| Objective:Precise Clinical Efficacy and Pre-experimental Study of tonifying kidney with promoting blood circulation method(Retention of Traditional Chinese Medicine Enema)for Pregnancy Administration of Renal Deficiency and Blood Stasis Infertility and IVF-ET Failure Basic,in order to further explore the mechanism of pre-pregnancy administration.This experimental study basic on an observation of Bushen Huoxue Prescription administration on embryo implantation disorder syndrome with kidney deficiency,blood stasis,and combining all the influences factors of the implantation window period from model rat for whole blood viscosity,plasma viscosity,Plasma DDimer(D-Dimer,D2),serum progesterone(P),wet uterine weight,uterine organ index,endometrial tissue morphology,number count of spiral arteries,vascular endothelial growth factor(VEGF)and its receptor of Fetal liver kinase-1(FLK-1).And influence factors on Angiopoietin 1(Ang-1),Angio-poietin 2(Ang-2)and their receptor tyrosine kinase-2(Tyrosine Effect of protein levels of kinase-2,Tie-2)on the expression levels of Ang-1,Ang-2,Tie-2,and progesterone receptor(PR)m RNAs in uterine tissues of the model rats during implantation period.Explore the mechanism of Bushen Huoxue Prescription regulate the Ang / Tie-2 signaling pathway and understanding the molecules mechanism of the tonifying kidney with promoting blood circulation method on promote kidney deficiency to blood stasis-embryo implantation disorder syndrome on model rat.Methods:Seventy SPF-grade non-mating adult female with weighing 220 ± 20 g SD rats were selected,and twenty healthy SPF-grade adult male with weighing 250 ± 20 g SD rats were selected.Sixty healthy female SD rats with regular estrous cycle were selected by vaginal smear HE staining,and randomly divided into 5 groups: blank group,model group,aspirin group,Bushen Huoxue Prescription low dose group,Bushen Huoxue Prescription high dose group were enrolled in the early stage of estrus.Except the blank group,each group was oral gavaged with hydroxyurea 450 mg /(kg · d)for every 9:00 am for 10 days to create a kidney deficiency model,and 0.3 mg /(kg · d)of epinephrine was added by subcutaneous injection on the 4th day for 7 days to create kidney deficiency with blood stasis model;For the blank group,an equal volume of saline solution was oral gavage for 10 days,and on the fourth day,an equal volume of saline solution was subcutaneous injected for 7 days.Observe the general changes of the rats after modeling and compare them with the blank group.At the same time as the start of modeling,except for the blank group and the model group,the other groups were treated with the corresponding drugs at 2:00 pm every day.The blank group and the model group were given the same amount of saline by oral gavage and enemas;the aspirin group were enemas by an equal volume of saline solution;Bushen Huoxue Prescription low-dose group and Bushen Huoxue Prescription high-dose group were oral gavage an equal volume of saline solution.On the 11 th day after successful modeling,vaginal smears were used to screen out model rats in estrus during each group and caged at 2: 1 every night at 18:00.The vaginal plug or vaginal smears were observed for sperm at 7:00 the next morning.The first day of pregnancy will be indicated when sperm was observed on the film.kidney deficiency and blood stasisembryo implantation disorder syndrome model was constructed by subcutaneous injection of mifepristone on the 4th day of pregnancy.On the 5th day of pregnancy,anesthesia with 1% sodium pentobarbital to the pregnant rat and stabilized for operation.After a quick laparotomy and blood collection from the abdominal aorta,the uterus was quickly dissected and made into slices.Each slice will be weighed with an electronic balance and recorded the uterine wet weight of each group.The endometrial morphology of each group of rats was observed with a light microscope.Hemorheology was used to detect the whole blood viscosity and plasma viscosity of rats in each group.The enzyme-linked immunosorbent assay(ELISA)was used to detect the plasma D2,VEGF,FLK-1 levels and serum P levels in each group.Immunohisto-chemical method(IHC)was used to detect the number of VEGF and spiral arteries in the uterine tissue of each group.Western-Blot was used to detect the Ang-1 and Ang-2 in the uterine tissue of the rats and Tie-2 protein expression levels.Real-Time PCR was used to detect the expression of Ang-1,Ang-2,Tie-2,PR m RNA in uterine tissue of each group.Results: 1.Enema with Bushen Huoxue Prescription for pre-pregnancy administration can increase kidney deficiency and blood stasis embryo implantation disorders in combination with serum progesterone levels and uterine tissue PR levels in model rats and reduce plasma D2 polymer levels in model rats.Serum P content and uterine tissue PR level in the implantation window of the model group were significantly reduced(P <0.01)compared with the blank group.and the plasma D2 aggregates were significantly increased(P <0.01).Serum P content and uterine tissue PR level in the Bushen Huoxue Prescription high dose group were significantly increased compared to the model group,and plasma D2 aggregates were significantly decreased(P <0.01).The PR level of uterine tissues in Bushen Huoxue Prescription low dose group was significantly increased,and plasma D2 aggregates were decreased(P <0.05).There is an increasing trend in serum P content.2.Enema with Bushen Huoxue Prescription for pre-pregnancy administration can increase the uterine wet weight and uterine organ index in the implantation window of model rats with kidney deficiency and blood stasis-embryo implantation disorder.The uterine wet weight and uterine organ index for the Bushen Huoxue Prescription high dose group,the Bushen Huoxue Prescription low dose group,and the aspirin group were all higher than those in the model group(P <0.05).There was no statistical difference between the high-dose group and the blank group(P> 0.05).3.Enema with Bushen Huoxue Prescription for pre-pregnancy administration can improve the endometrial tissue morphology during implantation window period with kidney deficiency and blood stasis embryo implantation disorder combined model ratsThe endometrium is poorly developed in the model group,there are few spiral arteries,and located at the base layer.The endometrial glands are small and straight.Cells are spindle-shaped,part of the interstitial is dense,and changes in the proliferative period;part of the interstitial is loose,with edema in the secretory phase and the development is asynchronous.Compared with the model group,the endometrium of the rats in the blank group,the Bushen Huoxue Prescription high,Bushen Huoxue Prescription low dose groups and the aspirin group developed better than the model group.The spiral arterioles proliferated,they were close to the functional layer,and the number of glands was more evenly distributed.The endometrial morphology of the Bushen Huoxue Prescription high dose group was similar to the blank group.4.Enema with Bushen Huoxue Prescription for pre-pregnancy administration can increase the expression levels of VEGF and FLK-1 in the implantation window stage of uterine tissue of kidney deficiency and blood stasis embryo implantation disorder combined model rats and promote spiral arterial hyperplasia.Compared with the blank group,the expression levels of VEGF and FLK-1 in the endometrium of the model group were significantly reduced(P <0.05),and the number of spiral arteries was also significantly reduced(P <0.01).Compared with the model group,the expression levels of VEGF,FLK-1 and the number of spiral arteries in the Bushen Huoxue Prescription high dose group were significantly increased(P <0.01);the number of spiral arteries in the Bushen Huoxue Prescription low dose group was significantly increased(P <0.01).The expression levels of VEGF and FLK-1 were also higher than those in the model group.5.Enema with Bushen Huoxue Prescription for pre-pregnancy administration can increase the expression levels of Ang-1,Ang-2,and Tie-2 proteins in the uterine tissue of kidney deficiency and blood stasis embryo implantation disorder combined model rats.Compared with the blank group,the expression level of Tie-2 protein in the uterine tissue of the model group was significantly reduced(P <0.01);compared with the model group,the Tie-2 protein expression level of the Bushen Huoxue Prescription group was higher(P <0.05);The levels of Ang-1 and Tie-2 protein expression in rats of Bushen Huoxue Prescription high dose group and aspirin group were significantly increased(P <0.01);the expression level of Ang-2 protein was not statistically different between the experimental groups,but the administration group had a tendency to increase.6.Enema with Bushen Huoxue Prescription for pre-pregnancy administration can increase the expression levels of Ang-1,Ang-2,and Tie-2 m RNA in uterine tissue of kidney deficiency and blood stasis embryo implantation disorder combined model rats.Compared with the blank group,the expression levels of Ang-1 and Tie-2 m RNA in the model group were significantly reduced(p <0.01),and the expression of Ang-2 m RNA was also lower(p <0.05).Compared with the model group,the expression levels of Ang-1 and Tie-2 m RNA in Bushen Huoxue prescription high dose group,Bushen Huoxue prescription low dose group and aspirin group were significantly increased(P <0.01),and the expression levels of Ang-1 and Tie-2 m RNA in the Bushen Huoxue prescription high dose group were higher than those in the blank group and no statistical difference between them(P> 0.05).Conclusion:1.Kidney deficiency and blood stasis-embryo implantation disorder combined model rats appeared with mental atrophy,arched back,sleepless,less active,slow response,sparse fur and even hair loss.Ear,tongue,claws and tail appeared in dark purple color refer to the TCM kidney deficiency and blood stasis symptom.Reducing both uterine wet weight and uterine index,poor endometrial development and decreased progesterone levels,including poor endometrial receptivity,indicate that the rat kidney deficiency blood stasis-embryo implantation disorder syndrome model was successfully modeled.2.Enema with Bushen Huoxue Prescription for pre-pregnancy administration can improve the endometrial blood flow,increases the expression levels of VEGF,FLK-1,Ang-1,and Tie-2 in the uterine tissue,maintains the normal expression level of Ang-2,promotes angiogenesis in the uterine tissue,and increases uterine tissue blood perfusion for energy supply and this mechanism may be one of the key to fertility.3.Enema with Bushen Huoxue Prescription for pre-pregnancy administration can increase the uterine wet weight and uterine organ index,increase uterine tissue blood perfusion,improve endometrial tissue morphology during implantation window period,and increase P and PR expression in order to improve endometrial receptivity for promoting Embryo implantation. |
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