IL-17/Th17-Treg Axis regulation by Huangqi Gancao decoction in immunosuppression: Integrating network pharmacology, metabolomics, and in vivo validation.

BACKGROUND: In this study, LC-MS/MS, network pharmacology, and molecular docking identified the key components and mechanisms of Huangqi Gancao Decoction (HQGCD) in enhancing immunity, further validated by in vivo experiments and Untargeted metabolomics.

METHODS: The active components of HQGCD and associated target proteins were identified using LC-MS/MS and the TCMSP database. UniProt IDs were validated through the UniProt database. Protein-protein interaction networks were constructed via STRING and visualized using Cytoscape. GO and KEGG pathway enrichment analyses were conducted using the DAVID database, followed by molecular docking with core proteins. Immunosuppressed mouse models were established through intraperitoneal injection of cyclophosphamide (Cy). Levels of IL-1β, IL-6, TNF-α, sIgA, and IL-17a in serum and tissues were quantified using ELISA. Protein expression levels of COX2 (PTGS2) and MCP1 (CCL2), associated with the IL-17 signaling pathway, were analyzed through Western blot. Gene expression was assessed via qPCR, and immunohistochemistry was used to evaluate the relative expression of COX2 and MCP1 in the spleen. Th17/Treg cell in spleen was assessed via flow cytometry. Untargeted metabolomics was applied to analyze changes in serum metabolites.

RESULTS: Network pharmacology combined with LC-MS/MS identified 13 active components in Astragali Radix (Astragalus membranaceus (Fisch.) Bunge) and 27 in Glycyrrhizae Radix et Rhizoma (Glycyrrhiza uralensis Fisch.). KEGG analysis hilighted key pathways, including TNF and IL-17 signaling, underlying HQGCD's anti-immunosuppressive effects. Molecular docking showed strong binding of ursolic acid to critical therapeutic targets such as COX2, MCP1, IL-1β, IL-6, and TNF-α. In vivo experiments revealed that HQGCD increased sIgA and IL-1β, IL-6, and TNF-α levels in serum, spleen, while upregulated COX2, MCP1, and IL-17a in the IL-17 pathway. Flow cytometry analysis revealed that HQGCD can regulate the balance between Treg and Th17. Metabolomic analysis identified significant alteration in pathways, including beta-alanine metabolism and aminoacyl-tRNA biosynthesis, as well as changes in aldosterone-regulated sodium reabsorption following HQGCD intervention.

CONCLUSION: This study reveals HQGCD's multi-target mechanisms in alleviating immunosuppression via IL-17-Th17/Treg regulation and metabolic shift, experimentally validated in Cy-treated mice. Metabolomics uncovered key serum changes, reinforcing HQGCD's therapeutic potential and providing a foundation for clinical translation.