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ObjectiveA good invasion ability of extravilloustrophoblas (EVTs) is the prerequisite for successful placental colonization and effective remodeling of the uterine spiral artery. This article aims to simulate the pathophysiological process of oxidative stress inducing trophoblasts to pyroptosis in vitro, exploring the correlation between trophoblasts pyroptosis and the pathogenesis of preeclampsia.MethodsTwenty-five patients with preeclampsia were selected from the Department of Obstetrics and Gynecology, Zhongda Hospital affiliated to Southeast University from September 2017 to January 2019. Among them, early-onset preeclampsia (gestational weeks<34) was early-onset group (n=17), late-onset preeclampsia (gestational weeks≥34) was late-onset group (n=8), and full-term pregnant women with normal blood pressure (39＜gestational weeks＞42) were selected as normal group (n=10). Human trophoblasts were cultured with HTR-8/SVneo for 12 hours, and then treated with H2O2 (100, 150, 200, 250μmol/L) (2, 4, 6, 12 h), to induce human trophoblast HTR-8/SVneo pyrolysis model; the control group was normal cultured cells of 1640+10% fetal bovine serum + 1% antibiotics. Placental specimens from 7 patients with preeclampsia were randomly selected, including 3 cases in early onset group, 4 cases in late onset group and 1 case in normal group. The total proteins of cells and placenta were extracted respectively, and the expression of scorch death-related molecular proteins was detected. The mRNA levels of pyroptosis related molecules in cells was detected by RT-qPCR, and the morphological changes of cells were observed by inverted phase contrast microscope.ResultsThe Western blot results showed that the activation of the key molecular activation form of the cell pyrogenesis pathway, Cleaved caspase1, could be detected in the placenta. When H2O2 was 150 mol/L for 2h, the mRNA levels of NLRP3 and IL-1, the key molecules of the upstream activation signal, were significantly up-regulated (8.680±0.481, 14.136±0.244) compared with the control group (1.00±0.00) (P<0.000). At 4h, mRNA levels of key molecule GSDMD and downstream inflammatory factor IL-18 (1.639±0.354 and 1.794±0.043) in the pyrogenesis pathway were significantly higher than those in the control group (1.00±0.00), with statistically significant differences (P<0.05). By reverse validation of the mRNA levels of the molecules associated with pyroptosis, the optimal conditions of the model induced by H2O2 were 150 mol/L and 4h, and the typical changes, such as cell swelling, fragmentation and plasma membrane bubble formation, could be seen under the light microscope.ConclusionThe pyroptosis model of trophoblast cells was successfully established, and the physiological process of oxidative stress inducing trophoblasts to pyroptosis in vitro was successfully simulated, providing new ideas and directions for the diagnosis and treatment of preeclampsia and the development of new drugs.
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OBJECTIVE@#To investigate the effect of vitamin D on microRNA-21(miR-21) expression and migration and invasion of human placental trophoblast cells.@*METHODS@#The changes in the expression of miR-21 were detected using RT-qPCR in HTR-8/SVneo cells following stimulation by vitamin D at different doses for 24, 48 and 72 h.HTR-8/SVneo cells transfected with miR-21 mimic or inhibitor with or without vitamin D treatment were examined for changes in cell migration and invasion abilities using Transwell assay, and Western blotting was used to detect protein expressions of E-cadherin, fibronectin, and MMP9.@*RESULTS@#Vitamin D obviously inhibited the expression of micoRNA-21 in HTR-8/SVneo cells in a concentration-and time-dependent manner.Transfection with the miR-21 mimic significantly inhibited the migration and invasion of HTR-8/SVneo cells, and this inhibitory effect was abolished by treatment with vitamin D; transfection with miR-21 inhibitor obviously promoted the migration and invasion of HTR-8/SVneo cells, and these effects were not significantly affected by vitamin D treatment.@*CONCLUSIONS@#Vitamin D may promote trophoblast cell migration and invasion to accelerate the development of preeclampsia by down-regulating the expression of miR-21.