Mechanism of Jianpi Shengxue Decoction in Alleviating Cyclophosphamide-Induced Myelosuppression in Mice

Summary

This protocol systematically elucidates how Jianpi Shengxue decoction mitigates cyclophosphamide-induced myelosuppression through an integrated network pharmacology and animal experiments approach, revealing its multi-target mechanisms and potential as an adjuvant therapy.

Abstract

The paper aims to investigate how Jianpi Shengxue Decoction (JPSXD) alleviates chemotherapy-induced myelosuppression through network pharmacology and animal experiments. To do this, key components and targets were analyzed, and Protein-Protein Interaction Network (PPI) and herb-component-target networks were constructed. Gene Ontology (GO) andKyoto Encyclopedia of Genes and Genomes (KEGG) enrichment identified major pathways. A cyclophosphamide (CTX)-induced myelosuppression model was established in 60 C57/BL6 mice. JPSXD was administered at different doses for 7 days. Peripheral blood counts, cytokines (thrombopoietin [TPO], erythropoietin [EPO], granulocyte-macrophage colony-stimulating factor [GM-CSF]), thymus index, bone marrow morphology, CD34+ cells, and AKT1, JAK2, and EGFR expression were evaluated. Network analysis identified 172 compounds and 454 targets, highlighting PI3K-Akt and JAK-STAT pathways. In vivo, CTX reduced leukocyte and signaling protein levels, while JPSXD restored counts, upregulated AKT1, JAK2, and EGFR, and improved thymus index (p < 0.05). Medium- and high-dose JPSXD increased TPO, EPO, GM-CSF, CD34⁺ cells, and improved bone marrow structure, with the high dose showing the strongest effect. Conclusively, JPSXD alleviates myelosuppression via multi-target, multi-pathway regulation, supporting its potential as an adjunctive therapy for chemotherapy-induced leukopenia.

Introduction

Chemotherapy is a cornerstone in the treatment of various malignancies, primarily by inhibiting cancer cell proliferation and survival^1,2, thereby extending patient survival. However, due to the lack of selectivity in targeting normal tissues, chemotherapy drugs often cause severe damage to healthy cells and tissues, leading to a range of adverse effects³. One of the most common and serious side effects of chemotherapy is myelosuppression, characterized by a reduction in the production of blood cells, including white blood cells (WBCs), red blood cells (RBCs), and platelets (PLTs)

Protocol

All animal experiments were conducted in strict accordance with the Regulations on the Administration of Laboratory Animals and the ethical guidelines of the Experimental Animal Center of Hunan Cancer Hospital. The study protocol was approved by the Animal Ethics Committee of Hunan Cancer Hospital (Approval No.: KNZY-202416). Throughout the study, researchers made every effort to minimize the number of animals used and to alleviate their suffering.

Animal preparation
A total of 60 10-week-old male C57/BL6 mice were obtained from Hunan Slack Jingda Laboratory Animal Co., Ltd. (License No.: SCXK [Hu] 2021-0002). All mic....

Results

Network pharmacology analysis
Identification of active compounds and targets
We first identified the primary marker compounds of each herb in JPSXD based on the literature to ensure comprehensive selection³⁴. Specifically, by consulting the pharmacopeial entries for relevant herbs (e.g., Panax ginseng, Astragalus membranaceus, Angelica sinensis), we compiled a list of the official marker constituents for all 15 .......

Discussion

Amid the escalating global incidence of cancer, the medical community is confronted with dual challenges: not only the treatment of cancer itself but also the management of associated side effects, among which myelosuppression is a particularly common and severe adverse reaction⁴³. This study systematically investigated the role and potential mechanisms of JPSXD in alleviating CTX-induced myelosuppression in mice, employing a combination of network pharmacology and animal experiments. The results .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% Paraformaldehyde (PFA)	Sigma-Aldrich	P6148
AKT (pan) Rabbit mAb	Cell Signaling Technology	#4691
Automatic Microplate Washer	BioTek (Agilent)	ELx405
BC-2800vet Auto Hematology Analyzer	Mindray	BC-2800vet
Cryostat Microtome	Thermo Fisher Scientific	CryoStar NX50
Cyclophosphamide Injection	Baxter Oncology GmbH	N/A (Batch: HJ20160467)
E100 Microscope	Nikon	E100
EGFR Antibody (A-10)	Santa Cruz Biotechnology	sc-373746
Embedding Machine	Leica	EG1150
EPO (Mouse) ELISA Kit	R&D Systems	MEP00B
GAPDH Rabbit Polyclonal Antibody	Proteintech	10494-1-AP
GM-CSF (Mouse) ELISA Kit	R&D Systems	MMC00B
Goat Anti-Rabbit IgG HRP-conjugated	Jackson ImmunoResearch	111-035-003
H&E Staining Kit	Sigma-Aldrich	HT10
High-Speed Refrigerated Centrifuge	Thermo Fisher Scientific	Sorvall ST 16R
High-Throughput Tissue Grinder	Servicebio	KZ-II
Immunohistochemistry Pen	Vector Laboratories	H-4000
JAK2 Rabbit mAb	Abcam	ab108596
Jianpi Shengxue Decoction (JPSXD)	Pharmacy of Hunan Cancer Hospital	Custom formulation
Microplate Reader	BioTek (Agilent)	Synergy H1
Normal Saline	Baxter	2B1324X
Rotary Microtome	Leica	RM2016
Tissue Processor	Leica	ASP300S
TPO (Mouse) ELISA Kit	R&D Systems	MTP00B
Vortex Mixer	Servicebio	MX-F