Network Pharmacology-based Study of Bushen Zhuyun Prescription in Treatment of Recurrent Spontaneous Abortion / 中国实验方剂学杂志

Objective:To explore the active components， targets， and signaling pathways responsible for Bushen Zhuyun prescription in treating the recurrent spontaneous abortion （RSA） based on network pharmacology and uncover its potential mechanism by molecular docking and in vitro cell experiments. Method:The active components of Bushen Zhuyun prescription were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， Traditional Chinese Medicine Integrated Database （TCMID） and the published articles， followed by the prediction of drug action targets based on such platforms as DrugBank and SwissTargetPrediction. GeneCards and Online Mendelian Inheritance in Man （OMIM） were searched to obtain the RSA targets， which were then intersected with the targets of Bushen Zhuyun Decoction. Following the plotting of Bushen Zhuyun prescription-compound-target-RSA network by Cytoscape 3.7.1， the protein-protein interaction （PPI） network was then constructed with STRING for screening the core network. The resulting common targets were then subjected to Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis using R software. Autodock Vina 1.1.2 was used for molecular docking. The activation of phosphatidylinositol 3-kinase/protein kinase B （PI3K/AKT） signaling pathway by Bushen Zhuyun prescription was verified in human umbilical vein endothelial cells （HUVEC） <italic>in vitro</italic>. Result:It was found that 49 potential active components of Bushen Zhuyun prescription might act on 133 RSA targets. GO enrichment analysis yielded 470 biological processes， with angiogenesis， vascular development， cellular proliferation， and oxidative activity mainly involved. KEGG enrichment analysis revealed 103 signaling pathways （<italic>P</italic><0.05）， and the PI3K/AKT signaling pathway， advanced glycation end product （AGE）/receptor for advanced glycation end product （RAGE） signaling pathway， and tumor necrosis factor （TNF） signaling pathway were the main ones. As indicated by molecular docking， the Vina scores of the main active component kaempferol with AKT1 and vascular endothelial growth factor A （VEGFA） were the lowest and similar. It was confirmed <italic>in vitro</italic> cell experiments that Bushen Zhuyun prescription activated the PI3K/AKT signaling pathway and up-regulated the expression of VEGFA and downstream AKT protein to promote angiogenesis. Conclusion:Bushen Zhuyun prescription promotes angiogenesis at the maternal-fetal interface by regulating angiogenesis and cellular proliferation， activating the PI3K/AKT pathway， and up-regulated the VEGFA expression， which is beneficial to the formation of placenta in early pregnancy and the maintenance of early pregnancy. This study has provided ideas for new drug development.

Effects of aspirin, vitamin D3, and progesterone on pregnancy outcomes in an autoimmune recurrent spontaneous abortion model

High proportions of placental lymphocytes expressing DX5+/CD25+/FOXP3+/CD45+/CD4+ are beneficial to maintain immune tolerance and improve pregnancy outcomes. This study aimed to compare and evaluate the therapeutic effects of aspirin, vitamin D3 (VitD3), and progesterone on the autoimmune recurrent spontaneous abortion (RSA) model. The autoimmune RSA mouse model was constructed, and the embryo loss rate was calculated for each group. Then, primary mouse placental lymphocytes were isolated, and the expression of DX5+/CD25+/FOXP3+/CD45+/CD4+ was detected through flow cytometry. The serum levels of anti-cardiolipin antibody (ACA), β2-GP1, CXCL6, IFN-γ, and IL-6 were measured by ELISA to evaluate the proportion of Th1 and Th2 cells. Autoimmune RSA significantly increased the embryo loss rate, which was improved by aspirin, VitD3, and progesterone treatment, and progesterone treatment had the best effect among the three treatments. The positive expression of DX5+/CD25+/FOXP3+/CD45+/CD4+ in the VitD3 and progesterone groups was significantly higher than that in the autoimmune RSA group, and the expression was highest in the progesterone treatment group. In the plasma of autoimmune RSA mice, the ACA, β2-GP1, CXCL6, and IFN-γ levels were significantly higher and the IL-6 level was lower than the levels in control mice. All these changes could be reversed by aspirin and progesterone treatment. In conclusion, aspirin, VitD3 and progesterone treatment improved pregnancy outcomes in autoimmune RSA mice by regulating the Th1/Th2 balance and cytokines, and progesterone had the best effect of the three treatments.

Intervention of Modified Yupingfeng San Combined with Aspirin on D-dimer， PagT Levels of RSA with Seronegative APS / 中国实验方剂学杂志

Objective:To observe the clinical efficacy of aspirin combined with modified Yupingfeng San on patients with recurrent spontaneous abortion and Seronegative antiphospholipid syndrome. Method:From December 2016 to March 2018, 109 cases of recurrent spontaneous abortion patients with seronegative antiphospholipid syndrome in Affiliated Hospital of Nanjing University of Chinese Medicine were selected as the study objects. According to the random number table, they were divided into three groups:blank group, control group and study group. The blank group was treated with folic acid tablets, 1 pill/time, qd, as placebo, the control group was treated with folic acid tablets, 1 pill/time, qd, Danshen pills, 4 pills/time, tid, and aspirin, 1 pill/time, tid, and the study group was treated with folic acid tablets, 1 pill/time, qd, aspirin, 1 pill/time, tid, and modified Yupingfeng San, 150 mL/time, bid. These patients were all asked for pregnancy preparation for 1 year and treated with aspirin, 1 pill/time, tid, and progesterone pills, 1 pill/time, tid, since the beginning of pregnancy until 13 weeks or abortion. The pregnancy rate, miscarriage rate, coagulation index before and after treatment, traditional Chinese medicine (TCM) symptom score as well as months of abortion were all observed and compared. Result:The coagulation index and TCM symptom score of the study group were significantly lower than those before treatment (PPPPPConclusion:The data proved that the combination of anti-coagulation medicine with either single herb or herbal compound can improve the curative effect of the patients, and traditional Chinese herbal compound can regulate the immune system, reduce the thrombosis and improve the symptoms of TCM, which made the curative efficacy more prominent. However, more samples were needed for further research. Because the time of fetal protection may not change the outcome of pregnancy, the therapy should be maintained at least until the end of the early pregnancy.

Professor LU Qibin's Clinical Experience in the Diagnosis and Treatment of Recurrent Spontaneous Abortion / 浙江中医药大学学报

[Objective]In this paper, valuable experience would be shared and the culture of Chinese medicine will be inherited through introducing the clinical experience of Professor LU Qibin in the diagnosis and treatment of RSA. [Methods]From the point of cause and pathology as well as therapeutic method of the disease, the author discussed the special treatment of Professor LU Qibin. At the same time, the author focuses on introducing the dialectics and characteristics of professor LU Qibin's treatment of complicated recurrent spontaneous abortion. In order to elaborate clearly, some clinical cases were reported. [Results]Professor LU Qibin has summarized that spleen-kidney deficiency is the foundation of recurrent spontaneous abortion. In consequence, invigorating the spleen and kidney is the basic treatment. For the complicated recurrent spontaneous abortion, Professor LU Qibin has many assistant methods on the basis of invigorating spleen and kidney. Such as clearing heat and activating blood, warming and invigorating the spleen and kidney and so on. Furthermore, Professor LU Qibin insists the treatment of traditional Chinese medicine coordinate with western medical therapies. In the reported cases, patients were successfully cured. [Conclusion]Professor LU Qibin is experienced and the clinical curative effect is remarkable. Hence, the clinical experience of Professor LU Qibin in the diagnosis and treatment of recurrent spontaneous abortion was worth of learning and wide application.

Women with recurrent spontaneous abortion have decreased 25(OH) vitamin D and VDR at the fetal-maternal interface

Immunological mechanisms have been proposed to underlie the pathogenesis of recurrent spontaneous abortion (RSA). Vitamin D has a potent immunomodulatory effect, which may affect pregnancy outcome. The objective of this study was to investigate 25-hydroxyvitamin D [25(OH) D] concentration and vitamin D receptor (VDR) expression in the decidual tissues of RSA patients. Thirty women with RSA (RSA group) and thirty women undergoing elective abortion (control group) were recruited during 2016 from gynecology outpatient clinics. We measured 25(OH) D, interleukin (IL)-17, IL-23, transforming growth factor β (TGF-β), VDR and 1-α-hydroxylase (CYP27B1) in decidual tissues collected during the abortion procedure. In the RSA group, 25(OH) D and TGF-β were significantly decreased while IL-17 and IL-23 were significantly increased compared with the control group. VDR expression was significantly decreased in the RSA group compared with the control group. Logistic regression analysis showed a significant negative correlation between 25(OH) D in decidual tissues and RSA. These results indicated that vitamin D concentrations in the decidua are associated with inflammatory cytokine production, suggesting that vitamin D and VDR may play a role in the etiology of RSA.

The single nucleotide polymorphism rs2305957 G/A is not associated with recurrent pregnancy loss.

Background: This study was conducted in order to investigate the association between the single nucleotide polymorphism (SNP) rs2305957 G/A and recurrent pregnancy loss (RPL) in a group of Palestinian women residing in Gaza strip. Methods: A retrospective case-control study was carried out during the period (May to August, 2015). A total of 380 females, 190 RPL patients and 190 control women without previous history of RPL, aged 20–35 years were included in the study. The SNP was analyzed by tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR). Results: No statistically significant difference existed between RPL cases and controls in terms of the allelic and genotypic distribution of rs2305957 G/A. Conclusions: SNP rs2305957 G/A does not represent a risk factor for RPL in the investigated population.

Reliability and validity of resilience scale for adults(RSA) in military students / 中华行为医学与脑科学杂志

Objective To research the reliability and validity of resilience scale for adults(RSA)in military students.Methods RSA, depression, anxiety and somatization of Self-report Symptom Checklist 90(SCL-90)were applied in this survey to 616 military students.Results ①A 5-factor structure of resilience was showed by explorative factor analysis.Each item had a factor loading between 0.423～0.834, and the total variance explained at 58.439%.②The correlations between the subscales of the RSA were all obvious positive, ranging from 0.228～0.580；and the correlations between subscales of the RSA and total resilience were all obvious positive, ranging from 0.565～0.789.③There was a obvious negative correlation between total depression, anxiety and somatization and total resilience, the correlation coefficient was-0.437(n=593, P=0.000).④Tbe reliability coefficient of the RSA was 0.91, and the respective dimensions had Cronbach's alphas between 0.52～0.86.Conclusion The results show that RSA has acceptable reliability and validity in military students.

Case-control study of the polymorphisms of CYP2E1-RsaⅠ and STT1 genes and susceptibility to pancreatic cancer / 西安交通大学学报(医学版)

Objective To investigate the correlation between the combination of smoking with CYP2E1-RsaⅠ and GSTT1 genes polymorphisms and pancreatic cancer. Methods The genetic polymorphisms of CYP2E1-RsaⅠ and GSTT1 were analyzed by polymorphism-polymerase chain reaction (PCR) technique in peripheral blood leukocytes of 150 pancreatic cancer cases and 150 non-cancer controls. Results The frequency of CYP2E1-RsaⅠ(c1/c1) and GSTT1(-) was 38.7% and 69.3% in pancreatic cancer cases and 20.7% and 44.7% in healthy controls, respectively. Statistical tests showed significant differences in the frequencies between the two groups (χ~2=15.75, P<0.01; χ~2=18.62, P<0.01). The risk of pancreatic cancer patients with CYP2E1-RsaⅠ(c1/c1) was significantly higher than that of controls (OR=3.19, 95% CI=2.53-4.26). The individuals who carried with GSTT1(-) had a higher risk of pancreatic cancer (OR=2.85, 95% CI=1.92-4.64). Combined analysis of the polymorphisms showed that the percentage of CYP2E1-RsaⅠ(c1/c1)/GSTT1(-) in pancreatic cancer and control groups was 30.7% and 6.7%, respectively (χ~2= 42.39, P<0.01). People who carried with CYP2E1-RsaⅠ(c1/c1)/GSTT1(-) had a higher risk of pancreatic cancer (OR=16.63, 95% CI=8.94-22.01). The smoking ratewas significantly higher in the case group than in the control group (OR=2.74, 95% CI=1.32-4.58, P<0.01), and statistical analysis suggested an interaction between smoking and CYP2E1-RsaⅠ(c1/c1) or GSTT1(-) genotypes polymorphisms which increased the risk of pancreatic cancer (OR=8.84, 95% CI=5. 51-11.62; OR=20.40, 95% CI=4.98-29.53). Conclusion CYP2E1-RsaⅠ(c1/c1) and GSTT1(-) are the risk factors in pancreatic cancer. Smoking is also related to the susceptibility to pancreatic cancer. There may be a synergetic interaction among CYP2E1-RsaⅠ(c1/c1) and GSTT1(-) and smoking on the elevated susceptibility of pancreatic cancer.

Preimplantation Genetic Diagnosis for Aneuploidy Screening in Patients with Poor Reproductive Outcome / 대한불임학회지

OBJECTIVES: The risk of aneuploidies of embryos increases in advanced maternal age or parental karyotype abnormality and it results in poor reproductive outcomes such as recurrent spontaneous abortion (RSA) or repeated implantation failure (RIF). Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) can be applied for better ART outcome by selecting chromosomally normal embryos. The aim of this study is to evaluate the clinical outcome of PGD-AS and which group can get much benefit from PGD-AS among the patients expected to have poor reproductive outcome. METHODS: In 42 patients, 77 PGD cycles were performed for aneuploidy screening. Patients were allocated to 3 groups according to the indication of PGD-AS: group I-patients with old age (> or =37) and RIF more than 3 times (n=11, mean age=42.2 yrs.), group II-patients with RSA (> or = 3 times) associated with aneuploid pregnancy (n=19, mean age=38.9 yrs.), group III-parental sex chromosome abnormality or mosaicism (n=18, mean age=29.6 yrs.) including Turner syndrome, Klinefelter syndrome and 47,XYY. PGD was performed by using FISH for chromosome 13, 16, 18, 21, X and Y in group I and II, and chromosome X, Y and 18 (or 17) in group III. RESULTS: Blastomere biopsy was successful in 530 embryos and FISH efficiency was 92.3%. The proportions of transferable embryos in each group were 32.5+/-17.5%, 23.0+/-21.7% and 52.6+/-29.2% (mean +/- SD), respectively, showing higher normal rate in group III (group II vs. III, p<0.05). The numbers of transferred embryos in each group were 3.9+/-1.5, 1.9+/-1.1 and 3.1+/-1.4 (mean +/- SD), respectively. The clinical pregnancy rates per transfer was 0%, 30.0% and 20.0%, and it was significantly higher in group II (group I vs. group II, p<0.05). The overall pregnancy rate per transfer was 19.6% (10/51) and the spontaneous abortion rate was 20% (2/10) of which karyotypes were euploid. Nine healthy babies (one twin pregnancy) were born with normal karyotype confirmed on amniocentesis. CONCLUSION: Our data suggests that PGD-AS provides advantages in patients with RSA associated with aneuploidy or sex chromosome abnormality, decreasing abortion rate and increasing ongoing pregnancy rate. It is not likely to be beneficial in RIF group due to other detrimental factors involved in implantation.

Peripheral Th1 and Th2 Lymphocytes Values by Intracellular Cytokine Staining for Interferon-and Interleukin-4 in the Patients with Repeated Spontaneous Abortion / 대한진단검사의학회지

BACKGROUND: In the women who experienced recurrent spontaneous abortions (RSA), cellular immune abnormalities of peripheral blood Natural Killer (NK) cell have been reported and the imbalance of Th1 and Th2 response to the trophoblasts may lead to pregnancy loss. This study was performed to evaluate the Th1 and Th2 lymphocytes as a more useful parameter for the impending status of RSA. METHODS: Peripheral blood samples from 17 women with a history of RSA of unknown etiology, 16 normal pregnant women, and 11 non-pregnant women with a history of RSA were studied. Peripheral Th1 and Th2 lymphocytes were measured by intracellular cytokines staining for interferon (IFN)-and Interleukin (IL)-4 using FACScan (Becton-Dickinson Co. San Jose, CA, USA) and FASTIMMUNE (TM) cytokine detection system. RESULTS: The percentage of Th1 cells (10.3+/-.9% and 13.3 +/-.0%) and Th2 cells (1.9+/-.5% and 7.2+/-.8%) measured at the second and fourth month of gestation, respectively, of the RSA patients was significantly higher compared with Th1 (8.1+/-.9%) and Th2 (0.5+/-.5%) cells among the non-pregnant control group with past abortion history. The ratio of the Th1/Th2 cells is not a useful predicting marker of impending abortion. CONCLUSIONS: Th1 and Th2 cell counts by flowcytometric staining of IFN-gamma /IL-4 intracellular cytokine detection may be a useful diagnostic tool and potential parameter to identify impending abortion involving abnormal immune alteration in reproductive processes.

Booster immunization with paternal lymphocytes to recurrent spontaneous abortors of less reaction to paternal lymphocyte immunization / 中国免疫学杂志

Objective:To evaluate curative impact of booster immunization with paternal lymphocytes on recurrent spontaneous abortors(RSA)with less reaction of primary patermal lymphocyte immunization.Methods:RSA patients with insufficient materno-fetal immuno-recognition were selected by flow cytometry of blocking antibody analysis and immunized with either induced paternal lymphocytes pretreated by IFN-? in vitro to those of anti-CD3-BE and anti-CD4-BE Ab lower than 0% or direct intradermal vaccination with their paternal lymphocytes without IFN-? pretreatment to those of anti-CD3-BE Ab beyond 0%.Reassessment of blocking antibodies was performed at the end of the second immnunization course.Results:Levels of blocking antibodies were significantly raised after the secondary booster immunization in RSA with insufficient materno-fetal immnuno-recognition whose blocking antibodies continuously decreased after being treated by the primary paternal lymphocyte immunization.No improvement of parameters was observed except the blocking effect in patients receiving secondary direct intradermal vaccination treatment.Conclusion:It is necessary for the RSA with insufficient materno-fetal immuno-recognition to experience secondary booster immunization preferably with paternal lymphocytes pretreated by IFN-? in vitro.

Study on association of HLA-E with recurrent spontaneous abortion / 中国免疫学杂志

Objective:To explore the mechanism of association between recurrent spontaneous abortion(RSA) and the polymorphism of HLA E,had detected the frequencies of five HLA E alleles in 53 women with RSA and 156 random Shanghai Chinese controls.Methods:PCR sequence specific oligonucleotide (PCR SSO) typing was carried to detect polymorphism in exon2 and exon3 of HLA E gene.Results:HLA E *0101 is the most common allele in both RSA patients and controls(50% and 43.09% respectively).HLA E *01031 and E *01032 were detected with allele freqencies of 23.79% and 33.12% in controls,20.75% and 29.25% in RSA.No HLA E *0102 and E *0104 alleles could be detected in both groups.Conclusion:Maternal HLA E polymorphism does not associated with pathogenesis of RSA.

Brain Stem Auditory Evoked Potential and R-R interval Analysis of the Workers Exposed to Trichloroethylene / 대한산업의학회지

In order to evaluate the effects of trichloroethylene (TCE) on the central and autonomic nervous system, the brain stem auditory evoked potentials (BAEPs) and the electrocardiographic R-R interval variability (CV(rr)) were measured in three groups of workers (11) working in TCE degreasing process, workers (4) exposed to lead and two groups of workers (12) not exposed to any kinds of neurotoxicants. Two components of the CVrr reflecting parasympathetic activity (C-CV(rsa)) and sympathetic and parasympathetic activities (CCV(mwsa)) were examined. The TCE workers were exposed to TCE at the level of less than 1 hour per week while the degreasing material was changed. There were no differences in the latencies of BAEPS and CV(rr) and the components of the CV(rr) between control and TCE and lead groups. However, two lead workers had the blood lead level of less than 60 g/dl recommended as present biological guideline by the Ministry of Labor, showed the delayed latencies of V5 and I-V in BAEP. This results suggested that intermittent TCE erposure didn't affect the auditory nervous pathway and the autonomic nervous system, however, lead exposure might affect the central nervous system at the level of less than 60 g/dl in blood lead.

Case-control study of the polymorphisms of CYP2E1-RsaⅠ and GSTT1 genes and susceptibility to pancreatic cancer / 西安交通大学学报(医学版)

Objective To investigate the correlation between the combination of smoking with CYP2E1-RsaⅠ and GSTT1 genes polymorphisms and pancreatic cancer.Methods The genetic polymorphisms of CYP2E1-RsaⅠ and GSTT1 were analyzed by polymorphism-polymerase chain reaction(PCR) technique in peripheral blood leukocytes of 150 pancreatic cancer cases and 150 non-cancer controls.Results The frequency of CYP2E1-RsaⅠ(c1/c1) and GSTT1(-) was 38.7% and 69.3% in pancreatic cancer cases and 20.7% and 44.7% in healthy controls,respectively.Statistical tests showed significant differences in the frequencies between the two groups(?2=15.75,P