| Animal research1 Objective:By using kidney deficiency abortion BMT mice with GFP cells as the object,to research the effect of reinforcing kidney and invigorating spleen on the number of EPCs and VEGF in mice model,from the bone marrow,peripheral blood and decidua.To reveal its mechanism of promoting the mobilization and homing of endothelial progenitor cells.,further explore the mechanism of preventing and treating abortion by reinforcing kidney and invigorating spleen,and provides a new basis for the clinical application of traditional Chinese medicine in the treatment of spontaneous abortion.2 Methods After a week adaptive feed,50 female BMT with GFP cells ICR mice models(bone marrow cells can stably express green fluorescent protein)and 25 male ICR mice were taken to close cage mate by 2:1 proportion every night.Female rats were randomly divided into 5 groups after pregnancy,one of the groups was used as the control group.The remaining 4 groups were fed hydroxyurea(450mg/kg.d)from day 1 to day 9 after gestation and mifepristone(4.0mg/kg.d)in day 10 after gestation.These four groups were considered to be kidney deficiency abortion models,The four groups were considered as kidney deficiency abortion model and were divided into: model group(normal saline),low-dose Chinese medicine group(Bushenantai Granule 3.0g/kg.d),medium dose Chinese medicine group(Bushen antai Granule 6.0g/kg.d),and high-dose Chinese medicine group(Bushenantai Granule 12.0g/kg.d).Meanwhile,the control group was also treated with normal saline.The above five groups were given intragastric administration for a total of 15 days.After 24 h of the last administration,collect blood samples and tissues.Use flow cytometry to detect the number of bone marrow EPCs,the number of peripheral blood EPCs,and the number of homing EPCs cells in decidual tissues,then VEGF and VEGFR-2 expression levels in peripheral blood and decidual tissues were detected by ELISA,The abortion rate and embryo loss rate of each group were also calculated.3 Results3.1 Flow cytometry detection of EPCs cells3.1.1 Numbers of bone marrow EPCs cells in pregnant mice in each group(1)GFP-labeled Flk1+/Sca1+ double-anti-positive expression cells were observed in the bone marrow of each group.(2)Compared with the control group,it was different in the number of EPCs cells in the bone marrow of the model group(P<0.05).(3)Compared with the model group,there was no significant difference in the low and medium-dose group of Bushenantai Granule(P>0.05),and the differences were statistically significant in the high-dose group(P<0.05).(4)The numbers of EPC in each group gradually increased from low,medium and high,and there had difference between high and medium(P<0.05).,but no difference between low and medium(P>0.05).3.1.2 Numbers of EPCs in peripheral blood of pregnant mice in each group(1)GFP-labeled Flk1+/Sca1+ double-anti-positive expression cells were observed in peripheral blood of pregnant mice in each group.(2)Compared with the control group,the numbers of EPCs cells in peripheral blood of the model group decreased significantly(P<0.05).(3)Compared with the model group,the levels of EPCs cells in the low,medium and high dose groups of Bushenantai Granule were no statistical differences(P>0.05).(4)The numbers of EPCs in each group gradually increased from low,medium and high,and there were significant differences among each group,with statistical significance(P<0.05).3.1.3 Numbers of EPCs cells homing in decidual tissues of pregnant mice in each group(1)GFP-labeled Flk1+/Sca1+ double-anti-positive expression cells were found in the decidual tissues of pregnant mice in each group.(2)Compared with the control group,the numbers of EPCs cells in peripheral blood of the model group decreased significantly(P<0.05).(3)Compared with the model group,the numbers of EPCs cells in the low,medium and high dose groups of Bushenantai Granule were significantly increased,with statistical significance(P<0.05).(4)The numbers of EPCs in each group gradually increased from low,medium and high,and there were significant differences among each group,with statistical significance(P<0.05).3.2 detection of VEGF expression level in pregnant mice in each group3.2.1 VEGF levels in peripheral blood of pregnant mice in each group(1)Compared with the control group,the peripheral blood VEGF level in the model group significantly decreased(P<0.05).(2)Compared with the model group,the levels of VEGF in the peripheral blood of the low,medium and high dose groups of Bushenantai Granule increased significantly,and the difference was statistically significant(P<0.05).(3)The levels of VEGF in each group gradually increased from low,medium and high,and there were significant differences between each group(P<0.05).3.2.2 VEGF levels in decidual tissues of pregnant mice in each group(1)Compared with the control group,the level of VEGF in the model group significantly decreased(P<0.05).(2)Compared with the model group,the levels of VEGF in decidual tissues of the low,medium and high dose groups of Bushenantai Granule increased significantly,and the differences were statistically significant(P<0.05).(3)The level of VEGF in each group gradually increased from low,medium and high,and there werea significant differences between each group(P<0.05).3.3 Abortion rate and embryo loss rate of pregnant mice in each group(1)The abortion rate of each group gradually increased from the control group,high-dose group,medium-dose group,low-dose group and model group.(2)Compared with the control group,the embryo loss rate in the model group increased significantly(P<0.05).(3)Compared with the model group,the embryo loss rate in the groups with high,medium and low doses of Bushenantai Granule decreased significantly,and the differences were statistically significant(P<0.05).(4)The embryo loss rate in each group decreased gradually from low,medium and high,and the difference between the low dose group and the medium dose group was statistically significant(P<0.05),while the difference between the medium dose group and the high dose group was not statistically significant(P>0.05).4 Conclusions4.1 There had difference in bone marrow EPCs between the control group and the model group.4.2 Low and medium-dose Bushenantai Granule has obvious effects on the number of EPCs in peripheral blood and decidual tissues of mice,but not on the number of EPCs in bone marrow,which may be caused by the low concentration has no ability to mobilize the precursor cells to transform into EPCs cells in bone marrow.The number of EPCs cells in bone marrow,peripheral blood and decidual membrane of aborted mice was significantly increased with the high dose of Bushenantai Granule.4.3 Bushenantai Granule can significantly improve the expression level of VEGF in peripheral blood and decidual membrane of aborted mice.4.4 Bushenantai Granule can effectively prevent embryo loss and reduce abortion rate.4.5 Bushenantai Granule can promote the mobilization and homing of EPCs,make them migrate to the ischemic site at the maternal and fetal interface of aborted mice,up-regulate the expression level of factors such as VEGF,and promote the formation of decidual blood vessels and the recasting of micro-blood supply.This promotes the development of embryos.4.6 This research provides theory basis for curing RSA by using reinforcing kidney and invigorating spleen medicine,has enriched the Chinese medicine "kidney advocate bone marrow "," the kidneys advocate reproduction ",“treated abortion from the spleen and kidney" scientific connotation,makes the research of TCM gynecology extends into cytology system,enrich the connotation of "holistic concept" of traditional Chinese medicine further.Clinical research1 Objective:To investigate the contents and clinical significance of vascular endothelial growth factor(VEGF)and vascular endothelial growth factor receptor-2(VEGFR-2)in peripheral blood of patients with different abortion types,and they were used as markers to explore the mobilization ability of EPCs with Bushenantai Granule.To provide clinical data support for the study of the role of reinforcing kidney and invigorating spleen medicine in the regulation mechanism of cell production in the treatment of abortion.2 Methods30 patients with pregnancy and 30 patients with threatened abortion were collected,who clinic or hospital in the department of gynaecology in the First Affiliated Hospital of Anhui University of Chinese Medicine between January 2019 to December 2019.Peripheral blood of patients with normal pregnancy and threatened abortion without medication was extracted,and peripheral blood of patients with threatened abortion was extracted again 14 days after taking traditional Chinese medicine of reinforcing kidney and invigorating spleen medicine for protecting pregnancy.The contents of VEGF and vegfr-2,considered the markers of EPCs,in peripheral blood of patients in each group were detected by Elisa and calculated the threatened abortion group’s Chinese medicine syndrome scores and efficacy evaluations before and after medication.then analyzed in parallel.3 Results3.1 General clinical characteristics of patients in the two groups There was no significant difference in age,gestational days and gestational times between the two groups(P>0.05).3.2 Differences of VEGF and VEGFR-2 in peripheral blood:3.2.1 Compared with the normal pregnancy group,the expression levels of VEGF and vegfr-2 in peripheral blood of threatened abortion without medication decreased(P<0.05).3.2.2 The levels of VEGF and VEGFR-2 in peripheral blood of threatened abortion after taking Chinese medicine for 14 d were significantly higher than those without the medicine,and the difference was statistically significant(P<0.05).3.2.3 Correlation analysis and results suggested that there was a positive correlation between VEGF and VEGFR-2 in the peripheral blood groups,and the regression equations of VEGF and VEGFR-2 were similar before and after the medication in the threatened abortion group,indicating that it was likely to be affected by a single factor.Therefore,the single cytokine level was used to analyze the correlation before and after medication in the threatened abortion group,showing that the correlation was strong,and all the results were P<0.05.3.2.4 The threatened abortion group’s TCM syndrome scores of kidney deficiency syndrome decreased significantly before and after taking the medicine(P<0.05),and the total effective rate was 96.67% after treatment.4.Conclusions4.1The decreased expression levels of VEGF and VEGFR-2 in peripheral blood of patients with threatened abortion suggested that the decrease of VEGF and vegfr-2might be related to the occurrence of abortion.4.2 The levels of VEGF and VEGFR-2 in peripheral blood of patients in the threatened abortion group after taking Chinese medicine for 14 days were higher than those before taking Chinese medicine,indicating that Chinese medicine for kidney and spleen can improve the expression level of cytokines related to promoting vascular development.4.3 Correlation analysis and results suggest that VEGF and VEGFR-2 are synchronously regulated by the traditional Chinese medicine for kidney and spleen strengthening,which is most likely the expression of the same group of EPCs cells after mobilization,indirectly reflecting the role of the traditional Chinese medicine for kidney and spleen strengthening in promoting the mobilization of EPCs cells into the peripheral blood.Combined with the preliminary study of the experimental group,it is suggested that the principle of the method of kidney strengthening and spleen strengthening to prevent and cure spontaneous abortion may be that it can mobilize EPCs to migrate to the ischemic site at the maternal and fetal interface,repair damaged blood supply and improve microcirculation.4.4 From the change of TCM syndrome score before and after taking medicine for patients with threatened abortion,it can be concluded that Bushenanzhen granule can effectively reduce the score of kidney deficiency syndrome,improve the symptoms of abortion,and provide a strong clinical basis for the treatment of threatened abortion with kidney deficiency by reinforcing kidney and invigorating spleen method. |
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