Clinical Study Of Peiyuan Bushen Antai Recipe In The Treatment Of RSA And Drug Mechanism Study Based On Endometrial Aging

Objectives:1 Observe the clinical efficacy of Peiyuan Bushen Antai Decoction in the treatment of LPD type RSA.2 To explore the mechanism of Peiyuan Bushen Antai Decoction in the treatment of RSA by means of network pharmacology and molecular docking.3 Explore the effect of progesterone-induced acute senescence of the endometrium in the"implant window period" on embryo implantation.4 Taking the acute aging of the endometrium during the "implant window period" as the starting point,explore the molecular mechanism of Peiyuan Bushen Antai Decoction in the treatment of RSA,and verify the above-mentioned network pharmacological prediction mechanism.Methods:1 Using the clinical randomized controlled study method,60 patients with luteal function insufficiency type RSA were collected and divided into treatment group with 30 cases and control group with 30 cases.The treatment group was treated with Peiyuan Bushen Antai Decoction and Dafutong,and the control group was treated with Dafutong treatment.Statistics of the general conditions of the two groups of patients,the outcome of pregnancy protection at 12 weeks,TCM syndrome scores before and after treatment,serum E2,P and?-HCG,pelvic ultrasound,liver and kidney function before and after treatment,blood routine and other safety indicators Analyze and discuss the clinical efficacy and safety of Peiyuan Bushen Antai Decoction.2 Using network pharmacology methods to predict the mechanism of Peiyuan Bushen Antai Decoction in the treatment of recurrent miscarriage,and use molecular docking to verify the results.The specific methods are as follows:By searching the TCM and TCMSP databases,the potential active ingredients of Peiyuan Bushen Antai Recipe were obtained;Obtain the corresponding target of the compound through TCMSP,Pubchem,and CHEMBL;collect RSA-related target genes through Genecards and OMIM database;use R language to obtain potential targets of Peiyuan Bushen Antai Decoction to treat RSA;obtain PPI network diagram from string database;application ClusterProfiler package common targets for GO and KEGG enrichment analysis;finally,MOE software molecular docking is used to evaluate the binding activity of important active ingredients of Peiyuan Bushen Antai Formula with key targets.3 Using a combination of in vivo and in vitro experiments,in vivo experiments:ICR female mice were randomly divided into PD0-PD9 group,anti-aging group,control group?,RU486 group,Control group?.The PD0-PD9 group was collected in the afternoon of PD0-9 days,and the control group?,the defatting group,the control group?,and the RU486 group were collected in the afternoon of PD5 and PD9 respectively.The number of embryo implantation was observed,and SA-?-gal staining was used for immunization Histochemistry and WB were used to detect the activity of SA-?-gal and the level of P16 protein in the endometrium.The PD0-PD9 group was collected in the afternoon of PD0-9 days,and the control group ?,the defatting group,the control group?,and the RU486 group were collected in the afternoon of PD5 and PD9 respectively.The number of embryo implantation was observed,and SA-?-gal staining was used for immunization Histochemistry and WB were used to detect the activity of SA-?-gal and the level of P16 protein in the endometrium.In vitro experiment:medroxyprogesterone(MPA)at different concentrations and time points was used to intervene in 2BS cells.WB was used to detect the expression of 2BS senescence marker proteins P53,P21,P16 in each group,and SA-?-gal staining to detect 2BS SA-?-a1 activity,optical microscope observation of 2BS morphological changes,EDU detection of 2BS proliferation function,RT-PCR detection of 2BS SASP,PRL mRNA expression.The senescence supernatant of 2BS induced by MPA was collected and co-cultured with HTR-8/SVneo,and the proliferation,migration and invasion functions of HTR-8/SVneo were detected by CCK8,EDU,scratch and Transwell experiments.4 Using Clark's classic recurrent miscarriage model mouse modeling method,the CBA/J female mice were randomly divided into 12 groups.Normal group B,model group B,traditional Chinese medicine low-dose group B,traditional Chinese medicine medium-dose group B,traditional Chinese medicine high-dose group B,western medicine group B,calculate the embryo loss rate at PD 14;normal group A,model group A,traditional Chinese medicine low-dose group A,Chinese medicine middle-dose group A,Chinese medicine high-dose group A,western medicine group A were collected in the afternoon of PD5,and HE staining was used to observe the area of mouse corpus luteum,Elisa to detect serum P level,and ?-gal stain to detect SA-?-gal in the inner membrane Activity,WB and RT-PCR detection of endometrial P16,P53,P21 protein and mRNA expression,IHC detection of uterine P16 protein location and expression.Results:1 Clinical effect of Peiyuan Bushen Antai Fang on LPD RSA1.1 Outcomes of fetal preservation:the treatment group's success rate of fetal preservation was 93.33%which is higher than the control group 70%(p<0.05).1.2 The efficacy of TCM syndromes:The total effective rate of the treatment group is 96.67%which is better than the control group 60%(p<0.01).1.3 TCM syndrome scores:The scores of TCM syndromes in the two sets were distinctly reduced after treatment.The treatment group is better than the control group in improving the syndrome of deficiency of both spleen and kidney in patients with LPD type RSA(p<0.05).1.4 Hemostasis time:The average hemostasis time in the treatment group was 4.29±0.46 days shorter than that of the control group 6.48±0.54 days(p<0.05).1.5 Abdominal pain disappearance time:The average abdominal pain disappearance time of the treatment group was 5.48±0.35 days shorter than that of the control group 8.63±0.84 days(p<0.01).1.6 Serum hormone levels:The serum E2 value of the treatment group was higher than the control group at 9,11,12 weeks of pregnancy(p<0.05);the serum P value of the treatment group was higher than the control at 6-7 weeks,9 weeks,and 11-12 weeks of pregnancy Group(p<0.05);the serum ?-HCG value of the treatment set was higher than that of the control set at 8-12 weeks of gestation(p<0.01).1.7 B-ultrasound examination:the treatment group had 22 cases of embryo development consistent with gestational age,6 cases of less than gestational age,and 2 cases of fetal arrest;control group had 14 cases of embryo development consistent with gestational age,7 cases of less than gestational age,and 9 cases of abortion.1.8 Adverse reactions:1 case of abnormal liver function in the treatment group,3 cases of abnormal liver function and 1 case of rash in the control group.There was no difference in safety between the two groups(p>0.05).2 Network pharmacology and molecular docking to explore the mechanism of Peiyuan Bushen Antai Decoction in the treatment of recurrent miscarriage2.1 Compound screening and prediction targets of Peiyuan Bushen Antai Recipe:186 potential compounds and 136 compound targets.2.2 RSA disease targets:1658 RSA targets were obtained after deduplication.2.3 Common target screening and interaction network construction:65 common targets,and potential targets are connected to at least two compounds respectively.2.4 Construction of PPI network and extraction of key targets:There are 65 nodes in the PPI network,of which VEGFA(47),IL6(46),EGFR(43),MPAK8(41),ESR1(39),etc.are the key targets.2.5 GO and KEGG enrichment:GO mainly involves transcription factor activity,monooxygenase activity and so on.KEGG mainly involves PI3K-Akt,MPAK,P53,apoptosis and so on.2.6 Molecular docking:The scores of core compound and potential target molecule docking are both ?-5.0 kcal/mol.3 Progesterone-induced senescence of endometrial cells in the "implantation window period" of embryo implantation3.1 The physiological manifestations of acute senescence in the inner membrane during the "implant window period":IHC showed that the expression of P16 in the PD0-PD5 group gradually increased with the increase in the number of days of pregnancy,and reached a peak at PD5,the expression of PD6-PD9 gradually decreased,and the P16 positive cells were mainly located Intimal mesenchymal fibroblasts,epithelial cells,etc.WB and ?-gal staining also gave the same result.3.2 Membrane senescence during the implantation window is conducive to embryo implantation:The number of embryo implantation,?-gal staining,and the expression of P16 protein in the inner membrane after removal of senescent cells are less than those of the control group(p<0.01).3.3 Progesterone can induce fibroblast senescence:Compared with the blank group,the?-gal positive rate of 2BS cells and the expression of senescence marker proteins P16,P21,P53 increased(p<0.01),and the cell morphology changed after MPA intervention in each group.Among them,MPA(8 ?M)was treated for 16 days,and the indexes changed significantly(p<0.01).EDU results showed that 2BS cell proliferation slowed down after MPA intervention(p<0.01).The results of RT-PCR experiments showed that the expressions of SASP and PRL mRNA such as CXCL-2 and CXCL-1 in the MPA group were higher than those in the blank group(p<0.01).3.4 Progesterone-induced cellular senescence is an important condition for embryo implantation:After RU486 blocked the effect of progesterone,the number of embryo implantation,the expression of intimal P16 protein and the positive rate of SA-?-gal staining were lower than those of the control group(p<0.01).3.5 The senescence microenvironment induced by progesterone is conducive to the function of trophoblast cells:the horizontal and vertical migration and invasion functions of HTR-8/SVneo cells co-cultured with Senescent CM are stronger than those of HTR-8/SVneo cells co-cultured with Young CM(p<0.01).4 Study on the effect of Peiyuan Bushen Antai Decoction in the treatment of recurrent abortion based on the aging of the inner membrane during the "implant window period"4.1 Embryo loss rate:the model group is more than the normal group(p<0.01),indicating that the model is successful.The traditional Chinese medicine group and the western medicine group were lower than the model group(p<0.05 or p<0.01).4.2 Serum P level:the model group is lower than the normal group(p<0.05).The traditional Chinese medicine dose group and western medicine group were higher than the model group(p<0.05 or p<0.01).4.3 The corpus luteum area of the ovary:the model group is less than the normal group(p<0.05).There were more Chinese medicine in the dose group and western medicine group than the model group(p<0.05 or p<0.01).4.4 SA-?-gal staining:the model group is less than the normal group(p<0.01).There were more Chinese medicine middle and high-dose groups and western medicine groups than the model group(p<0.01).4.5 The expression of P16,P53,P21 protein and mRNA in the inner membrane tissue:The expression of P16,P53,P21 protein and mRNA in the model group was lower than that of the normal group(p<0.05).The expression of P16,P53,P21 protein and mRNA in low and medium doses of traditional Chinese medicine and western medicine group is higher than that of model group(p<0.05).4.6 IHC detection of uterine P16 protein location and expression:P16 protein is mainly expressed in fibroblasts and epithelial cells.The mice in the model group were lower than those in the normal group(p<0.01),and the mice in the low,medium dose and western medicine groups were higher than the model group(p<0.01).Conclusion:1.Peiyuan Bushen Antai Decoction can significantly improve the deficiency of sp leen and kidney,relieve bleeding and abdominal pain,increase E2,P,?-HCG levels,p romote embryonic development,and increase the success rate of LPD RSA.2 Through network pharmacology,it is preliminarily determined that the pharmaco logical mechanism of Peiyuan Bushen Antai Decoction in the treatment of RSA may be through regulating the P53/P21 signal pathway and regulating cell senescence,there by treating RSA.3 Acute senescence of the endometrium during the "planting window period" is o ne of the necessary conditions for embryo implantation,and sufficient progesterone for a sufficient time is an important factor in inducing endometrium senescence.4 Insufficient P and insufficient intimal senescence in the "implant window period"may be the pathogenesis of LPD-type RSA.Peiyuan Bushen Antai Decoction may i ncrease the expression of P53,P21,and P16 in the endometrium during the "implant window period" by increasing the serum P and the area of ovarian corpus luteum,and promote the senescence of the endometrium during the "implant window period" to p lay a role in preventing pregnancy.