Combined analysis of bulk, single-cell RNA sequencing, and spatial transcriptomics reveals the expression patterns of lipid metabolism and ferroptosis in the immune microenvironment of metabolic-associated fatty liver disease

Aims: This study aims to identify key biomarkers associated with ferroptosis and lipid metabolism and investigate their roles in the progression of metabolic dysfunction-associated fatty liver disease (MAFLD). It further explores interactions between these biomarkers and the immune-infiltration environment, shedding light on how ferroptosis and lipid metabolism influence immune dynamics in MAFLD. Main methods: Single-cell RNA sequencing data from liver samples were analyzed to evaluate expression variations related to ferroptosis and lipid metabolism in MAFLD patients. Gene scores were assessed to explore their impact on the immune microenvironment, particularly hepatocyte-macrophage communication. Weighted Gene Co-expression Network Analysis (WGCNA) was applied to Bulk-RNA-Seq data to identify gene clusters associated with ferroptosis and lipid metabolism. The analyses were integrated into a risk assessment system and predictive model, with validation conducted through in vivo experiments. Key findings: Integration of single-cell and WGCNA data identified 11 key genes linked to ferroptosis and lipid metabolism (e.g., IER5L, SOCS2, KLF9), significantly influencing the liver's immune microenvironment. The risk assessment system and predictive model achieved an AUC of 0.92 and revealed distinct immune and biological characteristics in MAFLD patients across risk levels. The expression patterns and biological roles of these genes were confirmed in in vivo studies. Significance: This study establishes a strong link between ferroptosis- and lipid metabolism-related gene expression and MAFLD's complexity. It provides novel insights into disease mechanisms, supporting personalized prognosis and targeted therapeutic strategies for MAFLD patients.