Multi-omics integrated analysis reveals the therapeutic mechanism of 919 formula in metabolic dysfunction-associated steatotic liver disease.
Abstract
Background
Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease worldwide and is driven by metabolic stress-induced inflammation. Emerging evidence implicates cytotoxic CD8⁺ T cells as key contributors to the progression of MASLD. However, therapeutic strategies specifically targeting pathogenic CD8⁺ T cell responses in MASLD are still scarce. The clinical efficacy of the 919 formula in MASLD has been demonstrated, yet its cellular targets and molecular mechanisms remain unclear.
Methods
Single-cell transcriptomics profiled peripheral blood immune composition in 919 formula treated MASLD patients, and UPLC-QE-MS characterized bioactive constituents of the 919 formula. Efficacy and immune mechanisms were tested in HFD/MCD mouse models (AST/ALT and histology) and CD8⁺ T cell adoptive transfer. CD8⁺ T cells treated with control or 919 formula-containing serum were analyzed by flow cytometry, Seahorse assays, transcriptomics, immunoblotting, and molecular docking.
Results
Single-cell transcriptomic profiling showed that 919 formula treatment was associated with a selective reduction in circulating CD8⁺ GZMB⁺ T cells and an attenuation of CD8⁺ T-cell activation in patients with MASLD. In both HFD- and MCD-induced mouse models, the 919 formula markedly alleviated hepatic steatosis, inflammation, and fibrosis, which was accompanied by a decreased proportion of CD8⁺ GZMB⁺ T cells, reflecting reduced CD8⁺ T-cell activation. Furthermore, adoptive transfer experiments demonstrated that CD8⁺ T cells treated with 919 formula-containing serum were sufficient to confer protection against MCD-induced liver injury. Mechanistically, 919 formula treatment was associated with reduced PKM2-dependent glycolysis and STAT5 phosphorylation in CD8⁺ T cells, while pharmacological activation of PKM2 partially reversed these effects. Finally, Schizandrin C was identified as a representative bioactive component of the 919 formula. Molecular docking and SPR analyses supported its direct interaction with PKM2, while functional assays demonstrated its capacity to suppress CD8⁺ T-cell activation.
Conclusion
Together, these findings suggest that the 919 formula alleviates MASLD, at least in part, by attenuating pathological CD8⁺ T-cell activation and cytotoxicity, with PKM2-STAT5 signaling potentially contributing to this effect. Schizandrin C may contribute to the therapeutic effects of the 919 formula, although the overall efficacy of this complex herbal formulation is likely to involve coordinated actions of multiple bioactive constituents.