Mechanisms of Huatanqushihuoxue Formula (HQHF) in Ameliorating MAFLD 1 via Transcriptomic Analysis and Ferroptosis Regulation

Background: Metabolic dysfunction-associated fatty liver disease(MALD) is a prevalent condition with limited therapeutic options.Huatangushihuoxue Formula (HQHF) demonstrates efficacy against MAFLD, yet its broader clinical application is hindered by an unclear mechanism of action. Purpose:This study aimed to elucidate the therapeutic mechanisms and molecular targets of HQHF in MAFLD. Study design and Methods: MAFLD was induced in mice via a 26 high-fat diet (HFD). After HQHF intervention, systematic evaluations were conducted.Transcriptome analysis of liver tissues was performed, followed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of differentially expressed genes. Key targets were identified using Cytoscape.Experimental validations included protein immunoblotting, immunohistochemistry, quantitative PCR. and immunofluorescence.Finally, a preliminary analysis of the single-cell sequencing data was conducted. Results: Transcriptome analyses indicated that HQHF's therapeutic effects are centrally mediated through the regulation of oxidative stress and ferroptosis. Experimental validation confirmed that HQHF alleviates oxidative stress and inhibits ferroptosis to treat MALD. Furthermore, eight key target genes were identified: Tnfaip3, Cx3cll, ICAM-1, CCND1, MAPK13,VDAC2, ATF4, and c-Myc. HQHF was verified to regulate the expression of these genes, which are involved in MAFLD pathophysiology.