Da-Chai-Hu-Tang suppresses pancreatic cancer malignancy in association with HIF‒1α downregulation and modulation of mitochondrial dynamics through metabolism-related signaling

Background Pancreatic ductal adenocarcinoma is a highly lethal malignancy with a 5-year survival rate of only 13%, due to late diagnosis, aggressive progression, and chemoresistance. Da-Chai-Hu-Tang a classical formula in Treatise on Febrile Diseases , has shown clinical benefits in pancreatic cancer patients, but its pharmacological mechanisms and active constituents remain unclear. Methods An orthotopic PANC02-Luc model in C57BL/6J mice received DCHT (0.13, 0.26, 0.52 g·kg ‒1 ·d ‒1 ) or gemcitabine (20 mg·kg ‒1 , twice weekly) for 28 days. Tumor progression was monitored by bioluminescence imaging; organ coefficients, and histopathology were assessed. Tumor transcriptomics, tumor-targeted energy metabolomics, and serum untargeted metabolomics, were integrated to identify mechanistic pathways. In vitro , PANC02, PATU-8988T, and PANC1 cells were used to assess proliferation, colony formation, migration, reactive oxygen species (ROS) levels, mitochondrial membrane potential, and apoptosis. The expressions of HIF‒1α, VEGFA, mitophagy regulators (PINK1, Parkin, DRP1, MFN1/2), and apoptosis markers (Bcl-2, Bax) were examined by Western blotting, immunohistochemistry, and immunofluorescence. Chemical profiling combined with blood-absorbed component analysis identified Rhein as a representative compound underlying the anti-pancreatic cancer activity of DCHT. Results DCHT significantly inhibited orthotopic tumor growth, and alleviated multi-organ pathological damage without detectable toxicity. Multi-omics integration showed that DCHT predominantly modulated energy metabolism pathways, partially restoring TCA cycle intermediate balance by reversing an “upstream accumulation and downstream depletion” pattern (elevated citrate, isocitrate, cis-aconitate, and PEP; reduced α-KG, fumarate, and malate) in both tumor tissues and serum. DCHT downregulated HIF‒1α and VEGFA, suppressed PINK1/Parkin-mediated mitophagy, shifted mitochondrial dynamics toward fusion (decreased DRP1, increased MFN1/2), and promoted apoptosis (reduced Bcl-2/Bax ratio) both in vitro and in vivo . Integrative analysis of chemical profiling and blood-absorbed components pinpointed Rhein as one of the key active compounds which partly recapitulated the anti-proliferative, pro-apoptotic, and HIF‒1α–mitophagy modulating effects of the full formula. Conclusion These findings reveal that DCHT treatment is associated with concurrent changes in TCA-cycle metabolism, HIF‒1α, PINK1/Parkin-mediated mitophagy, and apoptosis. This study also identifies Rhein as one of the key pharmacologically active constituents. These findings provide mechanistic and experimental evidence supporting the potential clinical relevance of DCHT in pancreatic cancer therapy and offer a foundation for developing multi-target therapeutic strategies against this devastating disease.