Diosgenin

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.

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.

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.

Radiation nephropathy is a progressive inflammatory and fibrotic condition lacking effective therapies. The underlying cellular signalling mechanisms and their potential systemic involvement remain poorly defined.

We integrated single-cell transcriptomics, network pharmacology, molecular docking and dynamics simulations, and in vivo validation in a murine model of whole-body irradiation. RNA sequencing data from healthy and irradiated mouse kidneys were analyzed to map cell-type-specific signalling activities of mTORC1, endoplasmic reticulum (ER) stress, and inflammation, as well as intercellular communication. Mice received diosgenin at 30 or 100 mg/kg/d by orally or rapamycin at 2 mg/kg/d by intraperitoneal injection for 7 days before and 28 days after 5 Gy X-ray irradiation. Renal function, histopathology, oxidative stress, inflammatory cytokines, intestinal barrier integrity, hepatic inflammation, and gut microbiota were assessed.

Bulk transcriptomics and network pharmacology identified mTOR as a core target. Single-cell analysis revealed radiation-induced mTORC1 activation in proximal tubular cells and immune cells, coupled with ER stress and inflammation. Molecular docking predicted high binding affinity between diosgenin and mTOR. Molecular dynamics simulations confirmed stable diosgenin-mTOR binding over 100 ns. In vivo, diosgenin suppressed renal mTORC1 activity, reduced ER stress, and macrophage infiltration, lowered serum TNF-α, IL-1β, and IL-6, and decreased serum urea and creatinine by approximately 30.25% and 26.20%, respectively. Diosgenin alleviated renal fibrosis, restored colonic occludin expression by 1.5-fold, decreased hepatic F4/80-positive cells by 75.32%, reversed gut dysbiosis including suppression of Pseudomonadota, and these effects were similar to those of rapamycin, an mTOR inhibitor.

Diosgenin alleviates radiation nephropathy by suppressing mTORC1 signalling while exerting concomitant effects on the gut and liver. These findings establish a multi-omics and single-cell framework for radiation nephropathy and support diosgenin as a candidate for translational research.