EASL 2025 YAQ005: Toll-like receptor 4 inhibition restores cytochrome C oxidase

• Hyperammonemia has been demonstrated to induce mitochondrial dysfunction in hepatocytes, with a particular impact on oxidative phosphorylation (OXPHOS).1,2 • While inhibition of Toll-like receptor 4 (TLR4) has shown potential in preserving mitochondrial stability, supporting urea cycle enzyme (UCE) function, and reducing oxidative stress, the underlying mechanisms remain unclear. • This study investigates how TLR4 inhibition can restore mitochondrial function under hyperammonemic conditions. Contact informationReferences Introduction & Aim Methods Supachaya Sriphoosanaphan1,2,3, Annarein Kerbert1, Francesco De Chiara4, Abeba Habtesion1, Fausto Andreola1, Gautam Mehta1, Jan-Willem Taanman5 and Rajiv Jalan1 1) Institute for Liver and Digestive Health, Division of Medicine, University College London, United Kingdom; 2) Division of Gastroenterology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 3) Centre of Excellence in Liver Diseases, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand; 4) Institute for Bioengineering of Catalonia, Barcelona, Spain; 5) Department of Clinical and Movement Neurosciences, Queen Square Institute of Neurology, University College London, United Kingdom Toll-like receptor 4 inhibition restores cytochrome C oxidase mitigating hyperammonemia -induced hepatocyte mitochondrial dysfunction 1. Kerbert A et al. Sci. Adv. 2025;11(10):eado1648 2. Sriphoosanaphan S et al, EASL Congress 2024 Supachaya Sriphoosanaphan, M.D., M.Sc. Email: [email protected] Abbreviations: AA, amino acid; COX, Cytochrome C oxidase; KO, knock-out; NP, normal protein; OP, Ornithine Phenylacetate; TAK242, toll-like receptor 4 inhibitor; TLR4KO, TLR4 knock-out; WT, wild-type • Hyperammonemia was induced in wild-type (WT) and TLR4 knock-out (TLR4KO) mice via a 14-day amino acid (AA) diet. • WT-AA mice were treated with either the TLR4 antagonist, TAK-242 (10 mg/kg i.p.) or the ammonia scavenger Ornithine Phenylacetate (OP, 300 mg/kg i.p.) from days 10 to 14. • Liver transcriptomic analysis was performed using whole RNA sequencing, with specific focus on OXPHOS genes. Data were analysed with KEGG and Gene Ontology databases. • Quantitative activity of cytochrome C oxidase in isolated hepatocyte mitochondria was assessed using spectrophotometric enzyme assay. Among all OXPHOS complexes, only complex IV (Cytochrome C oxidase) was significantly affected by hyperammonemia, which was normalised following TAK-242 treatment. Mitochondrial cytochrome C oxidase activity assays further confirmed significantly elevated enzyme activity in WT-AA mice compared to controls, which was significantly reversed by TAK-242. TLR4 knock-out mice showed no changes in cytochrome C oxidase gene or protein expression, nor in enzyme activity, regardless of the AA diet. • Hyperammonemia drives mitochondrial dysfunction by selectively upregulating cytochrome C oxidase subunits, which are critical for mitochondrial OXPHOS function. • TLR4 inhibition effectively mitigates this dysregulation, stabilising mitochondrial bioenergetics and preserving UCE activity. • These results suggest that TLR4 antagonist may offer a promising therapeutic approach for hyperammonemia by restoring cytochrome C oxidase regulation. Main findings TLR4 inhibition protects against hyperammonemia. Liver transcriptomic analysis Gene expression of Cytochrome C oxidase subunits Protein expression of Cytochrome C oxidase subunits Cytochrome C oxidase enzyme activity (k/min/mg) Conclusions Log2 fold change heatmap for OXPHOS complex IV (Cytochrome C oxidase)

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