Multi-omics analysis reveals that Rabdosia rubescens aqueous extract inhibits esophageal squamous cell carcinoma by targeting the PI3K/AKT/mTOR pathway.

Esophageal squamous cell carcinoma (ESCC) is a refractory malignancy characterized by limited therapeutic options and poor prognosis. The aqueous extract of Rabdosia rubescens (R. rubescens), known as "Dong-Ling-Cao" (DLC), is traditionally administered as a decoction and exhibits anti-ESCC activity; however, It's pharmacodynamic material basis and mechanism of action remain unclear. This study aimed to systematically investigate the anti-ESCC efficacy and underlying mechanism of DLC. Serum samples collected from rats following intragastric administration of DLC were analyzed using UHPLC-Q Exactive MS to identify absorbed components. Network pharmacology was employed to predict potential targets and pathways of DLC. An in-vivo KYSE-510 xenograft model was established to evaluate the tumor-inhibitory effect of DLC. Untargeted metabolomics and 16S rDNA sequencing were performed to analyze changes in serum metabolites and gut microbiota, respectively. In vitro assays (including CCK-8, EdU, wound healing, flow cytometry, and Western blot) were conducted to verify the effects of DLC on cell proliferation, migration, apoptosis, and the PI3K/AKT/mTOR pathway. A total of 35 blood-entry constituents of DLC were identified, primarily flavonoids, glycosides, and organic acids. Network pharmacology screening revealed core targets (e.g., SRC, PIK3CA, and AKT1), which were notably enriched in the PI3K-Akt signaling pathway among others. In vivo, DLC dose-dependently inhibited tumor growth, induced apoptosis, and improved hepatic and renal function. Metabolomic analysis indicated that DLC influenced several metabolic pathways, including mTOR signaling, cAMP signaling, and oxidative phosphorylation. Gut microbiota analysis demonstrated that DLC modulated the relative abundance of genera such as Bacteroides and Lactobacillus. Furthermore, the in vitro experiment results confirmed that DLC suppressed cell proliferation, migration, and glycolysis by inhibiting the PI3K/AKT/mTOR pathway and its downstream glycolytic enzymes. DLC exerted anti-ESCC effects through multi-component and multi-target synergy, ultimately inhibiting the PI3K/AKT/mTOR signaling pathway and its associated metabolic reprogramming. The findings of this study may provide an experimental foundation for developing DLC as a potential therapeutic agent against ESCC.