Modified Bixuesan modulates inflammatory and apoptotic gene expression in post-radiotherapy chronic pharyngitis of laryngeal cancer: an integrative network pharmacology and in vitro validation study.

BACKGROUND: Post-radiotherapy chronic pharyngitis affects 60%-80% of laryngeal cancer survivors and is characterized by sustained elevation of pro-inflammatory cytokines and dysregulation of key apoptotic and oncogenic signaling pathways, including NF-κB/PI3K-AKT/EGFR and TP53/CASP3 axes. Modified Bixuesan (MBS), a classical Traditional Chinese Medicine formula with nearly 180 years of clinical use in pharyngolaryngeal disorders, has not been systematically characterized for its molecular mechanism of action in this context.

METHODS: We conducted an integrative study combining network pharmacology, multi-omics bioinformatics, and in vitro cell line validation. Active MBS compounds were screened from TCMSP and BATMAN-TCM databases using ADME thresholds. Single-cell transcriptomic data from laryngeal squamous cell carcinoma (LSCC; GSE127519) and bulk microarray data from radiation-induced pharyngitis (GSE103412) were analyzed to identify hub target genes. Molecular docking was performed using AutoDock Vina against crystal structures from RCSB PDB. In vitro RT-qPCR validation was conducted in Hep-2 and TU212 laryngeal cancer cell lines treated with MBS extract (0.5 mg/mL, 48 h), evaluating mRNA expression of four hub genes: TP53, EGFR, NF-κB1, and CASP3.

RESULTS: Network pharmacology identified 47 active MBS compounds and 89 putative therapeutic targets, with TP53, AKT1, EGFR, NF-κB1, CASP3, MMP9, and VEGFA as principal hub genes. Molecular docking revealed strong predicted binding of quercetin to TP53 (-9.4 kcal/mol) and kaempferol to EGFR (-8.7 kcal/mol). In vitro RT-qPCR in MBS-treated Hep-2 cells demonstrated significant upregulation of TP53 (1.72 ± 0.19-fold, p = 0.024) and CASP3 (1.89 ± 0.25-fold, p = 0.011), and significant downregulation of EGFR (0.46 ± 0.08-fold, p = 0.003) and NF-κB1 (0.43 ± 0.11-fold, p = 0.004). In TU212 cells, concordant and more pronounced changes were observed: TP53 (2.51 ± 0.31-fold, p = 0.001), CASP3 (2.24 ± 0.27-fold, p = 0.002), EGFR (0.38 ± 0.07-fold, p < 0.001), and NF-κB1 (0.36 ± 0.09-fold, p = 0.001).

CONCLUSION: This integrative study provides in vitro gene expression evidence suggesting that MBS may modulate inflammatory and apoptotic signaling associated with post-radiotherapy laryngeal mucosal injury, through predicted regulation of NF-κB/PI3K-AKT/EGFR pathway components and upregulation of TP53/CASP3 mRNA expression. These findings are exploratory; network pharmacology and docking analyses are hypothesis-generating only, and in vitro results require prospective mechanistic and clinical validation.