Faecal hsa-miR-7704 inhibits the growth and adhesion of Bifidobacterium longum by suppressing ProB and aggravates hepatic encephalopathy

**Background:** Hepatic encephalopathy (HE) is a severe neuropsychiatric complication of liver failure, associated with gut microbiota dysbiosis and systemic inflammation. MicroRNAs (miRNAs) in faeces can modulate gut microbes and influence disease, but their role in HE was unknown. This study aimed to identify faecal miRNAs that affect HE progression and to elucidate the underlying mechanisms.

**Methods:** Faecal samples were collected from 9 patients with HBV-ACLF with HE and 10 with chronic hepatitis B (CHB). Metagenomic sequencing and miRNA microarray were performed. A mouse model of HE was induced by thioacetamide (TAA) injection. Mice received oral gavage of human faeces, heat-inactivated faeces, purified faecal miRNAs, or synthetic miRNA mimics (including hsa-miR-7704). In vitro, Bifidobacterium longum was cultured with miR-7704 mimics; bacterial growth, adhesion to HT-29 cells, and gene expression were assessed. ProB knockout in E. coli was used to confirm function. Acetate levels, microglial activation (BV2 cells), and neuronal viability (HT-22 cells) were measured.

**Key Results:** Faecal microbiota composition differed between HE and CHB patients, with reduced Bifidobacterium (especially B. longum and B. pseudocatenulatum) in HE. Transfer of HE patient faeces to mice aggravated HE (higher serum ammonia, brain water content, TNF-α, IL-1β). Heat-inactivated faeces and purified faecal miRNAs had similar effects. miRNA microarray identified 10 upregulated miRNAs in HE faeces; hsa-miR-7704 was among the top six. Oral administration of synthetic hsa-miR-7704 to mice increased mortality (p<0.05), serum ALT (from ~100 to ~300 U/L), AST (from ~150 to ~400 U/L), ammonia (from ~100 to ~200 μmol/L), brain water content (from ~78% to ~80%), and neuroinflammation (TNF-α, IL-1β). Antibiotic treatment abrogated these effects. In vitro, miR-7704 specifically entered B. longum (not E. coli), inhibited its growth (OD600 reduced by ~40% at 24 h), and reduced adhesion to HT-29 cells (CFU count decreased by ~60%). miR-7704 suppressed proB gene expression (by ~50% by qPCR) and proline levels (from ~0.8 to ~0.4 μmol/g). ProB knockout in E. coli slowed growth and reduced adhesion. B. longum administration to mice alleviated HE (ammonia ~120 vs. ~200 μmol/L, brain water ~78% vs. ~80%, TNF-α and IL-1β reduced). Acetate, the major SCFA from B. longum, was lower in miR-7704-treated mice (serum acetate ~0.5 vs. ~1.0 mM). Acetate treatment inhibited LPS-induced microglial activation (TNF-α, IL-6, iNOS reduced; IL-10 increased) and protected HT-22 neuronal cells from TNF-α- and ammonia-induced toxicity.

**Clinical Implications:** This study identifies hsa-miR-7704 as a novel faecal miRNA that exacerbates HE by specifically targeting B. longum, suppressing proB, and reducing acetate production, leading to microglial activation and hyperammonemia. These findings highlight a miRNA–gut microbiota–brain axis in HE and suggest that restoring B. longum or acetate could be therapeutic strategies. The results also demonstrate that faecal miRNAs can survive digestion and modulate gut microbes, offering new avenues for microbiome-based interventions in liver disease.