Functional metagenomic reconstruction of microbial pathways altered by probiotic supplementation in liver failure.

Abstract

INTRODUCTION: Liver failure is a severe condition marked by circulatory failure, systemic inflammation, and gut microbial dysbiosis. This dysbiosis worsens liver damage by reducing beneficial metabolites and increasing harmful products. This study investigates the effects of probiotics on gut microbial functional pathways in liver failure. The aim is to link microbial metabolic reprogramming with host biochemical, inflammatory, and gut barrier responses through functional metagenomic reconstruction. METHODS: Acute liver failure was induced in male Wistar rats using D-galactosamine (700 mg/kg) and lipopolysaccharide (10 μg/kg). Probiotic treatment began 24 hours after induction and was administered daily for 14 consecutive days before euthanasia. Two doses were used: low (1×10⁸ CFU/day) and high (1×10⁹ CFU/day). Fecal samples underwent shotgun metagenomic sequencing, followed by functional pathway reconstruction. These predictions were validated using metabolite profiling, quantitative PCR of microbial genes, intestinal barrier assays, and immune cell cytokine analysis. Host phenotypic markers were correlated with microbial pathways. RESULTS AND DISCUSSION: Liver failure significantly elevated serum ALT (42.6±6.8 to 512.4±48.9 U/L), AST (78.3±9.5 to 684.7±62.1 U/L), and plasma ammonia (38.9±5.2 to 128.6±14.3 μmol/L). Probiotic supplementation showed a dose-dependent improvement. ALT dropped to 382.7±41.6 U/L (low dose) and 248.9±32.4 U/L (high dose). Ammonia levels decreased to 86.4±9.7 μmol/L and 59.8±7.6 μmol/L, respectively. Metagenomic analysis revealed a 1.7- and 2.6-fold increase in short-chain fatty acid (SCFA) biosynthesis pathways and a 38% and 61% decrease in urease-associated nitrogen metabolism. These changes were confirmed by higher fecal SCFAs (31.8±4.2 to 63.9±6.4 mM), lower ammonia (8.9±1.1 to 3.7±0.5 mM), improved intestinal barrier integrity (TEER: 462±38 to 721±44 Ω·cm²), and reduced TNF-α (214.6±22.8 to 74.9±12.3 pg/mL). Probiotic supplementation significantly reprogrammed the gut microbiome in liver failure. This highlights its potential as a therapeutic modulator of the gut-liver axis.