Integrative Analysis of Triphala's Therapeutic Mechanisms in Periodontitis: Network Pharmacology-guided Investigation with Experimental Validation in Cellular and Animal Models.

PURPOSE: Periodontitis is a chronic inflammatory disease characterised by progressive destruction of periodontal tissues and alveolar bone resorption. Triphala (TRP), a traditional Ayurvedic formulation comprising equal proportions of Terminalia chebula, Terminalia bellirica, and Phyllanthus emblica, has demonstrated anti-inflammatory and antioxidant properties. This study aimed to investigate the molecular mechanisms underlying TRP's therapeutic effects on periodontitis through an integrated approach combining network pharmacology with experimental validation, focusing on the PI3K/AKT signalling pathway. METHODS AND MATERIALS: Network pharmacology analysis was performed using TCMSP and TCM databases to identify active compounds and potential targets of TRP. The intersection between TRP targets and periodontitis-related genes was analysed. In vitro studies utilised lipopolysaccharide (LPS)-induced human periodontal ligament fibroblasts (hPDLFs) treated with various concentrations of TRP (5-40 μg/ml). Cell viability (CCK-8), reactive oxygen species (ROS) levels (flow cytometry), and expression of hub genes, oxidative stress markers, and PI3K/AKT pathway components were assessed via Western blotting and qPCR. Functional rescue experiments using PI3K activator (740Y-P) and inhibitor (LY294002) were conducted. In vivo validation employed a ligature-induced periodontitis rat model with TRP irrigation treatment, evaluated through micro-CT, histological staining (H&E and TRAP), and molecular analyses. RESULTS: Network pharmacology identified 129 potential targets of TRP for treating periodontitis, with PI3K/AKT emerging as a key signalling pathway. The top 10 hub genes included JUN, TP53, MYC, EGFR, and AKT1. TRP (20 μg/ml) significantly restored LPS-induced cell viability reduction (P 0.01) and decreased ROS levels (P 0.01). TRP downregulated the expression of hub genes (P53, MYC, EGFR, AKT1) and oxidative stress markers (SOD, CAT, Nrf2, HO-1) elevated by LPS (P 0.01). Mechanistically, TRP suppressed PI3K/AKT pathway activation, reducing phosphorylated PI3K, AKT1, and AKT2 levels while upregulating PTEN expression (P 0.01). These effects were reversed by the PI3K activator and enhanced by the PI3K inhibitor. TRP treatment significantly decreased inflammatory cytokines (IL-1β, IL-6, TNF-α) and MMP8 secretion (P 0.01). In vivo, TRP irrigation reduced alveolar bone loss (decreased ABC-CEJ distance, increased BV/TV ratio, P 0.05), decreased RANKL/OPG ratio (2.3 ± 0.242 vs 8.481 ± 1.56 in model group, P 0.05), reduced osteoclast numbers (P 0.05), and attenuated inflammatory cell infiltration in periodontal tissues. CONCLUSIONS: This study demonstrates that TRP exerts anti-inflammatory and anti-oxidative effects on LPS-induced periodontal inflammation through inhibition of the PI3K/AKT signalling pathway. The integration of network pharmacology with comprehensive experimental validation reveals TRP's multi-target therapeutic mechanisms in periodontitis. These findings provide scientific evidence supporting TRP as a promising natural therapeutic agent for periodontal disease management and suggest its potential for development as an adjunctive treatment in clinical periodontal therapy.