ABSTRACT
ObjectiveTo observe the effects of the kidney-tonifying and blood-activating prescription on the Wnt/β-catenin signaling pathway and uterine spiral artery remodeling in a mouse model of recurrent miscarriage and to explore its underlying mechanism. MethodA mouse model of normal pregnancy was established by mating CBA/J mice with BALB/c mice. A mouse model of recurrent miscarriage was established by mating CBA/J mice with DBA/2 mice. The modeled mice of recurrent miscarriage were randomized into model， dydrogesterone， and low- and high-dose Chinese medicine groups. The mice in normal pregnancy were used as the control group. Each group consisted of 10 mice， and the drug administration lasted for 14 days. After the treatment， the embryo absorption rate of each group was recorded. Hematoxylin-eosin （HE） staining was employed to observe the pathological morphology of the uterine decidua， and the physiological transformation rate of spiral arteries （SPA） was evaluated. Real-time polymerase chain reaction （Real-time PCR） and Western blot were performed to determine the mRNA and protein levels， respectively， of matrix metalloproteinases （MMP）-2， MMP-9， vascular endothelial growth factor （VEGF）， and Wnt/β-catenin signaling pathway. ResultCompared with the control group， the model group presented increased embryo absorption rate （P<0.05）， decreased physiological transformation rate of uterine SPA （P<0.05）， cellular swelling， degeneration， and disordered arrangement in the uterine decidua tissue， and down-regulated mRNA and protein levels of key factors involved in SPA remodeling （MMP-2， MMP-9， VEGF） and the Wnt/β-catenin signaling pathway （Wnt2， β-catenin， Cyclin D1， c-Myc） （P<0.05）. Compared with the model group， both the low- and high-dose Chinese medicine reduced embryo absorption rate （P<0.05）， increased SPA physiological transformation rate （P<0.05）， improved uterine decidua tissue morphology， and increased decidua vessel count. Furthermore， they up-regulated the mRNA and protein levels of MMP-2， MMP-9， VEGF， and proteins in the Wnt/β-catenin signaling pathway （P<0.05）. ConclusionRecurrent miscarriage is associated with impaired uterine spiral artery remodeling. The kidney-tonifying and blood-activating prescription can promote uterine spiral artery remodeling by activating the Wnt/β-catenin signaling pathway and promoting the expression of VEGF， MMP-2， and MMP-9， thus treating recurrent miscarriage.
ABSTRACT
SUMMARY: Trophoblasts perform different functions depending on their location. This study aimed to obtain structural clues about the functions of villous and extravillous trophoblasts by using light and electron microscopy. Term placenta samples were obtained from 10 healthy pregnant women following cesarean sections. Frozen sections were stained with hematoxylin-eosin, semi- thin sections were stained with toluidine blue and examined with a light microscope, while thin sections were contrasted using uranyl acetate-lead citrate and evaluated under an electron microscope. Fine structural features of villous trophoblasts overlapped some villous stromal cells. In addition to the usual appearance of mature capillaries in villous stroma, we demonstrated and reported maturational stages of angiogenetic sprouts in term placenta. Extravillous trophoblasts were classified according to their location: fibrinoid, chorion, trophoblastic, column, maternal vascular endothelium, or decidua. All of these trophoblasts shared some ultrastructural features but also were distinct from each other. In decidua, it was noted that the endothelial lining of some vessels was invaded by a few endovascular trophoblasts with irregular microvilli. These cells shared some ultrastructural properties with both villous trophoblasts and stromal cells. Examination showed that angiogenesis was still present in term placentas and that trophoblasts, endothelial and stromal cells have very similar properties ultrastructurally, suggesting they represent transformational forms.
RESUMEN: Los trofoblastos dependiendo de su ubicación realizan diferentes funciones. Este estudio tuvo como objetivo obtener pistas estructurales sobre las funciones de los trofoblastos vellosos y extravellosos mediante el uso de microscopía óptica y electrónica. Se obtuvieron muestras de placenta a término de 10 mujeres embarazadas sanas después de cesáreas. Las secciones congeladas se tiñeron con hematoxilina-eosina, las secciones semidelgadas se tiñeron con azul de toluidina y se examinaron con un microscopio óptico, mientras que las secciones delgadas se contrastaron con acetato de uranilo-citrato de plomo y se evaluaron con un microscopio electrónico. Las finas características estructurales de los trofoblastos vellosos se superponen a algunas células estromales vellosas. Además de la apariencia habitual de capilares maduros en el estroma velloso, demostramos e informamos etapas de maduración de brotes angiogenéticos en la placenta a término. Los trofoblastos extravellosos se clasificaron según su localización: fibrinoide, corion, trofoblástico, columna, endotelio vascular materno o decidua. Todos estos trofoblastos compartían algunas características ultraestructurales, pero también eran distintos entre sí. En decidua se observó que el revestimiento endotelial de algunos vasos estaba invadido por unos pocos trofoblastos endovasculares con microvellosidades irregulares. Estas células compartían algunas propiedades ultraestructurales tanto con los trofoblastos vellosos como con las células del estroma. El examen mostró que la angiogénesis todavía estaba presente en las placentas a término y que los trofoblastos, las células endoteliales y estromales tienen propiedades ultraestructurales muy similares, lo que sugiere que representan formas de transformación.
Subject(s)
Humans , Female , Placenta/ultrastructure , Trophoblasts/ultrastructure , Neovascularization, Physiologic , Microscopy, ElectronABSTRACT
Objective · To investigate the effects of growth arrest and DNA damage 45 alpha (Gadd45α) on the migration and invasion function of human extravillous trophoblast cells under hypoxia/re-oxygenation (H/R). Methods · Human extravillous trophoblast cells were infected by shRNA lentivirus targeting Gadd45α gene, to knock down Gadd45α gene expression. Then the oxidative stress model of preeclampsia was used in vitro to observe the changes of cell biological functions. The experiments were divided into 4 groups, nontreated group, hypoxia/re-oxygenation group, shRNA Gadd45α+H/R group and shRNA negative control+H/R group. Human villous explant experiments were used to determine the effects of silencing Gadd45α on human extravillous trophoblast cell under oxidative stress. Protein expression of Gadd45α was identified by Western blotting. Changes of cell migration and invasion were detected by transwell migration and Matrigel invasion assay. Gelatin zymography was used to detect the expression of matrix metalloproteinase (MMP) -2/9 in culture medium. Results · Hypoxia/re-oxygenation can increase the expression of Gadd45α in HTR8/SVneo cells and damage the trophoblast cell migration and invasion. Knocking down Gadd45α can increase the activities of MMP2/9, which can increase the cell migration and invasion. Conclusion · Knockdown of Gadd45α gene has promoted cell migration and invasion function of human extravillous trophobalst cells under oxidative stress.
ABSTRACT
Objective · To investigate the effects of a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 on biological function changes of human extravillous trophoblast cells induced by hypoxia/re-oxygenation (H/R). Methods · In-vitro cultured early pregnancy villus explants and human extravillous trophoblast cell line HTR8/SVneo were assigned to 4 groups according to different interventions, i.e. control group, SB203580 group (p38 MAPK inhibition), H/R group (simulation of preeclampsia by the oxidative stress model), and SB203580+H/R group. The effects of SB203580 on biological functions of human extravillous trophoblast cells under oxidative stress in early pregnancy villus explants were observed. Protein expression and phosphorylation of p38 MAPK in HTR8/SVneo cells were measured with Western blotting. Cell migration and invasion were observed with Transwell migration and Matrigel invasion assay, respectively. Gelatin zymography was used to measure the activity of matrix metalloproteinase (MMP) -2/9 in supernatant. ELISA was used to detect the levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng). Results · SB203580 could promote the exogenous migration of human extravillous trophoblast cells in early pregnancy villus explants under oxidative stress. H/R could decrease the migration and invasion of HTR8/SVneo cells , and increase the phosphorylation level of p38 MAPK in HTR8/SVneo cells and the secretion of sFlt-1 and sEng. SB203580 could increase the activity of MMP2/9 in supernatant and cell migration and invasion, decrease the phosphorylation level of p38 MAPK in HTR8/SVneo cells under oxidative stress and the secretion of sFlt-1 and sEng. Conclusion · SB203580 can protect biological functions of human extravillous trophobalst cells under oxidative stress.
ABSTRACT
The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated.Immunohistochemistry was used to determine the placental expression of leptin in first-trimester preg-nancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, inva-siveness and migration was assessed by MTT, Transwell invasion assay and migration assay respec-tively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that: (1) Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear mem-brane of cytotrophoblast; (2) Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly (P<0.01); (3) After culture with different concen-trations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls (P<0.05). It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotro-phoblast invasion and migration activity.
ABSTRACT
In this study, the expression of IL-24 at maternal-fetal interface and the roles in extravillous trophoblast (the TEV-1 cell line) invasion were examined. Immunohistochemistry was used to detect the expression of IL-24 in villi and decidual tissue. The proliferation of TEV-1 cells under the effect of IL-24 was measured by MTT assay. The invasiveness of TEV-1 cells under the effect of recombinant IL-24 (rhIL-24) was examined by transwell system. Immunohistochemical detection showed that IL-24 was expressed in the villi and decidual tissue, and distributed in villous column,trophoblasts, stroma and blood vessels. The proliferation of TEV-1 cells was not inhibited by rhIL-24of various concentrations. The examination of invasion in vitro showed that rhIL-24 could inhibit the invasion of TEV-1 cells in a concentration-dependent manner. The results suggested IL-24 could inhibit the invasion of TEV-1 cells. Therefore, IL-24 produced by maternal-fetal interface in human first trimester pregnancy may influence the invasion of trophoblasts and is involved in normal pregnancy.
ABSTRACT
OBJECTIVE: The aim of this study is to ascertain the differences in NF-kappaB (Nuclear Factor-kappa B : p50) activity between the placental tissues of preeclampsia and normal pregnancy, and to certify that the circulating lipid peroxides is increased in preeclamptic women. METHODS: Placental tissues were obtained from preeclamptic (n=33) and normal pregnancies (n=21) with no other medico-surgical illness or obstetric complications, delivered by cesarean section without labor. The activities of NF-kappaB and IkappaBalpha (Inhibitory factor kappaBalpha) on syncytiotrophoblast, cytotrophoblast, endothelium, extravillous cytotrophoblast, and decidua were separately measured by immunohistochemical staining using tissue microarray technique. Malondialdehyde assay was used to evaluate the oxidative stress, measuring lipid peroxide levels on each sample. Mann-Whitney test was done for statistical analysis of the data. RESULTS: Nuclear staining of NF-kappaB (p50) was seen more intensively within the extravillous cytotrophoblast of preeclampsia group compared with the control group (p<0.05). The immunoreactivity of NF-kappaB (p50) was also detected in cytotrophoblasts, syncytiotrophoblasts, endothelium, and decidua, but showing no statistical difference between two groups. IkappaBalpha was strongly expressed in both groups but there was no statistically significant between two gropups. Preeclamptic group showed significantly increased circulating lipid peroxide levels compared to normal pregnancy group (1.22+/-0.79 nmol/mL vs 0.41+/-0.12 nmol/mL, p<0.05). CONCLUSION: The expression of NF-kappaB is significantly increased in extravillous cytotrophoblast of preeclamptic women compared to normal pregnancy, and may be associated with increased levels of circulating lipid peroxide. These findings might help us to understand the pathologic mechanism of preeclampsia and further study should be done for effects of NF-kappaB on implantation.