Mechanistic Insights into Astragalus Membranaceus for Oral Submucosal Fibrosis: A Network Pharmacology and Experimental Approach

Background: Oral Submucosal Fibrosis (OSF) is a chronic progressive oral mucosal disease with a tendency to progress to cancer. Astragalus membranaceus (AST) is a traditional Chinese medicine used to invigorate Qi and strengthen the body, with anti-fibrosis properties. However, the effect and mechanism of AST on OSF remain unclear. Objective: This study aims to explore the mechanism of Astragalus membranaceus in OSF using network pharmacology and to validate its effects on oral mucosal fibroblasts through in-vitro experiments. Methods: Network pharmacology was employed to construct an "AST - ingredient - target - OSF" network and perform Protein-Protein Interaction (PPI) analysis. Molecular docking was used to confirm core interactions between key targets and ingredients, and all results met the criterion of a binding energy of <- -1.2 kcal/mol. In-vitro experiments were conducted to assess the cytotoxicity of arecoline (ARE) and Astragalus membranaceus injection (ASI) on Oral Mucosal Fibroblasts (OMF). method: First, genes related to AST and OSF were collected, and 68 intersection genes were identified as potential targets. Then, a protein-protein interaction (PPI) network of potential targets and an “AST-ingredient-target-OSF” network were constructed. In addition, KEGG and GO enrichment analyses identified 138 pathways and 178 biological processes. Subsequently, molecular docking was performed to pair the top potential targets with the ingredients to determine the core targets. In cellular experiments, CCK-8 assay was used to evaluate the cytotoxicity of arecoline (ARE) and astragalus injection (ASI) on oral mucosal fibroblasts (OMF). RT-qPCR was performed to detect the expression of ACTA2 in OMF induced by ARE, confirming the establishment of the OSF cell model. Finally, ASI intervention was conducted based on 30 µg/ml ARE to evaluate its inhibitory effects on the expression of ACTA2, EGFR, and VEGFA mRNA. Results: Analysis revealed 68 common targets between AST and OSF, and a corresponding PPI network was constructed. KEGG and GO enrichment analyses identified 138 pathways and 178 biological processes associated with these targets. Molecular docking confirmed core interactions between five key targets (EGFR, VEGFA, MAPK3, HRAS, JUN) and other ingredients. In-vitro experiments showed that ARE at concentrations of 20-40 μg/ml significantly upregulated ACTA2, EGFR, and VEGFA mRNA expression. ASI treatment at varying concentrations significantly inhibited these increases, with 100 mg/ml ASI downregulating EGFR and VEGFA mRNA, and 300-400 mg/ml ASI reducing ACTA2 expression. Conclusion: Astragalus membranaceus injection may suppress ARE-induced fibrosis by targeting EGFR and VEGFA, supporting its potential therapeutic role in the treatment of OSF.