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

Abstract

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. 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. 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). 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.