Therapeutic Mechanism of Huayu Zhitong Plaster in Acute Soft Tissue Injury Rats via the PI3K/Akt/NF-κB Signaling Pathway.

INTRODUCTION: To explore the therapeutic efficacy of external use of Huayu Zhitong Plaster (HZP) in treating Acute Soft Tissue Injury (ASTI) and its underlying mechanism in a rat model. METHODS: UPLC-Q-TOF-MS/MS was used to analyze HZP's chemical constituents, and network pharmacology was applied to predict its therapeutic targets against ASTI. An ASTI rat model was established by blunt trauma to the gastrocnemius muscle. Inflammatory mediators and oxidative stress indicators were quantified by ELISA; ASTI severity was evaluated via H&E staining; protein localization and expression were assessed by immunofluorescence and Western blotting, respectively, to investigate the mechanism of action. RESULTS: UPLC-Q-TOF-MS/MS identified 86 components in HZP, mainly flavonoids, alkaloids, cyclic enol ether terpenes, and phenolic acids. Network pharmacology analysis showed that ASTI- related genes were primarily associated with the PI3K-Akt and NF-κB pathways via PI3K subunit and AKT1. Animal experiments confirmed that HZP significantly inhibited p-PI3K, p- AKT, and p-NF-κB expression, blocking the PI3K/Akt/NF-κB pathway. It reduced the levels of proinflammatory mediators IL-6 and PGE2, and increased the activity of the oxidative stressrelated enzyme SOD, decreased the level of MDA and MPO, and alleviated tissue damage, edema, and inflammatory infiltration (H & E staining). DISCUSSION: Given that effective and well-tolerated treatments for ASTI are highly desirable, our experimental data offer compelling support for the curative efficacy of HZP, paving the path for the scientific standardization of this intricate TCM preparation and affirming its potential to serve as a valuable topical treatment agent. CONCLUSION: By suppressing the PI3K/Akt/NF-κB pathway, HZP exerts potent antiinflammatory and antioxidant effects in ASTI, thereby mitigating tissue damage and facilitating repair.