Background: Acute myeloid leukemia (AML) is a hematologic malignancy with a poor prognosis. In recent years, the clinical application of the Bcl-2 inhibitor Venetoclax (VEN) has significantly improved remission rates and short-term survival in AML patients. However, the increasing incidence of drug resistance has severely limited its long-term efficacy. Compound Zhebei Granule (CZBG), a traditional Chinese herbal formula, has demonstrated promising clinical potential in the treatment of relapsed and refractory (R/R) AML. Nevertheless, its underlying therapeutic mechanisms remain unclear and warrant further investigation.

Purpose: To investigate the therapeutic effects and potential molecular mechanisms of CZBG in the treatment of AML, with a particular focus on its efficacy in overcoming VEN resistance.Methods: The active components of CZBG were identified using UHPLC-MS/MS. Network pharmacology was employed to predict potential anti-AML targets, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to screen for key signaling pathways. An AML zebrafish model was established with three groups: a control group, a VEN-treated group, and a VEN combined with CZBG group, to evaluate tumor growth inhibition. Transcriptomic sequencing of zebrafish tissues was performed to identify differentially expressed genes and enriched core pathways, with selected results validated by qPCR. In the cellular experiments, a VEN-resistant AML cell line (THP-1 R) was established by low-dose VEN induction. The resistance index was determined using the CCK-8 assay, and the expression of resistance-related genes and proteins was analyzed by qPCR and western blot. THP-1 R cells were then treated with VEN, CZBG, or their combination to assess in vitro efficacy, including cell viability (AO/PI double staining), apoptosis (flow cytometry), and the expression changes of key signaling molecules, verified by qPCR and western blot.

Results: CZBG may exert its anti-AML effects by modulating the FOXO signaling pathway, thereby influencing cell proliferation and apoptosis. In the zebrafish in vivo model, the tumor fluorescence intensity in the CZBG combined with VEN group was significantly lower than that in both the model and VEN groups (P < 0.05), with reduced leukemic cell infiltration observed in tissues. The THP-1 R cells exhibited a 77.44-fold increase in resistance to VEN, accompanied by a marked upregulation of MCL-1 and significant downregulation of FOXO1 and FOXO3 expression (all P < 0.05). Following combination treatment, apoptosis levels increased, and the expression of FOXO1 and FOXO3 was significantly restored.

Conclusions: CZBG exhibits significant potential in overcoming VEN resistance in AML, and its mechanism may be closely associated with activation of the FOXO signaling pathway and induction of apoptosis. This study provides experimental evidence and new insights into the use of traditional Chinese medicine in combination with targeted therapies for the treatment of AML.

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2025
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