Multi-Target Anti-Steatotic Effects of Andrographolide in MAFLD: Insights from Network Pharmacology and in vitro Study

Abstract

Background Metabolic Associated Fatty Liver Disease (MAFLD) is a prevalent metabolic disorder with no effective approved pharmacotherapy. Andrographolide, a bioactive diterpenoid from Andrographis paniculata, has anti-inflammatory and antioxidant properties, but its mechanisms in MAFLD remain unclear. Methods An integrated computational and experimental approach was applied. Network pharmacology was used to identify shared targets between andrographolide and MAFLD using public drug-target and disease databases. Protein-protein interaction analysis, pathway enrichment, and molecular docking with hub proteins were then performed. For experimental validation, hepatic steatosis was induced in HUH7 cells with oleic acid (1 mM, 24 h) and treated with andrographolide (14 µM). Lipid accumulation was measured by Oil Red O staining, and hub gene expression was analyzed by qRT-PCR. Results In silico analysis identified 253 shared targets, with TNF, IL6, AKT1, TP53, IL1B, JUN, VEGFA, STAT3, CASP3, and EGFR as major hubs. Enrichment analysis highlighted lipid metabolism, and inflammation signaling pathways. Molecular docking showed strong binding to AKT1 (–7.7 kcal/mol), EGFR (–7.5 kcal/mol), and STAT3 (–7.2 kcal/mol). In vitro, andrographolide reduced oleic acid-induced lipid accumulation by 38% (p = 0.0032). qRT-PCR revealed significant downregulation of AKT1 (73.5%, p = 0.0159), EGFR (72.0%, p = 0.0137), and STAT3 (53.6%, p = 0.0220) mRNA expression in andrographolide-treated steatotic hepatocytes. These transcript-level changes are correlative observations and do not confirm direct inhibition of AKT1, EGFR, or STAT3 protein activity, which requires phosphorylation-specific validation. The mRNA findings suggest a potential association between andrographolide treatment and modulation of these signaling pathways at the transcriptional level. Conclusion Andrographolide alleviates hepatic steatosis and is associated with transcriptional modulation of AKT1, EGFR, and STAT3 mRNA. However, protein-level validation is absent, and these findings should be considered hypothesis-generating rather than mechanistic proof of target inhibition. The results support further investigation of andrographolide as a potential multi-target therapeutic candidate for MAFLD.