Identification of Metabolic Marker Profile and Study on Metabolic Pathway Regulation in the Progression of Non-Alcoholic Steatohepatitis to Hepatocellular Carcinoma

Objective: Non-alcoholic steatohepatitis (NASH) is a major risk factor for hepatocellular carcinoma (HCC), whereas the metabolic reprogramming features and regulatory mechanisms underlying disease progression remain unclear. This study combined human clinical specimens and staged SD rat NASH-HCC animal models to screen stage-specific metabolic markers separately from two cohorts and uncover core metabolic regulatory pathways, so as to supply metabolomic evidence for early diagnosis and targeted therapy of NASH-associated HCC.

Materials and Methods: Human samples including 32 simple steatosis, 45 fibrotic NASH, 28 early carcinogenesis and 50 HCC cases, alongside staged SD rat specimens (n=15 per subgroup) were enrolled. UPLC-MS/MS-based untargeted and targeted metabolomics was performed on human and rodent samples respectively. Differential metabolites were independently screened in human and animal cohorts via OPLS-DA and fold change analysis, and cross-species consistently altered metabolites were summarized. Pathway enrichment and PPI analysis were conducted separately for two datasets using MetaboAnalyst and STRING. An 8-biomarker diagnostic panel was built based on human metabolomic data and assessed with internal cross-validation. LO2 and HepG2 cell lines were used for functional verification of pivotal metabolites.

Results: A total of 127 differential metabolites were identified from combined human and rat data, among which 36 displayed consistent dynamic variation across full NASH-HCC progression in both cohorts, including sphingosine-1-phosphate, taurocholic acid and L-arginine. The diagnostic model based on eight human-derived metabolites yielded an AUC of 0.923 (95%CI: 0.876-0.961) after cross-validation to distinguish early carcinogenesis from fibrotic NASH. Parallel pathway analysis in two cohorts validated that disrupted lipid metabolism, abnormal amino acid metabolism and reprogrammed energy metabolism dominated disease progression. Cellular assays validated that sphingosine-1-phosphate facilitates hepatocyte proliferation and invasion via PI3K/Akt activation.

Conclusion: Using paired human and animal specimens, this study defines metabolic disturbance patterns during NASH-to-HCC transition and establishes a high-performance metabolic signature for staged diagnosis. The synergistic carcinogenic regulation of lipid-amino acid-energy metabolic network is clarified, providing novel metabolomic targets for risk stratification, early screening and precision intervention of NASH-related HCC.