Network Pharmacology, Molecular Docking, Molecular Dynamics Simulation and Experiment Verification Analysis to Reveal the Action Mechanism of RenShen Guipi Wan in treating Anaemia

Objective: To identify the possible phytochemical compounds of RGW that might treat anaemia through integrating network pharmacology, molecular docking, molecular dynamics simulation, and experiment verification to explore their potential mechanisms of action. Methods: UPLC-Q Exactive Orbitrap-HRMS was used to identify RGW components. The major chemical components and potential target genes of RGW were screened by bioinformatics. The key targets in anaemia were identified based on network modules. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. Pharmacodynamics was evaluated by establishing a micemodel of anaemia. Molecular docking, Molecular dynamics simulation, and Enzyme-linked immunosorbnent assay (ELISA) method were performed to confirm the effectiveness of targets in related pathways. Result: 171 compounds were identified in RGW using UPLC-Q Exactive Orbitrap-HRMS, eighty-two active ingredients in RGW were screened, 348 potential targets were identifed. In particular, Ginsenoside Rg4, Ginsenoside Rg1, 3,3',4,4'-Tetrahydroxy 2-methoxychalcone, Ginsenoside F1, Glycyrol, Chalconaringenin 4'-glucoside, Licochalcone B, 4',7-Dihydroxyflavone, Glycycoumarin, and Ginsenoside Rh1 were the core components and TP53, STAT3, PIK3R1, SRC, HIF-1α were the core targets. The GO and KEGG analyses indicated that RGW may modulate multiple biological processes and pathways, including the PI3K-Akt signaling pathway, HIF-1 signaling pathway, EGFR tyrosine kinase inhibitor resistance, and NF-kappa B signaling pathway. Molecular docking and molecular dynamics simulations showed good afnity between the active components and core targets of RGW, with stable binding within 100 nano seconds. Experiment verification revealed RGW could improve the routine blood markers of mice, and decrease the level of HIF-1α significantly. Conclusion: RGW might play a role by targeting the key target HIF-1α and regulating PI3K-Akt signaling pathway and HIF-1 signaling pathways. This study demonstrates the potential pharmacological mechanism of RGW in the treatment of anaemia and provides a reference for clinical application of the formula.