Network pharmacology, molecular docking, and experimental validation of the mechanisms of Withaferin a in treating periodontitis.

OBJECTIVE: To explore the function and therapeutic mechanism of Withaferin A (WFA) in treatment of periodontitis using network pharmacology analysis and experiment validation. METHODS: The optimal concentration of WFA was determined using CCK-8 assay. IL-6 and IL-1β protein levels were measured by ELISA. Osteogenic differentiation under inflammation was evaluated via ALP staining and activity, Alizarin Red staining, and mineralization quantification. Expression of osteogenic markers (RUNX2, OCN, ALP) was analyzed by Western blot and RT-qPCR. Network pharmacology and molecular docking were used to predict and validate WFA targets, followed by experimental verification with WB and RT-qPCR. Mitochondrial status was examined using JC-1 staining and TEM, while ROS levels were detected to assess oxidative stress. Expression of SLC7A11, GPX4, and OPA1 was measured by WB and RT-qPCR. WFA's therapeutic effect was further validated in a ligature-induced periodontitis (LIP) rat model using micro-CT, H&E staining, and immunohistochemistry. RESULTS: Our study demonstrated that WFA exerts multi-faceted therapeutic effects on periodontitis. It promoted osteogenesis by upregulating osteogenic markers (RUNX2, OCN, ALP) and counteracted Pg-LPS-induced inflammation, reducing IL-6 and IL-1β levels and attenuating bone resorption in vitro. Bioinformatics analysis identified 119 potential targets of WFA, with 100 overlapping periodontitis-related genes. GO and KEGG enrichment highlighted the AGE-RAGE, IL-17, and Relaxin signaling pathways. Molecular docking confirmed strong binding of WFA to JUN, FN1, and TGFβ, which were functionally validated. Mechanistically, WFA reduced ROS, preserved mitochondrial integrity, and upregulated SLC7A11 and GPX4, indicating anti-ferroptotic effects via the SLC7A11/GPX4 axis. In vivo, WFA alleviated Pg-LPS-induced bone loss. CONCLUSION: In conclusion, WFA exhibits therapeutic potential in periodontitis through suppressing ferroptosis via SLC7A11/GPX4 axis, which promotes osteogenesis and inhibits Pg-LPS-induced inflammation. CLINICAL RELEVANCE: WFA demonstrates significant potential as a therapeutic intervention for periodontitis.