Qingre huoxue formula and its key active components as associated with multi-targeted effects on human pancreatic cancer cells.

e16446 Background: Pancreatic cancer (PC) exhibits a high mortality rate, often diagnosed at advanced stages with limited therapeutic options. Traditional Chinese Medicine (TCM) may offer multi-target advantages. This study screened 1,314 TCM herbs against nine human PC cell lines, leading to a novel combination, Qingre Huoxue Formula (QHF), composed of Chun Pi ( Ailanthus altissima(Mill.) Swingle ), Bi Xie ( evenlobed Yam Rhizome ) , Fang Ji ( Stephaniae Tetrandrae Radix ), and Qian Cao ( Rubiae Radix et Rhizoma ) . Methods: MTT assays evaluated herb extracts for growth inhibition of PANC-1, PANC-1-GM, MIA PaCa-2, BxPC-3, AsPC-1, SW1990, Capan-2, CFPAC-1, and HPAF-II cells. The most potent extracts were combined into QHF. Transcriptomic analyses identified differentially expressed genes, followed by GO and GSEA to determine enriched pathways. A pharmacological gene network (PGN) predicted potential targets. Molecular docking (Vina), a neural network model (DrugBAN), and Amber simulations assessed compound–target interactions. QHF and key small molecules were validated via RTCA (migration/invasion), flow cytometry (cell cycle/apoptosis), and Western blot (PI3K/AKT/mTOR, TP53, glycolysis). SPR assays with recombinant proteins confirmed predicted binding interactions. Results: Twenty herbs showed broad-spectrum antiproliferative effects. QHF significantly inhibited viability and induced apoptosis in multiple PC lines. RTCA indicated reduced migration and invasion. Western blot revealed downregulation of PI3K/AKT/mTOR, upregulation of TP53, and changes in glycolytic enzymes. Key small molecules from QHF mirrored these effects, aligning with computational predictions. SPR confirmed the binding of kaempferol-3-O-glucoside to PLAU (Kd = 162 μM). Conclusions: QHF has multi-target mechanisms for treating PC, including modulation of PI3K/AKT/mTOR, TP53, and glycolytic pathways, present a promising strategy for PC therapy. Our comprehensive screening and network-based approach highlight the value of TCM in developing more effective interventions, providing a solid foundation for future precision treatments.