Single-nucleus and Spatial Transcriptomics Reveal Intestinal Cellular Heterogeneity, Differentiation, and Cell Communication Mechanisms in SAP-induced Intestinal Injury [Spatial Transcriptomics]
Background Acute pancreatitis (AP) is a severe inflammatory disease that frequently causes multi-organ damage, with intestinal injury being a major complication. The mechanisms underlying SAP-induced intestinal injury remain unclear, particularly regarding spatial cellular reorganization and functional interactions. Methods This study constructed a severe acute pancreatitis (SAP) rat model and employed single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptome sequencing (stRNA-seq) technologies to systematically analyze the dynamic changes in intestinal cellular composition, spatial distribution, and function during SAP-induced intestinal injury. Results snRNA-seq identified 18 major ileal cell populations spanning epithelial, immune, stem/TA, and stromal compartments. SAP was associated with compositional remodeling characterized by downward trends in Lgr5?/Olfm4? stem cells, TA1, goblet cells, and Paneth cells, with a reciprocal increase in enterocytes, although most proportion changes did not reach statistical significance at the animal level. Spatial transcriptomics independently captured SAP-associated tissue remodeling, including a significant reduction in Paneth cells accompanied by increases in fat cells, macrophages, goblet cells, and TA2 cells. Across epithelial lineages, SAP induced a transcriptional shift toward immune interaction with up-regulation of antigen presentation–related genes (e.g., Cd74, Apoa1) and down-regulation of antimicrobial/barrier effectors (e.g., Defa24, Pla2g2a, Dmbt1), which was corroborated by spatial expression patterns and spatially variable gene programs enriched for host defense responses. Pseudotime analysis suggested a redistribution of epithelial states along the stem/TA-to-enterocyte continuum, with relative depletion of early states and expansion of enterocyte-dominant states in SAP. CellChat analysis revealed globally intensified intercellular communication and nominated FN1 as the pathway with the highest differential information flow, with Lgr5? stem cells predicted as prominent FN1 senders targeting enterocytes and smooth muscle cells. SCENIC identified reduced activity and expression of Hmga2/Myb regulons in stem compartments, and immunofluorescence showed decreasing trends in Hmga2/Myb-positive Olfm4? and Lgr5? stem cells in SAP. Conclusions Integrated single-nucleus and spatial transcriptomics reveal that SAP is accompanied by spatially organized ileal remodeling, epithelial immune-interacting rewiring, and altered neighborhood architecture, together with an ECM-centered FN1 signaling axis and attenuated Hmga2/Myb-associated regulatory programs in stem compartments. These findings provide a spatially informed cellular framework and generate testable hypotheses for mechanisms underlying impaired epithelial regeneration during SAP-associated intestinal injury. Overall design: Severe acute pancreatitis (SAP) was induced in Sprague–Dawley rats and compared with control (CON) rats. Ileal tissues were collected and profiled using single-nucleus RNA sequencing (snRNA-seq; CON n=3, SAP n=3) and Stereo-seq spatial transcriptomics (CON n=2, SAP n=2) to characterize SAP-associated changes in cellular composition, differentiation trajectories, spatial organization, and cell–cell communication.