Objective To observe the effects of Bushen Antai Mixture on angiogenesis at the maternal fetal interface in recurrent spontaneous abortion(RSA)mice;To explore its mechanism in treating RSA based on the VEGF/Hippo/YAP pathway.Methods RSA mouse model and normal pregnancy mouse were constructed using Clark classic method.RSA model mice were randomly divided into model group,progesterone group,Bushen Antai Mixture low-,medium-and high-dosage groups,and normal pregnant mice were used as normal group,with 10 mice in each group.Starting from the first day of pregnancy,each medication group was given the corresponding medication by gavage for 15 days.The absorption rate of mouse embryos was calculated,the morphology of decidual tissue were observed by HE staining,the mRNA and protein expression of VEGF and Hippo/YAP pathway-related molecules of decidual tissue were detected by RT-qPCR and Western blot.Results Compared with the normal group,the absorption rate of embryos in the model group significantly increased(P<0.01),the arrangement of decidual tissue cells were disordered,edematous and degenerated,some nuclei disappeared,the vascular density decreased,the mRNA expressions of VEGF,Yes associated protein(YAP),large tumor suppressor factor(LATS)1 and LATS2 in decidual tissue significantly decreased(P<0.01),the protein expression of VEGF decreased(P<0.01),and the protein expressions of p-YAP/YAP and p-LATS/LATS significantly increased(P<0.01).Compared with the model group,the absorption rate of embryos in each dosage group of Bushen Antai Mixture was significantly reduced(P<0.05,P<0.01),the cells in the decidual tissue were arranged in an orderly manner,and edema degeneration was reduced,the mRNA expressions of VEGF,YAP,LATS1 and LATS2 in decidual tissue significantly increased(P<0.05,P<0.01),and the protein expression of VEGF significantly increased(P<0.05,P<0.01),the protein expressions of p-YAP/YAP and p-LATS/LATS decreased(P<0.05,P<0.01).Conclusion Bushen Antai Mixture can promote the expressions of VEGF,YAP,LATS by activating VEGF/Hippo/YAP signaling pathway,promote angiogenesis at the maternal-fetal interface,reduce the pregnancy loss rate,and play a therapeutic effect of RSA.

Objective: This study employs metabolomics to analyze the characteristic biomarkers and metabolic pathways in the serum of pregnant women with positive thyroid peroxidase antibodies(TPOAb) during early pregnancy. The objective is to explore the relationship between thyroid dysfunction and maternal-fetal health. Methods : Early-pregnancy women undergoing antenatal check-ups for thyroid function and antibody testing at our hospital were selected. According to the TPOAb results, participants were categorized into a TPOAb-positive group and a TPOAb-negative group. The serum metabolic profiles were analyzed using liquid chromatography-tandem mass spectrometry(LC-MS/MS) technology to identify differences between the two groups. The Kyoto Encyclopedia of Genes and Genomes(KEGG) database was utilized for metabolic pathway enrichment analysis of differential metabolites. Results : A total of 79 significantly different metabolites were identified in the serum of TPOAb-positive pregnant women compared to the control group, including 20 upregulated and 59 downregulated metabolites. KEGG enrichment analysis indicated that these differential metabolites were mainly involved in 21 key metabolic pathways. Among the metabolites associated with TPOAb, 31 were identified, with 6 showing positive correlation and 25 showing negative correlation. Metabolic pathway enrichment analysis revealed that these differential metabolites were closely related to Glycerophospholipid metabolism, Valine, leucine and isoleucine biosynthesis, Glycosylphosphatidylinositol(GPI)-anchor biosynthesis, and Glycerolipid metabolism pathways. Conclusion Significant differences in metabolites and their associated metabolic pathways are identified in the serum of TPOAb-positive pregnant women during early gestation, indicating that these metabolite alterations are closely linked to thyroid dysfunction and maternal-fetal health.

ObjectiveTo investigate the effect and mechanism of Yijingtang （YJT） in treating diminished ovarian reserve （DOR） in rats by regulating the Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor-κB （NF-κB） signaling pathway. MethodsFifty female SD rats with normal estrous cycles were randomly allocated into blank， model， low- and high-dose （12.579 and 25.158 g·kg^-1， respectively） YJT， and dehydroepiandrosterone （7.487 5 mg·kg^-1） groups， with 10 rats in each group. The rats in other groups except the blank group were administrated with the tripterygium glycosides tablet suspension （5 mg·kg^-1） by gavage for 14 days for the modeling of DOR. The rats in the drug treatment groups were administrated with corresponding drugs by gavage from day 15 for 30 consecutive days， and those in the blank and model groups received equal volumes of distilled water. The vaginal exfoliated cell smears were observed to assess the changes in the estrous cycle. The wet weight of bilateral ovaries was weighed for calculation of the ovarian index. Hematoxylin-eosin staining was performed to observe the histopathological changes in the ovaries and the proportions of follicles at various levels were calculated. The serum levels of sex hormones ［follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E₂）， and anti-Müllerian hormone （AMH）］ and inflammatory factors ［tumor necrosis factor-alpha （TNF-α）， interleukin-1beta （IL-1β）， and interleukin-10 （IL-10）］ were determined by enzyme-linked immunosorbent assay. Real-time quantitative polymerase chain reaction（Real-time PCR） was conducted to determine the mRNA levels of TLR4， MyD88， NF-κB profilin α （IκBα）， NF-κB and inflammatory factors in the ovarian tissue. Western blot was employed to measure the protein levels of factors related to the TLR4/MyD88/NF-κB signaling pathway in the ovarian tissue. Immunofluorescence （IF） was used to detect the nuclear translocation of NF-κB p65 in the ovarian tissue. ResultsCompared with the blank group， the model group showed disturbed estrous cycles， increased inflammatory infiltration in the ovarian tissue， decreases in ovarian index and proportion of presinusoidal follicles， and an increase in the proportion of atretic follicles （P<0.05， P<0.01）. In addition， the model group showed elevated serum levels of FSH， LH， TNF-α， and IL-1β， up-regulated mRNA levels of TLR4， MyD88， IκBα， NF-κB， TNF-α， and IL-1β and protein levels of TLR4， MyD88， p-IκBα， and p-NF-κB p65 （P<0.01）， lowered serum levels of AMH， E₂， and IL-10， down-regulated mRNA level of IL-10 （P<0.01）， and massive nuclear translocation of NF-κB p65 in the ovarian tissue. Compared with the model group， dehydroepiandrosterone and low and high doses of YJT restored the disturbed estrous cycle， reduced inflammatory infiltration in the ovarian tissue， increased the ovarian index （P<0.01）， and changed the follicular composition ratio （P<0.01）. Furthermore， the drugs lowered the serum levels of FSH， LH， TNF-α， and IL-1β， down-regulated the mRNA levels of TLR4， MyD88， IκBα， NF-κB， TNF-α， and IL-1β and the protein levels of TLR4， MyD88， p-IκBα， and p-NF-κB p65 （P<0.05， P<0.01）， raised the serum levels of AMH， E₂， and IL-10， up-regulated the mRNA level of IL-10 （P<0.05， P<0.01）， and reduced the nuclear translocation of NF-κB p65 in the ovarian tissue. ConclusionYJT may inhibit the release and expression of inflammatory factors by regulating the TLR4/MyD88/NF-κB signaling pathway to attenuate the inflammatory responses in the ovarian tissue， thereby improving the ovarian function in DOR rats.

ObjectiveTo investigate the effect and mechanism of Yijingtang （YJT） in treating diminished ovarian reserve （DOR） in rats by regulating the Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor-κB （NF-κB） signaling pathway. MethodsFifty female SD rats with normal estrous cycles were randomly allocated into blank， model， low- and high-dose （12.579 and 25.158 g·kg^-1， respectively） YJT， and dehydroepiandrosterone （7.487 5 mg·kg^-1） groups， with 10 rats in each group. The rats in other groups except the blank group were administrated with the tripterygium glycosides tablet suspension （5 mg·kg^-1） by gavage for 14 days for the modeling of DOR. The rats in the drug treatment groups were administrated with corresponding drugs by gavage from day 15 for 30 consecutive days， and those in the blank and model groups received equal volumes of distilled water. The vaginal exfoliated cell smears were observed to assess the changes in the estrous cycle. The wet weight of bilateral ovaries was weighed for calculation of the ovarian index. Hematoxylin-eosin staining was performed to observe the histopathological changes in the ovaries and the proportions of follicles at various levels were calculated. The serum levels of sex hormones ［follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E₂）， and anti-Müllerian hormone （AMH）］ and inflammatory factors ［tumor necrosis factor-alpha （TNF-α）， interleukin-1beta （IL-1β）， and interleukin-10 （IL-10）］ were determined by enzyme-linked immunosorbent assay. Real-time quantitative polymerase chain reaction（Real-time PCR） was conducted to determine the mRNA levels of TLR4， MyD88， NF-κB profilin α （IκBα）， NF-κB and inflammatory factors in the ovarian tissue. Western blot was employed to measure the protein levels of factors related to the TLR4/MyD88/NF-κB signaling pathway in the ovarian tissue. Immunofluorescence （IF） was used to detect the nuclear translocation of NF-κB p65 in the ovarian tissue. ResultsCompared with the blank group， the model group showed disturbed estrous cycles， increased inflammatory infiltration in the ovarian tissue， decreases in ovarian index and proportion of presinusoidal follicles， and an increase in the proportion of atretic follicles （P<0.05， P<0.01）. In addition， the model group showed elevated serum levels of FSH， LH， TNF-α， and IL-1β， up-regulated mRNA levels of TLR4， MyD88， IκBα， NF-κB， TNF-α， and IL-1β and protein levels of TLR4， MyD88， p-IκBα， and p-NF-κB p65 （P<0.01）， lowered serum levels of AMH， E₂， and IL-10， down-regulated mRNA level of IL-10 （P<0.01）， and massive nuclear translocation of NF-κB p65 in the ovarian tissue. Compared with the model group， dehydroepiandrosterone and low and high doses of YJT restored the disturbed estrous cycle， reduced inflammatory infiltration in the ovarian tissue， increased the ovarian index （P<0.01）， and changed the follicular composition ratio （P<0.01）. Furthermore， the drugs lowered the serum levels of FSH， LH， TNF-α， and IL-1β， down-regulated the mRNA levels of TLR4， MyD88， IκBα， NF-κB， TNF-α， and IL-1β and the protein levels of TLR4， MyD88， p-IκBα， and p-NF-κB p65 （P<0.05， P<0.01）， raised the serum levels of AMH， E₂， and IL-10， up-regulated the mRNA level of IL-10 （P<0.05， P<0.01）， and reduced the nuclear translocation of NF-κB p65 in the ovarian tissue. ConclusionYJT may inhibit the release and expression of inflammatory factors by regulating the TLR4/MyD88/NF-κB signaling pathway to attenuate the inflammatory responses in the ovarian tissue， thereby improving the ovarian function in DOR rats.

Objective To observe the effects of Bushen Antai Mixture on angiogenesis at the maternal fetal interface in recurrent spontaneous abortion(RSA)mice;To explore its mechanism in treating RSA based on the VEGF/Hippo/YAP pathway.Methods RSA mouse model and normal pregnancy mouse were constructed using Clark classic method.RSA model mice were randomly divided into model group,progesterone group,Bushen Antai Mixture low-,medium-and high-dosage groups,and normal pregnant mice were used as normal group,with 10 mice in each group.Starting from the first day of pregnancy,each medication group was given the corresponding medication by gavage for 15 days.The absorption rate of mouse embryos was calculated,the morphology of decidual tissue were observed by HE staining,the mRNA and protein expression of VEGF and Hippo/YAP pathway-related molecules of decidual tissue were detected by RT-qPCR and Western blot.Results Compared with the normal group,the absorption rate of embryos in the model group significantly increased(P<0.01),the arrangement of decidual tissue cells were disordered,edematous and degenerated,some nuclei disappeared,the vascular density decreased,the mRNA expressions of VEGF,Yes associated protein(YAP),large tumor suppressor factor(LATS)1 and LATS2 in decidual tissue significantly decreased(P<0.01),the protein expression of VEGF decreased(P<0.01),and the protein expressions of p-YAP/YAP and p-LATS/LATS significantly increased(P<0.01).Compared with the model group,the absorption rate of embryos in each dosage group of Bushen Antai Mixture was significantly reduced(P<0.05,P<0.01),the cells in the decidual tissue were arranged in an orderly manner,and edema degeneration was reduced,the mRNA expressions of VEGF,YAP,LATS1 and LATS2 in decidual tissue significantly increased(P<0.05,P<0.01),and the protein expression of VEGF significantly increased(P<0.05,P<0.01),the protein expressions of p-YAP/YAP and p-LATS/LATS decreased(P<0.05,P<0.01).Conclusion Bushen Antai Mixture can promote the expressions of VEGF,YAP,LATS by activating VEGF/Hippo/YAP signaling pathway,promote angiogenesis at the maternal-fetal interface,reduce the pregnancy loss rate,and play a therapeutic effect of RSA.

Objective To explore the effects and mechanism of Yijing Decoction containing serum on iron overload-induced oxidative stress of human ovarian granulosa cells.Methods SD rats were used to prepare medicated serum and blank serum.Iron dextran was used to induce oxidative damage of SVOG cells.The cells were divided into control group,model group,containing serum group,blank serum group,antioxidant group and iron chelating agent group.After corresponding intervention,cell viability was detected by CCK-8 method,estrogen(E2)and progesterone(P)content in supernatant were detected by ELISA,the contents of intracellular ferrous ion(Fe2+),malondialdehyde(MDA),adenosine triphosphate(ATP)and the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT)were detected by the kit,ROS content was detected by DCFH-DA fluorescence probe,the mitochondrial membrane potential was detected by JC-1 fluorescence probe,Western blot and RT-qPCR were used to detect the protein and mRNA expression of nuclear factor erythroid 2-related factor 2(Nrf2),transferrin(Tf),transferrin receptor 1(TfR1),ferritin heavy chain 1(FTH1)and ferritin light chain(FTL).Results Compared with the control group,the viability of SVOG cells decreased in the model group,the contents of E2 and P in cell supernatant decreased,the contents of Fe2+,MDA and ROS increased,the content of ATP and activities of SOD,GSH-Px and CAT decreased,the mitochondrial membrane potential decreased,the protein and mRNA expressions of Nrf2 and FTH1 decreased,and the protein and mRNA expressions of Tf,TfR1 and FTL increased,with statistical significance(P<0.05,P<0.01).Compared with the model group,the viability of SVOG cells in containing serum group,antioxidant group and iron chelating agent group increased,the contents of E2 and P in cell supernatant increased,the contents of Fe2+,MDA and ROS decreased,the content of ATP and activities of SOD,GSH-Px and CAT increased,the mitochondrial membrane potential increased,the protein and mRNA expressions of Nrf2 and FTH1 increased,and the protein and mRNA expressions of Tf,TfR1 and FTL decreased(P<0.05,P<0.01).Conclusion Yijing Decoction containing serum may relieve oxidative stress damage induced by iron overload,improve mitochondrial function,and restore granulosa cell function,thereby enhancing ovarian function,potentially through up-regulating Nrf2,FTH1 expression and down-regulating Tf,TfR1 and FTL expressions.

Objective:To observe the effects of Bushen Antai Mixture on nuclear transcription factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/nuclear transcription factor-κB (NF-κB) in placental tissue of recurrent spontaneous abortion (RSA) mice.Methods:The CBA/J mice (female) and DBA/2 (male), CBA/J mice (female) and BALB/c (male) were caged at a ratio of 2 : 1 to establish RSA and normal pregnant mice, respectively. The next morning, the vaginal plug was found or the sperm was seen under the microscope as the successful modeling, which was counted as the first day of pregnancy. CBA/J×BALB/c mice were set as normal group, and the RSA model pregnant mice were divided into model group, progesterone group, and Bushen Antai Mixture low-, medium-, and high-dosage groups using a random number table method, with 6 mice in each group. The normal group and the model group were given distilled water by gavage. Bushen Antai Mixture low-, medium-, and high-dosage groups were given 3.9 g/kg, 11.7 g/kg and 23.4 g/kg Bushen Antai Mixture by gavage. The progesterone group was given progesterone capsule aqueous solution 26 mg/kg by gavage for 14 consecutive days. The embryo loss rate of mice was calculated. The pathological changes of placental tissues were observed by HE staining. The expressions of Nrf2, HO-1 and NF-κB protein and mRNA in placental tissues were detected by Western blot and RT-qPCR.Results:Compared with the model group, the embryo loss rate of mice in the Bushen Antai Mixture low-, medium-, and high-dosage groups and the progesterone group significantly decreased ( P<0.05, P<0.01); the pathological morphology of placental tissue improved; the expressions of Nrf2, HO-1 protein and mRNA in placental tissue significantly increased ( P<0.05, P<0.01), and the expression of NF-κB protein and mRNA significantly decreased ( P<0.05, P<0.01). Conclusion:Bushen Antai Mixture may may treat RSA by activating the Nrf2/HO-1 pathway, inhibiting NF - κB expression, and improving oxidative stress and inflammatory response in the placental tissue of RSA pregnant mice.

Objective To explore the effects and mechanism of Yijing Decoction containing serum on iron overload-induced oxidative stress of human ovarian granulosa cells.Methods SD rats were used to prepare medicated serum and blank serum.Iron dextran was used to induce oxidative damage of SVOG cells.The cells were divided into control group,model group,containing serum group,blank serum group,antioxidant group and iron chelating agent group.After corresponding intervention,cell viability was detected by CCK-8 method,estrogen(E2)and progesterone(P)content in supernatant were detected by ELISA,the contents of intracellular ferrous ion(Fe2+),malondialdehyde(MDA),adenosine triphosphate(ATP)and the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT)were detected by the kit,ROS content was detected by DCFH-DA fluorescence probe,the mitochondrial membrane potential was detected by JC-1 fluorescence probe,Western blot and RT-qPCR were used to detect the protein and mRNA expression of nuclear factor erythroid 2-related factor 2(Nrf2),transferrin(Tf),transferrin receptor 1(TfR1),ferritin heavy chain 1(FTH1)and ferritin light chain(FTL).Results Compared with the control group,the viability of SVOG cells decreased in the model group,the contents of E2 and P in cell supernatant decreased,the contents of Fe2+,MDA and ROS increased,the content of ATP and activities of SOD,GSH-Px and CAT decreased,the mitochondrial membrane potential decreased,the protein and mRNA expressions of Nrf2 and FTH1 decreased,and the protein and mRNA expressions of Tf,TfR1 and FTL increased,with statistical significance(P<0.05,P<0.01).Compared with the model group,the viability of SVOG cells in containing serum group,antioxidant group and iron chelating agent group increased,the contents of E2 and P in cell supernatant increased,the contents of Fe2+,MDA and ROS decreased,the content of ATP and activities of SOD,GSH-Px and CAT increased,the mitochondrial membrane potential increased,the protein and mRNA expressions of Nrf2 and FTH1 increased,and the protein and mRNA expressions of Tf,TfR1 and FTL decreased(P<0.05,P<0.01).Conclusion Yijing Decoction containing serum may relieve oxidative stress damage induced by iron overload,improve mitochondrial function,and restore granulosa cell function,thereby enhancing ovarian function,potentially through up-regulating Nrf2,FTH1 expression and down-regulating Tf,TfR1 and FTL expressions.

Objective:To investigate the correlation between the imaging markers of cerebral small vessel disease (CSVD) and early hematoma expansion (HE) in patients with spontaneous intracerebral hemorrhage (sICH).Methods:Patients with sICH admitted to the Department of Neurology, the Affiliated Hospital of Qingdao University between January 1, 2015 and December 31, 2019 were enrolled retrospectively. All patients received noncontrast CT (NCCT) within 6 h after onset. Within 24 h after the initial NCCT examination, they were reexamed to determine whether HE occurred, and brain MRI examination was completed within 48 h after onset. HE was defined as the increase of hematoma volume on NCCT reexamination by >33% or >6 ml compared with the baseline. NCCT was used to evaluate the abnormal morphology and density signs, including blend sign, swirl sign, black hole sign, island sign, and satellite sign. MRI was used to evaluate CSVD imaging markers, including lacunar infarcts (LIs), enlarged perivascular space (EPVS), white matter hyperintensities (WMHs), cerebral microbleeds (CMBs), and cortical superficial siderosis (CSS). Multivariate logistic regression analysis was used to determine independent risk factors for HE. The receiver operator characteristic (ROC) curve was used to evaluate the predictive ability of imaging markers for HE in patients with sICH. Results:A total of 216 patients with sICH were included. Their age was 57±15 years, 113 (61.6%) were male, 88 (40.7%) had HE, 123 (56.9%) had NCCT signs, 122 (56.5%) had CMBs, 143 (66.2%) had WMHs, 44 (20.4%) had CSS, 25 (11.6%) had LIs, and 31 (14.4%) had EPVS. The baseline hematoma volume, blood calcium, the modified Rankin Scale score and the National Institutes of Health Stroke Scale score at admission, and detection rates of NCCT signs, CMBs, WMHs and CSS in the HE group were significantly higher than those in the non-HE group (all P<0.05). Multivariate logistic regression analysis showed that the blood calcium (odds ratio [ OR] 0.040, 95% confidence interval [ CI] 0.004-0.238; P=0.001), any NCCT signs ( OR 3.275, 95% CI 1.492-7.188; P=0.003), CMBs grade 4 ( OR 3.591, 95% CI 1.146-11.250; P=0.028), CSS ( OR 3.008, 95% CI 1.214-7.452; P=0.017), NCCT signs+ CMBs grade 3 ( OR 3.390, 95% CI 1.035-11.102; P=0.044), NCCT signs+ CMBs grade 4 ( OR 5.473, 95% CI 1.352-22.161; P=0.017), and NCCT signs+ CSS ( OR 3.544, 95% CI 1.215-10.336; P=0.021) were the independent risk factors for HE in patients with sICH. ROC curve analysis showed that the sensitivity of NCCT signs, CMBs and CSS for predicting HE were 81.8%, 64.8% and 34.1%, respectively, and the specificity were 60.2%, 60.9% and 89.1%, respectively. The predictive sensitivity of NCCT signs+ CMBs and NCCT signs+ CSS (59.1% and 30.7%, respectively) was lower than that of single imaging marker, while the specificity (78.1% and 93.7%, respectively) was higher than that of single imaging marker. Conclusions:The imaging markers of CSVD are closely associated with the risk of HE in patients with sICH. Severe CMBs and CSS are the independent risk factors for HE in patients with sICH. The specificity of NCCT signs combined with CSVD imaging markers for predicting HE is increased but the sensitivity decreased.