a known producer of the metabolite Equol from dietary precursors.
• This finding was integrated with metabolomics data and literature mining to identify Equol as a key downstream mediator.

HEG repaired the intestinal barrier, improved colon immunity, and ameliorated the tumor immune microenvironment via the gut microbiota-gut-liver axis.

• HEG exerted anti-HCC effects through repair of the intestinal barrier integrity.
• Improvement in colon immunity was observed in HEG-treated animals.
• Suppression of the MAPK signaling pathway was identified as the mechanistic link between gut microbiota modulation and immune microenvironment improvement.
• These effects operated through the gut-liver axis.

Equol, a metabolite produced by Adlercreutzia, was identified as a key metabolite through which HEG exerted its anti-HCC effects.

• Identification of Equol was achieved by integrating 16S rRNA sequencing with metabolomics data, supplemented by literature mining and in vitro validation.
• Equol was shown to be essential for the anti-HCC effects of HEG, indicating that its depletion or absence would compromise HEG's efficacy.
• Equol is produced by specific gut microbiota, particularly Adlercreutzia, linking microbiota composition to metabolite production.

Equol ameliorated the tumor immune microenvironment through inhibition of the MAPK signaling pathway.

• Equol's immunomodulatory effects in the tumor microenvironment were mediated via suppression of MAPK signaling.
• This finding was validated through in vitro experiments.
• The MAPK pathway suppression by Equol paralleled the pathway suppression observed with HEG treatment.

Equol inhibited the growth of HCC cells by inducing G0/G1 phase cell cycle blockade through suppression of the Cyclin E1-CDK2/Rb signaling pathway.

• Equol treatment resulted in G0/G1 phase arrest in HCC cells.
• The mechanism of cell cycle arrest involved suppression of the Cyclin E1-CDK2/Rb signaling pathway.
• This anti-proliferative effect was demonstrated in vitro.
• Equol was identified as a promising candidate for HCC treatment based on these findings.