The Underlying Pharmacological Mechanisms and Active Components of XZZTP in Modulating Bacterial Inflammation Elucidated by LC-MS/MS, Network Pharmacology, In Vitro Experiments, Molecular Docking, and Dynamics Simulations

Background: The Xiao Zhong Zhi Tong Patch (XZZTP) has been extensively utilized in China to alleviate many diseases associated with bacterial inflammation. However, its pharmacological mechanism and active components remain unclear. Methods: The anti-inflammatory effects of XZZTP were evaluated in vivo and in vitro models. The characterization of XZZTP and its transdermal components was performed using LC-MS/MS. The underlying pharmacological mechanism was predicted through network pharmacology using the identified transdermal components and verified by Western blotting. Molecular docking and molecular dynamics simulations were performed on key targets to screen active components. Results: XZZTP showed a swelling inhibition rate of 45.96% in xylene-induced ear edema mice in vivo. In vitro, the inflammatory mediators NO, TNF-α, and PGE2 were concentration-dependently reduced by XZZTP in the LPS-induced RAW 264.7 macrophages model, with inhibition rates of 56.53%, 53.75%, and 48.49% at 200 µg/mL, respectively. LC-MS/MS identified 126 chemical components (97 newly reported) in XZZTP, including 52 transdermal potential active components, among which a new iridoid and its isomer were reported for the first time. Network pharmacology analysis demonstrated that XZZTP mainly downregulated the PI3K/AKT/HIF-1 signaling pathway to alleviate bacterial inflammation. The protein expression of core targets p-PI3K, p-AKT, and HIF-1α in the LPS-induced RAW 264.7 macrophages was significantly reduced after XZZTP intervention. Eight active components were screened via molecular docking, and molecular dynamics simulations of three representative complexes validated stable binding interactions, supporting their therapeutic potential. Conclusions: These findings provide a theoretical basis for XZZTP as a potential agent to ameliorate bacterial inflammation-related diseases, serving as a reference for its further application.