Panax Notoginseng Saponins Regulate The Hematopoietic Stem Cells Differentiation In 4T1 Breast Cancer-Bearing Mouse

Objective Breast cancer is one of the most common malignancies in women.Hematopoietic stem cells have infinite proliferation and differentiation potential.It can differentiate into erythroid progenitor cells,granulocyte-monocyte progenitor cells,lymphocyte lines and megakaryocytic lines.Hematopoietic stem cell transplantation is an effective method for the treatment of hematological malignancies,immune system diseases and solid tumors.Late tumor can invade the bone marrow and spleen,affecting the differentiation of hematopoietic stem cells.Lymphocyte cell,granulocyte-monocyte cell line is an important inflammatory mediator cells that play an important role in regulating the immune system.Erythroid progenitor cells can differentiate into red blood cells,early red blood cells,reticulocytes,mature red blood cells,etc.Erythroid progenitor cells play an irreplaceable role in regulating hematopoietic function in vivo.The preliminary study found that the occurrence and development of tumor induced hematopoietic stem cell differentiation abnormalities.Eventually leading to tumor-bearing mice with thrombosis and acute anemia.Sanqi can improve hematopoietic cell differentiation abnormalities.Therefore,the effect of different saponins and their aglycones on the differentiation of tumor-associated hematopoietic stem cells was studied.Methods Section One:Forty-eight healthy BALB/c mice were selected and divided into 8 groups:control group,saponins group,panax notogysaccharide saponin group,panax notlaponin group,cyclophosphamide and recombinant human erythropoietin group.In addition to the normal group,in the mouse mammary fat pad injection of 1 million 4T1-Luc cell suspension to preparation of mouse 4T1 breast cancer tumor model.Respectively:MODE(0.9%physiological saline 0.2ml/only)、PNS(20mg/kg)、PDS(20mg/kg)、PTS(20mg/kg)、CTX(30mg/kg)、G-CSF(30μg/kg)、EPO(60U/kg).Continuous intraperitoneal injection of 25 days,CTX every other day,for 25 days.The tumor volume was measured every 5 days after administration,the tumor growth curve was drawn,and body weight was measured every 5 days.After 3 weeks of administration,tumor growth and metastasis were detected by injection of luciferase substrate,inhalation anesthesia and in vivo fluorescence imaging.After the last administration for 2h,blood was sacrificed.Cut the orthotopic tumor,lung,thymus and spleen,calculate the organ index.The number of lung metastases was recorded after perfusion with 10%formalin for 48 h.The spleen and tumor tissue were stained with hematoxylin-eosin(H&E)to observe the histopathological changes.Immunohistochemistry(IHC)was used to observe the positive expression of CD61 and MPO in spleen tissue.Blood routine detection of peripheral blood changes.CD71 and Ter119 antibodies,CD34,CD 115,CD4 and CD8,CD11b and Gr-1 antibodies were incubated with mouse bone marrow cells and spleen cells respectively.Effects of Panax Notoginseng Saponins on Distribution of erythroid and Myeloid Cells in 4T1 Metastatic Breast Cancer Mice.Section Two:There are many kinds of saponins in Panax notoginseng,but the main components are notoginsenoside R1,R2,R3,R4,R5,R6 and ginsenoside Ra,Rb,Rd,Re,Rgl and so on.These saponins in the body after the loss of all the ligand sugar chain,the final metabolism of saponin PPT,PPD,etc.Therefore,the second part we studied PPD,PPT on tumor-bearing mice spleen cells in vitro differentiation.The spleen cells of BALB/c mice were cultured in vitro.The effects of PPD and PPT on cell viability were measured by XTT.In vitro culture of 4T1 metastatic breast cancer tumor-bearing mice spleen cells,given different concentrations of PPD,PPT.The expression levels of PU.1,GATA-1,GATA-2,EBP/α,GM-CSF mRNA were detected by RT-PCR.The spleen cells of tumor-bearing mice were cultured with different concentrations of PPD and PPT on methylcellulose semi-solid medium.The number of colonies formed by BFU-E,CFU-GM and CFU-M was observed under microscope.Mouse splenocytes were labeled with CD71 and Ter119,CD34,CD 115,CD4 and CD8,CD11b and Gr-1 flow antibodies,respectively.To detection the effects of PPD and PPT on erythroid and myeloid differentiation of spleen hematopoietic stem cells in 4T1 metastatic breast cancer-bearing mice.The expression of EPO and G-CSF in PPT and PPT on 4T1 cells was detected by immunocytochemistry.Result Section One:PDS group began to decline on the 10th day after administration,there was significant difference(P<0.01～0.05).PTS group and PNS group in the administration of 15 days,20 days of weight decreased,there was a significant difference(P<0.05).There was a significant difference in tumor size between the PDS group and the 25th day after inoculation(P<0.05).PDS group had lower tumor index and higher lung index,and there was significant difference(P<0.05).Fluorescence in vivo imaging showed that the luminous intensity of PTS group and PNS group was lower than that of model group.The levels of RBC,HGB,HCT and NEUT%in PDS group were decreased,LYMPH%and EO%increased(P<0.05).The ratio of neutrophils to lymphocytes was positively correlated with the transfer rate.Peripheral blood pictures show breast cancer-bearing mice with mononuclear cells and lymphocytes increased,while erythrocyte reduction.PTS,PDS,PNS group neutropenia.The results of HE staining showed that the structure of thymus cells in PTS group was significantly higher than that in model group,and there were obvious dark spots in Ming area.After PTS,PDS treated,mice bone marrow cells CD34+,CD11b+/Gr-1+,CD71+/Ter119+,CD4+/CD8+ ratio increased,CD 115+,CD 11 b+/Gr-1-are reduced in proportion.The ratio of CD34+,CD11b+/Gr-1+ in spleen cells increased,CD71+/Ter119+,CD4+/CD8+,CD115+,CD11b+/Gr-1-were reduced.Spleen immunohistochemical experiments showed that:The positive expression of MPO in PDS,PTS and PNS group was lower than that in model group.The expression of CD61 positive cells in PDS and PTS group was lower than that in model group.Section Two:By XTT detection,PPD 10μM or lower concentration had no significant inhibitory effect on mouse spleen cells,PPT 30μM or lower concentration of mouse spleen cells did not significantly inhibit the proliferation of the role.PPT 10μM,1μM,0.1μM,PPD 10μM,1μM can inhibit the granulocyte-single progenitor cell formation,PPD inhibited the production of erythroid progenitor cells(P<0.01),Both PPT and PPD inhibited macrophage colony formation.The ratio of CD11b+/Gr-1-,CD71+/Ter119+cells in spleen cells of PPT and PPD group decreased,The ratio of CD 11b+/Gr-1-cells in PPD 10μM and PPT 10μM group was significantly decreased(P<0.01～0.05).PPT10μM,PPT 1μM,PPD 10μM CD11b+/Gr-1+ratio decreased,but no significant difference.The expression of GATA-1,GATA-2,GM-CSF,PU.1 and C/EBPa mRNA in PPD and PPT groups were decreased.The positive expression of EPO and G-CSF in PPD and PPT was lower than that in control group.Conclusion 1.Panax notoginseng saponin PDS has a certain anti-breast cancer effect,may be related to its regulation of hematopoietic stem cell differentiation,thus affecting the distribution of blood cells.2.Panax notoginseng saponins can increase the proportion of hematopoietic stem cells and erythroid progenitor cells in bone marrow and promote the hematopoietic capacity of tumor-bearing mice.3.PDS does not improve tumor-induced anemia,but can reduce the NLR to regulate the tumor immune environment.The role of PDS may be related to PDS to reduce the spleen coefficient of tumor-bearing mice and increase the thymus coefficient.4.PPD,PPT inhibition of spleen cell red line differentiation,may be associated with a decrease in the secretion of tumor EPO and thus reduce the expression of GATA-1 and GATA-2.5.PPD,PPT inhibits spleen cell granule-monocyte differentiation,may be associated with a decrease in the secretion of tumor G-CSF and thus reduce the expression of PU 1 and C/EBPα.