Moxa Cone Moxibustion at Zusanli(ST36) acupoint alleviate myelosuppression mouse induce by CTX and promote hematopoietic cell proliferation via HPA axis

Moxa Cone Moxibustion at Zusanli(ST36) acupoint alleviate myelosuppression mouse induce by CTX and promote hematopoietic cell proliferation via HPA axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Moxa Cone Moxibustion at Zusanli(ST36) acupoint alleviate myelosuppression mouse induce by CTX and promote hematopoietic cell proliferation via HPA axis Li Tan, Zhilin Li, Nan Cao, Jing Li, Zhiyu Ye, Hongda Chen, Ning Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2902015/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective. Clinical studies have reported that moxibustion alleviates the side effects of chemotherapy, such as myelosuppression; however, its therapeutic mechanisms remain unclear. We investigated whether moxa cone moxibustion (MCM) promotes recovery from chemotherapy-induced myelosuppression via the hypothalamus-pituitary adrenal (HPA) axis. Methods. A myelosuppression model was established in mice using cyclophosphamide (CTX), and they were grouped into control, model, and moxibustion groups. In the moxibustion group, the mice received MCM at ST36 for 7 days. The peripheral blood cells were detected using an automatic blood cell analyzer; serum levels of corticotropin-releasing hormone (CRH), corticotropin (ACTH), corticosterone (CORT) and glucocorticoid receptor (GRF) a/β were detected using enzyme-linked immunosorbent assay (ELISA); the expression and cell cycle of bone marrow hematopoietic cells were detected using flow cytometry (FC); and BGISEQ Instrument model-DNBseqTM Platform was used for RNA sequencing. The differentially expressed genes (DEGs) were subjected to gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results. Compared with the control group, the contents of the peripheral blood and LSK and HSC in the moxibustion group were significantly higher (P<0.05). Following moxibustion therapy, the S and G2 phases of bone marrow hematopoietic stem cells decreased significantly, while the G1 phase increased. The CRH, ACTRH, and CORT levels decreased (P<0.05), while GRF-a and GRFβ levels increased (P<0.05). Additionally, 193 DEGs were down-regulated in the control group and up-regulated in the moxibustion group, while 481 DEGs were up-regulated in the control group and down-regulated in the moxibustion group. GO analysis revealed that cross-DEGs were enriched in cell-cell junction, membrane raft, membrane microdomain, and T-cell receptor complex involved in T-cell activation, T-cell differentiation, and lymphocyte differentiation, which exerted the MF of cytokine binding, receptor activity, and activity. KEGG analysis revealed that hematopoietic cell lineage, primary immunodeficiency, cytokine-cytokine receptor interactions, and other pathways were significantly enriched in the moxibustion group. Conclusion. MCM could alleviate myelosuppression induced by CTX and promote hematopoietic cell proliferation via the HPA axis and promote the proliferation and differentiation of bone marrow hematopoietic cells by regulating gene expression. Moxibustion Myelosuppression HPA axis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Myelosuppression is a common adverse effect of chemotherapy for cancer. Approximately 80% of patients with cancer experience myelosuppression during chemotherapy [ 1 ]. Additionally, the immune and hematopoietic systems are the most obviously affected ones, and the most prominent manifestation is leukopenia. Clinical symptoms, such as severe infection and bleeding often occur and directly affect the treatment of such patients [ 2 ]. Therefore, addressing myelosuppression after chemotherapy plays an important role in cancer treatment. Moxibustion is an external treatment method of traditional Chinese medicine with a history of thousands of years in the prevention and treatment of diseases. Modern studies have confirmed that moxibustion can activate the hypothalamus-pituitary adrenal (HPA) axis and the sympathetic nervous system (SNS) [ 3 ] via thermal, radiation, and pharmacological effects, thus further regulating the immune and neurological functions [ 4 , 5 ]. Sun [ 6 ] reported that moxibustion has anti-myelosuppression effects in chemotherapy. In a multicenter randomized study [ 7 ], ginger-separated moxibustion was found to be reliable and better than oral proprietary Chinese medicine in the treatment of leukopenia caused by chemotherapy with good repeatability. A meta-analysis [ 8 ] found that moxibustion was more effective in increasing the white blood cell count than various types of control interventions. Although the level of evidence is low, it highlighted that moxibustion was superior to drug therapy in the treatment of chemotherapy-induced leukopenia. Studies have demonstrated that moxibustion could improve myelosuppression following chemotherapy [ 9 ]. ST36 was one of the most commonly used acupoints in the treatment of myelosuppression [ 10 , 11 ]. Some studies have found that activating the HPA axis could promote the proliferation of bone marrow hematopoietic cells and address the myelosuppression [ 12 ]. In this study, a mouse model of myelosuppression established using cyclophosphamide (CTX) was used to observe the effects of moxa cone moxibustion (MCM) on peripheral blood cells [ 13 , 14 ], bone marrow hematopoietic cells, and the cell cycle as well as the effects on the key parts of the HPA axis and explore the underlying mechanism by analyzing the differences in the genetic expressions of bone marrow cells between the groups. 2. Materials and Methods 2.1 Reagents The main reagents involved in the experiments are as follows: Cyclophosphamide was purchased from Baxter oncology GmbH (H20160467, Germany). Fluorescently labeled monoclonal antibodies MO HEMATOPTC LIN CKIL FITC (#22-7770-22, ), ANTI-MO CD150 MSHAD150 PE-CYN7 (#25-1502-82), ANTI-MO CD48 HM48-1 APC (#17–0481 -80), ANTI-MO CD34 RAM34 EF450 (#48-0341-80), ANTI-M LY-6A/E D7 PERCP-CYN5.5 (#45-5981-82), ANTI-MO CD117 2B8 PE(# 12-1171-81), Rat/ham ig Kpa Comp Bead set (#552845) and other flow-through antibodies were purchased from eBioscience (Thermo Fisher Scientific, USA). Elisa kits CRH (#MM-0509M2), ACTRH (#MM-0554M2), CORT (#MM-0061M2), GRFa (#MM-0296M2), GRFβ (#MM-0295M2) were purchased from Jiangsu MeiMian Industrial Co., Ltd. MolPure ® Cell/Tissue Total RNA Kit (#19221ES50, YEASEN). moxa (35:1), purchased from Beijing Guoyiyan Medical Equipment Co., Ltd. 2.2 Animals and group Male C57BL/6 mice (6–8 weeks of age) were procured from the Vital River Laboratory Animal Technology (Beijing, China). They were housed in a room maintained at 21 ± 2°C and 12-h light-dark cycle with free access of food and water. The experiments were approved by the ethics committee of Longhua District Central Hospital Affiliated to Guangdong Medical University (Shenzhen). The experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. After 1 week of acclimatization, mice were randomly divided into 3 groups with 5 mice in each group, namely control group, model group and moxibustion group. 2.3 CTX-induced Myelosuppression model established To establish the model, 15 mg of cyclophosphamide (CTX) was completely dissolved in 75 mL of PBS for a concentration of 5 mg/mL. The mice in the model and moxibustion groups were administered CTX solution (100 mg/kg) intraperitoneally once a day for five consecutive days followed by 50 mg/kg every other day until the 12th day of modeling. The mice in the control group received the same dose of PBS intraperitoneally. 2.4 Moxibustion and Acupiont We preferred the Zusanli (ST36) acupoints based on our previous work[ 11 ]. They are located at the posterolateral aspect of the knee joints of the hindlimbs of the mice approximately 5 mm below the capitellum of the fibula. A moxa cone is made of moxa velvet with a diameter of 1 ± 0.5 mm and a height of 2 mm. Mouse fur was shaved over a 1 × 1cm 2 area around ST36 to expose the skin (Fig. 1). On the 6th day, moxibustion was started on the ST36 of the mice in the moxibustion group, and the prepared moxa cone was placed on the acupoint for moxibustion. It was lit with incense sticks, and the moxa cone was replaced with the next one when it went out naturally for a total of three times per acupoint daily and continuously for 7 days. 2.5 Weight observation From the start of modeling, the changes in the body weight of mice in each group were observed daily. 2.6 Detection of peripheral blood cells in mice In EP tubes, 0.2 ml of blood was collected, and an automatic blood cell analyzer (Mindray veterinary automatic blood cell analyzer model: BC-2800vet) was used to analyze the following contents in the peripheral blood of mice in each group: white blood cell (WBC), lymphocyte (Lym), granulocyte (Gra), monocyte (Mon), red blood cell (RBC), hemoglobin (Hb), and platelets (PLT). 2.7 Mouse bone marrow hematopoietic cells and cell cycle using flow cytometry After the mice were euthanized with cervical dislocation, they were sterilized in 75% alcohol for 30 seconds. Subsequently, bilateral femur and tibia were removed, the closed ends on both sides of the bones were cut, and the bone marrow cells were flushed into a centrifuge tube. After filtration through a cell sieve, the samples were centrifuged at 1600 rpm and 4°C for 5 min, and the supernatant was discarded. The RBC lysate was added to lyse red blood cells at 37°C for 10 min, centrifuged at 1600 rpm for 10 min at 4°C, and the supernatant was discarded. Subsequently, 1 mL PBS containing 3% fetal bovine serum (PBS-F) was added to the tube to resuspend the bone marrow cells. Finally, antibodies (Lin, sca-1, c-kit, CD48, CD150, and CD34) were added to the samples for 30 min, and the cells were mixed using gentle shaking. In brief, bone marrow hematopoietic cells were fixed with cold 70% ethanol and stained with a PBS solution containing 50 lg/mL PI and 30 lg/mL RNase A. The prepared samples were analyzed using flow cytometry (BECKMA, Cytoflex LX) and FlowJo_V10 software. 2.8 Enzyme-linked immunosorbent assay(ELISA) For ELISA, 0.5 mL of blood was drawn from the retro-orbital space and collected in 1.5-mL EP tubes. It was allowed to stand at room temperature for 2 hours, and transferred to a refrigerator at 4°C for overnight storage. The serum was separated using centrifugation at 3000 rpm at 4 ℃ and transferred to a new centrifuge tube. The serum concentrations of corticotropin-releasing hormone (CRH), corticotropin (ACTH), corticosterone (CORT), and glucocorticoid receptor α and β (GRF-α, GRF-β) were estimated using ELISA. The procedures were performed according to the instructions of the manufacturers and analyzed using an enzyme labeling instrument (RaytoRT-6100). 2.9 RNA extraction and sequencing Two bone marrow samples were selected from each group, and RNA was extracted from the bone marrow cell suspension in strict accordance with the operating steps of the RNA Kit. Subsequently, RNA was quantified and identified using Agilent 2100 Bioanalyzer (Thermo Fisher Scientific, MA, USA). The extracted RNA was reverse-transcribed into the corresponding DNA fragments, and the exon region probes were used to hybridize and capture after polymerase chain reaction (PCR) amplification. The captured DNA was subsequently amplified using PCR and circularized to get single-stranded circular (ssCir) library. The ssCir library was then amplified using rolling circle amplification (RCA) to obtain DNA nanoball (DNB). The DNB was then loaded to a flow cell and sequenced using BGISEQ Platform (BGI, Shenzhen, China). 2.10 Differentially expressed genes (DEGs) analysis Sequencing data (raw data) was filtered using SOAPnuke (v1.5.2) and quality control (QC) was performed using FASTQ [ 16 ]. Subsequently, the obtained clean reads were mapped to the Mouse C57 BL/6 standard genome (GCF_000001635.27_GRCm39_genomic.fna) using Bowtie2 (v2.2.5) [ 17 ]. The expression levels of the genes were then calculated using RSEM (v1.2.8) [ 18 ]. Finally, DEG analysis was performed using DESeq2 (v1.4.5) with a Q value less than 0.05 [ 19 ]. 2.11 Functional enrichment analysis of differentially expressed genes The cluster Profiler function of the R package was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to better understand the function and pathway of MCM regulating DEGs in bone marrow hematopoietic cells. 2.12 Statistical analysis All data were analyzed with SPSS v23.0 (IBM Corp., Armonk, NY, USA). Mean ± standard deviation is used to express the data with normal distribution and homogeneity of variance. The differences between the groups were analyzed using one-way analysis of variance (ANOVA), and pairwise comparisons were conducted using the Bonferroni method. The ratio data and data of skewed distribution or variances were analyzed using non-parametric tests, and the median (P25-P75) was used with P < 0.05 considered statistically significant. All results were plotted using GraphPad Prism 8. 3. RESULTS 3.1 MCM treatment results in improvements to weight loss The weight of the mice in the control group gradually increased every day and the weights of mice in the model and moxibustion groups decreased from the second day of CTX. By the 12th day, the body weights of the mice in both groups were lower than that in the control group ( P < 0.05) (Fig. 2). 3.2 Changes in peripheral blood cell content At the end of the experiment, compared with the control group, the values of WBC, Lym, RBC, Hb, and PLT in the model group decreased ( P < 0.05) and that of Gra in the moxibustion group increased ( P < 0.05). Compared with the model group, the values of WBC, Gra, RBC, Hb, and PLT in the moxibustion group increased ( P < 0.05) and that of Lym increased; however, there was no statistically significant difference (Fig. 3). 3.3 Changes in bone marrow hematopoietic cells and cell cycle Compared with the control group, LSK (Lin − Sca-1 + c-kit + ), HSC (Lin − Sca-1 + c-kit + CD48 − CD150 + ), short-term hematopoietic stem cell (ST-HSC), and long-term hematopoietic stem cell (LT-HSC) in the model group were decreased ( P < 0.05); ST-HSC and LT-HSC in the moxibustion group were also decreased ( P < 0.05). Compared with the model group, LSK and HSC of the mice in the moxibustion group increased ( P < 0.05) (Figs. 4 and 5). After calculating the cell cycle subsets using the cell cycle module of FlowJo software, we found that after CTX modeling, the S phase of the hematopoietic stem cell cycle increased, while the G1 phase decreased ( P < 0.05). Following moxibustion treatment, the S and G2 phases were significantly decreased and the G1 phase increased ( P < 0.05) (Fig. 6). 3.4 Changes in CRH, ACTRH, CORT, GRF-a, and GRFβ in peripheral blood After the experiment, compared with the control group, the serum levels of CRH, ACTRH, and CORT in the peripheral blood in the model group were increased ( P < 0.05), while the levels of GRF-a and GRF-β had decreased ( P < 0.05). The serum levels of CRH, ACTRH, and CORT in the peripheral blood of mice in the group were decreased ( P < 0.05), while the contents of GRF-a and GRF-β had increased ( P < 0.05) (Fig. 7). 3.5 Gene expression analysis RNA sequencing of each sample was assessed using the BGISEQ platform. Subsequently, feature Counts v1.5.0-p3 was used to calculate the expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) of each sample, which represent the relative gene expression abundance. The boxplots revealed that the distribution of FPKM expression was roughly consistent across each sample, thus indicating that the RNA-seq data were reproducible and reliable (Fig. 8a). The principal component analysis (PCA)(Fig. 8b) and Pearson correlation heatmap visualization analysis (Fig. 8c) revealed that it can be found that the sample differences between the three groups became obvious. To further identify significant DEGs between the control, model, and moxibustion groups, DEGs were analyzed using the DESeq2 R package. |log2FC|>1 and p < 0.05 were considered criteria for searching for significant DEGs. Comparing the model and the control group, a total of 2977 DEGs were identified, including 1500 up-regulated genes and 1477 down-regulated genes. Comparing the model and moxibustion group, a total of 1137 DEGs were identified, including 293 up-regulated genes and 844 down-regulated genes. Among them, 193 genes were down-regulated in the model group and up-regulated in the moxibustion group; 481 genes were up-regulated in the model group and down-regulated in the moxibustion group (Fig. 8d). The 674 cross-DEGs were analyzed in the next step. Heatmap and hierarchical cluster analysis revealed that intragroup data were clustered together, and intergroup data were separated. The results revealed that moxibustion could restore the down-regulated gene levels in the myelosuppression group. These findings further suggest that the regulation of bone marrow hematopoietic cells by MCM in the CTX myelosuppression model can be accomplished by regulating the expression of related genes (Fig. 8e). 3.6 Go function and KEGG pathway enrichment analysis of DEGs GO functional enrichment analysis (including biological process [BP], cellular component [CC], and molecular function [MF]) was performed with 674 DEGs to predict the underlying biological function of DEGs between the MCM and model groups. The DEGs were enriched to 857 subclasses of GOs (742 BPs were enriched, 54 CCs were enriched, and 61 MFs were enriched). The top three enriched BPs were T-cell activation, T-cell differentiation, and lymphocyte differentiation; the top three CCs were cell-cell junction, membrane raft, and membrane microdomain; and the top three MFs included cytokine binding, cytokine receptor activity, and cytokine activity (Fig. 9). KEGG pathway analysis was conducted to analyze the DEGs between the MCM and model groups. The top KEGG pathways with the most enrichment were the hematopoietic cell lineage, primary immunodeficiency, and cytokine-cytokine receptor interaction. The top 20 enrichment pathways are shown in Fig. 10. 4. Discussion The results of the current study mainly illustrate that MCM can alleviate the degree of myelosuppression induced by chemotherapy in a mouse model. The potential underlying mechanism may be that MCM upregulates the expression of GFR, restores the negative feedback regulation mechanism of the HPA axis, and promotes normalization of bone marrow hematopoiesis. Additionally, MCM can modify the gene expression in the CTX model, thereby promoting the proliferation of hematopoietic cells. 4.1 Stability of CTX model As a commonly used alkylating agent in chemotherapy [ 20 ], CTX is used in establishing bone marrow suppression models mainly because of its stable effects, easy availability, and low cost. The mechanism of bone marrow suppression caused by it mainly includes destruction of the DNA of bone marrow hematopoietic stem cells, which results in irregular necrosis and apoptosis of hematopoietic cells and decreased activity of precursor cells and bone marrow suppression [ 21 – 25 ]. CTX-induced apoptosis in mouse bone marrow was one of the typical immunosuppression [ 26 ]. Some scholars [ 27 ] have investigated the doses and effects of cyclophosphamide modeling in recent years, and found that the number of bone marrow nucleated cells in mice treated with a total dose of 100–500 mg/kg of cyclophosphamide will rebound on the 5th–10th day. Subsequently, the number of white blood cells in the peripheral blood is also temporarily higher than the normal levels. However, after the modeling has stopped, the peripheral blood in the model group could gradually return to normal. Therefore, this experiment confirmed that 100 mg/kg of cyclophosphamide combined with 50 mg/kg maintenance dose every other day could well maintain the stability of the model. No recovery of peripheral blood cells and bone marrow hematopoietic cells was observed on the 7th day after the model was established. 4.2 MCM and hematopoietic stem cells Recent studies have demonstrated that moxibustion has a positive effect on protecting the blood, enhancing bone marrow hematopoiesis, and promoting cell cycle transformation. Similar results were noted in this experiment; moxa cone can promote the transformation of bone marrow hematopoietic cells from the G2 phase to M phase and promote the proliferation of ST-HSC, thereby promoting the mobilization of bone marrow hematopoietic cells and their migration to increase the peripheral blood leukocytes, RBC, Hb, and PLT. However, the currently available evidence of the mechanism of moxibustion in the treatment of myelosuppression following chemotherapy mainly focused on the following three aspects: (1) enhanced DNA repair and cell cycle transformation of bone marrow cells, which helps the proliferation and differentiation of hematopoietic cells[ 15 , 16 ]; (2) Regulation of bone marrow cell signaling pathways[ 17 ] and (3) improving the damaged hematopoietic microenvironment mainly by promoting the release of cytokines on the stromal cell membrane and the repair of microvascular damage[ 18 ]. However, these studies mainly focused on the molecular mechanisms of expression of related proteins in bone marrow cells. However, whether they were related to the regulation of other biologically active substances remains unclear. The HPA axis is an important part of the neuro-endocrine-immune network and mainly involved in regulating the body’s stress response. It is a complex system of direct and feedback interactions involving the hypothalamus, pituitary, and adrenal glands[ 19 ]. CRH in the hypothalamus serves as the starting point for the regulation of the HPA axis activity and is a key driving force of the axis that plays an important role in stress responses[ 20 ]. CRH from the hypothalamus stimulates the pituitary to release ACTH, which stimulates the adrenal gland to secrete glucocorticoids (GC). Under physiological conditions, excessively elevated GC inhibit the synthesis of ACTH and CRH through negative feedback regulation, thus maintaining a relatively stable and moderate stress response of GC. The negative feedback regulation of GC is mediated by the glucocorticoid receptor (GR) [ 21 ]. After binding to the ligands, GR enters the nucleus and specifically binds to the negative glucocorticoid response element (nGRE) in the promoter region of the CRH gene, thus inhibiting the gene expression of CRH in the hypothalamus [ 22 ]. If the negative feedback regulation mechanism of the HPA axis is damaged, it will eventually lead to hypersensitivity of the HPA axis [ 23 ]. Recent studies have demonstrated that neuroendocrine responses regulate the production of GC through the HPA axis under stress conditions (trauma, burn, pressure, and hemorrhagic shock), which plays a positive role in the regulation of bone marrow hematopoietic cells [ 24 – 26 ]. Kwan [ 27 ] found that the central nervous system regulates GR through the HPA axis in stress response, which plays a key role in the production of hematopoietic stem and progenitor cells (HSPC). GR agonists can enhance the formation of HSPC, while a lack of GR reduces the formation of HSPC. Long-term activation of GR plays a key role in the self-replication and proliferation of erythroid progenitor cells. Gao [ 28 ] also found that the activation of HPA axis promotes the release of glucocorticoids, which mainly regulated the directional migration of MSCs and EPCs through GR and promoted the mobilization of bone marrow stem cells. People with GC dysfunction have hematopoietic deficiency [ 29 ]. However, Pierce [ 30 ] suggested that GC was the key to the mobilization of HSC in the HPA axis, and the physiological level of cortisol promoted HSC migration through the GC receptor NR3C1. Too high or too low cortisol levels have a negative effect on the proliferation of HSC. It is worth noting that in this study, the serum levels of CRH, ACTH, and CORT in the model group were significantly higher than those in the control group and moxibustion group, while the GR level decreased significantly. This observation indicates that cyclophosphamide damaged the negative feedback regulation mechanism of the HPA axis and resulted in hyperfunction. Additionally, the hyperphysiological level of glucocorticoids did not play a positive role in the proliferation of bone marrow cells. However, after moxa moxibustion, the levels of GR increased, which restored the negative feedback regulation mechanism of the HPA axis and promoted the normalization of bone marrow hematopoiesis mechanisms. It was suggested that GR is still the key link in the regulation of bone marrow hematopoietic cells by the HPA axis. In order to determine whether MCM can promote the proliferation and differentiation of the hematopoietic cells at the genetic level, we analyzed 674 cross-DEGs between the model group and the control group as well as the moxibustion group and the model group using RNA-seq. The results demonstrated that MCM could significantly alter gene expression in the CTX model and consequently regulate the CTX-induced myelosuppression and promote the proliferation of hematopoietic cells. GO enrichment analysis revealed that the DEGs between moxibustion group and model group were related to the activities of cytokines, their receptors and ligands, and the proliferation and differentiation of nucleated cells (leukocytes, lymphocytes, and T-cells). These results are consistent with those of previous studies [ 31 – 33 ]. Therefore, MCM could promote the proliferation and differentiation of bone marrow hematopoietic cells by regulating gene expression. The most significant enrichment pathways in KEGG were the hematopoietic cell lineage, primary immunodeficiency, cytokine-cytokine receptor interaction, Th1 and Th2 cell differentiation, and Th17 cell differentiation. These pathways were more closely related to the proliferation and differentiation of hematopoietic and immune cells [ 34 – 36 ]. Hematopoietic cell lineage is a network of hierarchical differentiation of hematopoietic cells, which is very complex and huge with strong continuity, plasticity, and self-renewal ability [ 37 , 38 ]. In recent years, with the development of single-cell sequencing, the traditional hematopoiesis system has been continuously supplemented and improved [ 37 ]. The analysis of these KEGG pathways in this project could only reflect that MCM adjusted these networks. However, its complete role remains unclear and requires further investigations. 5. Conclusion MCM could promote the proliferation and differentiation of hematopoietic cells following CTX-induced myelosuppression by regulating the expression of genes, thus increasing the levels of peripheral blood cells. MCM is a non-drug therapy that acts on the body’s surface and plays a therapeutic role through stimuli, such as temperature and pain. The mechanism of its effects might be related to the negative feedback regulation of the HPA axis. However, due to the complexity of the hematopoietic system and the mechanism of action of MCM, we could not explore the deeper mechanisms. Declarations Disclosure Hongda Chen and Ning Wang are the co-corresponding author. Data Availability RNA-sequence data had been published in NCBI (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA859387). The data used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest The authors declare no conflicts of interest. 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Human & Experimental Toxicology. 2002;21(6):335-41. Salem ML, Al-Khami A, El-Nagaar SA, Zidan AA, Al-Sharkawi IM, Díaz-Montero C et al. Kinetics of rebounding of lymphoid and myeloid cells in mouse peripheral blood, spleen and bone marrow after treatment with cyclophosphamide. Cellular Immunology. 2012;276(1-2):67-74. Alyamkina EA, Nikolin VP, Popova NA, Dolgova EV, Shurdov MA. 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):1-10. Wang C, Zhao D, Yang Z, Jia Y, Chang J, Wang Y et al. Mouse Model of Cyclophosphamide-Induced Myelosuppression and Research Progress on Mechanism of Action of Chinese Meteria Medica in its treatment and prevention. World Chinese Medicine. 2017;12(9):2252-7. Mei L, Cao DM, Li DM, Zhao XX, Zhang HF. 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Influence of Acupuncture and Moxibustion on Bone Marrow Tissue Adhesion Molecule ICAM-1,VCAM-1 Protein Expression in Post Chemotherapy with CTX Mice. China Journal of Chinese Medicine. 2013;28(12):1840-2. doi:10.16368/j.issn.1674-8999.2013.12.030. Shao KH, Perry R, Ernst E. The effectiveness and efficacy of Rhodiola rosea L.: A systematic review of randomized clinical trials. Phytomedicine. 2011;18(4):235-44. Binder EB, Nemeroff CB. The CRF system, stress, depression and anxiety-insights from human genetic studies. Molecular Psychiatry. 2009;15(6):574-88. Oakley RH, Cidlowski JA. The biology of the glucocorticoid receptor: new signaling mechanisms in health and disease. The Journal of allergy and clinical immunology. 2013;132(5):1033-44. doi:10.1016/j.jaci.2013.09.007. Bali B, Ferenczi S, Kovács KJ. Direct inhibitory effect of glucocorticoids on corticotrophin-releasing hormone gene expression in neurones of the paraventricular nucleus in rat hypothalamic organotypic cultures. J Neuroendocrinol. 2008;20(9):1045-51. doi:10.1111/j.1365-2826.2008.01759.x. Adcock IM, Barnes PJ. Molecular mechanisms of corticosteroid resistance. Chest. 2008;134(2):394-401. doi:10.1378/chest.08-0440. Hannoush EJ, Sifri ZC, Elhassan IO, Mohr AM, Alzate WD, Offin M et al. Impact of enhanced mobilization of bone marrow derived cells to site of injury. Journal of Trauma & Acute Care Surgery. 2011;71(2):283-9. doi:10.1097/TA.0b013e318222f380. Hu C, Xin Y, Li C, Lü M, Liang G. CXCL12/CXCR4 axis promotes mesenchymal stem cell mobilization to burn wounds and contributes to wound repair. Journal of Surgical Research. 2013;183(1):427-34. doi:10.1016/j.jss.2013.01.019. Xiang M, Yuan Y, Fan L, Li Y, Li A, Yin L et al. Role of macrophages in mobilization of hematopoietic progenitor cells from bone marrow after hemorrhagic shock. Shock. 2012;37(5):518-23. doi:10.1097/SHK.0b013e318249b81d. Kwan W, Cortes M, Frost I, Esain V, Theodore L, Liu S et al. The Central Nervous System Regulates Embryonic HSPC Production via Stress-Responsive Glucocorticoid Receptor Signaling. Cell Stem Cell. 2016;19(3):370-82. doi:DOI: 10.1016/j.stem.2016.06.004. Gao W, Yang X, Du J, Wang H, Zhong H, Jiang J et al. Glucocorticoid guides mobilization of bone marrow stem/progenitor cells via FPR and CXCR4 coupling. Stem Cell Res Ther. 2021;12(1):16. doi:10.1186/s13287-020-02071-1. Masri-Iraqi H, Robenshtok E, Tzvetov G, Manistersky Y, Shimon I. Elevated white blood cell counts in Cushing's disease: association with hypercortisolism. Pituitary. 2014;17(5):436-40. doi:10.1007/s11102-013-0522-0. Pierce H, Zhang D, Magnon C, Lucas D, Frenette PS. Cholinergic Signals from the CNS Regulate G-CSF-Mediated HSC Mobilization from Bone Marrow via a Glucocorticoid Signaling Relay. Cell stem cell. 2017;20(5):648-58. doi:10.1016/j.stem.2017.01.002. Clapes T, Polyzou A, Prater P, Sagar, Morales-Hernández A, Ferrarini MG et al. Chemotherapy-induced transposable elements activate MDA5 to enhance haematopoietic regeneration. Nature cell biology. 2021;23(7):704-17. doi:10.1038/s41556-021-00707-9. Robb L. Cytokine receptors and hematopoietic differentiation. Oncogene. 2007;26(47):6715-23. doi:10.1038/sj.onc.1210756. Jackson M, Fidanza A, Taylor AH, Rybtsov S, Axton R, Kydonaki M et al. Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System. Stem cell reports. 2021;16(4):727-40. doi:10.1016/j.stemcr.2021.02.003. Melchers F. [From stem cells to lymphocytes]. Zeitschrift fur Rheumatologie. 2009;68(3):196-200, 2-4. doi:10.1007/s00393-008-0390-6. Liu C, Sun Y, Shao Z. Current Concepts of the Pathogenesis of Aplastic Anemia. Current pharmaceutical design. 2019;25(3):236-41. doi:10.2174/1381612825666190313113601. Dey R, Ji K, Liu Z, Chen L. A cytokine-cytokine interaction in the assembly of higher-order structure and activation of the interleukine-3:receptor complex. PloS one. 2009;4(4):e5188. doi:10.1371/journal.pone.0005188. Cheng H, Zheng Z, Cheng T. New paradigms on hematopoietic stem cell differentiation. Protein & cell. 2020;11(1):34-44. doi:10.1007/s13238-019-0633-0. Pouzolles M, Oburoglu L, Taylor N, Zimmermann VS. Hematopoietic stem cell lineage specification. Current opinion in hematology. 2016;23(4):311-7. doi:10.1097/moh.0000000000000260. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2902015","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":197945547,"identity":"c064fc5a-c94b-495e-9972-26e8a89e09b1","order_by":0,"name":"Li Tan","email":"","orcid":"https://orcid.org/0000-0002-3068-9078","institution":"Shenzhen Longhua District Central Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Tan","suffix":""},{"id":197945548,"identity":"93823c08-ad9e-4cc9-8d4e-3017b464cda0","order_by":1,"name":"Zhilin Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhilin","middleName":"","lastName":"Li","suffix":""},{"id":197945549,"identity":"50bb4161-c238-441c-bd1e-37d5f412ade3","order_by":2,"name":"Nan Cao","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Cao","suffix":""},{"id":197945550,"identity":"1086a0ff-3d5c-4139-af71-708fbc7b747b","order_by":3,"name":"Jing Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""},{"id":197945551,"identity":"bdf4472f-15c6-4537-b7c0-335cfe4e4b34","order_by":4,"name":"Zhiyu Ye","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhiyu","middleName":"","lastName":"Ye","suffix":""},{"id":197945552,"identity":"fb585de8-3a73-44f0-ad84-23dd5507f7fa","order_by":5,"name":"Hongda Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongda","middleName":"","lastName":"Chen","suffix":""},{"id":197945553,"identity":"b3d35383-f718-4058-a1ba-e7ca4d9055f1","order_by":6,"name":"Ning Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYLACxn82ckCy8QBMQIKwHrY0Y6CWBpK0HE5sAFLEaTE4fvbw6wKew+lr2w8Dbflz2N7gAPPB2zwMdnk4tZzJS7OeIZGeu+1MYsMBxrbDiRsOsCVb8zAkF+PSYnYgx8yYx8A6d9sBkJaGwwkGB3jMpHkYDoCdilXL+TdALQnM6WbnH8Icxv8Nv5YbOcaPeQ44J5jdANoCDAfGDQd42PBqsb/xxoyZtyHNcNsNoC2JbemJMw+zGVvOMUjGqUWyP8f4M2+DjbzZ+fSHDz78sbbnO9788MabCjucWoCADRELCQzNDAzMIJYBbvVAwPwBiVOHV+koGAWjYBSMTAAAMBpe1u07zcQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0972-2486","institution":"The Seventh Affiliated Hospital, Sun Yat-sen University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2023-05-06 14:18:39","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true,"coiExplicitlySet":false},"doi":"10.21203/rs.3.rs-2902015/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2902015/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36717404,"identity":"6c012739-1c22-4c74-8926-123185870197","added_by":"auto","created_at":"2023-05-08 21:15:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":150436,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of Moxa-cone moxibustion at ST36 in mice.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/ff3849693219c0cf830a812b.png"},{"id":36717045,"identity":"0d50d6d1-6024-417a-9189-c41eddec05e5","added_by":"auto","created_at":"2023-05-08 21:07:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86813,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in body weight of mice in each group during the experiment(Legend in all images, C represents control group,M represents model group, Mox represents moxibustion group).\u003csup\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e*P\u003c/em\u003e＜0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/9d626d9bac74f48a61012267.png"},{"id":36717043,"identity":"bd23cb0d-c65b-4e1b-870e-fe3d0c6820b5","added_by":"auto","created_at":"2023-05-08 21:07:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75571,"visible":true,"origin":"","legend":"\u003cp\u003eIntergroup comparison of peripheral blood cell content. Figures A-G represent the contents of WBC, Lym, Gra, Mon, RBC, Hb, and PLT in each group, respectively.\u003cem\u003e *P\u003c/em\u003e＜0.05.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/315713fcf0c08a9b87076c22.png"},{"id":36717047,"identity":"1dcceae4-3a35-4c40-a23c-31b7e87a83e5","added_by":"auto","created_at":"2023-05-08 21:07:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":696330,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the bone marrow hematopoietic cells by flow cytometry.\u0026nbsp;The LSK population was gated as (Lin\u003csup\u003e-\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003e/c-kit\u003csup\u003e+\u003c/sup\u003e) cells, the HSC population was gated as (LSK\u003csup\u003e-\u003c/sup\u003eCD48\u003csup\u003e-\u003c/sup\u003e/CD150\u003csup\u003e+\u003c/sup\u003e) cells, and the LT-HSC population was gated as (HSC\u003csup\u003e+\u003c/sup\u003eCD34\u003csup\u003e−\u003c/sup\u003e/SSC), the ST-HSC cell population was gated as (HSC\u003csup\u003e+\u003c/sup\u003eCD34\u003csup\u003e+\u003c/sup\u003e/SSC).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/54701d245bf824ad820ea620.png"},{"id":36717046,"identity":"397e6676-d967-493f-adbb-ab0c38b44f43","added_by":"auto","created_at":"2023-05-08 21:07:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":417158,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of bone marrow hematopoietic cells among groups.(A,B) Comparison of LSK cells between groups, and LSK increased after moxibustion compared with model group.(C,D) Comparison of HSC cells between groups, and HSC increased after moxibustion compared with model group.(E,F) Comparison of LT-HSC and ST-HSC cells between groups, and HSC increased after moxibustion compared with model group.\u003cem\u003e *P\u003c/em\u003e＜0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/483aa5048abed9f5a18e0f2d.png"},{"id":36717042,"identity":"303e2646-adab-4d26-ae22-301b160bc548","added_by":"auto","created_at":"2023-05-08 21:07:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":215194,"visible":true,"origin":"","legend":"\u003cp\u003eCell cycle analysis of hematopoietic stem cells. (A-C) Representing the cell cycle flow diagram of the control group, the model group and the moxibustion group. (D) Comparison of different cycles of hematopoietic stem cells in each group.\u003cem\u003e *P\u003c/em\u003e＜0.05.\u003c/p\u003e","description":"","filename":"FIgure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/a215d93d464c60d128137ec2.png"},{"id":36717041,"identity":"e7c0f080-60ab-420f-b3aa-2a1efb175a53","added_by":"auto","created_at":"2023-05-08 21:07:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":37142,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of CRH, ACTRH, CORT, GRFa and GRFβ in peripheral blood between groups.\u003cem\u003e *P\u003c/em\u003e＜0.05.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/d3a8e056a946bb29ded9050d.png"},{"id":36717405,"identity":"899169d2-86fc-413d-8726-1e9867722408","added_by":"auto","created_at":"2023-05-08 21:15:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":362838,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential gene expression analysis of peritoneal fluid cells in each group. (A) The overall range and distribution of FPKM values for gene expression across all samples are shown in Boxplot plots. (B) PCA analysis shows the differentiation among samples. (C)Pearson correlation coefficients of peritoneal fluid cells in each sample. Color scale represents Pearson correlation coefficients. (D) The up-regulated and down-regulated cross-differential genes of peritoneal fluid cells in each group. (E) Heatmap of the correlation of differential gene expression between each sample as shown in D).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/1ccbb33600e5a03bb97f3c90.png"},{"id":36717049,"identity":"6dcb9416-d055-450e-abce-88a557824329","added_by":"auto","created_at":"2023-05-08 21:07:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":265077,"visible":true,"origin":"","legend":"\u003cp\u003eGO functional enrichment analysis of DEGs. The size of the dots represents the number of enriched genes. The change in color reflects the size of the p-value.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/c8be10cfc1ad01fc13f40e6b.png"},{"id":36717403,"identity":"dc0507da-ca0b-4cd6-952e-481e11fcb6d9","added_by":"auto","created_at":"2023-05-08 21:15:26","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":247174,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG pathway enrichment analysis between MCM and model group. Bubble plots showing the top 20 significant pathways of DEGs. Larger bubbles illustrated higher number of genes. The color of each bubble represents the varied p-value.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/8a930dab34491a6bf1940741.png"},{"id":36717417,"identity":"6fd3634e-3cb6-41f6-9f58-8a4cf8222b1a","added_by":"auto","created_at":"2023-05-08 21:15:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2140627,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2902015/v1/bcb1ca59-5486-4d0a-80f7-7b1c201d0373.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eMoxa Cone Moxibustion at Zusanli(ST36) acupoint alleviate myelosuppression mouse induce by CTX and promote hematopoietic cell proliferation via HPA axis\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMyelosuppression is a common adverse effect of chemotherapy for cancer. Approximately 80% of patients with cancer experience myelosuppression during chemotherapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Additionally, the immune and hematopoietic systems are the most obviously affected ones, and the most prominent manifestation is leukopenia. Clinical symptoms, such as severe infection and bleeding often occur and directly affect the treatment of such patients [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, addressing myelosuppression after chemotherapy plays an important role in cancer treatment.\u003c/p\u003e \u003cp\u003eMoxibustion is an external treatment method of traditional Chinese medicine with a history of thousands of years in the prevention and treatment of diseases. Modern studies have confirmed that moxibustion can activate the hypothalamus-pituitary adrenal (HPA) axis and the sympathetic nervous system (SNS) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] via thermal, radiation, and pharmacological effects, thus further regulating the immune and neurological functions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSun [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] reported that moxibustion has anti-myelosuppression effects in chemotherapy. In a multicenter randomized study [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], ginger-separated moxibustion was found to be reliable and better than oral proprietary Chinese medicine in the treatment of leukopenia caused by chemotherapy with good repeatability. A meta-analysis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] found that moxibustion was more effective in increasing the white blood cell count than various types of control interventions. Although the level of evidence is low, it highlighted that moxibustion was superior to drug therapy in the treatment of chemotherapy-induced leukopenia. Studies have demonstrated that moxibustion could improve myelosuppression following chemotherapy [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. ST36 was one of the most commonly used acupoints in the treatment of myelosuppression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Some studies have found that activating the HPA axis could promote the proliferation of bone marrow hematopoietic cells and address the myelosuppression [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, a mouse model of myelosuppression established using cyclophosphamide (CTX) was used to observe the effects of moxa cone moxibustion (MCM) on peripheral blood cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], bone marrow hematopoietic cells, and the cell cycle as well as the effects on the key parts of the HPA axis and explore the underlying mechanism by analyzing the differences in the genetic expressions of bone marrow cells between the groups.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents\u003c/h2\u003e \u003cp\u003eThe main reagents involved in the experiments are as follows: Cyclophosphamide was purchased from Baxter oncology GmbH (H20160467, Germany). Fluorescently labeled monoclonal antibodies MO HEMATOPTC LIN CKIL FITC (#22-7770-22, ), ANTI-MO CD150 MSHAD150 PE-CYN7 (#25-1502-82), ANTI-MO CD48 HM48-1 APC (#17\u0026ndash;0481 -80), ANTI-MO CD34 RAM34 EF450 (#48-0341-80), ANTI-M LY-6A/E D7 PERCP-CYN5.5 (#45-5981-82), ANTI-MO CD117 2B8 PE(# 12-1171-81), Rat/ham ig Kpa Comp Bead set (#552845) and other flow-through antibodies were purchased from eBioscience (Thermo Fisher Scientific, USA). Elisa kits CRH (#MM-0509M2), ACTRH (#MM-0554M2), CORT (#MM-0061M2), GRFa (#MM-0296M2), GRFβ (#MM-0295M2) were purchased from Jiangsu MeiMian Industrial Co., Ltd. MolPure \u0026reg; Cell/Tissue Total RNA Kit (#19221ES50, YEASEN). moxa (35:1), purchased from Beijing Guoyiyan Medical Equipment Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animals and group\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (6\u0026ndash;8 weeks of age) were procured from the Vital River Laboratory Animal Technology (Beijing, China). They were housed in a room maintained at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 12-h light-dark cycle with free access of food and water. The experiments were approved by the ethics committee of Longhua District Central Hospital Affiliated to Guangdong Medical University (Shenzhen). The experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003cp\u003eAfter 1 week of acclimatization, mice were randomly divided into 3 groups with 5 mice in each group, namely control group, model group and moxibustion group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 CTX-induced Myelosuppression model established\u003c/h2\u003e \u003cp\u003eTo establish the model, 15 mg of cyclophosphamide (CTX) was completely dissolved in 75 mL of PBS for a concentration of 5 mg/mL. The mice in the model and moxibustion groups were administered CTX solution (100 mg/kg) intraperitoneally once a day for five consecutive days followed by 50 mg/kg every other day until the 12th day of modeling. The mice in the control group received the same dose of PBS intraperitoneally.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Moxibustion and Acupiont\u003c/h2\u003e \u003cp\u003eWe preferred the Zusanli (ST36) acupoints based on our previous work[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. They are located at the posterolateral aspect of the knee joints of the hindlimbs of the mice approximately 5 mm below the capitellum of the fibula. A moxa cone is made of moxa velvet with a diameter of 1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mm and a height of 2 mm. Mouse fur was shaved over a 1 \u0026times; 1cm\u003csup\u003e2\u003c/sup\u003e area around ST36 to expose the skin (Fig.\u0026nbsp;1). On the 6th day, moxibustion was started on the ST36 of the mice in the moxibustion group, and the prepared moxa cone was placed on the acupoint for moxibustion. It was lit with incense sticks, and the moxa cone was replaced with the next one when it went out naturally for a total of three times per acupoint daily and continuously for 7 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Weight observation\u003c/h2\u003e \u003cp\u003eFrom the start of modeling, the changes in the body weight of mice in each group were observed daily.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Detection of peripheral blood cells in mice\u003c/h2\u003e \u003cp\u003eIn EP tubes, 0.2 ml of blood was collected, and an automatic blood cell analyzer (Mindray veterinary automatic blood cell analyzer model: BC-2800vet) was used to analyze the following contents in the peripheral blood of mice in each group: white blood cell (WBC), lymphocyte (Lym), granulocyte (Gra), monocyte (Mon), red blood cell (RBC), hemoglobin (Hb), and platelets (PLT).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Mouse bone marrow hematopoietic cells and cell cycle using flow cytometry\u003c/h2\u003e \u003cp\u003eAfter the mice were euthanized with cervical dislocation, they were sterilized in 75% alcohol for 30 seconds. Subsequently, bilateral femur and tibia were removed, the closed ends on both sides of the bones were cut, and the bone marrow cells were flushed into a centrifuge tube. After filtration through a cell sieve, the samples were centrifuged at 1600 rpm and 4\u0026deg;C for 5 min, and the supernatant was discarded. The RBC lysate was added to lyse red blood cells at 37\u0026deg;C for 10 min, centrifuged at 1600 rpm for 10 min at 4\u0026deg;C, and the supernatant was discarded. Subsequently, 1 mL PBS containing 3% fetal bovine serum (PBS-F) was added to the tube to resuspend the bone marrow cells. Finally, antibodies (Lin, sca-1, c-kit, CD48, CD150, and CD34) were added to the samples for 30 min, and the cells were mixed using gentle shaking. In brief, bone marrow hematopoietic cells were fixed with cold 70% ethanol and stained with a PBS solution containing 50 lg/mL PI and 30 lg/mL RNase A. The prepared samples were analyzed using flow cytometry (BECKMA, Cytoflex LX) and FlowJo_V10 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Enzyme-linked immunosorbent assay(ELISA)\u003c/h2\u003e \u003cp\u003eFor ELISA, 0.5 mL of blood was drawn from the retro-orbital space and collected in 1.5-mL EP tubes. It was allowed to stand at room temperature for 2 hours, and transferred to a refrigerator at 4\u0026deg;C for overnight storage. The serum was separated using centrifugation at 3000 rpm at 4 ℃ and transferred to a new centrifuge tube. The serum concentrations of corticotropin-releasing hormone (CRH), corticotropin (ACTH), corticosterone (CORT), and glucocorticoid receptor α and β (GRF-α, GRF-β) were estimated using ELISA. The procedures were performed according to the instructions of the manufacturers and analyzed using an enzyme labeling instrument (RaytoRT-6100).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 RNA extraction and sequencing\u003c/h2\u003e \u003cp\u003eTwo bone marrow samples were selected from each group, and RNA was extracted from the bone marrow cell suspension in strict accordance with the operating steps of the RNA Kit. Subsequently, RNA was quantified and identified using Agilent 2100 Bioanalyzer (Thermo Fisher Scientific, MA, USA). The extracted RNA was reverse-transcribed into the corresponding DNA fragments, and the exon region probes were used to hybridize and capture after polymerase chain reaction (PCR) amplification. The captured DNA was subsequently amplified using PCR and circularized to get single-stranded circular (ssCir) library. The ssCir library was then amplified using rolling circle amplification (RCA) to obtain DNA nanoball (DNB). The DNB was then loaded to a flow cell and sequenced using BGISEQ Platform (BGI, Shenzhen, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Differentially expressed genes (DEGs) analysis\u003c/h2\u003e \u003cp\u003eSequencing data (raw data) was filtered using SOAPnuke (v1.5.2) and quality control (QC) was performed using FASTQ [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Subsequently, the obtained clean reads were mapped to the Mouse C57 BL/6 standard genome (GCF_000001635.27_GRCm39_genomic.fna) using Bowtie2 (v2.2.5) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The expression levels of the genes were then calculated using RSEM (v1.2.8) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Finally, DEG analysis was performed using DESeq2 (v1.4.5) with a Q value less than 0.05 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Functional enrichment analysis of differentially expressed genes\u003c/h2\u003e \u003cp\u003eThe cluster Profiler function of the R package was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to better understand the function and pathway of MCM regulating DEGs in bone marrow hematopoietic cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were analyzed with SPSS v23.0 (IBM Corp., Armonk, NY, USA). Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation is used to express the data with normal distribution and homogeneity of variance. The differences between the groups were analyzed using one-way analysis of variance (ANOVA), and pairwise comparisons were conducted using the Bonferroni method. The ratio data and data of skewed distribution or variances were analyzed using non-parametric tests, and the median (P25-P75) was used with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. All results were plotted using GraphPad Prism 8.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 MCM treatment results in improvements to weight loss\u003c/h2\u003e \u003cp\u003eThe weight of the mice in the control group gradually increased every day and the weights of mice in the model and moxibustion groups decreased from the second day of CTX. By the 12th day, the body weights of the mice in both groups were lower than that in the control group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) (Fig.\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Changes in peripheral blood cell content\u003c/h2\u003e \u003cp\u003eAt the end of the experiment, compared with the control group, the values of WBC, Lym, RBC, Hb, and PLT in the model group decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and that of Gra in the moxibustion group increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with the model group, the values of WBC, Gra, RBC, Hb, and PLT in the moxibustion group increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and that of Lym increased; however, there was no statistically significant difference (Fig.\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Changes in bone marrow hematopoietic cells and cell cycle\u003c/h2\u003e \u003cp\u003eCompared with the control group, LSK (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003ec-kit\u003csup\u003e+\u003c/sup\u003e), HSC (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003ec-kit\u003csup\u003e+\u003c/sup\u003eCD48\u003csup\u003e\u0026minus;\u003c/sup\u003eCD150\u003csup\u003e+\u003c/sup\u003e), short-term hematopoietic stem cell (ST-HSC), and long-term hematopoietic stem cell (LT-HSC) in the model group were decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); ST-HSC and LT-HSC in the moxibustion group were also decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with the model group, LSK and HSC of the mice in the moxibustion group increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;4 and 5).\u003c/p\u003e \u003cp\u003eAfter calculating the cell cycle subsets using the cell cycle module of FlowJo software, we found that after CTX modeling, the S phase of the hematopoietic stem cell cycle increased, while the G1 phase decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Following moxibustion treatment, the S and G2 phases were significantly decreased and the G1 phase increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Changes in CRH, ACTRH, CORT, GRF-a, and GRFβ in peripheral blood\u003c/h2\u003e \u003cp\u003eAfter the experiment, compared with the control group, the serum levels of CRH, ACTRH, and CORT in the peripheral blood in the model group were increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the levels of GRF-a and GRF-β had decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The serum levels of CRH, ACTRH, and CORT in the peripheral blood of mice in the group were decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the contents of GRF-a and GRF-β had increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;7).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Gene expression analysis\u003c/h2\u003e \u003cp\u003eRNA sequencing of each sample was assessed using the BGISEQ platform. Subsequently, feature Counts v1.5.0-p3 was used to calculate the expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) of each sample, which represent the relative gene expression abundance. The boxplots revealed that the distribution of FPKM expression was roughly consistent across each sample, thus indicating that the RNA-seq data were reproducible and reliable (Fig.\u0026nbsp;8a). The principal component analysis (PCA)(Fig.\u0026nbsp;8b) and Pearson correlation heatmap visualization analysis (Fig.\u0026nbsp;8c) revealed that it can be found that the sample differences between the three groups became obvious.\u003c/p\u003e \u003cp\u003eTo further identify significant DEGs between the control, model, and moxibustion groups, DEGs were analyzed using the DESeq2 R package. |log2FC|\u0026gt;1 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered criteria for searching for significant DEGs. Comparing the model and the control group, a total of 2977 DEGs were identified, including 1500 up-regulated genes and 1477 down-regulated genes. Comparing the model and moxibustion group, a total of 1137 DEGs were identified, including 293 up-regulated genes and 844 down-regulated genes. Among them, 193 genes were down-regulated in the model group and up-regulated in the moxibustion group; 481 genes were up-regulated in the model group and down-regulated in the moxibustion group (Fig.\u0026nbsp;8d). The 674 cross-DEGs were analyzed in the next step.\u003c/p\u003e \u003cp\u003eHeatmap and hierarchical cluster analysis revealed that intragroup data were clustered together, and intergroup data were separated. The results revealed that moxibustion could restore the down-regulated gene levels in the myelosuppression group. These findings further suggest that the regulation of bone marrow hematopoietic cells by MCM in the CTX myelosuppression model can be accomplished by regulating the expression of related genes (Fig.\u0026nbsp;8e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Go function and KEGG pathway enrichment analysis of DEGs\u003c/h2\u003e \u003cp\u003eGO functional enrichment analysis (including biological process [BP], cellular component [CC], and molecular function [MF]) was performed with 674 DEGs to predict the underlying biological function of DEGs between the MCM and model groups. The DEGs were enriched to 857 subclasses of GOs (742 BPs were enriched, 54 CCs were enriched, and 61 MFs were enriched). The top three enriched BPs were T-cell activation, T-cell differentiation, and lymphocyte differentiation; the top three CCs were cell-cell junction, membrane raft, and membrane microdomain; and the top three MFs included cytokine binding, cytokine receptor activity, and cytokine activity (Fig.\u0026nbsp;9). KEGG pathway analysis was conducted to analyze the DEGs between the MCM and model groups. The top KEGG pathways with the most enrichment were the hematopoietic cell lineage, primary immunodeficiency, and cytokine-cytokine receptor interaction. The top 20 enrichment pathways are shown in Fig.\u0026nbsp;10.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe results of the current study mainly illustrate that MCM can alleviate the degree of myelosuppression induced by chemotherapy in a mouse model. The potential underlying mechanism may be that MCM upregulates the expression of GFR, restores the negative feedback regulation mechanism of the HPA axis, and promotes normalization of bone marrow hematopoiesis. Additionally, MCM can modify the gene expression in the CTX model, thereby promoting the proliferation of hematopoietic cells.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Stability of CTX model\u003c/h2\u003e \u003cp\u003eAs a commonly used alkylating agent in chemotherapy [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], CTX is used in establishing bone marrow suppression models mainly because of its stable effects, easy availability, and low cost. The mechanism of bone marrow suppression caused by it mainly includes destruction of the DNA of bone marrow hematopoietic stem cells, which results in irregular necrosis and apoptosis of hematopoietic cells and decreased activity of precursor cells and bone marrow suppression [\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. CTX-induced apoptosis in mouse bone marrow was one of the typical immunosuppression [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Some scholars [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] have investigated the doses and effects of cyclophosphamide modeling in recent years, and found that the number of bone marrow nucleated cells in mice treated with a total dose of 100\u0026ndash;500 mg/kg of cyclophosphamide will rebound on the 5th\u0026ndash;10th day. Subsequently, the number of white blood cells in the peripheral blood is also temporarily higher than the normal levels. However, after the modeling has stopped, the peripheral blood in the model group could gradually return to normal. Therefore, this experiment confirmed that 100 mg/kg of cyclophosphamide combined with 50 mg/kg maintenance dose every other day could well maintain the stability of the model. No recovery of peripheral blood cells and bone marrow hematopoietic cells was observed on the 7th day after the model was established.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.2 MCM and hematopoietic stem cells\u003c/h2\u003e \u003cp\u003eRecent studies have demonstrated that moxibustion has a positive effect on protecting the blood, enhancing bone marrow hematopoiesis, and promoting cell cycle transformation. Similar results were noted in this experiment; moxa cone can promote the transformation of bone marrow hematopoietic cells from the G2 phase to M phase and promote the proliferation of ST-HSC, thereby promoting the mobilization of bone marrow hematopoietic cells and their migration to increase the peripheral blood leukocytes, RBC, Hb, and PLT. However, the currently available evidence of the mechanism of moxibustion in the treatment of myelosuppression following chemotherapy mainly focused on the following three aspects: (1) enhanced DNA repair and cell cycle transformation of bone marrow cells, which helps the proliferation and differentiation of hematopoietic cells[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]; (2) Regulation of bone marrow cell signaling pathways[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and (3) improving the damaged hematopoietic microenvironment mainly by promoting the release of cytokines on the stromal cell membrane and the repair of microvascular damage[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, these studies mainly focused on the molecular mechanisms of expression of related proteins in bone marrow cells. However, whether they were related to the regulation of other biologically active substances remains unclear.\u003c/p\u003e \u003cp\u003eThe HPA axis is an important part of the neuro-endocrine-immune network and mainly involved in regulating the body\u0026rsquo;s stress response. It is a complex system of direct and feedback interactions involving the hypothalamus, pituitary, and adrenal glands[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. CRH in the hypothalamus serves as the starting point for the regulation of the HPA axis activity and is a key driving force of the axis that plays an important role in stress responses[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. CRH from the hypothalamus stimulates the pituitary to release ACTH, which stimulates the adrenal gland to secrete glucocorticoids (GC). Under physiological conditions, excessively elevated GC inhibit the synthesis of ACTH and CRH through negative feedback regulation, thus maintaining a relatively stable and moderate stress response of GC. The negative feedback regulation of GC is mediated by the glucocorticoid receptor (GR) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. After binding to the ligands, GR enters the nucleus and specifically binds to the negative glucocorticoid response element (nGRE) in the promoter region of the CRH gene, thus inhibiting the gene expression of CRH in the hypothalamus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. If the negative feedback regulation mechanism of the HPA axis is damaged, it will eventually lead to hypersensitivity of the HPA axis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have demonstrated that neuroendocrine responses regulate the production of GC through the HPA axis under stress conditions (trauma, burn, pressure, and hemorrhagic shock), which plays a positive role in the regulation of bone marrow hematopoietic cells [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Kwan [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] found that the central nervous system regulates GR through the HPA axis in stress response, which plays a key role in the production of hematopoietic stem and progenitor cells (HSPC). GR agonists can enhance the formation of HSPC, while a lack of GR reduces the formation of HSPC. Long-term activation of GR plays a key role in the self-replication and proliferation of erythroid progenitor cells. Gao [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] also found that the activation of HPA axis promotes the release of glucocorticoids, which mainly regulated the directional migration of MSCs and EPCs through GR and promoted the mobilization of bone marrow stem cells. People with GC dysfunction have hematopoietic deficiency [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, Pierce [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] suggested that GC was the key to the mobilization of HSC in the HPA axis, and the physiological level of cortisol promoted HSC migration through the GC receptor NR3C1. Too high or too low cortisol levels have a negative effect on the proliferation of HSC. It is worth noting that in this study, the serum levels of CRH, ACTH, and CORT in the model group were significantly higher than those in the control group and moxibustion group, while the GR level decreased significantly. This observation indicates that cyclophosphamide damaged the negative feedback regulation mechanism of the HPA axis and resulted in hyperfunction. Additionally, the hyperphysiological level of glucocorticoids did not play a positive role in the proliferation of bone marrow cells. However, after moxa moxibustion, the levels of GR increased, which restored the negative feedback regulation mechanism of the HPA axis and promoted the normalization of bone marrow hematopoiesis mechanisms. It was suggested that GR is still the key link in the regulation of bone marrow hematopoietic cells by the HPA axis.\u003c/p\u003e \u003cp\u003eIn order to determine whether MCM can promote the proliferation and differentiation of the hematopoietic cells at the genetic level, we analyzed 674 cross-DEGs between the model group and the control group as well as the moxibustion group and the model group using RNA-seq.\u0026nbsp;The results demonstrated that MCM could significantly alter gene expression in the CTX model and consequently regulate the CTX-induced myelosuppression and promote the proliferation of hematopoietic cells. GO enrichment analysis revealed that the DEGs between moxibustion group and model group were related to the activities of cytokines, their receptors and ligands, and the proliferation and differentiation of nucleated cells (leukocytes, lymphocytes, and T-cells). These results are consistent with those of previous studies [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, MCM could promote the proliferation and differentiation of bone marrow hematopoietic cells by regulating gene expression. The most significant enrichment pathways in KEGG were the hematopoietic cell lineage, primary immunodeficiency, cytokine-cytokine receptor interaction, Th1 and Th2 cell differentiation, and Th17 cell differentiation. These pathways were more closely related to the proliferation and differentiation of hematopoietic and immune cells [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Hematopoietic cell lineage is a network of hierarchical differentiation of hematopoietic cells, which is very complex and huge with strong continuity, plasticity, and self-renewal ability [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In recent years, with the development of single-cell sequencing, the traditional hematopoiesis system has been continuously supplemented and improved [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The analysis of these KEGG pathways in this project could only reflect that MCM adjusted these networks. However, its complete role remains unclear and requires further investigations.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":" \u003cp\u003eMCM could promote the proliferation and differentiation of hematopoietic cells following CTX-induced myelosuppression by regulating the expression of genes, thus increasing the levels of peripheral blood cells. MCM is a non-drug therapy that acts on the body\u0026rsquo;s surface and plays a therapeutic role through stimuli, such as temperature and pain. The mechanism of its effects might be related to the negative feedback regulation of the HPA axis. However, due to the complexity of the hematopoietic system and the mechanism of action of MCM, we could not explore the deeper mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHongda Chen and Ning Wang are the co-corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-sequence data had been published in NCBI (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA859387). The data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the support of the National Natural Science Foundation of China (No. 81804171).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFan K, Dai LM, Feng WZ, Yang GY, Yang M, Pharmacy CO. Advances in chemotherapy-induced myelosuppression. China Journal of Traditional Chinese Medicine and Pharmacy. 2017;1(32):210-4. \u003c/li\u003e\n\u003cli\u003eAhlmann M, Hempel G. The effect of cyclophosphamide on the immune system: implications for clinical cancer therapy. 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Current opinion in hematology. 2016;23(4):311-7. doi:10.1097/moh.0000000000000260.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"ca031ac4-809a-42d0-9ace-87db67dc9ee1","identifier":"10.13039/501100001809","name":"National Natural Science Foundation of China","awardNumber":"No. 81804171","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Shenzhen Longhua District Central Hospital","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Moxibustion, Myelosuppression, HPA axis","lastPublishedDoi":"10.21203/rs.3.rs-2902015/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2902015/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eObjective.\u003c/strong\u003e\u003c/em\u003e Clinical studies have reported that moxibustion alleviates the side effects of chemotherapy, such as myelosuppression; however, its therapeutic mechanisms remain unclear. We investigated whether moxa cone moxibustion (MCM) promotes recovery from chemotherapy-induced myelosuppression via the hypothalamus-pituitary adrenal (HPA) axis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eA myelosuppression model was established in mice using cyclophosphamide (CTX), and they were grouped into control, model, and moxibustion groups. In the moxibustion group, the mice received MCM at ST36 for 7 days. The peripheral blood cells were detected using an automatic blood cell analyzer; serum levels of corticotropin-releasing hormone (CRH), corticotropin (ACTH), corticosterone (CORT) and glucocorticoid receptor (GRF) a/β were detected using enzyme-linked immunosorbent assay (ELISA); the expression and cell cycle of bone marrow hematopoietic cells were detected using flow cytometry (FC); and BGISEQ Instrument model-DNBseqTM Platform was used for RNA sequencing. The differentially expressed genes (DEGs) were subjected to gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults. \u003c/strong\u003e\u003c/em\u003eCompared with the control group, the contents of the peripheral blood and LSK and HSC in the moxibustion group were significantly higher (P\u0026lt;0.05). Following moxibustion therapy, the S and G2 phases of bone marrow hematopoietic stem cells decreased significantly, while the G1 phase increased. The CRH, ACTRH, and CORT levels decreased (P\u0026lt;0.05), while GRF-a and GRFβ levels increased (P\u0026lt;0.05). Additionally, 193 DEGs were down-regulated in the control group and up-regulated in the moxibustion group, while 481 DEGs were up-regulated in the control group and down-regulated in the moxibustion group. GO analysis revealed that cross-DEGs were enriched in cell-cell junction, membrane raft, membrane microdomain, and T-cell receptor complex involved in T-cell activation, T-cell differentiation, and lymphocyte differentiation, which exerted the MF of cytokine binding, receptor activity, and activity. KEGG analysis revealed that hematopoietic cell lineage, primary immunodeficiency, cytokine-cytokine receptor interactions, and other pathways were significantly enriched in the moxibustion group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusion.\u003c/strong\u003e\u003c/em\u003e MCM could alleviate myelosuppression induced by CTX and promote hematopoietic cell proliferation via the HPA axis and promote the proliferation and differentiation of bone marrow hematopoietic cells by regulating gene expression.\u003c/p\u003e","manuscriptTitle":"Moxa Cone Moxibustion at Zusanli(ST36) acupoint alleviate myelosuppression mouse induce by CTX and promote hematopoietic cell proliferation via HPA axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-08 21:07:21","doi":"10.21203/rs.3.rs-2902015/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"87649b2a-1fe4-4549-ba1f-6dd40dca9110","owner":[],"postedDate":"May 8th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-05-08T21:07:22+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-08 21:07:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2902015","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2902015","identity":"rs-2902015","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}