Mechanistic Investigation of Astragalus Root in the Management of T2DM-NAFLD Comorbidity: An Integrated Network Pharmacology, Molecular Docking, Molecular Dynamics Simulation, and In Vitro Study

Background/Objectives: Astragalus root is a classical qi-tonifying traditional Chinese medicine that has demonstrated potential therapeutic efficacy in T2DM and 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 IL-6, AKT1, JUN, TNF, CASP3, and ESR1. KEGG analysis further identified the PI3K-AKT as the most significantly en-riched pathway. Molecular docking of the principal bioactive constituent formononetin from astragalus root with the six core targets was conducted using AutoDock4 software. Molecular dynamics simulations verified the stability of the interactions between for-mononetin and each of the six core target proteins. In vitro experiments demonstrated that formononetin obviously decreased lipid droplet accumulation, downregulated TC and TG levels, suppressed the expression of TNF-α, IL-6, and IL-1β, decreased ROS and MDA levels, and enhanced GSH content and SOD activity. These therapeutical effects were achieved through inhibition of protein expression within the PI3K/AKT/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.