Unraveling the Therapeutic Mechanisms of Shanzhen Mingmu Pill in Diabetic Retinopathy: An Integration of Serum Pharmacochemistry, Network Pharmacology, and Experimental Validation

PURPOSE: This study aimed to identify the bioactive ingredients of Shanzhen Mingmu Pill (SMP) and investigate its mechanisms of action against diabetic retinopathy (DR). METHODS: We performed a qualitative analysis of SMP and drug-containing serum using UPLC-Q-Exactive MS/MS. Network pharmacology was applied to predict core targets and signaling pathways, followed by the construction of a "formula-component-target-pathway-disease" topological network. Molecular docking was employed to predict the binding capabilities between core targets and active constituents, followed by molecular dynamics simulations to validate the docking results. Furthermore, in vitro experiments using high glucose-induced human retinal microvascular endothelial cells (HRMECs) were performed to validate the anti-DR efficacy and mechanism. RESULTS: We identified 159 chemical components in SMP, along with 16 prototype components and 11 metabolites in serum. The core targets, including TP53, SRC, STAT3, ESR1, and AKT1, were identified through network pharmacology, and strong binding affinities between the active components and these targets were confirmed by molecular docking. In vitro, SMP significantly attenuated high glucose-induced apoptosis and reduced intracellular reactive oxygen species (ROS) levels in HRMECs. Western blot showed that this protective effect was mediated through the PI3K-Akt signaling pathway. CONCLUSION: The chemical components, blood-absorbed components, potential targets, and mechanistic pathways of SMP in DR treatment were systematically elucidated. This study preliminarily determined the material basis and pharmacological mechanisms of SMP, laying a foundation for further translational research and clinical application.