Multiomics, multi-species profiling, and animal validation identify mitoxyperilysis as a conserved therapeutic target in metabolic dysfunction-associated steatohepatitis

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) is driven by complex intercellular communication and regulated cell death pathways. Mitoxyperilysis, a recently characterized form of regulated lytic cell death, remains poorly defined in MASH. Here, we integrated multiomics, cross-species transcriptomics, machine learning, and in vivo models to dissect mitoxyperilysis activation and its therapeutic relevance in MASH. METHODS: Single‑cell and spatial transcriptomic datasets from mouse and human MASH livers were analyzed. Cell-cell communication was inferred using CellChat, and metabolite‑mediated cell-cell communication (mCCC) was assessed with MEBOCOST. Mitoxyperilysis activity was scored via AUCell and AddModuleScore. Eleven machine learning classifiers identified transcriptomic signatures distinguishing MASH from controls, and key regulators were subjected to in silico knockout (scTenifoldKnk). A choline‑deficient, L‑amino acid‑defined high‑fat diet (CDAHFD) mouse model was used for in vivo validation. RESULTS: Single‑cell profiling revealed that mitoxyperilysis activity was highly enriched in myeloid cells, particularly monocyte‑derived macrophages (Mo‑Macrophages) and neutrophils, and was strongly induced by high‑fat high‑sucrose diet. CellChat and mCCC analyses demonstrated that Mo‑Macrophages served as central hubs of rewired inflammatory and metabolic crosstalk in MASH. Spatial transcriptomics confirmed the expansion of pro‑inflammatory hepatocyte subsets and conserved upregulation of mitoxyperilysis components (e.g. BAX, TLR4, NINJ1) in both mouse and human MASH. Cross‑species profiling across mouse, rat, hamster, non‑human primate, and human datasets demonstrated evolutionarily conserved activation of the entire mitoxyperilysis pathway. Machine learning identified Bax as the sole overlapping driver gene linking diet‑induced transcriptomic signatures to mitoxyperilysis. In silico Bax ablation perturbed lipid metabolism and immune pathways. In CDAHFD-induced MASH mice with typical pathological phenotypes, core mitoxyperilysis genes were transcriptionally upregulated, and Bax/Bid/Bak1 upregulation was validated at the protein level. While RhoA mRNA increased, total RhoA protein remained stable, with its ubiquitination elevated and activity suppressed. CONCLUSIONS: Mitoxyperilysis is an evolutionarily conserved, myeloid‑enriched pathway activated in MASH. Bax acts as a critical regulatory node, while RhoA is modulated via ubiquitination and functional suppression. Targeting this axis may provide a novel therapeutic strategy for MASH.