| Objective The dysvascularization of the maternal fetal interface has attracted much attention in recurrent spontaneous abortion(RSA).The absorption rate of embryo,pathological morphology of decidual tissue,the expression of rat sarcoma(RAS),mitogen activated protein kinase(MAPK)in serum and the expression of Ras and MAPK protein expression in placenta microvascular endothelial cells(PMVEC),placental vascular smooth muscle cells(PVSMC)and placental fibroblast(PF)were observed in RSA pregnant rats treated with Bushen Antai granule.The effects of were evaluated to promote the angiogenesis,development and remodeling of RSA maternal fetal interface,and the mechanism of fetal protection was further elaborated from the molecular and cellular level to provide scientific basis for clinical application.Methods Ten normal pregnant mice were established by cage the healthy female CBA / J mice and the healthy male BALB / c mice according to the ratio of 2:1.50 pregnant rats of RSA model were established by combining healthy female CBA / J mice and male DBA/ 2 mice in a ratio of 2:1.When female mice detected negative thrombus or found a large number of sperm on the vaginal smear of female mice under microscope,it was regarded as the day 0 of pregnancy.According to the order of pregnancy,they were randomly divided into model group,progesterone group,Bushen Antai granule high,medium and low dose group,10 mice in each group.On the first day of pregnancy,the pregnant rats in normal pregnancy group and model group were given distilled water,while those in medicine progesterone group and high,medium and low dose Bushen Antai granule group were given corresponding drugs respectively.Each group was continuously intervened for 15 days.PMVEC,PVSMC and PF were extracted and cultured from serum and decidua of each group.The embryo loss of each group of pregnant rats was observed.The histomorphology of decidua of each group of pregnant rats was analyzed by hematoxylin eosin staining(HE).The enzyme-linked immuno sorbent assay(ELISA)was used determine the content of Ras and MAPK in the serum of pregnant rats in each group.The expression of Ras and MAPK in PMVEC,PVSMC and PF were detected by immunofluorescence and Western blot.Results1.Comparison of embryo absorption rate of pregnant rats in each group The rate of embryo loss in normal group was significantly lower than that in model group(P<0.01).Compared with the model group,the embryo absorption rate in the high,middle and low dose groups of Bushen Antai granule and the progesterone group decreased significantly(P<0.05).Compared with the high dose group of Bushen Antai granule,there was no significant difference in embryo absorption rate between the progesterone group(P > 0.05),but the embryo absorption rate of the middle and low dose group of Bushen Antai granule was significantly lower than that of the high dose group of Bushen Antai granule(P<0.01).2.Histomorphological changes of decidua in pregnant rats of each group In the normal group,the decidual cells were regular in shape,arranged tightly and orderly,with large nucleolus,oval shape,normal cytoplasm,abundant blood vessels and complete tube wall.However,in the model group,the number of decidual cells was significantly reduced,the shape was different and the arrangement was loose,the nucleus was pyknosis and fragmentation,and even a few of them appeared apoptosis and disappearance,cytoplasmic edema,the number of blood vessels was significantly reduced,the lumen was narrow,and the wall was incomplete.Compared with the model group,the decidual cell structure,cell number,vascular morphology and quantity of the pregnant rats were improved after the drug intervention,among which the high dose group of Bushen Antai granule and the progesterone group had the most significant effect,the cell morphology was more regular,the arrangement was still neat andcompact,a small number of micronuclei were pyknosis,and the blood vessels were more abundant.3.Comparison of Ras and MAPK contents in serum of pregnant rats in each group The content of Ras and MAPK in serum of model group was significantly lower than that of normal group(P<0.01);compared with model group,the content of Ras and MAPK in serum of progesterone group and low,middle and high dose Bushen Antai granule group was significantly higher(P<0.05);compared with high dose Bushen Antai granule group,the content of Ras and MAPK in serum of progesterone group was not significantly different,there was no statistical significance(P>0.05).There was significant difference between the low and middle dose groups of Bushen Antai granule(P<0.01).4.The expression of Ras and MAPK in PMVEC,PVSMC and PF was detected by immunofluorescence The positive expression of Ras and MAPK in PMVEC,PVSMC and PF was the strongest in normal group,while that in model group was significantly weakened.The positive expression of Bushen Antai granule in low,medium and high dose groups and progesterone group was stronger than that in model group,especially in high dose group and progesterone group.Compared with the normal group,the expression of Ras and MAPK in PMVEC,PVSMC and PF in the model control group decreased significantly(P<0.01);Compared with the model group,the expression of Ras and MAPK in the PMVEC,PVSMC and PF in the low,medium and high dose groups of Bushen Antai granule and the progesterone group were all increased(P<0.01);Compared with high dose of Bushen Antai granule,the expression of Ras and MAPK protein in PMVEC,PVSMC and PF in low and medium dose groups of Bushen Antai granule were decreased(P<0.01),There was no significant difference between the expression of Ras and MAPK protein in PMVEC in progesterone group(P>0.05);the expression of Ras protein in PVSMC was significantly lower than that in Bushen Antai granule high dose group(P < 0.01),there was no significant difference in MAPK protein expression between the high dose group(P>0.05);The expression of MAPK protein in PF decreased significantly(P<0.01),but there was no significant of Ras protein difference between high dose of Bushen Antai granule(P>0.05).5.Western blot was used to detect the expression of Ras and MAPK in PMVEC,PVSMC and PF Compared with the normal group,the expression level of Ras and MAPK protein in PMVEC,PVSMC and PF in the model group decreased significantly(P<0.01).Compared with the model group,the expression level of Ras and MAPK protein in PVSMC and PF in the progesterone group and low,medium and high dose Bushen Antai granule group were significantly increased(P<0.01).Compared with the high dose group,the expression level of Ras and MAPK protein in the PMVEC,PVSMC and PF of pregnant rats in the middle and low dose group of Bushen Antai granule decreased significantly(P<0.01);the expression level of MAPK protein in the PMVEC of pregnant rats in the progesterone group decreased significantly(P<0.01),but the expression level of Ras protein did not decrease significantly(P < 0.01);There was no significant difference in the expression of Ras and MAPK protein in the PVSMC and PF in progesterone group(P > 0.05).Conclusion1.Bushen Antai granule can improve the pathological changes of uterine decidua,improve the vascular morphology and promote the angiogenesis of uterine decidua,so as to reduce the embryo loss rate of RSA pregnant rats.2.The antiabortifacient mechanism of Bushen Antai granule may be related to the increase of Ras and MAPK protein expression in PMVEC,pvsmc and placental fibroblasts of RSA pregnant rats,so as to promote the angiogenesis at the maternal fetal interface and provide sufficient blood supply for the growth and development of embryos. |
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