Ziyuglycoside II ameliorates chemotherapy-induced neutropenia by promoting neutrophil differentiation and functional recovery via SPI1 and C/EBPε transcriptional regulation

Lingdi Li¹Huan Lei¹Luqi Chen¹Chunye Cao¹Haolin He¹Yanfeng Zhang¹Lin Zhang¹Liang Peng¹Yuxiu Yang¹Yulin Feng²Haihong Fang^1*

• ¹Jiangxi Science and Technology Normal University, Nanchang, China
• ²Jiangxi University of Chinese Medicine, Nanchang, China

Abstract Background: Chemotherapy-induced neutropenia (CIN) remains a major dose-limiting toxicity associated with myelosuppressive chemotherapy regimens. The development of therapeutic strategies capable of effectively restoring neutrophil production and function could address a critical unmet clinical issue. ZGSII, a bioactive compound derived from Sanguisorba officinalis, has shown potential in ameliorating leukopenia. To further evaluate its therapeutic applicability for CIN, a comprehensive understanding of its underlying mechanisms is essential. This study aims to assess the efficacy of ZGSII in mitigating cyclophosphamide-induced neutropenia and myelosuppression and to elucidate the underlying mechanism involved through transcriptome sequencing, protein-protein interaction network construction, and functional validation assays. Methods: A murine model of cyclophosphamide-induced neutropenia and the human promyelocytic leukemia cell line NB4 were employed to evaluate the effects of ZGSII alleviating chemotherapy-induced neutropenia and promoting neutrophil differentiation, through blood cell count, flow cytometry analysis, and Wright-Giemsa staining. The underlying molecular mechanisms of ZGSII in treatment CIN were systematically investigated through integrated approaches including transcriptomics profiling, computational simulations, and in vivo function validation. Results: This study demonstrated that ZGSII effectively alleviates cyclophosphamide-induced neutropenia and bone marrow suppression in murine models, promoting neutrophil reconstitution without inducing excessive bone marrow mobilization. Transcriptomic analysis revealed that ZGSII restores neutrophil-related transcriptional programs, thereby enhancing pathways associated with leukocyte migration, myeloid cell activation, and inflammatory regulation. Integration of publicly granulopoiesis datasets enabled the identification of 37 key genes associated with neutrophil differentiation and maturation. Mechanistically, computational modeling suggests potential interaction of ZGSII with SPI1 and C/EBPε, restoring their protein expression and driving granulocytic differentiation. Functional assays further confirmed that ZGSII enhances neutrophil phagocytosis activity, reactive oxygen species (ROS) production, and cytokine homeostasis. Notably, administration of ZGSII conferred significant survival advantages in neutropenic mice following challenge with Staphylococcus aureus. Conclusions: ZGSII alleviates CIN by regulating SPI1 and C/EBPε transcriptional activity to promote neutrophil differentiation and functional recovery. These findings provides a preclinical proof for ZGSII as a therapeutic adjuvant or alternative treatment option for CIN.