| 1 Objective To study the expressions and Clinical signiscance of vascular endothelial growth factor(VEGF)、vascular endothelial growth factor receptor 2(VEGFR-2)、estrogen receptor(ER)、 progesterone receptor(PR)and hypoxia-inducible factor(HIF-1a)in the decidual tissue of patients with recurrent spontaneous abortion(RSA),explore the influence of regulatory factors effect on angiogenesis of maternal-fetal interface in RSA,for the experimental research on the kidney and invigorate the spleen medicine regulation mechanism of angiogenesis in abortion mice to provide theoretical support.2 Methods10 patients of RSA were collected and categorized into research group from January2017 and December 2017 in the first affiliated hospital of anhui medical university;meanwhile,10 patiens of early pregpancy with artificial surgery were categorized into control group.To detect the expression of VEGF,VEGFR-2,ER,PR,HIF-1ain the decidual tissue of researth group and conrtol group by Immunohistochcmistry,and the results were statistical analysis.3 Results3.1 Comparision of Characteristics between two groups Age and gestational age has no statistical significance between two groups,Comparision of gestational times between two groups,the difference was statistically significantly(P <0.01).3.2 Two groups of decidual tissue in patients with pathological morphological changes Decidual tissue of RSA Patients,We use light microscope to observe,cell arranged irregularly shaped,by extrusion deformation,or fusion phenomenon,gland structure disorder,most of the decidual cell cytoplasm severe edema,glandular structof nuclear disappear,vacuolated,nucleus pycnosis,the number of vessels decreased,blood vessel walls not complete.Decidual tissue in patients with early pregpancy with artificial surgeryt,then see decidual cells are arranged or polygon round each other embedded tight,rendering bricklaying,intercellular space is larger,abundant cytoplasm and small nucleus,nucleus shows ovoid,located in the central,blood vessels,hemal wall integrity.3.3 Immunohistochemical results3.3.1 Expression of VEGF in the two groups of decidual tissue The expression of VEGF was expressed the two groups,expression in endothelial cells,glandular epithelial cells in the cytoplasm and nucleus not seen obvious color.Compared with the control group,VEGF level was significantly decreased in researth group,the difference was statistically significantly(P <0.05).3.3.2 Expression of VEGFR-2 in the two groups of decidual tissue The expression of VEGFR-2 was expressed the two groups,expression in vascular endothelial cells,and glandular epithelial cells in the cytoplasm.Compared with the control group,VEGFR-2 level was significantly decreased in researth group,the difference was statistically significantly(P <0.05).3.3.3 Expression of ER in the two groups of decidual tissue The expression of ER was expressed the two groups,expression in the nucleus,very little expression in decidual cell cytoplasm.Compared with the control group,ER level was significantly decreased in researth group,the difference was statistically significantly(P <0.01).3.3.4 Expression of PR in the two groups of decidual tissue The expression of PR was expressed the two groups,expression in the nucleus,very little expression in decidual cell cytoplasm.Compared with the control group,PR level was significantly decreased in researth group,the difference was statistically significantly(P <0.01).3.3.5 Expression of HIF-1a in the two groups of decidual tissue The expression of HIF-1awas expressed the two groups,expression in decidual cell cytoplasm,a few expression in the nucleus.Compared with the control group,HIF-1a level was significantly decreased in researth group,the difference was statistically significantly(P <0.05).3.4 Correlation analysis of the expression of decidua tissue VEGF and VEGFR-2、ER、PR and HIF-1a between two groups3.4.1 In the research group of decidual tissue Correlation analysis showed that,VEGF positively correlated with VEGFR-2(P<0.05),positively correlated with ER,PR and HIF-1a(P<0.01).3.4.2 In the control group of decidual tissue Correlation analysis showed that,VEGF positively correlated with VEGFR-2(P<0.05),positively correlated with ER,PR and HIF-1a(P<0.01).4 Conclusion4.1 The difference of decidual tissue pathology morphological between patients with recurrent spontaneous abortion and patiens of early pregpancy with artificial surgery.4.2 There exists vascular dysplasia and the number of vessels decreased in patients with recurrent spontaneous abortion of decidual tissue.4.3 Compared with the early pregpancy with artificial surgery,and find that the expression of VEGF,VEGFR-2,ER,PR,HIF-1a decreased in patients with recurrent spontaneous abortion.Suggest the decreased of VEGF,VEGFR-2,ER,PR,HIF-1a are associated with RSA,which induced the decidual and placental vascular dysplasia,decrease of angiogenesis,which nourish inadequate biood supply to trophoblasts,causing embryonic development stop,the occurrence of abortion.4.4 Expression of VEGF has positively correlation with VEGFR-2,ER,PR,HIF-1a in patients with RSA.Suggest promote angiogenesis factor of VEGF is affected by the regulatory factors of ER,PR,HIF-1a,the abnormal expression of regulatory factors would impact VEGF protein genes transcription,the imbalance of the relationship between VEGF and VEGFR-2,ER,PR,HIF-1a may give rise to vascular dysplasia,angiogenesis obstacles,which may be the important cause of RSA and closely related with RSA.1 Objective The detection of reinforce the kidney and invigorate the spleen medicine(Bushenantai Granule)in high,medium and low three groups of recurrent spontaneous abortion mice in the peripheral blood vascular endothelial growth factor(VEGF),estrogen(E2),progesterone(P),hypoxia-inducible factor(HIF-1a)and recurrent spontaneous abortion mice decidual tissues of VEGF,VEGFR-2,ER,PR,HIF-1a,protein expression of Bushenantai granule in each dose;Explore Bushenantai granule impaction of vessels regulatory factors of recurrent spontaneous abortion mice,reveal the mechanism and significance of reinforce the kidney and invigorate the spleen medicine for recurrent spontaneous abortion mice in the regulation of angiogenesis,the recasting of maternal-fetal interface blood vessels;As a result,it is supposed to provide a theoretical basis for reinforce the kidney and invigorate the spleen medicine in the intervention of RSA.2 Methods The mouse model of recurrent spontaneous abortion was established by female mice CBA/J and male mice DBA/2,the mouse model of normal pregnancye stablished by female mice CBA/J and male mice BALB/c,with the female and the male mated by the ratio of 2:1.We took the positive detecting of pessary as the gestation day 0.After one week of adaptive feed,60 pregnant mice as models were selected,all of which divided into six groups: the normal pregnancy group,the pregnancy model group of recurrent spontaneous abortion,the western medicine group,the Chinese medicine group of low dose,the Chinese medicine group of medium dose,the Chinese medicine group of high dose.The gavage for mice started from the first day of pregnancy: the normal pregnancy group and the pregnancy model group of recurrent spontaneous abortion were gavaged with diluted distilled water;the western medicine group were given progesterone capsules with the dose of 26mg/kg.d;the Chinese medicine group of low dose/medium dose/high dose,i.e.,the group of low-dose/ medium-dose/high-dose Bushenantai Granule,would be respectively administered with Bushenantai Granule,the dose of which was 3.9g/kg.d/11.7g/kg.d/35.1g/kg.d.The successive administration was given 15 days.The mice were executed in 24 h after the last administration,weighing,pick the eyeball blood,whose uteri decidua were then pulled out.The next step was to calculate the organ weight index,observe the uterine and embryonic development after dissection,calculate the abortion rate and embryonic loss rate,observe the pathologic changes of decidual tissues,and detect the expression of VEGF,E2,P,HIF-1a by Elisa,protein expression of VEGF,VEGFR-2,ER,PR,HIF-1a in the decidual tissue by SP Immunohistochemistry.3 Results3.1 Uterus and embryonic development situation of every group pregnancy mice The observation finds that the uteri and embryos in the normal pregnancy group are well-developed,equal volume,being reddish,uterine form bulky like beads.Besides,there are almost no notable points of blood stasis and evident bleeding in the uteri.The development of the uteri and embryos in the model group being dark brown,shaped in a bamboo spine,embryo volume uneven development or disappear or incomplete.There are evident blood stasis in the uteri.As for the western medicine group and high-dose Bushenantai Granule groups,uteri and embryos are well-development,being reddish,with no obvious blood stasis points and bleeding in the uteri.The low-dose Bushenantai Granule groups,uteri and embryos volume uneven development,being Light dark,with scattered point of blood stasis in the uteri.The medium-dose Bushenantai Granule groups,the uterus and embryonic development is relatively good,and in a relatively uniform size,there are very small amounts points of blood stasis and ibleeding in the uteri and implantation sites.3.2 Comparison of abortion rate of every group pregnancy mice Compared with the normal group,abortion rate was significantly increased in the model group of recurrent spontaneous abortion mice(P<0.01).Compared with the model group,abortion rate was significantly decreased in the medium/high-dose Bushenantai Granule groups of recurrent spontaneous abortion mice,the difference was statistically significantly(P<0.01);the low-dose Bushenantai Granule group of recurrent spontaneous abortion,which abortion rate is lower than the model group,the difference was statistically(P<0.05).Compared with the western medicine group,the low-dose Bushenantai Granule group abortion rate increased,the difference was statistically(P <0.05).However,abortion rate was not significantly different between the medium/high-dose Bushenantai Granule groups(P>0.05).3.3 Comparison of embryonic loss rate of every group pregnancy mice Compared with the normal group,embryonic loss rate was significantly increased in the model group of recurrent spontaneous abortion mice(P<0.01).Compared with the model group,embryonic loss rate was significantly decreased in the western medicine group,medium/high-dose Bushenantai Granule groups of recurrent spontaneous abortion mice,the difference was statistically significantly(P<0.01);the low-dose Bushenantai Granule group of recurrent spontaneous abortion mice,which embryonic loss rate is lower than the model group,the difference was statistically(P<0.05).Compared with the western medicine group,the low-dose Bushenantai Granule group embryonic loss rate reduced,the difference was statistically(P<0.05).However,embryonic loss rate was not significantly different between the medium/high-dose Bushenantai Granule groups(P>0.05).3.4 Comparison of the organ weight index of every group pregnancy mice Compared with the normal group,organ weight index was significantly decreased in the model group of recurrent spontaneous abortion mice(P<0.01).Compared with the model group,organ weight index was significantly increased in the western medicine group,medium/high-dose Bushenantai Granule groups of recurrent spontaneous abortion mice,the difference was statistically significantly(P<0.01);the low-dose Bushenantai Granule group of recurrent spontaneous abortion mice,which organ weight index is higher than the model group,the difference was statistically(P<0.05).Compared with the western medicine group,the difference of organ weight index in the low/medium-dose Bushenantai Granule group was statistically significantly(P<0.01).However,organ weight index was not significantly different in the high-dose Bushenantai Granule group(P>0.05).3.5 Comparison the expression of VEGF,E2,P,HIF-1a in every group pregnancy mice Compared with the normal group,the expression of VEGF,E2,P,HIF-1a were significantly decreased in the model group of recurrent spontaneous abortion mice(P<0.01).Compared with the model group,the expression of VEGF,E2,P,HIF-1a were significantly increased in the western medicine group,low/medium/high-dose Bushenantai Granule groups of recurrent spontaneous abortion mice,the difference was statistically significantly(P<0.01).Compared with the western medicine group,the expression of VEGF,P,HIF-1a reduced in the low/medium-dose Bushenantai Granule group,the difference was statistically significantly(P<0.01).However,the expression of VEGF,P,HIF-1a was not significantly different in the high-dose Bushenantai Granule group(P>0.05).The expression of E2 lower in the low/medium-dose Bushenantai Granule group than the western medicine group,higher in the high-dose Bushenantai Granule group than the western medicine group,the differences were both statistically significantly(P<0.01).3.6 The observation of decidual tissue pathology morphological in every group pregnancy mice In the normal group,we use light microscope to observe,decidual cells are fold the brick sample each other embedded tight,cell arranged regularly shaped,abundant cytoplasm,and nucleus located in the centra,nuclear dyed blue,blood vessel number is normal,blood vessel wall is complete,no obvious blood stasis in the pipe wall.In the model group,decidual cells arranged irregularly,cytoplasm edema,most of the nuclear disappear,necrosis,nuclear pyknosis,even vacuoles;The number of blood vessel decreased significantly,blood vessel walls not completed,there is obvious blood stasis in tube wall.In the western medicine group,decidual cells arranged neatly,cytoplasm is rich,a small number of cell cytoplasm edema,the most part the nucleus is located in the central,a few nuclear disappeared,pyknosis,cavitation;Blood vessels is rich,the wall is full,multiple surrounded by vascular endothelial cells,it is not obvious blood stasis in blood vessel wall.In the low-dose Bushenantai Granule,the most of decidual cells arranged irregularly,cytoplasm edema,most of the nuclear disappear,pyknosis,vacuoles obivious;Vascular quantity is less,blood vessel walls not complete,it is blood stasis in blood vessel wall.In the medium-dose Bushenantai Granule,the part of decidual cells arranged irregularly,cytoplasm edema,part of the nuclear disappear,pyknosis;more vascular quantity,vascular endothelial cells arranged neatly,it is very few blood stasis in blood vessel wall.In the high-dose Bushenantai Granule,a small number of decidual cells arranged irregularly,a small number of cytoplasm edema,basic no nucleus pycnosis,disappear,necrosis;blood vessel number and tube wall is complete,multiple vascular endothelial cells around,it is no blood stasis in blood vessel wall.3.7 Comparison the protein expression of VEGF,VEGFR-2,ER,PR,HIF-1a in every group pregnancy mice(1)Compared with the normal group,the protein expression of VEGF,VEGFR-2,ER,PR,HIF-1a were significantly decreased in the model group of recurrent spontaneous abortion mice(P<0.01).(2)Compared with the model group,the protein expression of VEGF,VEGFR-2,ER,PR,HIF-1a were significantly increased in the western medicine group,low/medium/high-dose Bushenantai Granule groups of recurrent spontaneous abortion mice,the difference was statistically significantly(P<0.01).(3)Compared with the western medicine group,the protein expression of VEGF,HIF-1a reduced in the low/medium-dose Bushenantai Granule group.However,the protein expression of VEGF,HIF-1a higher than it,the differences were both statistically significantly(P<0.01).(4)Compared with the western medicine group,the protein expression of VEGFR-2,reduced in the low/medium-dose Bushenantai Granule group,the differences were statistically significantly(P<0.01,P<0.05).However,the protein expression of VEGFR-2 higher than it,the difference was statistically(P<0.05).(5)Compared with the western medicine group,the protein expression of ER,PR lower in the low/medium/high-dose Bushenantai Granule group have statistically difference(P<0.01,P<0.05),and the protein expression of ER,PR in the high-dose Bushenantai Granule group higher than it.4 Conclusion4.1 Bushenantai Granule can improve uterus and embryonic development situation of recurrent spontaneous abortion mice.4.2 Bushenantai Granule can reduce the abortion rate,embryonic loss ratethe and the organ weight index of recurrent spontaneous abortion mice.4.3 Bushenantai Granule can reduce the damagement of decidual tissue pathological of recurrent spontaneous abortion mice.4.4 Bushenantai Granule can improve the maternal-fetal angiogenesis interface and developmental disorder,which has the effect of prevention and control the occurrence of abortion.It is supposed to provide a theoretical basis for reinforce the kidney and invigorate the spleen medicine in the intervention of RSA,enrich the scientific connotation of "cell venation in kidney",treating RSA through “spleen and kidney from the perspective” of traditional Chinese theories.4.5 Bushenantai Granule improved the maternal-fetal angiogenesis interface of RSA mce from the following tips:(1)Bushenantai Granule enhanced the maternal-fetal angiogenesis and embryonic development in RSA mice by up-regulation of the expression of VEGF,Combining with VEGFR-2 and producing biological activities,regulating our previous studies of the RAS/MARK、PI3K/AKT signal pathway in cells.(2)Bushenantai Granule enhanced the maternal-fetal angiogenesis in RSA mice by increasing of the expression of E2,P,Combining with ER and PR respectively and up-regulation of the expression of VEGF,starting the VEGF mediated angiogenesis signaling pathways.(3)Bushenantai Granule enhanced the maternal-fetal angiogenesis in RSA mice by increasing of the expression of HIF-1a,up-regulating the target gene transcription of VEGF. |
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