Triptolide induces ferroptosis in lung adenocarcinoma cell by modulating lipid metabolism pathways

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To investigate the mechanism by which Triptolide (TPL) regulates ferroptosis in lung cancer cells through a comprehensive approach integrating network pharmacology, molecular docking, molecular dynamics simulations, and in vitro validation. The CCK-8 assay was initially used to examine the inhibitory effects of varying concentrations of TPL on the proliferation of human lung adenocarcinoma (LAD) cells. The objective is to identify ferroptosis-related pathway targets from the FerrDb database. Then, the intersection of the three should be determined to obtain potential ferroptosis targets regulated by TPL treatment in LUAD. Secondly, a protein-protein interaction network was constructed using the STRING database and Cytoscape software. Subsequently, network submodule analysis was performed based on the MCODE algorithm. Enrichment analysis revealed the key regulatory pathways through which TPL combats lung cancer. Molecular docking and kinetic simulations provided a detailed exploration of the potential mechanism by which TPL inhibits lung cancer via the ferroptosis pathway, with in vitro experiments providing validation. Finally, ADMET analysis was conducted to simulate drug metabolism processes, thereby providing a foundation for its clinical application. The specific experimental workflow is illustrated in Fig. 1. TPL inhibited the proliferation and migration of A549 cells in a concentration‑dependent manner. PPI network and KEGG enrichment analysis identified ferroptosis as the primary pathway targeted by TPL. Molecular docking and MD simulations revealed stable interactions between TPL and GPX4, SLC7A11, and ACSL4. Western blot, flow cytometry, and CETSA validated that TPL directly targets GPX4, SLC7A11, and ACSL4 in A549 cells. ADMET analysis indicated favorable physicochemical properties, oral absorption, and acceptable safety profiles of the ferroptosis pathway regulated by TPL in the context of lung adenocarcinoma. Molecular docking and molecular dynamics simulations revealed stable interactions between TPL and GPX4, SLC7A11, and ACSL4. In vitro studies employing Western Blot, flow cytometry, and Cell Thermal Shift Assay (CETSA) have validated the molecular mechanism by which TPL directly targets GPX4, SLC7A11, and ACSL4. A comprehensive physicochemical and pharmacokinetic analysis was conducted on TPL, revealing its favorable physicochemical properties, drug-like characteristics, and synthetic feasibility. The study further demonstrated significant advantages in oral absorption. Its metabolic pathways are well-defined, posing a low risk of drug interactions. While demonstrating adequate cardiac and hepatic safety, it exhibits a relatively low maximum tolerated dose. TPL inhibits GPX4 and SLC7A11 expression while upregulating ACSL4 in A549 cells, and directly binds to these three proteins. By modulating lipid metabolism, TPL induces ferroptosis, positioning it as a promising therapeutic candidate for LUAD.

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