Association Between Active DNA Demethylation and Liver Fibrosis in Individuals with Metabolic‐ Associated Steatotic Liver Disease (MASLD)

Background: Metabolic-associated steatotic liver disease (MASLD) represents the most common chronic hepatopathy worldwide and an independent risk factor for cardiovascular disease and mortality, particularly when liver fibrosis occurs. Epigenetic alterations, as DNA methylation, may influence MASLD susceptibility and progression; yet mechanisms underlying this process are limited. This study aimed to investigate active DNA demethylation in MASLD, alongside the methylation and mRNA levels of inflammation- and fibrosis-related candidate genes. Methods: Global demethylation intermediates (5-hydroxymethylcytosine[5hmC], 5-formylcytosine[5fC]) were quantified in peripheral blood mononuclear cells (PBMCs) from 89 individuals with/without MASLD using ELISA. Site-specific DNA methylation of SOCS3, SREBF1 and TXNIP was analyzed by mass spectrometry-based bisulfite sequencing; mRNA expression was assessed via rt-PCR. Results: Individuals with MASLD and moderate-to-high fibrosis risk (estimated by Fibrosis NASH Index, FNI) progressively exhibited greater global 5hmC and 5fC levels. FNI inversely correlated with methylation levels of SOCS3 gene promoter, and linearly with SOCS3, TXNIP, IL-6 and MCP-1 mRNA expression. Conclusions: Elevated fibrosis risk in MASLD is associated with active global DNA demethylation, as well as differential methylation and expression patterns of genes which are key regulators of inflammation and fibrosis. These epigenetic alterations may potentially contribute to fibrogenesis and represent novel biomarkers for MASLD progression toward fibrosis.