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)^2,4. Myelosuppression severely impairs the immune defense system, increasing the risk of infections, bleeding, anemia, and other complications. It also significantly diminishes patients' quality of life and can even be life-threatening⁵. Thus, alleviating and preventing chemotherapy-induced myelosuppression is critical to improving patients' tolerance to chemotherapy and overall survival.

Current clinical treatments for myelosuppression primarily involve hematopoietic growth factors such as granulocyte colony-stimulating factor [G-CSF], erythropoietin [EPO]) and transfusion therapy^6,7. However, these approaches are often associated with high costs, potential side effects, and limited efficacy. As a result, exploring safer and more effective methods for preventing and alleviating myelosuppression remains a major research focus. In recent years, traditional Chinese medicine (TCM) has garnered significant attention for its role in cancer treatment and managing chemotherapy-induced side effects^8,9. Many TCM formulations are believed to have tonifying and immune-regulating effects, such as Shengxuebao decoction¹⁰, Dihuang Shengbai tablets¹¹, and ginsenoside compounds¹², which not only protect normal cells and reduce toxic side effects but also enhance anti-tumor efficacy.

Jianpi Shengxue Decoction (JPSXD) is a classical TCM formula that exemplifies the therapeutic principles of tonifying Qi and nourishing blood¹³. This formula is a sophisticated modification of the Sijunzi Decoction combined with Er Zhi Wan, integrating the synergistic effects of multiple herbs to address Qi and blood deficiency¹⁴. The formula comprises of ginseng (Ren Shen), astragalus (Huang Qi), epimedium (Yin Yang Huo), atractylodes (Bai Zhu), yam (Shan Yao), salvia (Dan Shen), prepared rehmannia (Shu Di Huang), dodder seed (Tu Si Zi), wolfberry (Gou Qi Zi), ligustrum (Nv Zhen Zi), angelica (Dang Gui), fried barley (Chao Mai Ya), fried chicken gizzard (Chao Ji Nei Jin), dried tangerine peel (Chen Pi), coix seed (Yi Yi Ren), and several other herbs. Each herb plays a specific role in the formula, adhering to the TCM principle of sovereign, minister, assistant, and guide herbs, which ensures a balanced and targeted therapeutic effect.

The Sijunzi Decoction, a foundational formula in TCM, is renowned for its ability to tonify Qi and strengthen the spleen. It has been widely used to treat conditions associated with Qi and blood deficiency, such as fatigue, poor appetite, and weakened immunity. Modern pharmacological studies have demonstrated that Sijunzi Decoction enhances immune function, promotes hematopoiesis, and improves the body's self-repair capabilities^15,16. Building on this foundation, JPSXD incorporates additional herbs to further enhance its efficacy in nourishing blood and address more complex deficiencies. For instance, prepared rehmannia and angelica are key blood-nourishing herbs¹⁷, while astragalus and ginseng work synergistically to boost Qi and improve overall vitality¹⁸.

Recent studies have highlighted the potential anti-tumor effects of JPSXD, suggesting that its multi-component, multi-target approach may inhibit tumor growth and enhance the efficacy of conventional cancer treatments^19,20. The formula's ability to modulate immune responses and improve hematopoietic function makes it particularly valuable in supporting patients undergoing chemotherapy or radiation therapy, who often experience Qi and blood deficiency as a side effect of treatment²¹. Furthermore, the inclusion of herbs like salvia and epimedium has been shown to improve microcirculation and enhance cellular repair mechanisms, contributing to the formula's overall therapeutic benefits²².

Modern pharmacological studies have demonstrated that each individual herb in JPSXD possesses pharmacological activities related to hematopoietic function, anti-myelosuppression, immunomodulation, and antioxidation, as well as microcirculatory improvement, providing indirect research support for the use of JPSXD in alleviating chemotherapy-induced myelosuppression. Table 1 summarizes the primary pharmacological effects of each herb along with the corresponding references, with a focus on studies investigating hematopoiesis, anti-myelosuppressive activity, immunomodulation, and antioxidative or microcirculation-enhancing properties. The complex synergy of components in JPSXD involves both cooperative and antagonistic interactions among the herbs, targeting multiple pathways to achieve a holistic therapeutic effect. For example, while ginseng and astragalus work together to tonify Qi, salvia and prepared rehmannia collaborate to nourish blood and improve circulation. At the same time, herbs like dried tangerine peel and fried barley help regulate digestion and prevent stagnation, ensuring that the tonifying effects of the formula are effectively absorbed and utilized by body²³. This multi-faceted approach aligns with the TCM philosophy of treating the root cause of disease while addressing its symptoms.

In conclusion, JPSXD is a well-balanced and scientifically supported TCM formula that embodies the principles of Qi and blood detoxification. Its potential applications in enhancing immunity, promoting hematopoiesis, and supporting anti-tumor therapy make it a valuable addition to both traditional and modern medical practices. However, high-quality clinical evidence directly evaluating the efficacy of JPSXD in alleviating chemotherapy-induced myelosuppression remains lacking. Further research is needed to elucidate its underlying mechanisms and to optimize its clinical application strategies. Therefore, this study aims to provide proof of concept for the potential therapeutic effects of JPSXD from a preclinical mechanistic perspective.

Network pharmacology, with its systemic and integrative nature, has been widely used to identify active ingredients of Chinese medicines and elucidate their mechanisms of action^24,25. In this study, network pharmacology was employed to analyze the potential signaling pathways and key targets affected by JPSXD in the context of myelosuppression. Additionally, a cyclophosphamide (CTX)-induced myelosuppression mouse model was established to assess relevant indicators and evaluate the effects of JPSXD on myelosuppression. Preliminary validation of the key targets and pathways identified through network pharmacology was also conducted. This is the first study to systematically integrate network pharmacology with animal experiments to elucidate the molecular mechanism by which JPSXD alleviates chemotherapy-induced myelosuppression through the multi-target regulation of signaling pathways, such as the PI3K-AKT pathway, thereby filling a gap in the mechanistic understanding of the hematopoietic protective effects of this compound formula.

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 mice were housed in the Experimental Animal Center of Hunan Cancer Hospital under controlled environmental conditions: temperature: 23 ± 2 °C, humidity: 50% ± 10%, and a 12 h light/dark cycle (8:30-20:30). Mice had ad libitum access to food and water. Prior to experimentation, they were acclimated to the environment through daily handling for 5 min over a 3 day period. Following acclimatization, all 60 mice were stratified by body weight and randomly assigned into six groups (n = 10 per group) using a computer-generated random number table. The groups included: blank control, model, normal saline (NS) control, high-dose JPSXD (40 mg/kg), medium-dose JPSXD (30 mg/kg), and low-dose JPSXD (20 mg/kg). The randomization process ensured no significant differences in mean body weight across groups (p > 0.05). The randomization sequence was generated and executed by researchers not involved in subsequent experimental procedures to minimize bias.

Material preparation
Safety and waste disposal: This study involved CTX, a highly toxic chemotherapeutic agent. All procedures were conducted in strict accordance with the Regulations on the Safety Management of Hazardous Chemicals and Biosafety Level 2 laboratory standards. Specific precautions included the use of disposable protective clothing, nitrile gloves, N95 respirators, and protective goggles by all personnel. CTX preparation and administration were performed inside a Class II biosafety cabinet. Preparation of the CTX injection solution was carried out in a fume hood using dedicated instruments to prevent the formation of aerosols. Sharps and items contaminated with CTX, such as syringes and gloves, were discarded in puncture-resistant sharps containers. Other contaminated materials (e.g., bedding, animal tissues) were inactivated by immersion in 10% sodium hypochlorite for 24 h and subsequently incinerated by a certified medical waste disposal provider. In the event of CTX spillage, the area was immediately covered with absorbent cotton and neutralized with a 1% sodium thiosulfate solution. The incident was then reported to the safety supervisor.

Drugs and reagents: CTX injection was obtained from Baxter Oncology GmbH, Germany (Batch No.: HJ20160467). JPSXD was prepared by the Department of Traditional Chinese Medicine at Hunan Cancer Hospital. It consisted of 15 traditional herbs -- Panax ginseng, Astragalus membranaceus, Epimedium brevicornum, Atractylodes macrocephala, Dioscorea opposita, Salvia miltiorrhiza, Rehmannia glutinosa, Cuscuta chinensis, Lycium barbarum, Ligustrum lucidum, Angelica sinensis, fried malt, chicken gizzard lining, dried tangerine peel, and Coix seed -- combined according to classical compatibility ratios²⁰. The decoction was concentrated to a final concentration of 3 kg/L (i.e., 3 g/mL). Before gavage, the concentrate was diluted with NS to achieve the required concentrations for each dose group: 4 mg/mL for the high-dose group (40 mg/kg), 3 mg/mL for the medium-dose group (30 mg/kg), and 2 mg/mL for the low-dose group (20 mg/kg)²⁶. Gavage volume was calculated as 0.1 mL per 10 g of body weight, resulting in a final administration volume of 0.2-0.25 mL per mouse (based on a weight range of 20-25 g)²⁷.

ELISA kits: Commercially available ELISA kits for mouse Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), EPO, and Thrombopoietin (TPO) were used. Serum samples were diluted 1:5 in PBS buffer (pH 7.4) prior to testing. Standards were reconstituted in 100 µL per well.

Secondary antibody: HRP-conjugated goat anti-rabbit IgG was diluted 1:5000 (final concentration: 0.04 µg/mL) in TBST buffer containing 5% non-fat dry milk and 0.05% Tween-20. The solution was freshly prepared and incubated at room temperature for 1 h.

Primary antibodies for Western blot: GAPDH (rabbit polyclonal), AKT (rabbit polyclonal), JAK2 (rabbit monoclonal), and EGFR (mouse monoclonal) were used. All antibodies were diluted in TBST buffer containing 5% BSA: GAPDH (1:1000), AKT (1:1000), JAK2 (1:800), and EGFR (1:800). The incubation volume was 100 µL per membrane.

Tissue Fixative: A 4% paraformaldehyde (PFA) solution was prepared by dissolving PFA powder in PBS (pH 7.4). Tissues were fixed for 24 h.

Hematoxylin and eosin (H&E) staining solution: Hematoxylin solution (0.1% Harris hematoxylin) and eosin Y solution (0.5%) were prepared according to standard pathology protocols²⁸. Staining durations were 5 min for hematoxylin and 3 min for eosin.

Immunofluorescence blocking buffer: PBS buffer containing 10% goat serum and 1% BSA was used for blocking, with a 30 min incubation.

Antibody dilution buffer for immunofluorescence: Primary antibodies (CD34+, TPO, EPO, GM-CSF) for immunofluorescence were diluted in PBS buffer containing 1% BSA, following the manufacturer's recommended dilution ratio (1:200), yielding a final concentration of 2.5 µg/mL. The incubation volume was 50 µL per tissue section.

Induction of an animal model
60 mice were randomly divided into six groups (n = 10 per group): blank control, model, high-dose, medium-dose, low-dose, and NS control. Except for the blank control group, all mice received intraperitoneal injections of CTX (20 mg/kg) 1x daily for 3 consecutive days to establish a myelosuppression model. All model mice exhibited WBC < 1.8 × 10⁹/L, PLT < 85 × 10⁹/L, and >55% reduction in bone marrow nucleated cells at 24 h post-final injection. This protocol aligns with established CTX-induced myelosuppression models^29,30. Following injection, mice were monitored daily for general condition, including activity level, coat appearance, and changes in body weight. From the onset of modeling until the end of the experiment, body weight was recorded at the same time each day using an electronic balance. The body weight change rate was calculated as follows: (final weight / initial weight) x 100%, to evaluate the general physiological status and the potential protective effect of JPSXD. Post-injection, mice were housed in individually ventilated cages (IVCs), each clearly labeled as CTX-contaminated. Bedding was changed every 2 days, and all used bedding was disposed of in accordance with hazardous waste protocols described above.

Intragastric administration
Mice in the high-, medium-, and low-dose groups received JPSXD via gavage at doses of 40 mg/kg, 30 mg/kg, and 20 mg/kg, respectively. The saline control group received an equal volume of NS. Gavage solutions were prepared at concentrations of 4 mg/mL, 3 mg/mL, and 2 mg/mL, respectively, and administered at a volume of 0.1 mL per 10 g of body weight. All mice were gavaged 2x daily for 7 consecutive days. The saline control group received 0.2-0.25 mL per mouse of NS. No treatment was given to the blank and model groups. Absence of coughing or struggling during gavage indicated correct administration technique.

Blood collection and serum preparation
On the 3^rd day after CTX injection, 0.2 mL of blood was collected from the tail vein to evaluate the success of model establishment by measuring peripheral blood cell counts using a fully automated hematology analyzer. At 24 h after the final gavage of JPSXD, blood was collected from the orbital sinus of randomly selected mice (n = 10 per group). A portion of the blood was used for complete blood count analysis, while the remainder was processed for serum preparation. Samples were centrifuged at 3,000 x g for 15 min at 4 °C. The supernatant (a clear, pale-yellow upper layer) was collected as serum and aliquoted for storage at -80 °C for subsequent ELISA assays and liver/kidney function tests. The serum layer was observed to be clearly separated from the blood cell layer, with no signs of hemolysis or turbidity. Hematological parameters measured included WBC, RBC, PLT, and hemoglobin (Hb) levels.

Liver and kidney function assessment
A fully automated biochemical analyzer was used to measure serum liver function markers -- alanine aminotransferase (ALT) and aspartate aminotransferase (AST) -- and kidney function markers-blood urea nitrogen (BUN) and creatinine (Cre). All procedures followed the instrument's standard operating protocol. Specifically, 100 µL of serum was mixed with the corresponding assay kits (ALT, AST, BUN, and Cre) and incubated at 37 °C for 5 min. Absorbance was measured at 340 nm. Each sample was tested in duplicate, and the average value was used for analysis.

Organ index calculation
At 24 h after the final gavage administration of JPSXD, mice were euthanized by gradual-fill carbon dioxide (CO₂) inhalation (flow rate: 20% chamber volume/min) followed by cervical dislocation as a secondary method³¹. Immediately after euthanasia, the liver, kidneys, spleen, and thymus were aseptically harvested through a ventral midline incision. Organs were gently dissected free from surrounding connective tissues and fat, then rinsed in ice-cold normal saline (NS) to remove residual blood. Organs were blotted dry with pre-weighed filter paper under standardized pressure (3 gentle presses) and weighed immediately on a calibrated electronic balance (accuracy: ±0.1 mg) at room temperature (22 ± 1°C).

All procedures were performed on a pre-chilled stainless-steel tray to minimize autolysis. The organ index was calculated as organ weight (mg) divided by body weight (g) to evaluate the effects of JPSXD on major organs. Portions of each tissue were reserved for Western blot analysis, while the remaining samples were fixed in 4% PFA for subsequent histological evaluation.

ELISA
Serum concentrations of TPO, EPO, and GM-CSF were measured using ELISA kits according to the manufacturers' instructions. All centrifugation steps, including sample washing and reagent reconstitution, were performed at room temperature at 1,000 x g for 5 min. Washing steps were conducted using PBST buffer (PBS containing 0.05% Tween-20), with 300 µL of wash solution added per well and repeated 3x. After washing, no residual liquid remained at the bottom of the wells. Following color development, a distinct blue precipitate was visible. Absorbance was measured at 450 nm using a microplate reader after the reaction was terminated.

H&E staining
Following euthanasia, the thymus and femur were harvested. The thymus was weighed to calculate the thymus index (thymus weight [mg]/body weight [g]). After weighing, part of the thymus was used for Western blot analysis, while the remainder, along with the femur, was fixed in 4% PFA. Femur samples underwent graded ethanol dehydration (70%, 80%, 95%, and 100%, each for 1 h). Proper dehydration was indicated by a uniformly white appearance, without softening or deformation. Paraffin-embedded tissue blocks were sectioned at 4 µm thickness using a rotary microtome fitted with high-precision tungsten-carbide blades (Type H31). Sections were floated on a 40 °C water bath containing 0.5% gelatin solution to eliminate wrinkles, then mounted on microscope slides. All sections exhibited intact morphology, smooth edges, and uniform translucency under light microscopy, confirming optimal sectioning quality. H&E staining was performed, followed by mounting with an aqueous mounting medium²⁸. After staining, nuclei appeared blue and cytoplasm pink, with well-defined cellular architecture under microscopic observation using an inverted microscope. Histomorphological analysis was conducted using software. For each H&E-stained section, three random fields were selected at 200x magnification. The software automatically quantified the number of nucleated cells per unit area after manual delineation of the region of interest, and results were expressed as mean ± standard deviation (x̄ ± s).

Western blot
Thymus tissues (50 ± 2 mg/sample) were homogenized in ice-cold RIPA buffer containing 1x protease inhibitor cocktail at a precise ratio of 100 mg tissue per 1 mL buffer, using a motorized pellet pestle homogenizer fitted with RNase-free pestles. Homogenization was performed on ice with 15 pulses (1 s pulse/2 s pause), followed by 30 min incubation on ice with vortexing every 10 min. The homogenate was centrifuged at 12,000 x g for 20 min at 4 °C, and the resulting supernatant was collected for protein quantification. The supernatant appeared clear and transparent, free of precipitates or lipid layers. Protein concentration was measured using a BCA protein assay kit, with a standard curve range of 0-2000 µg/mL. Western blot was performed to assess the expression levels of AKT1, JAK2, and EGFR in thymus tissue, using GAPDH as the internal control. After electrophoresis and membrane transfer, the protein marker (10-200 kDa) displayed clear bands, and the target bands aligned with the expected molecular weights (AKT1: 60 kDa, JAK2: 130 kDa, EGFR: 170 kDa, and GAPDH: 37 kDa).

Densitometric analysis of Western blot bands was conducted using software. Prior to analysis, bands were visually inspected to confirm the absence of smearing or diffusion. The analysis procedure included: Manual selection of target band regions following image import, with automated edge detection by the software, followed by local background subtraction using regions adjacent to the bands to correct gray values. Then, the calculation of the gray value ratio between the target protein and internal control for relative quantification was done. The normalization formula used was:

Relative expression of target protein = (gray value of target band −local background) / (gray value of internal control −local background)

Immunohistochemistry
Immunohistochemistry staining was performed on femur sections. Slices were permeabilized with 0.1% Triton X-100 for 10 min, followed by blocking for 30 min. Primary antibodies were incubated overnight at 4 °C, and secondary antibodies were incubated for 1 h at room temperature. After staining, distinct positive signals with low background and no non-specific staining were observed under a light microscope. Slides were mounted using an anti-fade medium containing DAPI, which stained nuclei blue. The expression of CD34+, TPO, EPO, and GM-CSF was then evaluated. Immunohistochemistry images were analyzed using digital pathology software (Version 2.1.0). Prior to analysis, images were verified to be free of overexposure or signal degradation. The analysis procedure³²included:

Model Selection: A pre-trained bone marrow segmentation model (U-Net-based) was used to automatically identify tissue regions³³.
Threshold Setting: Positive signal thresholds were defined based on the negative control group. Regions with chromogenic intensity exceeding the background mean by three standard deviations were classified as positive.
ROI Definition: The software automatically divided each field into 500 × 500 µm grid regions and calculated the number of positive cells per unit area (positive cell count/field area).
Data Export: A statistical report of positive cell density was automatically generated.

Statistical analysis
Statistical analysis was performed using SPSS software (Version 22.0). Data were expressed as x̄ ± s. Prior to intergroup comparisons, normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was evaluated with Levene's test. If both normal distribution (p > 0.05) and variance homogeneity (p > 0.05) were confirmed, one-way ANOVA was used for group comparisons. Post hoc multiple comparisons were conducted using the Least Significant Difference (LSD) method; to control for Type I error, all post hoc P-values were adjusted using the Bonferroni correction, with adjusted p < 0.05 considered statistically significant.

If data failed to meet the assumptions of normality or homogeneity of variance (p ≤0.05 in either test), the Kruskal-Wallis test was applied, followed by Dunn's test for post hoc analysis with Bonferroni correction. All statistical graphs and visualizations were generated using GraphPad Prism (Version 9.0). The significance level was set at α = 0.05. All reported p-values were adjusted values. Outliers were identified using Grubbs' test (α = 0.05) and excluded only if clearly attributable to experimental error. All remaining data were included in the final analysis. Results are presented as x̄ ± s.

Network pharmacology methodology
This study employed a network pharmacology approach to explore the potential mechanisms by which JPSXD alleviates chemotherapy-induced myelosuppression. First, the chemical constituents of JPSXD and their corresponding targets were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https://old.tcmsp-e.com/tcmsp.php). The screening criteria were set as follows: oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18. For compounds not included in the TCMSP database, target prediction was conducted using the SwissTargetPrediction platform (https://www.swisstargetprediction.ch/).

To ensure the selected compounds covered the major bioactive constituents of JPSXD, reference was made to the Pharmacopoeia of the People's Republic of China³⁴. Specifically, we identified marker compounds such as ginsenosides, astragaloside IV, icariin, and salvianolic acids by reviewing the Identification and Assay sections for each herb. These compounds, commonly used as quality control markers³⁵, have defined chemical structures and quantitative requirements. For accuracy, we cross-referenced the General Notices and Appendices of the Pharmacopoeia to confirm naming conventions and analytical methods (e.g., high-performance liquid chromatography, HPLC). Analysis confirmed that the screening criteria (OB ≥30%, DL ≥0.18) effectively captured the core compounds of the formula³⁶, including key pharmacopeial constituents such as ginsenosides Rg1 and Rb1 (from Panax ginseng), astragaloside IV (from Astragalus membranaceus), and icariin (from Epimedium brevicornum), indicating that the selected compounds are representative and encompass the primary active ingredients responsible for JPSXD's qi- and blood-tonifying effects.

Subsequently, gene targets associated with myelosuppression were retrieved from the GeneCards database (https://www.genecards.org/), and the results were downloaded into a spreadsheet. Genes with a relevance score ≥44 were selected for further analysis. The intersection of drug targets and disease-related genes was visualized using a Venn diagram, and the overlapping genes were imported into the STRING database (https://string-db.org/). The organism was set to human, and interaction sources were limited to Experiments and Databases with a minimum required interaction score > 0.7. The resulting protein-protein interaction (PPI) network was then analyzed for topological characteristics to identify core targets and visualized using Cytoscape 3.10.2.

Based on the identified core targets, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the DAVID database (https://david.ncifcrf.gov/). The parameters were set as: Count ≥5, EASE score ≥0.05, and p-value < 0.01. Enrichment results were visualized through the Microbiological Letter platform (http://www.bioinfo-cloud.org/). Pathway analysis further clarified the key signaling routes and regulatory markers involved, thereby revealing the potential mechanisms through which JPSXD mitigates chemotherapy-induced myelosuppression.

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 herbal co...

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