A fusion strategy of network pharmacology and targeted metabolomics revealed anti-esophageal squamous cell carcinoma effect of Isodon rubescens and its heterogeneity

Ethnopharmacological relevance Isodon rubescens (Hemsl.) Hara, a traditional Chinese medicinal herb, demonstrates tumor inhibiting properties and therapeutic effecacy in treating esophageal squamous cell carcinoma (ESCC). However, its mechanism of action and the differential effects on varying ESCC differentiation grades remain unclear, despite the direct correlation between pathological grade and patient prognosis. Aim of the study To elucidate the effect heterogeneity of I. rubescens aqueous extracts on well-differentiated and poorly differentiated ESCC cell lines at metabolic level. Materials and methods A fusion strategy of network pharmacology combining targeted metabolomics was developed, focusing on key metabolic pathways through a “chemical components – targets – ESCC” network. Then, a targeted analysis method utilizing ultra-high performance liquid chromatography-multiple reaction monitoring tandem mass spectrometry (UPLC-MRM MS/MS) technology was established for core metabolites. The metabolic regulatory patterns of I. rubescens on two ESCC cell lines were determined through cytological experiments and targeted metabolomics analysis, with regulatory target enzymes verified through ELISA experiments. Molecular docking technology identified effective components acting on the regulatory enzymes. Results The primary regulatory pathways of I. rubescens in ESCC treatment encompassed central carbon metabolism, pyruvate metabolism, choline metabolism and retinol metabolism. A targeted metabolomics method analyzing 74 core metabolites in these pathways was established that had high accuracy and precision. The study confirmed heterogeneous effects of I. rubescens on well-differentiated KYSE-30 and poorly differentiated KYSE-150 cell lines. Citrate synthase (CS) expression varied significantly different between the two cell lines, showing upregulation after I. rubescens administration, albeit with varying degree. Molecular docking analysis revealed strong binding affinity between CS and four compounds: oridonin, quercetin, isorhamnetin, and ponicidin. Conclusions I. rubescens exhibits heterogeneous metabolic regulation in ESCC at different differentiation levels. CS was identified and confirmed as a crucial regulatory target, with oridonin, quercetin, isorhamnetin, and ponicidin serving as the primary active components.