Impairment of Regenerative and Metabolic Pathways in Intestinal Organoids From Patients With MASLD‐Cirrhosis

Background and Aims: Gut-liver axis has been implicated in the pathophysiology of cirrhosis due to metabolic dysfunction-associated steatotic liver disease (MASLD), an in vitro model for studying epithelial gut dysfunction in MASLD is lacking. In this study, we aimed to characterise intestinal organoids derived from subjects with MASLD. Materials and Methods: Intestinal organoids were obtained from duodenal samples of individuals with non-fibrotic MASLD and with MASLD-cirrhosis. After 7 to 10 days of culture, RNA extraction, transcriptomic analysis and real-time PCR were performed. Energetic and redox status of organoids were assessed by chromatographic analysis. Results: Microarray analysis showed approximately 600 dysregulated transcripts in organoids isolated from patients with MASLD-cirrhosis compared to non-fibrotic MASLD. Bioinformatic analysis indicated that dysregulated transcripts were involved in pathways related to regeneration and pluripotency of stem cells and regulating mitochondrial metabolism and hypoxia. Overall, chromatographic analysis demonstrated that energetic status was remarkably impaired in both differentiated and undifferentiated organoids from cirrhotic patients compared to steatotic subjects. In either undifferentiated or differentiated organoids from cirrhotic subjects, the triphosphate sum, the ATP/ADP and NAD+/NADH ratios were lower in MASLD-cirrhosis, indicating a decline in the phosphorylating capacity and a mitochondrial derangement of duodenal cells. In addition, we found lower levels of reduced glutathione, NADPH and UDP-glucuronic acid in undifferentiated and differentiated cells from cirrhotics indicating impairment of antioxidant defence and of response to xenobiotics. Conclusions: Our study provides a comprehensive view of intestinal epithelial dysfunction in MASLD-cirrhosis, characterised by a cluster of regenerative and metabolic disturbances.