Network pharmacology, molecular docking, and experimental validation-based approach to explore the mechanism of action of ginsenoside Rh4 on acute myeloid leukemia cells.

Acute Myeloid Leukemia (AML) is a type of leukemia caused by the abnormal proliferation and arrest of differentiation of primitive myeloid cells. Despite the increasing number of treatment options, the prognosis for AML patients remains poor. Ginsenosides have potent anti-inflammatory, anti-neoplastic, and neuroprotective properties. However, the specific effects of ginsenoside Rh4 on AML remain unknown. We used network pharmacology, molecular docking, and in vitro experiments to study the effects of ginsenoside Rh4 on AML. Potential targets of ginsenoside Rh4 were predicted using Swiss Target Prediction and other databases, while AML associated targets were collected from GeneCards, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Database (TTD). Common targets were identified and analyzed via Protein-Protein Interaction (PPI) network, Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and molecular docking. For in vitro validation, the anti-leukemic activity of ginsenoside Rh4 was assessed by measuring cell viability in both HL-60 and U-937 cell lines using the Cell Counting Kit-8 (CCK-8) assay. Apoptosis was evaluated using the Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) assay in both HL-60 and U-937 cells, while Western Blot analysis was performed specifically in HL-60 cells to investigate the underlying molecular mechanisms. PPI analysis revealed that ginsenoside Rh4 shares 75 targets with AML. Target pathway analysis revealed that these targets are primarily mediated by genes associated with apoptosis, protein phosphorylation, and the Phosphatidylinositol 3-Kinase-Protein Kinase B (PI3K-Akt) signaling pathway. Molecular docking results showed that Signal Transducer and Activator of Transcription 3 (STAT3), Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1), and Mechanistic Target of Rapamycin (mTOR) have a high binding affinity with target proteins. Experimental results demonstrated that ginsenoside Rh4 significantly inhibits leukemia cell proliferation and induces apoptosis. Mechanistic investigations revealed that ginsenoside Rh4 exerts anti-leukemic effects by downregulating phosphorylated Phosphatidylinositol 3-Kinase (p-PI3K), phosphorylated Protein Kinase B (p-AKT), phosphorylated Mechanistic Target of Rapamycin (p-mTOR), phosphorylated Janus Kinase 2 (p-JAK2), and phosphorylated Signal Transducer and Activator of Transcription 3 (p-STAT3) expression and upregulating Bcl-2-Associated X Protein (Bax) and Caspase-3 expression while suppressing B-cell Lymphoma 2 (Bcl-2) expression. These findings suggest that ginsenoside Rh4 may inhibit leukemia cell growth by modulating multiple signaling pathways. Our preliminary elucidation of the potential targets and molecular mechanisms of ginsenoside Rh4 in the treatment of AML provides important data for pharmacological research on ginsenosides and offers new insights into AML treatment.