Identification of key genes related to phagocytic regulatory factors in nonalcoholic fatty liver disease: transcriptome and single cell combined analysis

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease. Disturbed phagocytic regulation may affect lipid clearance, inflammatory balance, and cell-cell communication during disease progression, but phagocytosis regulatory factors (PRFs) in NAFLD have not been systematically defined. Public bulk transcriptomic datasets were analyzed to identify differentially expressed PRFs. LASSO and XGBoost were then combined with expression validation and ROC analysis to screen key genes. Immune infiltration, GSVA/GSEA, transcription factor and drug prediction, and single-cell RNA sequencing analyses were further used to characterize biological function, cellular localization, intercellular communication, and pseudotime dynamics. Zebrafish larvae were used for in vivo validation at 7 and 15 days. PTX3 and WASF3 were identified as key NAFLD-related genes and showed stable diagnostic value across the training and validation cohorts. PTX3 was positively correlated with neutrophils, whereas WASF3 was positively correlated with activated CD8 T cells. Both genes shared 19 enriched pathways, including the spliceosome and insulin signaling. Single-cell analysis identified cholangiocytes and hepatocytes as key cell populations, and cell-cell communication analysis highlighted the cholangiocyte-hepatocyte axis, including ANGPTL1-(ITGA1 + ITGB1), during NAFLD progression. Pseudotime analysis further showed dynamic changes in PTX3 and WASF3 expression during hepatocyte and cholangiocyte differentiation. Zebrafish experiments supported dynamic pathological and transcriptional changes during disease progression. PTX3 and WASF3 are associated with NAFLD progression and may reflect alterations in immune signatures, transcriptional pathways, and hepatobiliary cellular states. These findings provide candidate biomarkers, provide insights into NAFLD-associated molecular and immune alterration, and establish a basis for future mechanistic inveatigation.