ObjectiveTo observe the effect of Cuscutae Semen total flavonoids combined with Tripterygium wilfordii polyglycoside tablets （TWPT） on ovarian germline stem cells of female physiological mice through neurogenic locus notch homolog （Notch） signaling pathway. MethodSixty female Kunming mice （5 weeks old） were randomly divided into normal group， Tripterygium wilfordii polyglycoside tablets low-， high-dose groups （13.65 mg·kg^-1·d^-1 and 27.3 mg·kg^-1·d^-1， 1 and 2 times clinical equivalent dose）， Cuscutae Semen total flavonoids low- and high-dose groups （150 mg·kg^-1·d^-1 and 300 mg·kg^-1·d^-1）， and combination group （13.65 mg·kg^-1·d^-1 TWPT and 150 mg·kg^-1·d^-1 Cuscutae Semen total flavonoids）， with 10 in each group. After 3 weeks of continuous administration， the uterus/brain and ovarian/brain indexes were calculated， and the pathological changes of ovarian tissue were observed under light microscope. The content of estradiol in serum was determined by enzyme linked immunosorbent assay （ELISA）. Immunofluorescence assay was performed to observe the expressions of germline stem cell markers in ovarian epithelium， including mouse vasa homologue （Mvh）， octamer-binding transcription factor 4 （Oct4）， tyrosine-protein kinase receptor （c-kit）， Nanog， Notch signaling pathway molecules， neurogenic locus notch homolog protein 1 （Notch1）， hes family BHLH transcription factor 1（Hes1）， and jagged canonical Notch ligand 1 （JAG1）. ResultCompared with the normal group， low and high doses of TWPT had no significant effect on the uterus/brain and ovary/brain indexes and the uterus and ovary morphologies of mice， while only the number of atretic follicles was increased （P<0.01）. The expressions of ovarian germline stem cell markers and Notch signaling pathway molecules had a decreasing trend in TWPT low-dose group， while the expressions of Mvh， c-kit， and Nanog were down-regulated （P<0.05， P<0.01） and the expressions of Notch1 and Hes1 were also reduced （P<0.01） in TWPT high-dose group. However， the above indexes were increased in Cuscutae Semen total flavonoids low-dose group （P<0.05， P<0.01）. Compared with the low does of TWPT group， the combination group had a decrease in the increased number of atretic follicles （P<0.01）， an improvement in the down-regulated expressions of Mvh and Nanog （P<0.01）， and an increase in the expressions of Notch1 and Hes1 （P<0.05， P<0.01）. ConclusionOvarian germline stem cells are the source target of the reproductive toxicity of TWPT. Cuscutae Semen total flavonoids participate in the regulation of the germline stem cell pathways to alleviate the reproductive toxicity caused by TWPT， and its mechanism of action may be related to the Notch signaling pathway.

Objective:To explore the value of karyotyping and chromosomal microarray analysis (CMA) in the prenatal diagnosis of balanced translocation/inversion carriers.Methods:This was a retrospective study involving 117 balanced translocation/inversion carrier couples. Among them, 90 women had a history of spontaneous abortion(≥2 times), stillbirth, fetal multiple malformations, or giving birth to children with chromosome abnormality disease and the peripheral blood karyotyping and fluorescence in situ hybridization testing confirmed that one partner was balanced translocation/inversion carrier. The present pregnancies of these cases were spontaneous and lasted until 18-25 weeks. The other 27 cases were confirmed by chromosome examination at the present pregnancy after the indication of fetal structural abnormalities by fetal karyotyping due to advanced maternal age and abnormal ultrasound and prenatal screening results. The results of karyotyping and CMA by amniocentesis during 18 to 25 gestational weeks were all summarized and described. Results:The successful rate of both methods was 100.0% (117/117). Unbalanced and balanced translocation/inversion were detected in seven (6.0%) and 39 (33.3%) fetuses by karyotyping, respectively. CMA revealed 14 fetuses with pathogenic copy number variation (CNV) and one with variants of uncertain significance(VUS), with an anomaly detection rate of 12.8% (15/117). Among the 15 cases with CNV, 13 were related to the parental translocation/inversion, one with de novo mutation (22q11.2 microdeletion syndrome), and one Duchenne muscular dystrophy mutation carrier. Based on the results of karyotype and CMA, there were 12 fetuses with unbalanced chromosomal fragments (10.3%), 37 fetuses with balanced translocation/inversion (31.6%), and 68 fetuses with normal chromosomes (58.1%). Conclusions:The combination of karyotyping and CMA can provide more accurate prenatal genetic diagnosis when one of a couple carries balanced chromosomal translocations/inversion.

ObjectiveTo study the constituents migrating to blood of Qingyan formula by serum pharmacochemistry， and investigate the binding energy between these constituents and estrogen receptor （ER）， so as to confirm the pharmacodynamic material basis of Qingyan formula in rats. MethodUltra-high performance liquid chromatography-quadrupole electrostatic field-orbital trap high resolution mass spectrometry （UPLC-Q-Orbitrap-MS） was used to determine the constituents migrating to blood of Qingyan formula in rats by comparing the fingerprint differences of 70% ethanol extract of Qingyan formula， 70% ethanol extract of each single drug in this formula， blank serum and serum after administration of 70% ethanol extract of Qingyan formula， according to the retention time， relative molecular weight and the primary and secondary ion fragments provided by MS. Mobile phase was 0.1% formic acid aqueous solution （A）-acetonitrile（B） for gradient elution （0-5 min， 2%-20%B； 5-10 min； 20%-50%B； 10-15 min， 50%-80%B； 15-25 min， 80%-95%B； 25-26 min， 95%-2%B； 26-30 min， 2%B）， the flow rate was 0.3 mL·min^-1 and the injection volume was 5 μL， electrospray ionization was used with detection range of m/z 150-2 000， positive and negative ion scanning modes. Molecular docking technology was used to characterize the binding energy of constituents migrating to blood with ERα and ERβ， and to confirm the material basis of this formula. ResultAfter oral administration of Qingyan formula， 30 components were detected in serum， of which 9 were prototype components and 21 were metabolites. Nine prototype components were identified as monotropein， asperuloside， verbascoside， β-ecdysone， allantoin， deacetyl asperuloside acid， echinacoside， betaine and caffeic acid， 21 metabolites mainly included organic acids， amino acids， cholines and so on. The binding energies of the above 9 prototype components with ERα were -6.7， -8.9， -6.0， -5.7， -5.3， -4.9， -7.3， -3.3， -6.3 kcal·mol^-1 （1 kcal≈4 184 J）， and the binding energies of them with ERβ were -6.6， -7.2， -7.7， 8.0， -7.4， -5.5， -6.9， -3.6， -6.4 kcal·mol^-1， respectively. ConclusionThese nine prototype components into blood are the active ingredients of Qingyan formula that play estrogen-like role in the body， which can provide experimental basis for the formulation of quality standards and subsequent research and development of Qingyan formula.

Objective To explore the mutation characteristics of DMD gene in patients with Duchenne or Becker muscular dystrophy and female carriers, to provide effective prenatal diagnosis. Methods Samples were collected from 94 male patients clinically diagnosed with Duchenne or Becker muscular dystrophy and 121 corresponding female relatives from Qingdao Women and Children′s Hospital from June 2011 to October 2018. Multiplex ligation-dependent probe amplification (MLPA) was used to detect their DMD gene, and 23 high risk pregnants were performed prenatal diagnosis. Any candidate of DMD gene single-exon deletion was validated by further PCR amplification. The sample with whole DMD gene deletion was confirmed by chromosomal microarray analysis (CMA) to detect copy number variations and break site. Results Among 94 clinical Duchenne or Becker muscular dystrophy patients, 66(70.2%, 66/94) were detected gene mutation; 56 cases were exon deletion mutation and 10 cases were duplication mutation. In 121 female relatives, 48 cases (39.7%, 48/121) were diagnosed as carriers. The mutation carrying rate, was 64.5% (40/62) identified in 62 mothers of Duchenne or Becker muscular dystrophy patients. Five Duchenne or Becker muscular dystrophy fetuses and 5 carrier fetuses were prenatally diagnosed in 23 high risk pregnants. Two children with the entire DMD gene deletion were identified more deletions at Xp21, with deletions of 6.66 Mb and 10.64 Mb respectively. Conclusions MLPA may be an important method to detect DMD gene mutation of deletion and duplication. Therefore, the diagnosis of probands, female carriers and making an effective prenatal diagnosis are essential to reduce the birth of children with Duchenne or Becker muscular dystrophy.

OBJECTIVE: To assess the value of chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) for the analysis of abortic tissues. METHODS: A total of 242 samples of spontaneous abortion were collected and tested by CMA or NGS. RESULTS: The detection was successfully in 238 cases (98.35%). In total 143 cases of chromosomal abnormalities were detected, which accounted for 60.08% of all cases. Numerical chromosomal abnormalities were found in 133 cases(93.01%), structural abnormalities were found in 9 cases (6.29%), and uniparental disomy was found in 1 case(0.70%). CONCLUSION Both CMA and NGS have the advantages of high-throughput, good coverage, high resolution and rapid analysis. They can be used for the detection of the causes of spontaneous abortions. CMA is more useful for the detection of aneuploidies and uniparental disomy, while NGS has advantages in its throughput, capacity in detecting low percentage chimerism and cost, which can provide more options for clinicians.
Abortion, Spontaneous ; genetics ; Chromosome Aberrations ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Microarray Analysis ; Pregnancy

ABSTRACT:Objective To explore role of U0126,the specific inhibitor of ERK signaling pathway,in early brain injury (EBI)and the autophagy of nerve cells in hippocampus area in subarachnoid hemorrhage (SAH). Methods A total of 48 male adult SD rats were randomly divided into control group,SAH group,DMSO+SAH group,and U0126+SAH group,with 12 in each.We established SAH rat model by the puncture of internal carotid artery.The same amount of saline water,DMSO and U0126 solution of 0.5 mL per rat was injected respectively into the rats of different groups 30 min before modeling.The rats were killed at 24 h.To measure brain water content by Wet and dry method after 24 h,the morphological changes of hippocampus CA1 neural cells were observed by microscopy;the expression levels of ERK,Beclin-1 and LC3 were detected by using immunohistochemical method. Results Compared with that in sham group,brain water content increased obviously in SAH model group.The density of surviving neurons in SAH group was significantly lower than that in control group (P<0 .0 5 ).ERK signaling pathway was activated obviously,the expressions of Beclin 1 and LC3-Ⅱ were significantly higher than those in control group (P<0.05).Compared with SAH model group,in U0126 group brain water content increased obviously.Compared with those in SAH group,the density of surviving neurons was significantly lower (P<0.05), ERK signaling pathway was suppressed,the expressions of Beclin-1 and LC3-Ⅱ were significantly lower (P<0.05). Conclusion The U0126,the ERK signaling pathway inhibitor,can inhibit neuron autophagy and increase EBR of SAH.

Objective To explore the effect of extracellular regulated protein kinases (ERK) signaling pathway on early brain injury and autophagy of nerve cell in hippocampus area in rats with subarachnoid hemorrhage (SAH). Methods Forty-eight adult male Sprague-Daw-ley rats were randomly divided into sham group, SAH group, SAH+dimethyl sulfoxide (DMSO) group and SAH+U0126 group, with 12 rats in each group. The SAH model was established with puncture of internal carotid artery. The SAH+U0126 group was injected with U0126 0.05 mg/kg;the sham group and SAH group were injected with normal saline, and the SAH+DMSO group was injected with DMSO 30 min-utes before modeling. They were sacrificed 24 hours after modeling. The brain water content was measured with wet and dry method. The morphology changes of neural cells in hippocampus CA1 were observed by HE staining. The expression of phosphorylation ERK (p-ERK), Beclin-1 and LC3-Ⅱwere detected with immunohistochemical method and Western blotting. Results Compared with the sham group, the brain water content increased (P<0.05), the number of survival neurons decreased (P<0.05), the expression of p-ERK, Beclin-1 and LC3-Ⅱincreased in SAH group (P<0.05). Compared with SAH group, the brain water content increased, the number of survival neurons decreased (P<0.05), the expression of p-ERK, Beclin-1 and LC3-Ⅱ decreased in SAH+U0126 group (P<0.05); and no significant difference was found in SAH+DMSO group (P>0.05). Conclusion The activation of ERK signaling pathway may alleviate early brain injury after SAH by regulation of autophagy.

Objective To compare the cumulative analgesic effects of electroacupuncture at Sanyinjiao (SP6), Xuanzhong (GB39) and non-acupoint in treating primary dysmenorrhea. Method By adopting a multi-centered randomized controlled study method, 501 patients recruited from Dongzhimen Hospital of Beijing University of Chinese Medicine, China-Japan Friendship Hospital, Beijing Hospital of Traditional Chinese Medicine of Capital Medical University, First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Huguosi Hospital of Chinese Medicine of Beijing University of Chinese Medicine and the Outpatient of Shandong University of Traditional Chinese Medicine were randomized into a Sanyinjiao group, a Xuanzhong group, and a non-acupoint group, 167 subjects in each group. The electroacupuncture intervention was applied when dysmenorrhea flared up and the Visual Analogue Scale (VAS) ≥40 mm, with frequency at 2/100 Hz and intensity during patient’s endurance, 30 min each time, once a day, and for successive 3 d. Before the first treatment, 30 min after the first treatment, and respectively prior to the second and third treatment, VAS was used to measure the pain intensity. Meanwhile, the Retrospective Symptom Scale (RSS-COX 2) was investigated before the first treatment, right after the removal of needles for the first treatment, before the second and third treatment. Result The decrease of VAS in Sanyinjiao group was more significant than that in Xuanzhong group and non-acupoint group (MD=﹣2.92 mm, P=0.028; MD=﹣3.47 mm, P=0.009), while there was no significant difference between Xuanzhong group and non-acupoint group (MD=﹣0.56 mm, P=0.674); there were no significant differences in comparing the RSS-COX2 total score among the three groups (P=0.086). Conclusion Sanyinjiao (SP6) can produce a more significant cumulative analgesic effect for primary dysmenorrhea patient than Xuanzhong and non-acupoint, and the effects of Xuanzhong and non-acupoit are equivalent.

ObjectiveTo study the anatomical abnormalities of basal ganglia and research their influence on depression status in patients with post stroke depression (PSD)by diffusion tensor imaging (DTI) of MRI.MethodsPatients with basal ganglia infarction were recruited,and divided into groups of PSD and non depression control group by Hamilton Depression Rating Scale (HAMD) assessment. All the patients were evaluated with National Institute of Health Stroke Scale ( NIHSS). And the patients were checked by DTI sequence.Fractional anisotropy (FA),average diffusion coefficient (ADC) values and the number of nerve fiber were measured in bilateral caudatum,pallidum,putamen and thalamus.ResultsThe score of NIHSS (6.29 ± 3.45 ) was significantly higher in PSD group than that in non-depression group (3.95 ± 1.90 ;t =2.219,P =0.036). No significant difference was found between the two groups for the DTI data of the basal ganglia nuclei ( t =0.056-1.618,all P ＞ 0.05 ). Compared with contralateral construction (0.40 ± 0.02 ),the FA value decreased in the left putamen ( 0.37 ± 0.03 ) in the PSD group ( t =2.243,P =0.045 ).By Spearman correlations analysis,the HAMD score was positively correlated with NIHSS score ( r =0.464,P =0.017 ),and negatively correlated with the FA values of left pallidum (r=-0.563,P=0.005),right pallidum (r=-0.416,P=0.035) and left putamen (r =-0.428,P =0.029).Conclusions The occurrence of PSD was associated with neurological functional deficit following basal ganglia infarction.The depression level was correlated with the increasing of NIHSS score,the reductions in bilateral pallidum and left putamen FA values.This research contributes to evaluation of the PSD status in patients with basal ganglia infarction.

In this study we implanted magnetically labeled neural stem cells (NSCs) in PD rats and then monitored their survival and migration in the host brain by magnetic resonance imaging (MRI). The mesencephalic NSCs were obtained from the brain of SD rats. Superparamagnetic iron oxide (SPIO) was transferred to NSCs by Lipofectamine transfection. Eighteen PD lesioned rats were selected for transplantation by evaluation of their rotational behavior in response to amphetamine and randomly assigned to 3 groups, i.e., sham group, PBS group and NSCs transplanted group, with 6 rats in each group. MR scanning was performed at 1, 2, 4, 6, 8 and 10 week(s) following transplantation. At the meantime, rotational behavior was assessed in each group. Our results showed that SPIO particles were clearly visible with Prissian blue staining in neurospheres and cells derived from NSCs. The rotational behavior of the NSCs transplanted group was remarkably improved compared with that of sham group and PBS group (P < 0.05). In vivo MR tracking of NSCs showed that SPIO labeling led to a strong susceptibility change of signal 1 week after transplantation on T2 weighted images. And a large circular hypointense signal appeared in the transplanted area on T2* gradient echo images. Ten weeks following transplantation, the hypointense signal on T2 weighted and T2* gradient echo images was still displayed. It is concluded that SPIO particles could label NSCs effectively, and MRI detection of SPIO labeled cells is a promising method and novel approach to analyzing the NSCs following transplantation in the treatment of PD.