ABSTRACT: The aim of this research is to reveal the cellular crosstalk in fibrosis liver using transgenic pigs (TG) expressing humanized risk genes (PNPLA3I148M-GIPRdn-hIAPP) as a MAFLD model. The study uses single-nucleus sequencing to reveal the differentiation and interaction characteristics of various cell populations in the liver during the development of MAFLD. Compared to wild-type pigs (WT), the model pigs showed significantly increased plasma levels of neurotensin and phosphatidylserine, along with insulin resistance and significantly decreased HDL-c levels. After 6 months of high-fat high-sucrose diet induction, the model pigs exhibited obvious liver pathological features, including fat deposition, inflammatory cell aggregation, fibrosis, and blocked insulin signaling pathways. Single-nucleusl transcriptome sequencing identified six main cell types in the liver, and correlation analysis showed similar liver cell clusters, zones, and functions between pigs and humans. Liver cells near the central vein and liver sinusoidal endothelial cells (LSECs) were sensitive to metabolic changes and exhibited impaired function and reduced numbers first. Fibrosis-related pathways and the Rap1 pathway were activated in hepatic stellate cells of the model pigs, while retinol metabolism decreased, and the number of activated hepatic stellate cells increased. The differentiation direction of macrophages in the model pig's liver was markedly different from that in the WT pigs; the former differentiated toward the M1 phenotype, showing high expression of MHC-II antigen presentation molecules, various cytokines, phagosomes, and lysosomal-related genes. In contrast, the macrophages in the WT pig's liver primarily differentiated toward the M2 phenotype and expressed genes related to immune regulation and injury repair. There was active cell interaction between hepatic stellate cells, endothelial cells, and M1-type macrophages, promoting the development of chronic inflammation and fibrosis through interactions of receptors such as FGFs-FGFRs, PDGFs-PDGFRs, EFNA1-EPHRs, and CXCL12-CXCR4/CXCR7.In conclusion, the transgenic pig model of MAFLD exhibits liver pathological features consistent with MAFLD patients. The receptor interactions between hepatic stellate cells, endothelial cells, and macrophages play a key role in regulating metabolic inflammation and fibrosis development. Remarkable changes in receptor interactions hold promise for application in MAFLD drug development.
