Wuse-oil Compound 03 mitigates radiation-induced oral mucositis by modulating oral microbiota to mediate TLR4/NF-κB pathway and inhibit M1 macrophage polarization

BACKGROUND: Radiation-induced oral mucositis (RIOM) is a prevalent condition among patients with head and neck cancer undergoing radiotherapy. It is characterized by a high incidence and severe symptoms that significantly compromise patients' quality of life. Currently, there exists no consensus regarding an effective treatment modality. Wuse-oil Compound 03 (WC03), a Traditional Chinese Herbal Medicine, demonstrates potential in managing RIOM. PURPOSE: This study aims to investigate the efficacy of WC03 in treating RIOM through the modulation of oral microbiota and associated biological pathways. STUDY DESIGN: Mechanism study experimental design. METHODS: A rat model of RIOM was established via radiation exposure. WC03 was topically administered to the oral mucosa of the rats twice daily for seven days subsequent to irradiation. The effectiveness of WC03 was evaluated using hematoxylin and eosin (H&E) staining and the Oral Mucositis Index (OMI). Oral microbiota samples were analyzed through 16S rRNA sequencing. RNA sequencing and pathway analysis were conducted on the buccal mucosa. Differentially expressed genes were validated through immunohistochemistry and protein blotting techniques. The polarization of inflammatory M1 macrophages was assessed using immunofluorescence. The active components of WC03 were identified through mass spectrometry and examined using network pharmacology. The role of Emodin was investigated via Transwell assays, flow cytometry, and Western blotting to elucidate its impact on suppressing the inflammatory response and macrophage polarization. RESULTS: The topical application of WC03 significantly mitigated the loss of basal layer epithelial cell density, mucosal thickness, and epithelial cell proliferation in instances of RIOM. Treatment with WC03 facilitated the restoration of oral microbiota alterations induced by radiotherapy and attenuated pro-inflammatory factors, including IL-2, IL-4, IL-6, IL-13, MCP-1, TNF-α, along with proteins implicated in the TLR4/NF-κB signaling pathway. The active compound, Emodin, was observed to suppress M1 macrophage polarization and inhibit the activation of the TLR4/NF-κB pathway. CONCLUSION: WC03 preserves the oral microbiota in RIOM rats. Our findings demonstrate for the first time that Emodin acts as a key active compound by inhibiting the TLR4/NF-κB pathway and M1 macrophage polarization, thus alleviating RIOM symptoms.