Activating NK cell immunity against prostate cancer with Hedysarum polybotrys Hand.-Mazz.: A multi-omics integration of computational prediction and experimental validation on PI3K/AKT and PD-1/PD-L1 axes

Ethnopharmacological relevance Hedysarum polybotrys Hand.-Mazz. (Hongqi, HQ) is a prized Qi-tonifying herb in Traditional Chinese Medicine (TCM), historically employed to enhance the body's resistance and modulate immune function. While its anti-tumor properties are recognized, the specific immunomodulatory mechanisms by which HQ empowers Natural Killer (NK) cells to combat prostate cancer (PCa) remain under-explored. Aim of the study To systematically elucidate the material basis and molecular mechanisms through which the aqueous extract of HQ enhances NK cell-mediated cytotoxicity against PCa cells, utilizing an integrated approach of multi-omics and experimental validation. Methods The chemical profile of HQ aqueous extract was characterized using UHPLC-MS/MS untargeted metabolomics. Network pharmacology and transcriptomic analyses (RNA-seq) were integrated to predict core targets and signaling pathways. Specific interactions were simulated via molecular docking. Validations were performed using an in vitro co-culture model of NK-92 and PC-3 cells. Assays included CCK-8, LDH release, flow cytometry (apoptosis, surface markers), ELISA (cytokines), and Western blot. Results A total of 69 compounds were identified in HQ, with Genistein and Isoliquiritigenin highlighted as key active constituents. HQ treatment significantly enhanced NK-92 cell cytotoxicity against PC-3 cells, evidenced by increased LDH release and PC-3 apoptosis. Mechanistically, HQ upregulated the expression of cytolytic effectors (Perforin, Granzyme B) and pro-inflammatory cytokines (IFN-γ, TNF-α, IL-17A). Crucially, HQ treatment achieved a "dual-effect": it reactivated the PI3K/AKT signaling pathway in NK cells (confirmed by increased p-PI3K and p-AKT levels) while concurrently downregulating the immunosuppressive PD-1/PD-L1 checkpoint axis. Conclusion This study demonstrates that HQ potentiates NK cell anti-tumor immunity against prostate cancer through a dual mechanism involving PI3K/AKT pathway activation and PD-1/PD-L1 axis inhibition. These findings provide modern pharmacological evidence supporting the traditional use of HQ for enhancing immune surveillance in cancer therapy.