Integrated Multi-Omics Reveals BCAA Metabolic Remodeling Associated with the Antifibrotic Effects of Ku-Cai-Gao in Liver Fibrosis.

INTRODUCTION: Ku-Cai-Gao (KCG) is a traditional Chinese medicinal preparation with both therapeutic and dietary functions. Preliminary clinical observations suggest that KCG may alleviate liver fibrosis-related manifestations and improve liver function, but whether its antifibrotic effects involve hepatic amino acid metabolic remodeling remains unclear. This study investigated the hepatoprotective pathways of KCG using an integrated multi-omics strategy. METHODS: A carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis was established and treated with KCG. KCG constituents were profiled by ultra-performance liquid chromatography-tandem mass spectrometry. Liver histopathology and collagen deposition were evaluated, and serum transaminases, oxidative stress markers, and inflammatory cytokines were measured. Fibrosis- and apoptosis-related proteins were assessed by immunohistochemistry/immunofluorescence and Western blotting. Transcriptomics and proteomics were integrated to identify key pathways, with selected targets validated by reverse transcription-quantitative polymerase chain reaction and Western blotting. Targeted metabolomics assessed Branched-Chain Amino Acid (BCAA) metabolism, and 16S ribosomal RNA sequencing characterized gut microbiota changes. RESULTS: KCG markedly ameliorated CCl4-induced liver injury, as shown by improved liver morphology, reduced liver injury scores, decreased collagen deposition, and attenuation of inflammatory cell infiltration. KCG treatment reduced serum ALT, AST, ALP, and T-BIL levels, decreased MDA, IL-6, and TNF-α levels, and restored SOD activity. Histological and molecular analyses showed that KCG reduced α-SMA, collagen I, TGF-β1, PCNA, and caspase-3 expression, indicating attenuation of hepatic stellate cell activation, extracellular matrix accumulation, inflammation, and apoptosis. Integrated transcriptomic and proteomic analyses converged on the valine, leucine, and isoleucine degradation pathway, and targeted metabolomics showed decreased hepatic Branched-Chain Amino Acid (BCAA) levels after KCG treatment. These changes were accompanied by upregulation of Peroxisome Proliferator-Activated Receptor Alpha (PPARα)-related signaling. In addition, KCG treatment was associated with changes in gut microbiota composition, including alterations in Firmicutes, Bacteroidetes, and Muribaculaceae. DISCUSSION: These findings suggest that the antifibrotic effects of KCG are associated with PPARα-related signaling, BCAA metabolic remodeling, and improvement of inflammatory and metabolic disturbances. However, the causal roles of PPARα signaling, BCAA metabolism, and gut microbiota changes require further validation. CONCLUSION: KCG alleviated CCl4-induced liver fibrosis in mice, and its protective effects were associated with BCAA metabolic remodeling, PPARα-related signaling, and changes in gut microbiota composition.