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ORIGINAL ARTICLE
Year : 2022  |  Volume : 18  |  Issue : 79  |  Page : 685-691  

Protective effect of 20(R)-ginsenoside Rg3 on chemotherapy-induced myelosuppression in mice


1 Chongqing Three Gorges Medical College, Baianba, Wanzhou District, Chongqing, China
2 College of Chinese Medicinal Material, Jilin Agricultural University, Changchun, Jilin Province, China

Date of Submission02-Oct-2021
Date of Decision19-Dec-2021
Date of Acceptance27-Apr-2022
Date of Web Publication19-Sep-2022

Correspondence Address:
Enbo Cai
College of Chinese Medicinal Material, Jilin Agricultural University, Xincheng Street No. 2888, Changchun, Jilin Province - 130118
China
Xiuyong Yue
Chongqing Three Gorges Medical College, 366 Tianxing Street, Baianba, Wanzhou District, Chongqing - 404120
China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_451_21

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   Abstract 


Background: 20(R)-ginsenoside Rg3 (R-Rg3) is a kind of ginseng glycol tetracyclic triterpenoid saponin, which is a recognized traditional Chinese medicine monomer and has the action of inhibiting the proliferation of tumor cells. Objectives: The goal of our experiment was to investigate the protective function of R-Rg3 on chemotherapy-induced myelosuppression in mice. Materials and Methods: Cyclophosphamide (CTX) was injected into the intraperitoneal (i.p.) to establish the mice myelosuppression model. We measured the number of peripheral blood cells (PBCs), and the number of bone marrow nucleated cells (BMNCs) was counted. Hematopoietic progenitor cells (HPCs) were cultured in vitro, and the amount of cell colonies was recorded at different times. Then ELISA was used to detect the levels of hematopoietic-related cytokines and flow cytometry was employed to test cell cycle. The network pharmacology was used to predict the main pathways of action. Furthermore, the expression of p-JAK2 and p-STAT5 was detected via western blotting. Results: The experimental outcomes showed that R-Rg3 could improve the amount of PBCs in mice with myelosuppression, increase the quantity of karyota cells in bone marrow, and enhance the proliferation of HPCs and adjust the content of hematopoietic-related cytokines. Moreover, the JAK–STAT signaling pathway may be the key to the role of R-Rg3. Conclusion: All of these results implied that R-Rg3 might be a therapeutic agent for myelosuppression after chemotherapy.

Keywords: 20(R)-ginsenoside Rg3, chemotherapy, cyclophosphamide, myelosuppression, network pharmacology


How to cite this article:
Fu Z, Sun N, Li S, Han J, Lang S, Yue X, Cai E. Protective effect of 20(R)-ginsenoside Rg3 on chemotherapy-induced myelosuppression in mice. Phcog Mag 2022;18:685-91

How to cite this URL:
Fu Z, Sun N, Li S, Han J, Lang S, Yue X, Cai E. Protective effect of 20(R)-ginsenoside Rg3 on chemotherapy-induced myelosuppression in mice. Phcog Mag [serial online] 2022 [cited 2022 Sep 26];18:685-91. Available from: http://www.phcog.com/text.asp?2022/18/79/685/356397



SUMMARY

  • The experiment was to investigate the protective function of R-Rg3 on chemotherapy-induced myelosuppression in mice. The method of intraperitoneal injection of CTX was used to establish the mice myelosuppression model. The numbers of PBCs and BMNCs were detected, and thymus and spleen indices were measured. Through examining the changes in hematopoietic-related cytokines and the cell cycle, we researched the impacts of drugs on the proliferation and differentiation of HPCs. The network pharmacology was used to predict R-Rg3's main action pathways, and the expression levels of p-JAK2 and p-STAT5 were tested. Finally, it was concluded that R-Rg3 could improve the quality of life of the myelosuppressed mice.




Abbreviations used: CTX: Cyclophosphamide; BMNCs: Bone marrow nucleated cells; HPCs: Hematopoietic progenitor cells; GM-CSF: Granulocyte-macrophage colony-stimulating factor; PBS: Phosphate buffer saline; CFU-E: Colony-forming unit erythroid; BFU-E: Burst-forming unit erythroid; CFU-GM: Colony-forming unit granulocyte macrophages; CFU-Meg: Colony-forming unit megakaryocyte; DAVID: Database for Annotation, Visualization and Integrated Discovery; KEGG: Kyoto Encyclopedia of Genes and Genomes; WBC: White blood cell; RBC: Red Blood Cell; PLT: Platelet.


   Introduction Top


Cancer has become a serious disease that harms human health in today's society. Chemotherapy is one of the commonly used treatments for malignant tumors at this stage. However, due to the lack of selectivity and more toxic side effects of chemotherapy drugs, myelosuppression often occurs after chemotherapy.[1] Cyclophosphamide (CTX) is the most widely used alkylating agent in clinical practice.[2] Relevant studies have shown that after the action of CTX, the division and proliferation of normal hematopoietic cells were blocked, resulting in the damage of bone marrow regeneration or hematopoietic system.[3] It can cause various symptoms such as anemia, hemorrhage and low immunity. It is prone to serious infections and even death,[4] which seriously affects the patient's quality of life. Therefore, the treatment of myelosuppression can greatly ameliorate the effect of chemotherapy and improve the quality of life of cancer patients.

Ginseng (Panax ginseng C. A. Mey.), which is a traditional Chinese herbal medicine in China, holds an important position in traditional Chinese medicine. Modern research reports that various active ingredients in ginseng, such as ginsenosides and ginseng polysaccharides, have pharmacological effects such as improving immunity, assisting tumor treatment, enhancing bone marrow hematopoietic function, and regulating the nervous system.[5–9] Ginseng also is the “Chinese medicine and food homologous” Chinese medicine approved by the Ministry of Health. Up to now, a variety of ginsenosides have been isolated and identified from ginseng, and each ginsenoside has its own physiological activity.[10] R-Rg3 is a kind of ginseng glycol tetracyclic triterpenoid saponin which is one of the main active components of ginseng. Its chemical composition is complex, has multi-target and multi-channel pharmacological activity, and has less toxicity than chemical drugs. It is recognized as a monomer of Chinese medicine with the effect of inhibiting the proliferation of tumor cells,[11],[12] and also has many functions such as anti-diabetes,[13] anti-depression,[14] anti-inflammatory[15] effects, and so on. It has been developed and used all over the world, but no one has studied its therapeutic effect on myelosuppression.

In this study, we applied the method of intraperitoneal injection of CTX to establish the mice myelosuppression model. We detected the number of peripheral blood cells (PBCs) and bone marrow nucleated cells (BMNCs) in mice. After dissecting the mice, we measured their thymus and spleen index. In order to research the impacts of drugs on the proliferation and differentiation of hematopoietic progenitor cells (HPCs), we also carried out in vitro culture of HPCs. Additionally, we examined the changes in hematopoietic-related cytokines and cell cycle. Finally, network pharmacology was used to analyze the potential targets of R-Rg3 and predict its main action pathways, and the expression levels of p-JAK2 and p-STAT5 were tested.


   Materials and Methods Top


Materials

R-Rg3 was obtained from the Jilin University (Changchun, China). It was 99.5% pure, as confirmed by HPLC. Cyclophosphamide (CTX) was obtained from Baxter Oncology GmbH (Germany). Mouse granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin (TPO), erythropoietin (EPO) and interleukin-3 (IL-3) were purchased from Novoprotein Scientific Inc. (Shanghai, China).

Experimental Animal

Male Balb/c mice aged 6 to 8 weeks old were obtained from the Laboratory Animal Quality Testing Center of Jilin Province (Certificate no. SCXK-2016-0003). The mice were allowed to live in a temperature of 22 ± 2°C and humidity of 50 ± 10% with a 12 hr/12 hr light/dark cycle. All efforts were made to decrease the pain of the animals. The handling of animals was carried out in accordance with the rules of the National Institute of Health Laboratory Animal Care and Use Guidelines. And our research had been approved by the Animal Ethics Committee of the Chinese Academy of Sciences.[16]

After seven days of acclimatization, all mice were randomly divided into five groups (10 per group): (1) control group (control), (2) CTX treatment group (model), (3) rhG-CSF group (positive), (4) high-dose R-Rg3 treatment group (R-Rg3-H), and (5) light-dose R-Rg3 treatment group (R-Rg3-L). Except for the control group (given an equal amount of physiological saline 0.9% NaCl aq), the other groups were given CTX 100 mg/kg/d for 3 days. After modeling, the mice in group 3 were handled with rhG-CSF (11.25 μg/kg/d), the mice in groups 4 and 5 were handled with R-Rg3 (10 mg/kg/d, 5 mg/kg/d), the mice in groups 1 and 2 were handled with equivalent 0.9% NaCl aq for seven days.

Detection of peripheral blood cells

After the last administration of the drug for 24 hr, blood was collected from the orbital venous plexus. The number of blood cells was diluted and measured using a CX3 automatic biochemical analyzer.

Determination of the amount of karyota cells in bone marrow

Mice were euthanized by cervical dislocation and then immersed in 75% ethanol solution. Under sterile conditions, bilateral femurs were taken out and bone marrow cells were rinsed out with phosphate buffer saline (PBS). Then the single bone marrow cell suspension was prepared by filtration using a 4-gauge needle and centrifuged for 10 min at 1200 rpm. After taking out, the liquid was drained by using a pipette, and 0.5 mL of red blood cell (RBC) lysis buffer was added. After standing for 3 min, it was centrifuged for 10 min at 1200 r/min. Then the liquid was drained by using a pipette, the sediment was flushed twice with 1 mL PBS for 10 min at 1200 rpm and then resuspended in PBS. Next a pipette was use to drain the liquid and 1 mL of PBS was added to the mix. The number of bone marrow nucleus cells was calculated under an inverted microscope.

Determinations of thymus/spleen index

The mice were dissected to remove the thymus and spleen. After removing the adipose tissue, the weight was weighed using a precision electronic balance. The thymus and spleen index of the mice were calculated as follows:

Organ index (%) = (weight of organ/mouse body weight) ×100%

Hematopoietic progenitor cells culture

IMDM medium was plated with bone marrow cells at a concentration of 105/mL, then supplemented with horse serum, 10− 4 M 2-mercaptoethanol, 2 mM 3% L-glutamine, 20 ng/mL (rm) IL-3, 20 U/mL (rh) EPO, 50 ng/mL (rm) GM-CSF, and 5 ng/mL (rm) TPO.

The culture environment of plates was 37°C with 5% CO2. Colony-forming unit erythroid (CFU-E) was counted after 3 days of culture. Burst-forming unit erythroid (BFU-E), colony-forming unit granulocyte macrophages (CFU-GM), and colony-forming unit-megakaryocyte (CFU-Meg) were counted after 7 days of culture.[16]

Detection of cytokines in serum

Blood samples were taken under the 37°C water-bath for 30 min and centrifuged at 4000 rpm for 10 min to gain serum. After separating the serum, the concentrations of GM-CSF, EPO and TPO in the serum of each group were tested according to the enzyme-linked immunosorbent assay (ELISA) method.

Determination of the cell cycle

A single bone marrow cell suspension was centrifuged (1000 rpm, 10 min), then the supernatant was discarded. 2 mL 70% cold ethanol was added to fix the cells overnight at 4°C. The Propidium iodide dye (30 min avoid light) was added to stain. The flow cytometry was used for testing the changes in the cell cycle. The cell proliferation index (PI) was calculated according to the following formula:

PI = (S + G2/M)/(G0/G1 + S + G2/M) × 100%

Network pharmacology

The main targets of R-Rg3 were predicted and screened by Swiss Target Prediction database and UniProt database. Cytoscape 3.2.1 software was used to establish the “component-target” network. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for pathway prediction. The acquired pathways were enriched and analyzed through OmicShare database. The database for Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to further analyze the resulting pathways.

Western blotting analysis

The determination of protein content was done using western blot analysis. Bradford method was applied to determine protein concentration. SDS-PAGE electrophoresis was employed to separate denatured protein samples. Then the samples were transferred to PVDF membrane by semi-dry electrophoresis, closed in 5% skimmed milk liquid for 1 hr, and added primary antibody, 4°C overnight. After adding a secondary antibody (1:3000) and incubating for 1 hr, chemiluminescence was used to develop the color. β-actin was regarded as an internal reference. The protein bands were quantitatively analyzed, and the relative expression level of each target protein was calculated.[16]

Statistical analysis

All analyses were performed using the Statistical Pathway for the Social Sciences (SPSS) version 17.0 software, and experimental data were expressed as mean ± standard deviation. All data were analyzed by one-way analysis of variance (ANOVA). And P < 0.05 was considered to be a statistically significant difference.


   Results Top


Effects of R-Rg3 on WBC, RBC and PLT

[Table 1] showed that contrasted with the control group, the number of white blood cells (WBCs) in the model group increased markedly (P < 0.01), and the number of RBC and PLT decreased significantly (P < 0.01). Contrasted with the number of WBCs in the model group, the R-Rg3-H group showed a significant decrease (P < 0.01), and there was no obvious difference in the R-Rg3-L group. There was no significant difference between the R-Rg3-L group and the R-Rg3-H group compared with the number of RBCs in the model group. Contrasted with the model group, the number of platelet (PLT) in the R-Rg3-L group and the R-Rg3-H group was markedly increased (P < 0.01).
Table 1: Effects of R -Rg3 on WBC, RBC and PLT

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Effect of R-Rg3 on the number of BMNCs

As shown in [Figure 1], the number of BMNCs in the model group was markedly reduced in contrast to the control group (P < 0.01). Compared with the model group, the number of BMNCs in R-Rg3-L group and R-Rg3-H group was significantly increased (P < 0.01 or P < 0.05), and the effect of R-Rg3-H group was stronger than that in the R-Rg3-L group (P < 0.01).
Figure 1: Effect of R-Rg3 on the number of BMNC (n = 6). ## P < 0.01 as compared with the control group. * P < 0.05 as compared with the model group. ** P < 0.01 as compared with the model group. aa P < 0.01 as compared with the R-Rg3-L group

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Effects of R-Rg3 on thymus and spleen index

It can be seen from [Table 2] that the thymus index of the model group decreased significantly (P < 0.01) and the spleen index increased significantly (P < 0.01) when compared with the control group. Compared with the model group, the thymus index of R-Rg3-L group and R-Rg3-H group were markedly increased (P < 0.01), and the effect of R-Rg3-H group was higher than that of R-Rg3-L group (P < 0.01). The spleen index of R-Rg3-L group and R-Rg3-H group were lower than that of the model group, but the effects were not obvious.
Table 2: Effects of R -Rg3 on Thymus Index and Spleen Index

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Effects of R-Rg3 on colony yield of HPC in vitro

[Figure 2] shows that the number of cell colonies of CFU-GM, CFU-E, BFU-E and CFU-Meg in the model group was markedly lower than that in the control group (P < 0.01). Compared with the model group, the number of cell colonies of CFU-GM, CFU-E, BFU-E and CFU-Meg in R-Rg3-H group was markedly higher (P < 0.01). Besides, the number of cell colonies of CFU-GM and CFU-Meg in R-Rg3-L group was markedly higher (P < 0.05). The colony numbers of R-Rg3-H group in CFU-GM, CFU-E, BFU-E and CFU-Meg were markedly higher than those in the R-Rg3-L group (P < 0.01).
Figure 2: Effects of R-Rg3 on cell colony number in vitro of hematopoietic progenitor cells (n = 6). ## P < 0.01 as compared with the control group. *P < 0.05 as compared with the model group. ** P < 0.01 as compared with the model group. aa P < 0.01 as compared with the R-Rg3-L group

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Effects of R-Rg3 on hematopoietic-related cytokines

We measured the levels of hematopoietic-related cytokines GM-CSF, EPO and TPO. As shown in [Figure 3], the levels of GM-CSF, EPO and TPO in the model group markedly increased compared with the control group (P < 0.01). The levels of GM-CSF and TPO in the R-Rg3-H group were significantly lower than those in the model group (P < 0.01 or P < 0.05). The levels of GM-CSF, EPO and TPO in the R-Rg3-L group were lower than those in the model group, but not obvious. The number of hematopoietic-related cytokines of R-Rg3-H group in GM-CSF and TPO were markedly lower than those in the R-Rg3-L group (P < 0.01 or P < 0.05).
Figure 3: Effects of R-Rg3 on hematopoietic-related cytokines (n = 10). ## P < 0.01 as compared with the control group. * P < 0.05 as compared with the model group. ** P < 0.01 as compared with the model group. aa P < 0.01 as compared with the R-Rg3-L group.a P < 0.05 as compared with the R-Rg3-L group

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Effects of R-Rg3 on cell cycle

The distribution of each stage of the cell cycle is shown in [Figure 4]. In contrast with the control group, the bone marrow cell of mice in the model group were blocked in the G0/G1 phase, and the proliferation index (PI) of mice was reduced significantly (P < 0.01). The number of bone marrow cells in G0/G1 phase decreased, but that in G2/M and S phases, it increased after R-Rg3 administration.
Figure 4: Effects of R-Rg3 on cell cycle (n = 6). ## P < 0.01 as compared with the control group. ** P < 0.01 as compared with the model group

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Results predicted by network pharmacology

Analysis of C-T network

R-Rg3 was imported into the Swiss Target Prediction database to obtain potential targets (Top 15). Targets were imported into UniProt database for calibration. Cytoscape 3.2.1 software was used to construct the “component-target” (C-T) network, as shown in [Figure 5], which contained 16 nodes (15 potential targets and 1 chemical components) and 15 edges.
Figure 5: Compound-target network of R-Rg3

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Analysis of T-P network

Through the functional annotation tool in the DAVID, the potential targets of R-Rg3 were analyzed. Finally, a total of 7 pathways were obtained (P < 0.05). Cytoscape 3.2.1 software was employed to construct the target-pathway (T-P) network, as can be seen in [Figure 6]. Among them, there were 3 signal transduction pathways, 3 cancer-related pathways, and 1 immune disease-related pathway. Enrichment analysis of 7 pathways is shown in [Figure 7]. Among these 7 pathways, multiple pathways contained the JAK-STAT signaling pathway. Therefore, it was speculated that the JAK-STAT signaling pathway may be the key to the role of R-Rg3. The analysis methods were the same as the quoted article.[17]
Figure 6: Target-pathway network of R-Rg3

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Figure 7: Pathway enriching of targets of R-Rg3

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Effects of R-Rg3 on the expression of p-JAK2 and p-STAT5

[Figure 8] shows the expression levels of p-JAK2 and p-STAT5 in different experimental groups. The expression levels of p-JAK2 and p-STAT5 in the model group significantly decreased (P < 0.01) when compared with the control group. Compared with the model group, the expression levels of p-JAK2 and p-STAT5 in the R-Rg3-H group significantly increased (P < 0.01).
Figure 8: The expression levels of p-JAK2 and p-STAT5 in different experimental groups. (n = 3). (A) Use β-actin as an internal refere nce. After the film was scanned, the protein bands were quantitatively analyzed. (B (a)) The expression levels of p-JAK2 in different experimental groups. (B (b)) The expression levels of p-STAT5 in different experimental groups. ## P < 0.01 as compared with the control group. ** P < 0.01 as compared with the model group

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   Discussion Top


Chemotherapy is one of the first choices for the treatment of malignant tumors. However, chemotherapeutic drugs have the disadvantage of poor selectivity, and can cause killing effects on tumor cells and normal human cells. The side effects of bone marrow suppression are often the important reasons for patients not being able to complete chemotherapy on time.[18] Therefore, the presence of myelosuppression is one of the biggest obstacles in the treatment of cancer patients. By reducing the bone marrow suppression caused by chemotherapy and promoting the recovery of bone marrow hematopoietic function, the therapeutic effect of chemotherapy can be significantly improved, and the cancer patient's quality of life can be ameliorated.

In this study, we found that mice in the control group had better mental state, sensitive response, and increased body weight. The mice developed weight loss, poor mental state, lethargy, massive hair loss and reduced diet after intraperitoneal injection of CTX. After administration of the drug, the state of the mice recovered to varying degrees over time. Among them, the R-Rg3-H group recovered better than the R-Rg3-L group.

PBCs can indirectly reflect the hematopoietic function of the bone marrow, which plays an important role in maintaining life. WBCs are relatively large in volume and mainly play a role in phagocytosis. RBCs are the highest number of blood cells in the blood. It can transport oxygen to various tissues through hemoglobin. In addition, the carbon dioxide produced by the metabolism of various tissues in the body is also transported to the lungs through the RBCs and excreted. Platelets (PLTs) are biologically active, small cytoplasm whose main function is coagulation and hemostasis. The average survival time of WBCs is the shortest, RBCs is about 120 days and PLTs is about 5–7 days. Therefore, myelosuppression has the greatest impact on WBCs, and the effect on RBCs is usually small. In our research, the number of WBCs in the model group increased markedly. We speculated that this was a positive feedback effect of the body on the missing WBCs, resulting in a large number of differentiated WBCs released into the blood. However, these WBCs were incomplete or weakly immune, and the number of WBCs decrease rapidly after a brief increase, which was confirmed in our preliminary experiments. R-Rg3 could alleviate this mechanism to some extent. Additionally, R-Rg3 could significantly increase the amount of PLTs in the blood of myelosuppressed mice, which is very beneficial for the adjuvant treatment of chemotherapy.

BMNCs can roughly represent the division and proliferation of bone marrow hematopoietic cells, which can directly reflect the hematopoietic function of bone marrow. The large number of BMNCs indicates that there are more immature blood cells, which reflects that the bone marrow has a better hematopoietic function.[19] The BMNC data of this experiment showed that R-Rg3 could significantly increase the number of BMNCs, which proved its good role in improving the myelosuppressed mice.

Thymus and spleen are two main immune organs. The thymus itself secretes thymosin as a mature organ of T cells and plays a key role in immune system.[20] The protection of the thymus can undoubtedly protect cellular immunity, humoral immunity and non-specific cellular immune function.[21] According to reports in the literature, CTX causes apoptosis in thymocytes.[22],[23] The outcomes of our research showed that CTX caused severe atrophy of the thymus. R-Rg3 could improve this phenomenon by increasing the thymus index, which reversed thymus atrophy. The spleen is the largest peripheral lymphoid organ in the human body. And it has many important immune active cells and immune cytokines. When the bone marrow function is impaired, the spleen can play a role in compensating for hematopoiesis.[24] Injection of CTX in our experiment caused splenomegaly, which we speculated was due to spleen compensatory hematopoiesis. R-Rg3 could alleviate splenomegaly by reducing spleen index.

During bone marrow hematopoietic stem cells cultured in vitro, the cells were stimulated by different colony-stimulating factors to differentiate into different colonies. The proliferation and differentiation ability of bone marrow hematopoietic cells are reflected by the production of these colonies.[25] Compared with the control group, the colony yields of CFU-GM, CFU-E, BFU-E and CFU-Meg in the model group were markedly reduced, which indicated that the ability of HPCs to proliferate and differentiate in myelosuppressed mice was severely impaired. However, R-Rg3 could stimulate the formation of CFU-GM, CFU-E, BFU-E and CFU-Meg cell colonies, which could promote the recovery of hematopoietic function in myelosuppressed mice. Among them, the effect of high dose was more obvious.

In order to explore the mechanism of R-Rg3, we examined the levels of hematopoietic-related cytokines. HPCs are stimulated by different cytokines to differentiate into various morphologically identifiable blood cells.[26] GM-CSF is a polypeptide growth factor, which has a broad-spectrum effect. Bone marrow stromal cells are important sites for secreting GM-CSF.[27],[28] It binds to GM-CSF receptors and promotes various hematopoietic cell proliferation and differentiation. Erythropoietin (EPO) is mainly produced by the kidneys and is one of the main cytokines for promoting the production of erythroid blood cells in mammals. TPO is mainly produced by the liver and kidneys. It is recognized as a specific positive regulator of megakaryocyte system.[29],[30] Our study found that the levels of GM-CSF, EPO and TPO in myelosuppressed mice increased, which should be the feedback mechanism of the body for various cell reduction. In addition, the results of the HPC culture showed that the ability of cell proliferation and differentiation of myelosuppressed mice had weakened. We speculated that hematopoietic progenitors are less sensitive to these hematopoietic factors, or hematopoietic progenitors are difficult to complete normal proliferation and differentiation after stimulation with these factors, and the body thus produces more cytokines. After treatment with R-Rg3, the ability of HPCs to proliferate and differentiate was improved, so the various hematopoietic cytokines secreted by the body were correspondingly reduced.

Cell proliferation is achieved through the operation of the cell cycle. The bone marrow cell cycle is the main indicator reflecting the hematopoietic action. The state of proliferation of the bone marrow cells can be reflected by the distribution of various phases of the cell cycle. There are two important checkpoints in the cell cycle regulation mechanism which are between G1 and S, and G2 and M.[31],[32] The former is entering the DNA synthesis phase, and the latter is entering the mitosis phase.[25] In our experiment, after intraperitoneal injection of CTX, the percentage of G0/G1 phase cells in the model group was markedly higher than that in the control group, and the percentage of G2/M phase cells was significantly lower than that in the control group. This suggested that CTX could cause G1 phase arrest, which in turn led to S phase arrest. After treatment with R-Rg3, the percentage of G0/G1 cells was markedly lower than that of the model group, and the percentage of cells in G2/M phase had significantly increased. This indicated that R-Rg3 can promote the completion of DNA synthesis and promote the cell into the mitosis stage, thereby enhancing the ability of cell proliferation and differentiation.

JAK-STAT signaling pathway was first discovered in a study of signal molecules required for target gene activation after interferon action.[33] This signaling pathway directly transmits the received extracellular signals to the nuclear target gene promoter through a transmembrane receptor. It plays a very important regulatory role in the survival, proliferation, differentiation and apoptosis of hematopoietic cells.[34–36] JAK is a soluble cytosolic tyrosine protein kinase, and JAK2 is a member of the JAK family. It has seven domains, such as kinase homology domain 1, kinase homology domain 2 and amino-terminal FERM domain.[37] It plays an important role in cell survival and proliferation.[38] Existing studies have shown that the absence of JAK2 can cause severe HPC dysfunction and severe anemia in adult animals.[39] STAT is a direct substrate of JAK. It exists in the cytoplasm in a resting state. STAT5 is a member of the STAT family with seven domains.[40] A study by Lin et al.[41] found that if the STAT5 protein of mice was inhibited, the natural killer cells in the body were greatly reduced. Natural killer cells are important immune cells in the body. STAT5 maintains the survival and function of natural killer cells by promoting the expression of anti-apoptotic proteins and inhibiting the expression of pro-apoptotic proteins. A study by Wierenga ATJ et al.[42] found that STAT5 not only participated in hematopoietic differentiation, but also strengthened the self-renewal and erythroid differentiation of HPCs. Therefore, we speculated that the myelosuppression caused by CTX may be related to the inhibition of the JAK–STAT signaling pathway. Regulating the expression levels of JAK2 and STAT5 on the JAK–STAT signaling pathway may be one of the important mechanisms of R-Rg3.


   Conclusion Top


R-Rg3 could restore the number of blood cells, reverse thymus atrophy, relieve splenomegaly, enhance the proliferation and differentiation of bone marrow hematopoietic stem cells, and promote the secretion of hematopoietic cell-associated cytokines, thereby improving the hematopoietic and immune functions of myelosuppressed mice to improve their quality of life.

Acknowledgements

We thank the Chongqing Education Commission provided the funds, Chongqing Three Gorges Medical College and Jilin Agricultural University provided the experimental environment, and we are also very grateful to all members for their contributions to this project.

Financial support and sponsorship

This work was financially supported by the Scientific and Technological Research Projects of Chongqing Education Commission (grant No. KJQN201902712). Construction Project of Key Disciplines of Traditional Chinese Medicine (Basic Theory of Traditional Chinese Medicine) in Chongqing (Chongqing Traditional Chinese Medicine (2021] No. 16).

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Yang YY, Xu SP, Xu QX, Liu XM, Gao Y, Steinmetz A, et al. Protective effect of Dammarane Sapogenins against chemotherapy-induced myelosuppression in mice. Exp Biol Med 2011;236:729-35.  Back to cited text no. 1
    
2.
Alyamkina E, Nikolin V, Popova N, Dolgova E, Proskurina A, Orishchenko K, et al. A strategy of tumor treatment in mice with doxorubicin-cyclophosphamide combination based on dendritic cell activation by human double-stranded DNA preparation. Genet Vaccines Ther 2010;8:1-10.  Back to cited text no. 2
    
3.
Yin JH, Shen XH. Advances in animal experimental study of intervention with TCM to myelosuppression caused by chemotherapy of malignant tumor. J Shanghai University Tradit Chin Med 2010;24:78-80.  Back to cited text no. 3
    
4.
Chen ZG, Zheng QH. Research status of TCM and Western Medicine on bone marrow suppression after chemotherapy. World Latest Med Inform 2018;18:27-28.  Back to cited text no. 4
    
5.
Irfan M, Kwak YS, Han CK, Hyun SH, Man HR. Adaptogenic effects of Panax ginseng on modulation of cardiovascular functions. J Ginseng Res 2021;45:32-40.  Back to cited text no. 5
    
6.
Wang CZ, Hou L, Wan JY, Yao HQ, Yuan J, Zeng J, et al. Ginseng berry polysaccharides on inflammation-associated colon cancer: Inhibiting T-cell differentiation, promoting apoptosis, and enhancing the effects of 5-fluorouracil. J Ginseng Res 2020;44:282-90.  Back to cited text no. 6
    
7.
He MJ, Wang N, Zheng WX, Cai XQ, Di DM, Zhang YQ, et al. Ameliorative effects of ginsenosides on myelosuppression induced by chemotherapy or radiotherapy. J Ethnopharmacol 2020;268:113581.  Back to cited text no. 7
    
8.
Jiao LL, Zhang XY, Li B, Liu Z, Wang MZ, Liu SY. Anti-tumour and immunomodulatory activities of oligosaccharides isolated from Panax ginseng C. A. Meyer. Int J Biol Macromol 2014;65:229-33.  Back to cited text no. 8
    
9.
Kim M, Mok H, Yeo WS, Ahn JH, Choi YK. Role of ginseng in the neurovascular unit of neuroinflammatory diseases focused on the blood-brain barrier. J Ginseng Res 2021;45:599-609.  Back to cited text no. 9
    
10.
Lin MY, Liu HY, Liu JP, Lu D, Li YP. Progress in chemical research of ginsenosides. Ginseng Res 2011;23:43.  Back to cited text no. 10
    
11.
An N, Zhu W, Feng ZH, Ye SJ, Yu CJ, Cai CJ. Effect of 20(R) ginsenoside Rg3 on protein expression of lung cancer cell line. Chin J Lung Cancer 2008;11:311-20.  Back to cited text no. 11
    
12.
Zhao Y. Research progress of 20 (R) -ginsenoside Rg3 on antitumor effect. Chin Clin Oncol 2001;6:81-2.  Back to cited text no. 12
    
13.
Kim KS, Yang HJ, Lee IS, Jang HJ. The aglycone of ginsenoside Rg3 enables glucagon-like peptid-1 secretion in enteroendocrine cells and alleviates hyperglycemia in type 2 diabetic mice. Sci Rep 2015;5:18325.  Back to cited text no. 13
    
14.
Zhang H, Zhou Z, Chen Z, Zhong Z, Li Z. Ginsenoside Rg3 exerts anti-depressive effect on an NMDA-treated cell model and a chronic mild stress animal model. J Pharmacol Sci 2017;134:45-54.  Back to cited text no. 14
    
15.
Shin YM, Jung HJ, Choi WY, Lim CJ. Antioxidative, anti-inflammatory, and matrix metalloproteinase inhibitory activities of 20(s)-ginsenoside Rg3 in cultured mammalian cell lines. Mol Biol Reports 2013;40:269-79.  Back to cited text no. 15
    
16.
Han JH, Xia J, Zhang LX, Cai EB, Zhao Y, Fei X, et al. Studies of the effects and mechanisms of ginsenoside Re and Rk3 on myelosuppression induced by cyclophosphamide. J Ginseng Res 2019;43:618-24.  Back to cited text no. 16
    
17.
Han M, Jia XH, Cai EB, Yang LM, Dai M, Sun N, et al. The effects of Arctigenin-Valine ester on chemotherapy-induced myelosuppression in mice. Bioorgan Med Chem 2019;27:2480-6.  Back to cited text no. 17
    
18.
Song XH, Hongl in situ. Research progress of traditional Chinese medicine for bone marrow suppression after chemotherapy. Zhejiang J Integr Tradit Chin Western Med 2009;19:326-8.  Back to cited text no. 18
    
19.
Gai Yun, Gao RL, Niu YP, Jin JH, Shi YQ. Effect of Panax Notoginsenosides on the Proliferation of haematopoietic Progenitor Cells in Mice with Immunemediated Aplastic Anemia. Chin J Integr Med 2003;23:680-3.  Back to cited text no. 19
    
20.
Turrini P, Tirassa P, Vigneti E, Aloe L. A role of the thymus and thymosin-alpha1 in brain NGF levels and NGF receptor expression. J Neuroimmunol 1998;82:64-72.  Back to cited text no. 20
    
21.
Ge MZ, Zhao YL, Ren SL. Protective effects of polysaccharides from Adenophora flavescens on radiation damage of immune organs in mice. Chin Tradit Herbal Drugs 1996;27:673-5.  Back to cited text no. 21
    
22.
Wang GJ, Li W, Cai L. Induction of apoptosis in rat thymus by cyclophosphamide. Chin J Immunol 1999;15:359-60.  Back to cited text no. 22
    
23.
Jing H, Bai XZ, Liu YL, Li YQ. Experimental study on effect of eclipta prostrata L on thymocyte apoptosis induced with cyclophosphamide. J Jinzhou Med College 2004;25:22-4.  Back to cited text no. 23
    
24.
Jiang HC, Qiao HQ. Spleen function and reserved splenic surgery. Chin J Pract Surg 1999;19:709-10.  Back to cited text no. 24
    
25.
Xiao WC. Comparative Study on Effects of Liuwei Dihuang Pill and Guifu Dihuang Pill on haematopoietic Mice Induced by Chemoradiation. Chengdu University TCM. (2009).  Back to cited text no. 25
    
26.
Li W, Zhao Y, Li XN. Effect of Zishenshengxue capsule on myelosuppression in mice induced by cyclophosphamide. J Tradit Chin Med 2013;33:233-7.  Back to cited text no. 26
    
27.
Huang XQ, Jiang YZR, Zhu BD. Study on the changes of EPO receptor in kidney, EPO receptor in spleen and GM-CSF mRNA expression in bone marrow cells of mice with blood deficiency syndrome induced by combined radiation and chemotherapy. J Chengdu University Tradit Chin Med 2009;32:56-58.  Back to cited text no. 27
    
28.
Patra K, Bose S, Sarkar S, Rakshit J, Jana S, Mukherjee A, et al. Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid. Chem Biol Interact 2012;195:231-9.  Back to cited text no. 28
    
29.
Sherr CJ. Cancer cell cycles. Sci 1996;274:1672-7.  Back to cited text no. 29
    
30.
Sun HP, Shen ZX, Qian LM. The effects of several recombinant cytokines on human megakaryocytopoiesis. Acta Universitatis Med Secondae Shanghai 1997;17:170-2.  Back to cited text no. 30
    
31.
Sun WW, Sun Q, Sun D, Sun WZ. Effects of different doses of Busuishengxue Granule on proliferation of haematopoietic progenitor cells in aplastic anemia model Rats. Acta Chin Med Pharmacology 2009;37:12-4.  Back to cited text no. 31
    
32.
Thalmeier K, Meissner P, Reisbach G, Hültner L, Mortensen BT, Brechtel IA, et al. Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines. Exp Hematol 1996;24:1.  Back to cited text no. 32
    
33.
Darnell JE. Studies of IFN-induced transcriptional activation uncover the Jak-Stat pathway. J Interf Cytok Res 1998;18:549-4.  Back to cited text no. 33
    
34.
Xiao MF, Zhang LM, Zhou YZ, Rajoria P, Wang CF. Pyrvinium selectively induces apoptosis of lymphoma cells through impairing mitochondrial functions and JAK2/STAT5. Biochem Biophys Res Commun 2015;469:716-22.  Back to cited text no. 34
    
35.
Peter V. Targeting the JAK2-STAT5 pathway in CML. Blood 2014;124:1386-8.  Back to cited text no. 35
    
36.
Heltemes-Harris LM, Willette MJL, Vang KB, Farrar MIA. The role of STAT5 in the development, function, and transformation of B and T lymphocytes. Ann N Y Acad Sci 2011;1217:18-31.  Back to cited text no. 36
    
37.
Chen YX, Li Y, Zhang LY, Liu X, Shan NN. JAK2V617F mutation and p-STAT5 protein expression in peripheral blood cells of patients with myeloproliferative neoplasm and their relations with clinical features. Chin J Exp Hematol 2012;20:1398-404.  Back to cited text no. 37
    
38.
Gnanasambandan K, Sayeski PP. A structure-function perspective of Jak2 mutations and implications for alternate drug design strategies: The road not taken. Curr Med Chem 2011;18:4659-73.  Back to cited text no. 38
    
39.
Akada H, Akada S, Hutchison RE, Sakamoto K, Wagner KU, Mohi G. Critical role of Jak2 in the maintenance and function of adult haematopoietic stem cells. Stem cells 2014;32:1878-89.  Back to cited text no. 39
    
40.
Levy DE, Darnell JE. STATS: Transcriptional control and biological impact. Nat Rev Mol Cell Biol 2002;3:651-62.  Back to cited text no. 40
    
41.
Lin JX, Du N, Li P, Kazemian M, Gebregiorgis T, Spolski R, et al. Critical functions for STAT5 tetramers in the maturation and survival of natural killer cells. Nat Commun 2017;8:1320.  Back to cited text no. 41
    
42.
Wierenga ATJ, Schepers H, Moore MAS, Vellenga E, Schuringa JJ. STAT5-Induced self-renewal and impaired myelopoiesis of human haematopoietic stem/progenitor cells involves downmodulation of C/EBP alpha. Blood 2006;107:4326-33.  Back to cited text no. 42
    


    Figures

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  [Table 1], [Table 2]



 

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