Altered cholangiocyte subsets with neural pathway enrichment in metabolic dysfunction-associated steatotic liver disease.

Hepatocytes have been extensively studied in metabolic dysfunction-associated steatotic liver disease (MASLD), yet the role of cholangiocytes remains underexplored. We utilized a comprehensive approach that integrates high-dimensional weighted gene co-expression network analysis (hdWGCNA), cellular interaction evaluation, machine learning techniques, immune cell infiltration analysis, and Mendelian randomization to investigate both single-cell sequencing data and bulk sequencing datasets. Single-cell analysis identified significant alterations in cholangiocyte subpopulations in metabolic dysfunction-associated steatohepatitis (MASH) patients, including an increase in cholangiocytes with elevated SEMA3 pathway expression. The hdWGCNA analysis identified the green module, which is enriched with genes associated with neural pathways pertinent to MASH. Seven machine learning models assessed the diagnostic feasibility of key genes in the green module for MASLD spectrum. Immune infiltration analysis highlighted complex interactions between cholangiocytes enriched for neural-related pathways and immune cells, particularly neutrophils and monocytes. Patient stratification identified two distinct subtypes within the MASLD spectrum, with subtype 2 exhibiting enhanced activity in neural-related pathways. Mendelian randomization analysis identified PLEKHA5 as a candidate causal gene for MASLD, suggesting a potential role in neural mechanisms (SEL1L3 showed a suggestive association, P = 0.0413). Collectively, this hypothesis-generating bioinformatics study elucidates distinct characteristics of cholangiocyte subpopulations in MASH, revealing neural pathway alterations that may contribute to disease pathogenesis and providing a foundation for future investigation into cholangiocyte-related mechanisms in MASLD.