Mechanism Study on Tuoli Tounong Decoction Regulating Macrophage Polarization Based on Network Pharmacology and Cell Experiment Verification

Objective To investigate the mechanism of action of the active components of Tuoli Tounong Decoction in regulating macrophages for the treatment of postoperative wounds of perianal abscess through network pharmacology, molecular docking, and cell-based experimental verification.Methods The active components and their corresponding targets of Tuoli Tounong Decoction were screened and identified using the TCMSP and UniProt databases. Potential therapeutic targets for postoperative wounds of perianal abscess were collected from the OMIM, GeneCards, DrugBank, and TTD databases. By constructing a protein-protein interaction (PPI) network diagram and performing topological analysis, potential core targets of Tuoli Tounong Decoction in treating postoperative wounds of perianal abscess were identified. Meanwhile, the DAVID database was used to conduct enrichment analysis on the screened intersecting targets. Based on these results, AutoDock software was employed for molecular docking verification, and the data were then imported into PyMOL software for visualization. Finally, CCK-8 assays, ELISA assays, and Western blotting were used to evaluate the effects of the active components of Tuoli Tounong Decoction on macrophages (RAW264.7 cells), and to observe their impacts on the AKT/PI3K pathway and the expression of related proteins.Results Through network pharmacology analysis, a total of 170 active components of Tuoli Tounong Decoction and 438 intersection targets were identified, with AKT1 emerging as the core target following screening. KEGG enrichment analysis revealed that the PI3K-Akt signaling pathway was the most relevant pathway associated with the treatment of post-operative wounds of perianal abscess. Molecular docking results indicated tight binding of AKT1 to astragaloside IV, luteolin, and quercetin. CCK-8 assay results demonstrated that the active components of Tuoli Tounong Decoction exhibited no toxicity toward RAW264.7 cells. ELISA results showed that these components regulated the expression levels of inflammatory factors IL-1β and TNF-α, as well as the growth factor TGF-β. Western Blot results showed thatthese components could all activate PI3K and AKT1 proteins, down regulate the expression levels of the M1-type macrophage marker iNOS, and upregulate the expression level of the M2-type macrophage marker Arg-1. Through these effects, they regulate the release of cytokines and exert anti-inflammatory and wound-healing promoting effects.Conclusion Tuoli Tounong Decoction may regulate macrophage polarization by modulating the PI3K-Akt signaling pathway, reducing the expression of downstream inflammatory factors IL-1β and TNF-α, and upregulating the expression of growth factor TGF-β, thereby promoting the healing of post-operative wounds of perianal abscess.