Effect And Mechanism Of Direct Moxibustion At Zusanli On Myelosuppression Mice

ObjectiveBone marrow suppression is a common and predictable adverse reaction in cancer chemotherapy.Leukopenia,red blood cells and thrombocytopenia lead to infection,fatigue,bleeding and other clinical manifestations associated with bone marrow suppression are often the main reasons affecting the follow-up anti-tumor treatment.Therefore,improving bone marrow suppression after chemotherapy plays a positive role in tumor treatment.In this study,cyclophosphamide was used to induce bone marrow suppression in mice,and direct moxibustion at Zusanli(ST 36)was used for therapeutic intervention.To investigate the therapeutic effect of direct moxibustion and explore its possible mechanism,the general state of mice,changes of body weight and organ index,blood routine test,statistics of total number of nucleated cells in bone marrow,tissue section staining,expression of peripheral serum related hematopoietic factors,and detection of cell cycle and hematopoietic cell content in bone marrow cells were used.Methods1.Construction and treatment of mouse myelosuppression modelMethods:Eighteen healthy male C57/BL mice with SPF grade of 10-12 weeks were randomly divided into three groups:Vehicle Group,model group and moxibustion groupCytoxan(CTX)was prepared into a solution of 5 mg/ml with PBS.Mice in the model group and moxibustion group were injected intraperitoneally at a dose of 40 mg/(kg·d),once a day for 5 consecutive days.Zusanli point:it is located in the muscle groove 0.3cm below the head of fibula under the knee joint of mice,with one point on each side.After shaving the mouse hair in the acupoint area to expose the skin,place the prepared moxa stick on the acupoint in advance,and then ignite it with thread incense.When moxa stick burns to the mouse struggling,use tweezers to quickly replace moxa stick.From Zusanli on the left to Zusanli on the right,each point is 3 strong/time,once a day,a total of 10 days.2.Detection of body weight and organ index of miceFrom the first day of modeling and treatment to the end of 10 days of treatment,the weight of mice in each group was detected at the same time every day.The thymus index,spleen index,liver index and kidney index were calculated.3.Detection of peripheral blood cells in miceThe blood from the posterior orbital venous plexus of mice was collected and put into the EDTA anticoagulant tube.The number of white blood cells,platelets,red blood cells and hemoglobin of mice in each group was detected and counted by small animal automatic hematology analyzer.4.The number of nucleated cells in mouse bone marrowAfter disinfection with alcohol,the two ends of the right femur of mice were cut open,pbsf was extracted by syringe,bone marrow cells were flushed until the bone marrow cavity changed from red to white,and then bone marrow single cell suspension was made by repeated and slow aspiration with No.4 injection needle.After centrifugation,the red blood cells were lysed,and the bone marrow cells were washed and detected by cell counter.5.Detection of cell cycle of mouse bone marrow stromal cellsAfter centrifugation,red blood cell lysis and PBS suspension,the cells were fixed with 70%ethanol pre cooled by ice bath.After centrifugation and PBS suspension,propidium iodide staining solution was added into each flow cytometer tube to take a 37℃ dark bath.The red fluorescence was detected by flow cytometry at the excitation wavelength of 488 nm.6.Histomorphological observation of miceThe bone marrow,spleen and kidney were fixed,dehydrated and sectioned for 30 μm roughing 4μm.The samples were sliced at 65℃ for 2 hours.After dewaxing and rewatering,the pathological sections were stained with hematoxylin staining solution,and the differentiation solution was stained with eosin staining solution.After washing,the sections were dehydrated,permeabilized and sealed.7.Detection of serum hematopoietic factors in miceThe whole blood of mice was naturally coagulated at room temperature for 10-20 minutes.The supernatant was collected by centrifugation.Enzyme labeled reagent was added into the standard well and sample well,and the plate was sealed with membrane.Wash with detergent for 6 times and pat dry.After the reaction was terminated,the absorbance of each pore was measured.8.Detection of bone marrow nucleated cells hematopoietic stem cells and hematopoietic progenitor cells in miceThe bone marrow single cell suspension was blown and centrifuged.After lysed red blood cells and pbsf were resuspended,the antibody was added and incubated in dark.The supernatant was centrifuged.4%paraformaldehyde was added while shaking,and the indicators were detected by flow cytometry.Results1.Direct moxibustion at Zusanli can effectively increase the weight and spleen index of myelosuppression mice.2.Direct moxibustion at Zusanli can increase the contents of peripheral blood leukocytes,platelets,red blood cells and hemoglobin,and increase the number of nucleated cells in bone marrow.3.Direct moxibustion at Zusanli can improve the histomorphological structure of bone marrow,spleen and kidney in myelosuppression mice,increase the area of bone marrow hematopoietic tissue,increase the number of megakaryocytes,and widen the renal capsule space.4.Direct moxibustion at Zusanli(ST36)can down regulate the contents of EPO,TPO and GM-CSF in serum of myelosuppression mice,and improve the hematopoietic microenvironment.5.Direct moxibustion at Zusanli can promote the transformation of bone marrow cells from G1 phase to S phase and S phase to G2 phase,and promote the proliferation of bone marrow cells.6.Direct moxibustion at Zusanli can increase the content of HSCs and HPCS in bone marrow cells,increase the mobilization of bone marrow stem cells,and improve the damage of hematopoietic system in myelosuppression mice.ConclusionDirect moxibustion at Zusanli(ST 36)can effectively alleviate cytoxan induced hematopoiesis injury.The mechanism may be to increase the number of nucleated cells in bone marrow cells;Regulate the cell cycle of bone marrow cells and promote the proliferation and differentiation of cells;Improve the hematopoietic microenvironment of bone marrow and promote the expression of hematopoietic growth factor;It improves the activity of some HSCs and HPCS,improves the hematopoietic function of bone marrow cells,and then participates in the process of active mobilization of hematopoietic system.