The role and mechanism of diosgenin glucoside in modulating megakaryopoiesis through promotion of autophagy via the AMPK/mTOR/ULK1 signaling pathway.

BACKGROUND: Radiation-induced thrombocytopenia (RIT) is a severe, dose-limiting complication of cancer radiotherapy with limited clinical management options. This study investigated the therapeutic efficacy and molecular mechanism of Diosgenin glucoside (DG), a natural constituent uniquely prioritized through a graph-based deep learning virtual screening pipeline utilizing the directed graph intersection network (DGIN) architecture. METHODS AND RESULTS: In vitro evaluations in Meg-01 and K562 cells demonstrated that non-cytotoxic concentrations of DG effectively promoted megakaryocytic lineage commitment and terminal polyploidization (≥4 N and ≥ 8 N). This maturation process was accompanied by intensified filamentous actin (F-actin) cytoskeletal assembly-validated via quantitative mean fluorescence intensity (MFI) analysis-and the upregulation of surface markers CD41 and CD42b. Mechanistically, DG treatment was associated with the active engagement of the canonical AMPK/mTOR/ULK1 autophagic axis and the mobilization of a core transcriptional program governed by GATA1, NF-E2, and p-STAT3. Pharmacological interception using Compound C and siRNA-mediated knockdown of AMPK suppressed these pro-maturation shifts, confirming pathway involvement within the megakaryocytic lineage. In vivo, daily administration of DG accelerated peripheral platelet recovery in a murine model of RIT, displaying a non-monotonic, bell-shaped dose-response profile where the 2.5 mg/kg dosage exhibited superior efficacy over higher cohorts-a phenomenon postulated to reflect target receptor saturation or counter-regulatory feedback at elevated concentrations. DG treatment restored functional hemostasis, including tail bleeding duration and ex vivo clot retraction kinetics, by expanding bone marrow megakaryocyte progenitors and stimulating compensatory splenic extramedullary hematopoiesis. Furthermore, DG treatment was accompanied by a proposed cytoprotective effect on the normal hematopoietic niche, suppressing radiation-induced DNA damage γ-H2AX and apoptotic markers (Bax and Caspase-3), while maintaining a favorable preliminary safety profile. CONCLUSION: Collectively, our findings demonstrate that DG promotes megakaryopoiesis in functional coordination with the activation of the AMPK/mTOR/ULK1-autophagic cascade, offering a promising therapeutic scaffold for myelosuppressive thrombocytopenia.