Integrated multi-omics analysis identifies and validates endoplasmic reticulum stress and mitophagy-related biomarkers in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is one of the most prevalent chronic liver diseases worldwide. Growing evidence indicates that endoplasmic reticulum stress (ERS) and mitophagy play critical roles in MASLD progression. However, the specific mechanism by which ERS and mitophagy participate in MASLD is not clear, and there is still a lack of treatment strategies for these pathways. Therefore, this study aims to integrate GEO data mining, machine learning, and scRNA-seq analysis to elucidate potential mechanisms by which ERS and mitophagy mediate MASLD progression, and to identify candidate therapeutic agents targeting these pathways through high-throughput virtual screening. Nuclear receptor subfamily 4 group A member 1 (NR4A1) was identified as a key target involved in the progression of MASLD mediated by ERS and mitophagy. Functional enrichment and immune infiltration analyses suggested that NR4A1 participates in immunoregulatory and adaptive responses under metabolic stress. scRNA-seq analysis confirmed that the expression of NR4A1 decreases gradually during the progression of MASLD, particularly throughout macrophage activation and differentiation. Experimental validation further demonstrated that NR4A1 was downregulated in FFA-treated hepatocytes, MASLD mice, and human liver tissues. A ceRNA network centered on NR4A1 was constructed, and potential therapeutic compounds were identified via molecular docking. Taken together, this study highlights NR4A1 as a key target in ERS- and mitophagy-mediated MASLD progression and provides novel insights into therapeutic strategies for alleviating MASLD by targeting ERS and mitophagy pathways.