Mechanism of Qilu Buxue granules in alleviating chemotherapy-induced leukopenia: Insights from network pharmacology, integrated omics, and experimental validation.

Leukopenia is a prevalent side effect of cancer chemotherapy, characterized by fever, fatigue, weakness and a significantly elevated risk of life-threatening infection. Qilu buxue granules (QBG) are clinically utilized to mitigate cyclophosphamide-induced leukopenia (CTX-IL), but the precise molecular mechanism underlying its therapeutic effects remain unclear. of study: This study aims to elucidate the therapeutic mechanisms of QBG against CTX-IL and identify its active compounds and targets, using an integrated approach of network pharmacology, pharmacodynamics, metabolomics, proteomics and experimental validation. UHPLC-qTOF-MS/MS approach was utilized to determine the chemical composition of QBG. Network pharmacology was applied to predict therapeutic targets of QBG against leukopenia. Pharmacodynamic experiments were conducted to evaluate efficacy of QBG in mice. Serum metabolomic and spleen proteomic were applied to characterize the altered metabolomic profile and differentially expressed proteins (DEPs). Furthermore, western blotting was carried out to validate the expression levels of key proteins in mice spleen. A total of 33 bioactive chemical components of QBG were identified from UHPLC-qTOF-MS/MS analysis. 444 QBG-related and 969 leukopenia-related targets were obtained from SwissTarget Prediction database and BATMAN-TCM, PharmGkb, DisGeNET, and OMIM databases. Drug target disease network identifies the top 7 bioactive components including apigenin, genkwanin, acacetin, formononetin, oleuropein, diphyllin and rubiadin 1-methyl ether. Protein-protein interaction (PPI) identifies PI3KR1, AKT1, STAT3, EGFR, PIK3CD, PIK3CA, and HRAS as core target proteins. GO and KEGG enrichment analysis revealed PI3K/AKT, JAK/STAT signaling, inflammatory, and immune response pathways are the enriched terms. In vivo studies showed that QBG significantly improved white blood cell counts in the CTX-IL mouse model. QBG also normalized the levels of serum cytokines. Flow cytometric analysis and immunofluorescence staining revealed that CTX suppressed the growth of bone marrow hematopoietic stem cells while, QBG reversed these effects. Metabolomic results highlights that QBG normalize the levels of citric acid, succinic acid, D-sphingolipids, L-arginine and other metabolites in CTX-IL model group. Additionally, proteomic analysis finds key significant DEPs whose level alter after QBG administration. Finally, the protein expression of key signaling molecules was analysed via western blotting. Overall, this study identified the multi-components, multi-target therapeutic mechanism of QBG against CTX-IL using integrated omics and network pharmacology. These findings provide scientific evidence supporting the clinical relevance of QBG as an effective intervention for chemotherapy related leukopenia.