| Objective:The current study evaluated the clinical efficacy of Moxibustion in the prevention and treatment of neutropenia after chemotherapy in patients with malignant tumors through retrospective clinical research and meta-analysis.The effect of moxibustion on bone marrow suppression of peripheral blood neutrophils in tumor bearing mice was clarified through experimental research;To investigate the effect of moxibustion on bone marrow suppression of bone marrow granulocyte lineage development in tumor bearing mice;The differentially expressed genes related to moxibustion intervention were screened based on eukaryotic transcriptome sequencing,and the molecular mechanism of moxibustion promoting neutrophil maturation was explored.Methods:1.Clinical study:According to the inclusion and exclusion criteria,the retrospective clinical study continuously collected the malignant tumor patients hospitalized with chemotherapy,extracted the clinical data and test results,statistically analyzed the blood routine results before the last chemotherapy and before and after moxibustion treatment,and evaluated the clinical efficacy of Moxibustion in the treatment of neutropenia.Meta analysis searched the randomized controlled clinical trial of moxibustion for the prevention and treatment of neutropenia in patients with malignant tumors.As of February 28,2022,the data were extracted by two independent researchers and evaluated by evidence-based methodology.The data results were meta-analyzed by Stata 14.0.The effect quantity was counted by relative risk(RR)for binary variable data,and the effect quantity was counted by mean difference(MD)for continuous variable data,95%confidence interval was used for interval estimation.Revman 5.3 was used to evaluate the quality of literature.2.Experimental study:A 4T1 breast cancer mouse model of bone marrow suppression was established.The first experiment was divided into tumor bearing group,model group,Ji Lifen group and moxibustion group.The tumor bearing group was observed without intervention;The model group was accompanied by grasping and fixed observation without intervention;Ji Lifen group received intraperitoneal injection of Ji Lifen 11.25 for 3 days μg/kg,once a day;In the moxibustion group,the points were Qihai,Guanyuan and ZuSanli.Each point was treated with moxibustion for 5 minutes,once a day,and the materials were taken after 4 days of intervention;Blood routine and blood smear were detected before,after and 4 days after intervention to dynamically observe the changes of blood cell count and blood cell morphology.The test samples of Experiment 2 were from Experiment 1.Through he,organ coefficient,flow cytometry and immunohistochemistry,the hematopoietic structure of bone marrow,organ coefficient,the proportion of cells in each stage of myeloid differentiation to granulocyte lineage and the expression of CXCR2/CXCR4 protein were observed.Experiment 3 was divided into model group and moxibustion group.The intervention method was the same as experiment 1.Neutrophils were extracted after taking materials.A total of 12 samples(6 in each group)were used for eukaryotic transcriptome sequencing.Two groups of differentially expressed genes were screened through biological information analysis and verified at mRNA level and protein level.Results:Clinical study:A total of 58 patients were included in the retrospective clinical study.The results showed that the counts of leukocytes and neutrophils increased after moxibustion and the difference was statistically significant,indicating that moxibustion can increase the number of leukocytes and neutrophils in patients with malignant tumors.15 studies were included in the meta-analysis.7,13 and 5 studies reported the neutrophil and leukocyte counts of 7 days,10-14 days and 21-30 days of treatment.It was suggested that the neutrophil and leukocyte count of the experimental group were higher than those of the control group,and the difference was statistically significant,indicating that moxibustion can increase the neutrophil and leukocyte counts of patients with bone marrow suppression.Nine studies reported KPS score results.The experimental group was higher than the control group,and the difference was statistically significant,indicating that moxibustion can improve the quality of life of patients.Due to the low overall quality and high heterogeneity of the included studies,Begg’s and egger’s tests suggest that there may be a risk of publication bias.Experimental study:1.Effect of moxibustion on peripheral blood of bone marrow suppression mice bearing tumor:1)General conditions:Before the establishment of bone marrow suppression model,mice in each group had vivid eyes,active spirit,quick movement,strong jumping,rapid response to external stimuli,smooth hair,bright and shiny hair color,normal diet and drinking water,consistent baseline body weight,clear and pale yellow urine color,and normal stool formation.After modeling,the mice in the tumor bearing blank group had little change,while the mice in the other three groups were reduced in dietary water,weight loss,poor mental state,dry hair,abnormal bowel and stool,and the above states were improved after moxibustion.2)Before modeling,blood cell baseline levels of the four groups were consistent;After chemotherapy modeling,white blood cells and neutrophils decreased by more than 50%,while red blood cells and platelets decreased by less than 50%in the model group,Ji Lifen group and moxibustion group.After 4 days of moxibustion treatment,compared with the model group,white blood cell and neutrophil count increased in the Ji Lifen group and the moxibustion group,and the difference was statistically significant.Erythrocyte count decreased slightly,platelet count decreased significantly,but no significant difference.3)The morphology of peripheral blood cells in each group was observed under oil microscope.The proportion of lymphocytes in tumor-bearing mice was lower than that of neutrophils,and lobulated neutrophils were higher than rod-shaped neutrophils.After modeling and intervention,the proportion of neutrophil increased and lymphocyte decreased in tumor-bearing mice.After modeling,the proportion of lobulated neutrophils increased,the proportion of rod-shaped nuclear cells decreased,and the proportion of lymphocytes decreased in model group,Ji Lifen group and moxibustion group.After 4 days of treatment,the proportion of neutrophils in the leaf nucleus of the model group decreased,while the proportion of neutrophils in the rod-shaped nucleus increased.Compared with the model group,the proportion of the leaf nucleus in the Ji Lifen group and the moxibustion group increased,while the proportion of the rod-shaped nucleus decreased,and the differences were statistically significant.The proportion of lymphocytes decreased,and there was no significant difference among the three groups.2.Effect of moxibustion on the development of granulocyte lineage in bone marrow suppression mice bearing tumor:1)HE staining:In the tumor-bearing group,the hematopoietic structure of the bone marrow of mice was intact,the cells were evenly arranged,and there were a great number of inflammatory cells.In the model group,the hematopoietic structure of bone marrow was destroyed,adipocytes increased,the morphology of bone marrow cells was abnormal,the distribution and arrangement of various lines of cells were uneven,and the cell density was reduced,mainly the number of nucleated cells was reduced,the cell space was widened,and the phenomenon of nuclear pyknosis appeared.The hematopoietic structure of bone marrow of mice in the Ji Lifen group and the moxibustion group was generally normal,the edge of blood sinusoid was regular,no obvious adipocytes were observed,the cell morphology of each line was normal,the number of nucleated cells increased,and no nuclear pyknosis was observed.2)Organ coefficient:Spleen coefficient in tumor bearing group was significantly higher than that in the other three groups,and the difference was statistically significant;Compared with model group,spleen coefficient of moxibustion group and Ji Lifen group decreased,but there was no significant difference.The thymus coefficient of the tumor-bearing group was higher than that of the model group,the Ji Lifen group and the moxibustion group,and the difference was statistically significant.Compared with the model group,the thymus coefficient of moxibustion group and Ji Lifen group increased,but there was no significant difference.3)There was no significant difference in the proportion of hematopoietic stem cells,hematopoietic progenitor cells,common myeloid progenitor cells and immature neutrophils between the moxibustion group and the model group.The proportion of neutrophil progenitor in moxibustion group was lower than that in model group,and the proportion of mature neutrophil was higher than that in model group,with statistical significance.Mature neutrophils can be divided into two large subpopulations,which can be further divided into seven small subpopulations.4)Compared with the model group,the expression level of CXCR2 protein in the moxibustion group increased,while the expression level of CXCR4 protein decreased,and the difference was statistically significant.3.Eukaryotic reference transcriptome sequencing to explore the mechanism of moxibustion promoting neutrophil maturation:Compared with the model group,a total of 270 differentially expressed genes were screened out in the moxibustion group,including 92 up-regulated genes and 178 down-regulated genes.GO enrichment analysis indicated that 105 differentially expressed genes were enriched in the items of "positive regulation of biological functions".Among them,Cebpβ,GATA1,Irf8 and Spib can regulate the development of neutrophils.KEGG enrichment analysis showed that Cebpβ was significantly enriched in TNF signaling Pathway,Transcriptional MIS-regulation in cancer and IL-17 signaling pathway.By predicting transcription factors,Cebpβ,GATA1,Irf8 and Spib were found to belong to TF-bZIP,zf-GATA,IRF and ETS transcription factor families,respectively.PPI protein interaction network analysis showed that Cebpβ,GATA1,Irf8 and Spib interacted with each other.Rt-PCR and immunohistochemistry were used to verify mRNA level and protein level,respectively.This study suggests that moxibustion improves bone marrow suppression and promotes neutrophil maturation by regulating transcription factors Cebpβ,GATA1,Irf8 and Spib.Conclusion:1.Moxibustion Qihai,Guanyuan and ZuSanli can improve bone marrow suppression after chemotherapy in malignant tumors,and promote the recovery of bone marrow hematopoietic structure and reconstruction of hematopoietic function.2.Moxibustion Qihai,Guanyuan and ZuSanli can promote the development and differentiation of bone marrow hematopoietic cells into granulocyte lineages,promote the development and maturity of neutrophils,and regulate the circulating release of neutrophils into peripheral blood by up-regulating the expression of CXCR2 protein and down-regulating the expression of CXCR4 protein.3.Neutrophils are not a homogenous group of cells.Mature neutrophils have a relatively complex structure with certain heterogeneity.4.Differentially expressed genes were screened by eukaryotic reference transcriptome sequencing,and GO and KEGG functional enrichment analysis was performed to identify the four transcription factors involved in neutrophil regulation.Moxibustion may promote neutrophil maturation by up-regulating Cebpβ expression and down-regulating GATA1,Irf8 and Spib expression. |
PDF Full Text Request