An Integrated Network Toxicology and Machine Learning Framework for Deciphering the Gastrointestinal Toxicity of Ginkgo biloba Seeds

Background : Ginkgo biloba seeds (GBS), a dual-use food and medicine, can cause severe gastrointestinal toxicity, but the underlying mechanisms remain unclear. Purpose : This study aims to elucidate the integrated mechanisms of GBS toxicity by combining network toxicology, machine learning, and experimental validation. Methods : A network toxicology approach was employed to map the interaction between GBS toxicants and gastrointestinal toxicity-related targets. The presence of the three major toxic constituents—ginkgolic acids (GAs), 4'-O-methylpyridoxine (MPN), and 4'-O-methylpyridoxine-5'-glucoside (MPNG)—was experimentally confirmed in both the raw GBS powder and the GBS-containing serum using UHPLC-Q Exactive HFX-MS, verifying their systemic bioavailability. An integrated machine learning framework, combining LASSO, Random Forest, and SVM-RFE, was used to prioritize key genes, with SHAP analysis enhancing model interpretability. Molecular docking and molecular dynamics (MD) simulations were performed to assess the potential direct interaction between GBS constituents and the MMP9 protein. In vitro assays (CCK-8, EdU, LDH, ROS, apoptosis) evaluated cytotoxicity, while qPCR, WB, IF, and CETSA assessed MMP9 expression and target engagement. Results : The integrated analysis, initiated by UHPLC-Q Exactive HFX confirmation of GAs, MPN, and MPNG in both raw GBS and GBS-containing serum, identified MMP9 as the top key gene associated with toxicity through a combined network toxicology and machine learning approach. MD revealed stable binding conformations between these toxicants and the MMP9 protein. GBS-containing serum significantly inhibited cell proliferation, induced oxidative stress, promoted apoptosis, and triggered an inflammatory response. MMP9 expression was markedly elevated at both the mRNA and protein levels, with CETSA confirming direct target engagement. Conclusion : This study establishes a novel framework for investigating herbal toxicity and identifies MMP9 as a central mediator of GBS-induced gastrointestinal injury. The direct interaction between GBS constituents and MMP9, along with the activation of the IL-17/NF-κB/IL-6 inflammatory axis and the Bax/Bcl-2/Caspase-3 apoptotic pathway, provides a systems-level understanding of GBS toxicity and highlights MMP9 as a potential biomarker for safety assessment.