Targeting Ezrin-Driven Oxidative Stress in Bile Reflux Carcinogenesis: Therapeutic Inhibition by Chaihu Shugan San and Xuanfu Daizhe Tang Combined

Objective To investigate the protective effects and underlying mechanisms of a combined decoction of Chaihu Shugan San and Xuanfu Daizhe Tang (hereafter abbreviated as GWTZ decoction) against ezrin-mediated oxidative stress during bile reflux-induced esophagitis-carcinoma transformation. Methods The absorbed bioactive components of GWTZ decoction in serum were identified by UPLC-QE-MS/MS and analyzed via network pharmacology. Sprague-Dawley (SD) rats underwent esophagogastroduodenal anastomosis (EGDA) to establish the bile reflux model. Tissues collected at 4-20 weeks post-surgery were analyzed by H&E staining, RNA-seq, and ELISA for TNF-α/IL-1β. In Vivo Intervention: From week 2 post-modeling, GWTZ decoction (10mL/kg/day) was administered intragastrically for 6 weeks. Tissues were subsequently evaluated via H&E staining, IHC (P53/ezrin/p-ezrin), RT-qPCR ( TNF-α/IL-1β ), and Western blot (ezrin/p-ezrin). In Vitro Model: a deoxycholic acid (DCA)-induced human esophageal epithelial cell (HEEC) injury model was used to assess the effects of GWTZ decoction on ezrin phosphorylation at Thr567 and mitochondrial superoxide production. Results Network pharmacology revealed that the bioactive components of GWTZ decoction are predominantly enriched in reactive oxygen species (ROS)-responsive pathways. In vivo, the bile reflux-induced rat model showed a time-dependent progression from esophagitis to carcinoma. GWTZ decoction intervention markedly alleviated esophageal pathological injury, reduced the incidence of precancerous lesions and carcinoma, and downregulated P53 protein expression and TNF-α mRNA levels. In vitro, GWTZ decoction substantially suppressed ezrin phosphorylation at Thr567 and attenuated DCA-induced proliferation and oxidative stress, with efficacy comparable to the ezrin inhibitor NSC668394. Conclusion GWTZ decoction effectively prevents and mitigates bile reflux-induced esophageal carcinogenesis by inhibiting ezrin phosphorylation at Thr567 and reducing oxidative stress.