| Objective Based on previous study, this paper aimed to reveal the molecular biology mechanism of Bushen Antai Granule for improving vascular remodeling at the maternal-fetal interface and securing pregnancy. The research regarded murine models of RSA as the object, VEGF/PI3K/AKT signal pathway as the pointcut, and detected the expression of VEGF, VEGF receptor angiogenic factors, anti angiogenic factors, PI3 K, AKT and p-AKT by monitoring the changes of decidual VEGF, VEGFR-2, s VEGFR-1, MAPK, HIF-1α、ES、RAS, MVD expression and tissue morphology. This study will enrich the scientific connotation of spleen-strengthening and kidney-reinforcing medicine in RSA treatment.Method Through literature study, this study has summarized and analyzed the relationship between RSA angiogenesis of maternal-fetal interface and spleen and kidney. We also illuminated the mechanism of treating RSA mice decidual tissue angiogenesis through spleen and kidney from the perspective of traditional Chinese medicine theories. Fifty female CBA/J mice were mated in a 2:1 ratio with twenty male DBA/2 mice and five male BALB/c mice. We took the positive detecting of pessary as the gestation day 0 and established RSA CBA/J×DBA/2 mice model and normal pregnant CBA×BALB/c mice model, which were randomly divided into 4 groups(normal group, model group and progesterone capsules control group, low and high dose group of Bu Shen An tai granule) with 8 mice for each according to the order of gestation. The normal pregnancy group and model control group were given distilled water, and Bu Shen An tai granule low dose group as well as high dose group were fed progesterone capsules, by gavages constantly for 15 days. 24 h after the last administration, we decapitated the pregnant mice, collected statistics of the loss rate of mouse embryo uterine section, separated the decidual tissues, observed the pathologic changes of them, and detected the protein expression of VEGF, VEGFR-2, s VEGFR-1, MAPK, HIF-1α, ES, Ras and MVD, by SP munohistochemistry. The expression of MVD, HIF-1, VEGF, ES, PI3 K, AKT, and p-AKT were examined by immunoblotting technique(Western Blotting) in decidual tissue. Additionally, the RNA expression level of VEGF, VEGFR-2, s VEGFR-1, Ras, MAPK, HIF-1α, and ES in decidual tissue were detected by QPCR.Result Theoretical results: 1. Maternal-fetal interface vascular remodeling is the key to normal pregnancy maintenance. The normal growth of fetus and placenta is mainly determined by the differentiation of the trophoblast cells and the construction of the uterine placental vascular network. Trophoblast cells invaded endometria producing angiogenic factors, and invaded the spiral arteries to replace the endothelial cells, leading to the dilatation of spiral arteries and increasing blood flow at the implant site, which accelerated the decidualization of endometrial. A large number of new blood vessels were formed when a variety of vascular growth factors occurred. 2. Angiogenesis is the essence of promoting maternal-fetal interface vascular remodeling. Due to the strict control of high levels of vascular formation inhibitory factor and the lack of angiogenesis stimulating factor or angiogenesis stimulating factor in normal tissues, angiogenesis and inhibiting angiogenesis were kept in balance. However, this balance would be broken when the concentration of angiogenic growth factor increased or the concentration of angiogenesis inhibition factor decreased. In general, the dominant state of angiogenic factors would trigger the angiogenesis process. 3. The regulation of VEGF/PI3K/AKT signal pathway can promote the vascular remodeling of maternal-fetal interface. Activating the PI3K/AKT signaling pathway will promote the secretion of VEGF. The mechanism is to upregulate the expression of hypoxia inducing factor-1a(HIF-1α) through a variety of methods, which would initiate the transcription of VEGF gene and promote the expression of VEGF. 4. The primary pathogeny of RSA is deficiency of kidney and spleen. During the process from implantation to the form of placental, the rich blood supplement of the gestation and the changes about the angiogenesis and permeability were indispensably occurred to the embryo, the growth of fetus, placenta and decidua vessels were essentially necessary for the growth of embryonic. Successful pregnancy requires full development of placental vascular network to keep the necessary exchange of gas and nutrient between the maternal and embyro, and to eliminate the metabolic waste excretion of fetal. These were closely related to the theories of “spleen governing movement and transformation”, “spleen governing ascending clear”, “kidney essence and reproduction” in TCM. 5. Strenthening the spleen and tonifying the kidney is vastly important to promote the remodeling of vascular in maternal-fetal interface of RSA. The therapy for Tonifying the kidney and strenthening the spleen has an exact curative effect in preventing abortion. Bushen Antai granules is one of the typic traditional Chinese medicine Baotai methods, which can promote angiogenesis and growth, in favour of maintain normal gestation. Experimental results: 1. Comparing with normal pregnant mice, the rate of RSA embryonic loss in model group mice increased significantly(P<0.01). Besides, compared with the model group, the rate of RSA embryonic loss decreased distinctly in low dose group, high dose group and positive drug group(P<0.01). 2. Compared with normal pregnant mice, the model group RSA mice were variable in size and shape of villus, and the cells of syncytiotrophoblast and cytotrophoblast were decreased significantly. These cells were obviously atrophied and disordered. Apoptotic cells and inflammatory cells were also visible, and the nuclei were pyknosised and deeply stained. Mesenchymal and matrix fibrinoid degenerated, as well as, interstitial blood vessels decreased. Degeneration happened to the decidual tissue, epitheliums were incomplete, cells arranged irregularly, glandulas shrinked, and vessel occurred to be congested. With the administration of Bushen Antai granules and progesterone capsules, the vascular morphology of decidual tissue of RSA mice presented a trend of improvement. 3. In general, VEGF mainly expressed in cytoplasm of decidual cells, glandular epithelial cells and endothelial cells of decidual tissue and their nucleus showed no significant staining and strong positive staining showed in decidual cells; VEGFR-2 appeared the positive expression in nucleus and cytoplasm of majority of decidual cells and only a few in glandular epithelial cells; s VEGFR-1 expressed abundantly in vascular endothelial cells. In RSA group mice, the expression of VEGF and VEGFR-2 is lower than the normal group, low dose group, high dose group and positive drug group; The expression of s VEGFR-1 was higher than normal group, low dose group, high dose group and positive drug group. 4. Compared with the normal group, the protein expression of HIF-1α, VEGF, MVD was significantly decreased in RSA group mice, while ES increased significantly(P<0.01); compared to the model group, the protein expression of HIF-1α, VEGF and MVD were marked increased in the low dose group, high dose group and positive control group. Inversely, ES was obviously reduced(P<0.05). 5. Compared with the normal group, the expression of PI3 K, AKT, p-AKT showed decreased obviously in model group(P<0.01); and increased significantly in the low dose group, high dose group and positive control group(P<0.05). 6. Compared with the normal group, m RNA expression of HIF-1α and VEGF in RSA model group was remarkablely reduced, while the ES m RNA was obviously increased(P<0.05). In the low dose group, high dose group and positive control group, m RNA expression of HIF-1α and VEGF was significantly increased, ES was obviously declined in comparison with the model group(P<0.05). 7. Compared with the normal group, the m RNA expression of RAS, MAPK and VEGF-R2 was decreased obviousely in RSA model group. While the m RNA expression of SVEGF-R1 was significantly increased(P<0.05). In low dose group, high dose group and positive control group, it showed a significantly increased in m RNA expression of the Ras MAPK and VEGF-R2, compared with the model group. Inversely, the m RNA expression of SVEGF-R1 was significantly decreased(P<0.05).Conclusion There is a obstacles of decidual angiogenesis in RSA mice, which correlated with VEGF/PI3K/AKT expression. Bushen Antai granule can significant improve the rate of embryonic loss and promote the decidual angiogenesis in RSA mice. The mechanism of Bushen Antai granules strengthen decidual angiogenesis in RSA mouse as follows: 1. Bushen Antai granule would certainly in favour of the decidua vessels angiogenesis of RSA mice through regulating the expression of the decidual tissue cytokine, which was up-regulation of the expression of VEGF, VEGFR-2, MAPK, HIF-1α, RAS, and MVD in decidua vessels and down-regulation of the expression of endothelial cell growth factor ES and s VEGFR-1. 2. Bushen Antai granule enhanced the decidua vessels angiogenesis in RSA mice by regulating the VEGF/PI3K/AKT signal pathway in decidual tissue: the up-regulation of PI3 K, AKT, p-AKT protein expression in decidual vessels and the regulation of VEGF/PI3K/AKT pathway, which promoted the loss rate of embryo and decidual angiogenesis in RSA mice. 3. Bushen Antai granule improved the decidua vessels angiogenesis in RSA mice through regulating the levels of HIF-1α, VEGF, ES m RNA in decidua vessels: the up-regulation of m RNA expression of HIF-1α, VEGF and the down-regulation of ES m RNA levels, which improved the rate of embryo loss and decidual angiogenesis in RSA mice. 4. Bushen Antai granule improve the decidua vessels angiogenesis in RSA mice through regulating the level of RAS, MAPK, VEGF-R2, SVEGF-R1 m RNA in decidual tissue: up-regulating the expression of Ras, MAPK, VEGF-R2 m RNA and down-regulating the levels of SVEGF-R1 m RNA, which improved the rate of embryo loss and decidual angiogenesis in RSA mice. |
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