Investigating the mechanism of Tryptanthrin, a bioactive constituent of Yinhuang Gargle, in treating periodontitis based on network pharmacology, molecular docking, molecular dynamics simulation and in vitro experiments

Periodontitis is a chronic inflammatory disease induced by microbial biofilm dysbiosis and aberrant host immune responses, leading to progressive alveolar bone resorption. Traditional Chinese Medicine (TCM) offers multi-target therapeutic strategies for managing inflammatory diseases. This study aimed to investigate the anti-inflammatory potential of Tryptanthrin, a candidate bioactive constituent of Yinhuang Gargle, our hospital’s empirical formula (composed of Scutellariae Radix (10 g), Phellodendri Chinrnsis Cortex (10 g), Lonicerae Japonicae Flos (5 g), Isatidis Folium (8 g), Agrimonia Eupatoria (5 g), and licorice (3 g)), for treating periodontitis using network pharmacology, molecular docking, molecular dynamics simulation, and in vitro experimental methods. Pharmacological databases were utilized to screen the active components of the formula. Potential action targets were predicted and intersected with targets from periodontitis-related disease databases. A protein-protein interaction (PPI) network was constructed to identify core targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Molecular docking and molecular dynamics simulation was employed to verify the interactions between key active compounds and core targets. Finally, the anti-inflammatory effects of the core compound Tryptanthrin were validated through in vitro cell experiments. Network pharmacology analysis revealed that the formula likely exerts its effects by modulating key targets such as TNF-α, AKT1, BCL2, IL-6, and NFKB1, which are involved in inflammatory responses, oxidative stress, and osteoclastogenesis. Inflammatory signaling pathways including the TNF-α/NF-κB network were identified as core pathways. Molecular docking confirmed strong binding affinity between the bioactive compounds and these core targets. Molecular dynamics simulations revealed the multifaceted binding stability between Tryptanthrin and BCL2. In vitro experiments further validated that Tryptanthrin not only reduces the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) but also downregulates LPS‑induced upregulation of iNOS and COX‑2 at the protein level, further supporting its anti‑inflammatory mechanism. This study identifies Tryptanthrin as a promising antiinflammatory candidate for periodontitis, and offers a valuable paradigm for mechanismbased discovery of bioactive constituents from TCM formulas through the integration of network pharmacology, molecular docking, and in vitro experimental.