Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.

Background and Purpose: Chronic liver disease and hepatic fibrosis constitute a threat to global health. Clinical translation of preclinical research has been limited, highlighting an urgent need for novel treatments. Cyclophilin inhibitors have shown beneficial effects in liver disease; however, the underlying mechanism of action and the effect across different aetiologies remain elusive. Here, we investigate the impact of a pan‐cyclophilin inhibitor (rencofilstat, RCF) in human models of fibrosis and alcohol‐related liver disease. Experimental Approach: RCF was tested in human precision‐cut liver slices (PCLS) and primary human hepatic stellate cells (HSCs). Fibrosis and cell activation were assessed using transcriptomic and protein analysis. A comprehensive characterisation of changes in extracellular matrix (ECM) biochemical and structural composition was performed in PCLS and HSC‐derived matrix using proteomics, imaging and bioinformatic tools to study ECM alignment. PCLS stiffness upon treatment was assessed by atomic force microscopy. Key Results: Transcriptomic and proteomic analyses of PCLS revealed a dramatic impact of RCF on ECM organisation and remodelling. Biochemical composition and fibre alignment analysis of the ECM obtained from HSCs showed a reduction in the amount of ECM core proteins and associated enzymes by RCF, reshaping the architecture of matrix fibres without affecting the HSC activation. The disordered matrix detected in RCF‐treated HSC cultures reflected a less‐stiff ECM, which was confirmed in the PCLS. Conclusions and Implications: This work provides evidence for a novel mechanism linking cyclophilins and ECM remodelling in advanced 3D models of liver disease, with potential applications in therapeutic development.