Abstract
Background/Objectives: Astragalus root is a classical qi-tonifying traditional Chinese medicine that has demonstrated potential therapeutic efficacy in type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). However, the precise mechanisms underlying its effects on the comorbidity of these two disorders remain unclear. This study investigated the molecular mechanisms by which Astragalus root ameliorated T2DM-NAFLD comorbidity. Methods: Network pharmacology, molecular docking, molecular dynamics simulation, and in vitro experiments were employed to elucidate the potential roles and mechanisms of Astragalus root in the management of T2DM-NAFLD comorbidity. Results: A total of 25 bioactive constituents and 152 corresponding targets associated with Astragalus root were identified. PPI network analysis revealed the top ten core candidate targets, among which six possessed suitable crystal structures for molecular docking, including interleukin-6 (IL-6), threonine-protein kinase 1(AKT1), transcription factor AP-1(JUN), tumor necrosis factor (TNF), cysteine-dependent aspartate-specific protease 3 (CASP3), and estrogen Receptor 1(ESR1). Kyoto encyclopedia of genes and genomes (KEGG) analysis further identified the phosphatidylinositol 3-kinase (PI3K)-AKT as the most significantly enriched pathway. Molecular docking validated the potential binding modes of formononetin to the six core targets, a finding that was further confirmed by molecular dynamics simulations, which proved the stability of the resulting complexes. In vitro experiments demonstrated that formononetin obviously decreased lipid droplet accumulation, downregulated total cholesterol (TC) and triglyceride (TG) levels, suppressed the expression of TNF-α, IL-6, and interleukin-1β (IL-1β), decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and enhanced glutathione (GSH) content and superoxide dismutase (SOD) activity. These therapeutic effects were achieved through inhibition of protein expression within the PI3K/AKT/mechanistic target of rapamycin (mTOR) signaling pathway. Conclusions: This study determined the potential therapeutic targets and underlying mechanisms of formononetin derived from Astragalus root in the T2DM-NAFLD management, thereby providing a scientific basis for its clinical application.