Using an integrative multi-omics and in vitro approach to investigate the role of tris(2-butoxyethyl) phosphate in promoting hepatic steatosis.

Abstract

Objective: To investigate the potential role of the environmental pollutant tris(2-butoxyethyl) phosphate (TBOEP) in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) and to elucidate its underlying molecular mechanisms.

Methods: We employed an integrated multi-omics approach. First, a multidatabase strategy identified overlapping targets between TBOEP and MASLD, which were subjected to functional enrichment and protein-protein interaction (PPI) network analysis. Molecular docking was then used to predict binding affinities. Second, transcriptomic profiling (RNA sequencing (RNA-seq)) was performed on TBOEP-treated AML-12 hepatocytes to identify differentially expressed genes and pathways. Finally, key findings were validated in vitro using Oil Red O staining, triglyceride quantification and western blot analysis.

Results: We identified 154 common targets between TBOEP and MASLD, with enrichment analysis pointing to pathways in non-alcoholic fatty liver disease and insulin resistance. PPI analysis highlighted ACTB, IL6, PPARG and PPARA as pivotal hub proteins. RNA-seq revealed 326 differentially expressed genes, with significant alterations in metabolic pathways. Cross-referencing pathways predicted from target genes with RNA-seq data revealed 10 pathways potentially impacted by TBOEP, emphasising its multifaceted effects on hepatocyte function. In vitro studies showed that TBOEP promotes lipid accumulation in hepatocytes by increasing PPARγ expression.

Conclusion: Our study provides evidence that TBOEP acts as a potential environmental risk factor for MASLD. We demonstrate that TBOEP promotes hepatocyte steatosis, likely through the dysregulation of metabolic pathways centred on PPARγ signalling. These findings provide new insights into the pathogenesis of MASLD.