| Objective:The effectiveness of Bushen Huoxue(BSHX)Decoction was verified from both clinical research and animal experimental research,to explore the mechanism of BSHX Decoction in the treatment of recurrent spontaneous abortion(RSA)patients in prethrombotic state.Methods:Part 1: Clinical study of Bushen Huoxue Decoction in the treatment of RSA in prethrombotic stateFrom March 2019 to February 2021,98 patients who met the inclusion criteria in the outpatient department of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine and the First Maternity and Child Health Hospital Affiliated to Tongji University were selected and randomly divided into 49 cases in the control group and 49 cases in the test group.In the same period,30 early-pregnant women and 30 non-pregnant women each had a history of at least one live birth were included.The study was carried out in three stages: In the first stage,the control group was treated with aspirin+dydrogesterone.The experimental group was treated with BSHX Decoction I on the basis of the treatment of the control group for a total of 3 months,strictly contraception during the treatment.In the second stage,self-pregnancy is tried for 6months after treatment,and drug treatment is suspended during the trial period.In the third stage,36 cases were included in the control group of confirmed intrauterine pregnancy patients,and 39 cases in the test group were treated.The control group was treated with low-molecular-weight heparin+aspirin+dydrogesterone,and the test group was treated with BSHX Decoction II on the basis of the control group.The observation time is up to 12 weeks of gestation.If the embryo stops developing,stop the observation and medication.The study observed the 12-week pass rate(main curative effect index)of the control group and the test group,and the secondary efficacy index mainly included(1)TCM syndrome scores,D-Dimer,PAGT,anti-β2GP1 antibody and ACA levels of the two groups before and after the first stage treatment;(2)6-7 weeks,10-11 weeks’ gestation,B ultrasound uterine artery blood flow(PI,RI,S/D)of the two groups;(3)Pregnancy 6-7 weeks,8-9 weeks,10-11 weeks embryo development(abnormal rate of gestational sac,embryo,sac bud)of the two groups;(4)Control group,test group,normal group before the first stage treatment and 6 weeks of pregnancy HA,VEGF,NO content.Using SPSS23.0 software for statistics,P<0.05 was considered statistically significant.Part II: Experimental Study of Bushen Huoxue Decoction on Vascular Endothelial Barrier and Blood Flow in RSA Model MiceCBA/J♀ mice were mated with DBA/2J♂ mice to establish a spontaneous abortion model,and BALB/c♀ mice were mated with DBA/2J♂ mice to establish a RSA model.The spontaneous abortion models were randomly divided into traditional Chinese medicine group,western medicine group,and model group.The traditional Chinese medicine group was given 15.0g/kg/bottle/day of Bushen Huoxue Decoction from the0.5th day of pregnancy,and the western medicine group was given 3.1 mg/kg/bottle/day of Dydrogesterone water solution from the 0.5th day of pregnancy.The normal group and the model group an equal volume of distilled water was administered on the 0.5th day of pregnancy,and each group was given intragastric administration for 13 days.Placental tissue was taken after anesthesia on the 13.5th day of pregnancy,the embryo absorption rate of each group was observed,and the placental decidua of each group was observed by HE staining method.And trophoblast cell tissue morphology changes,ELISA method to detect peripheral blood TXA2 and PGI2 content,nitrate reductase method to detect placental tissue NO content,Western Blot method to detect placental tissue VEGF and VE-cadherin protein content,RT-PCR method to detect placenta Tissue HA synthase HAS-1,HAS-2,HAS-3 and HA enzyme Hyal-1,Hyal-2,Hyal-3 m RNA content.Results:Part 1:1.The pass rate at 12 weeks of pregnancy in the control group was 75%,and the pass rate at 12 weeks in the test group was 94.87%.The difference between the two groups was statistically significant(P<0.05).2.After the first stage of treatment,the scores of TCM syndromes in the control group and the experimental group decreased(P<0.001).The comparison between the groups showed that the experimental group was better than the control group(P<0.01);The contents of PAGT,ACA,and anti-β2GP1 antibodies of the two groups all decreased(P<0.05).The anti-β2GP1 antibody of the experimental group decreased more significantly than that of the control group,and the difference was statistically significant(P<0.05).There was no significant difference between the PAGT and ACA content groups(P>0.05);There was no significant decrease in D-Dimer content in the two groups(P>0.05),and the difference between the groups was not statistically significant(P>0.05).3.Before the first stage of treatment,the content of HA in the normal group was significantly lower than that of the control group and the test group(P<0.001),and the levels of NO and VEGF in the normal group were significantly higher than the control group and the test group(P<0.001);at 6 weeks gestation,the HA content of the control group was significantly higher than that of the normal group and the experimental group(P<0.001),there was no difference between the normal group and the test group(P>0.05),and there was no difference in the levels of NO and VEGF at 6 weeks of gestation among the three groups(P>0.05).4.At 6-7 weeks and 10-11 weeks of gestation,the PI of the test group was lower than that of the control group(P<0.05);the RI of the two groups was only different at 10-11 weeks of gestation,and the RI of the test group was lower than the control group(P<0.05);There was no difference in S/D values between the two groups at 6-7 weeks and 10-11 weeks of gestation(P>0.05).5.At 6-7 weeks of gestation,the gestational sac and embryo in the test group were significantly larger than those in the control group(P<0.01);at 8-9 weeks of gestation,the embryo in the test group was significantly larger than the control group(P<0.05);at10-11 weeks of gestation,two There was no significant difference in the size of gestational sac and embryo length between the two groups(P>0.05).Part 2:1.HE staining of placental tissue:(1)Normal group: The decidual cells were densely arranged in oval shape,with rich cytoplasm,large and obvious nucleoli,located in the center of the cell body,stained light blue,with intact blood vessel walls;labyrinthine trophoblast cells were in a network and arranged in a shape,rich in cytoplasm,clear nucleus,rich in maternal blood sinusoids and clear tissue structure.(2)Model group: The decidual cells fragmentation,cytoplasmic edema,vacuolation,loss of nucleus,decreased blood vessel number;labyrinthous trophoblast cells hyperplasia and hypertrophy,extensive vacuolation of cytoplasm,thickening of interstitial membrane,maternal blood sinusoid.There was fibrin deposition,clusters of red blood cells and microthrombosis,degeneration,necrosis,and unclear structure in some areas.(3)Traditional Chinese medicine group: some decidual cells have cytoplasmic edema,vacuole-like changes,deep staining of nuclei,and inflammatory exudates can be seen in the vascular cavity;labyrinthous trophoblast cells were arranged in a network with clear nuclei,abundant cytoplasm,maternal blood sinusoids Relatively rich,the overall structure was clear.(4)In the western medicine group,some decidual cells had cytoplasmic edema,vacuole-like changes,and deepened nuclear staining,vascular endothelial cells were incomplete,and inflammatory cells infiltration could be seen in the lumen;labyrinthous trophoblast cells were arranged in a network,and some nuclear staining was deepened.The morphology was not clear,some cytoplasmic vacuoles change,maternal blood sinusoids were more abundant,and the structure of some areas was unclear.2.Embryo loss rate: normal group 5.88%,model group 22.72%,traditional Chinese medicine group 9.88%,western medicine group 11.96%.The embryo loss rate of the normal group,the traditional Chinese medicine group and the western medicine group was lower than that of the model group,and the difference was statistically significant(P<0.05);the difference between three of them were not statistically significant(P>0.05).3.The content of TXA2 in peripheral blood of pregnant mice was not statistically different among the four groups(P>0.05).The level of PGI2 in the peripheral blood of pregnant rats was significantly lower in the model group than the normal group,traditional Chinese medicine group,and western medicine group(P<0.001);except for the model group,there was no difference between the other three groups(P>0.05).4.The content of NO in placenta tissue was significantly higher than the model group in the normal group and the Chinese medicine group(P<0.01),and the Chinese medicine group was significantly higher than the western medicine group(P<0.01).5.The content of VEGF in placenta tissue the normal group is higher than that of the model group(P<0.05);there is no difference between the normal group and the traditional Chinese medicine group(P>0.05);the normal group is lower than the western medicine group(P<0.01);the traditional Chinese medicine group is lower than the western medicine group(P<0.05).6.The content of VE-cadherin in placenta tissue was higher in the normal group than in the model group(P<0.05);there was no difference between the normal group and the traditional Chinese medicine group(P>0.05);the normal group was lower than the western medicine group(P<0.001),and the traditional Chinese medicine group was significantly Lower than the western medicine group,the difference was statistically significant P<0.01.7.The content of HA synthase m RNA HAS-1 in the placenta tissue had not difference between the normal group,the traditional Chinese medicine group,and the western medicine group(P>0.05);the normal group was significantly higher than the model group(P<0.001);the model group was significantly lower than the Chinese medicine group(P<0.001);the model group was lower than the western medicine group(P<0.05).8.The content of HA synthase m RNA HAS-2 in the placenta tissue was not different between the normal group,the traditional Chinese medicine group,and the western medicine group(P>0.05);the normal group was significantly higher than the model group(P<0.001);the model group was significantly lower than the Chinese medicine group(P<0.01);the model group is lower than the western medicine group(P<0.05).9.The content of HA synthase m RNA HAS-3 in placenta tissue was significantly higher in the normal group than in the model group(P<0.01);there was no difference between the normal group and the traditional Chinese medicine group(P>0.05);the normal group was significantly lower than the western medicine group(P<0.001);the model group was significantly lower than the traditional Chinese medicine group and the western medicine group(P<0.001);the traditional Chinese medicine group was lower than the western medicine group(P<0.01).10.There was no significant difference in the levels of HA enzyme m RNA Hyal-1,m RNA Hyal-2 and m RNA Hyal-3 in the placental tissues(P>0.05).Conclusions:1.Bushen Huoxue Decoction can significantly increase the pass rate of patients with RSA in the prethrombotic state at 12 weeks of pregnancy and improve the syndrome of kidney deficiency and blood stasis in patients.2.Bushen Huoxue Decoction can significantly reduce the level of anti-β2GP1 antibody in the peripheral blood of patients with RSA in the prethrombotic state,while inhibiting the decomposition of HA,and has the effect of maintaining the integrity of the vascular barrier before and after pregnancy.3.Bushen Huoxue Decoction can improve the uterine artery blood flow pulsation index and blood flow resistance index in prethrombotic RSA patients,which was beneficial to the blood supply of early embryos.4.Bushen Huoxue Decoction can balance the content of VEGF and VE-cadherin at the maternal-fetal interface of RSA mice,promote the release of NO,inhibit vascular endothelial cell dysfunction,and promote vascular remodeling.5.Bushen Huoxue Decoction can increase the transcription of m RNA HAS-1 and m RNA HAS-2,promote the expression of HA in polymer form,and specifically inhibit the inflammatory reaction of the maternal-fetal interface. |
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