Integrated Metabolomics, Network Pharmacology, and Experimental Validation Elucidate the Mechanisms Underlying Dingxiang-Guanshitong Decoction in the Treatment of Esophageal Squamous Cell Carcinoma.

INTRODUCTION: Esophageal Squamous Cell Carcinoma (ESCC) is a highly aggressive malignancy with limited therapeutic options and poor prognosis, particularly in advanced stages. Dingxiang Guan Shitong Decoction (DGD) is a traditional Chinese medicine preparation that can alleviate symptoms in patients with advanced ESCC. However, the chemical composition, pharmacological effects, and molecular mechanisms of action of DGD remain unclear. METHODS: This study evaluated the efficacy of DGD in advanced ESCC by conducting a clinical trial. Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) was employed to identify the chemical constituents of DGD. The interaction between the bioactive compounds in DGD and potential targets of ESCC was predicted using network pharmacology analysis, and the predictions were verified by molecular docking. The effects of DGD on cell biology and the expression of predicted target genes were confirmed in vitro using the ESCC cell line. RESULTS: DGD treatment significantly improved quality of life and TCM syndrome scores in patients with advanced ESCC. The UPLC-MS analysis identified 54 bioactive components in DGD. Network pharmacological analysis indicated that these bioactive components in DGD could target 129 potential ESCC-related genes. Functional analysis demonstrated that these core target genes were enriched in tumor-related pathways, including the p53 signaling pathway, apoptosis, and the IL-17 signaling pathway. Molecular docking experiments showed that the core DGD constituents (quercetin, kaempferol, and β-sitosterol) exhibited strong binding affinities for the protein products of key target genes (such as TP53 and AKT1). Moreover, in vitro experiments demonstrated that DGD inhibited the proliferation and migration of Eca-109 cells and simultaneously induced apoptosis in a dose-dependent manner. In addition, DGD treatment upregulated the expression of Bax, cleaved caspase-3, and p53 at the protein level in Eca-109 cells. DISCUSSION: The integration of clinical observation, metabolomics, network prediction, and experimental validation provides a multi-level understanding of DGD's therapeutic mechanism. The convergence of computational predictions and in vitro experimental data strongly supports the p53/caspase-3 axis as a key mediator of DGD-induced apoptosis. These findings position DGD as a promising adjunctive therapy for advanced ESCC, particularly for patients who are ineligible for or intolerant of aggressive conventional treatments. CONCLUSION: Our study provides evidence that DGD leads to significant improvements in the quality of life for individuals diagnosed with advanced ESCC. Its active ingredients may exert anti-tumor effects by inducing apoptosis through the p53/caspase-3 signaling pathway. This study provides foundational data for the clinical application of DGD and a basis for further investigation of its pharmacological mechanism.