Integrating metabolomics and molecular dynamics simulation to elucidate the anti-atherosclerotic mechanisms of Gynostemma pentaphyllum.

Atherosclerosis (AS) is a leading global cause of cardiovascular mortality. Gynostemma pentaphyllum (GP), a well-recognized traditional Chinese medicine with a long history of medicinal application, has emerged as a promising candidate for anti-atherosclerotic intervention due to its well-documented potential in regulating cardiovascular homeostasis. This study aimed to systematically elucidate the anti-atherosclerotic mechanisms of GP using metabolomics, network pharmacology, molecular docking, molecular dynamics (MD) simulations, and in vitro validation. Leaf and root samples from five-leaf and seven-leaf gynostemma were analyzed by UHPLC-MS/MS for metabolite profiling. Active components and targets of GP were retrieved from TCMSP and PharmMapper, while AS-related targets were collected from GeneCards, OMIM, DrugBank, and DisGeNET. PPI networks were constructed using Cytoscape, and functional enrichment was analyzed via GO and KEGG. Molecular docking and MD simulations assessed binding affinities between GP components and core targets. Based on the bioinformatics analysis, PPARγ was chosen as a key therapeutic target for further in vitro experimental validation. Metabolomics identified 1898 compounds, with 208 differentially accumulated metabolites between GP varieties. Network pharmacology revealed 24 active components, 168 potential targets, and 69 AS-overlapping targets. Twelve core genes were identified, including AKT1, ALB, PPARγ, ESR1, CASP3, MMP9, EGFR, SRC, MMP2, MAPK1, MPO, and MAPK8. Enrichment analysis linked these targets to lipid metabolism, efferocytosis, and inflammation pathways. Molecular docking and MD simulations confirmed strong and stable binding of Gypenoside XL to PPARγ. Gypenoside A, one of the relatively abundant constituents among gypenosides (GPs), was also directly bound to PPARγ. In vitro, GPs reduced lipid accumulation, upregulated PPARγ and LXRα, suppressed NF-κB phosphorylation and nuclear translocation and attenuated oxidative stress. These coordinated regulations on lipid metabolism, inflammation, and oxidative stress collectively mitigate key pathological processes of AS, including foam cell formation and atherosclerotic plaque progression. GP exerts anti-atherosclerotic effects through a multi-component, multi-target mechanism, with activation of the PPARγ-LXRα pathway and inhibition of NF-κB driven inflammation being central to its therapeutic action.