The Mechanism Of Maternal Cadmium Exposure During Pregnancy Impairing Placental Angiogenesis Through Active Glucocorticoid Overexposure

Objective: It is known that maternal environmental cadmium(Cd)exposure during pregnancy impair placental vascular development.This study aims to investigate the role of active glucocorticoid overexposure in environmental Cd damage placental blood vessels and its mechanism.Methods: In this study,animal and cell experiments were conducted to explore the mechanism of environmental cadmium(Cd)exposure to damage the development of placental blood vessels,and a case-control study was conducted to verify it in a population.(1)Animal studies.(1)Pregnant mice were given with CdCl2(4.5 mg/kg,i.p.)on gestational day(GD)8,and the mice were dissected at GD12 or GD16.Maternal sera,placentae and amniotic fluid were collected to observe the effect of Cd exposure during pregnancy on angiogenesis of fetal mice,and to detect the expression of vascular endothelial growth factor(VEGF-A)and active glucocorticoid(GC)and glucocorticoid receptor(GR)levels.(2)Pregnant mice were given with Cd Cl2(1.0mg/kg/day,i.p.)from GD12 to GD17.At the same time,the mice in the NAC+Cd group were orally given 300 mg/kg,400 mg/kg and 300 mg/kg NAC treatment 8 h before and 0.5 h after the Cd Cl2 treatment and 8 h after the Cd treatment every day.Pregnant mice were dissected at GD12 or GD16,and maternal sera and placentae were collected.Detection of active glucocorticoid level and detection of 11β-HSD2,p-PERK and p-e IF2α and other protein expression.(2)Cell studies.(1)Human placental primary trophoblast cells and human JEG-3 cells were given with Cd Cl2(20μM)for 0-24 h to collect supernatant and protein to detect the active GC level,GR and VEGF-A and other proteins expression.(2)Human JEG-3 cells were pretreated with GR si RNA and PERK si RNA for 1 h before Cd Cl2(20 μM)treatment.Cell supernatant and cell protein were collected,and the expression of GR,VEGF-A and11β-HSD2 and other proteins were detected.(3)After pretreatment with NAC(4 m M)for 1 h,human JEG-3 cells were given with Cd Cl2(20 μM)for 6 h or 12 h,and cell supernatant and cell protein were collected,and 11β-HSD2,p-PERK and p-e IF2α and other protein expression were detected.(3)Case-control study: The placentae of 101full-term pregnant women were collected and divided into appropriate-for-gestational-age placentae and small-for-gestational-age placentae based on birth weight,and detect the level of placental active glucocorticoid.Results: The results of HE staining and immunohistochemistry showed that maternal environmental cadmium(Cd)exposure during pregnancy significantly reduced the area of red blood cells in the sinusoidal area of the mouse placentae and the number of placental vessels labeled with CD34.The expression of VEGF-A protein in human placental trophoblast cells was also down-regulated.These results revealed that environmental Cd exposure inhibited VEGF-A-regulated placental angiogenesis.Western blot results showed that environmental cadmium exposure increased GR protein levels in human placental trophoblast cells.Pretreatment with dexamethasone,an active inducer of GR,increased the down-regulation effect of Cd on VEGF-A protein expression in human placental trophoblast cells,while GR si RNA inhibited the down-regulation effect of Cd on VEGF-A protein expression in human placental trophoblast cells.ELISA results showed that maternal Cd exposure during pregnancy increased the active GC levels of placenta,amniotic fluid and cell culture media.This suggested that Cd exposure activated GC/GR signaling in placental trophoblasts.WB results showed that Cd exposure significantly reduced 11β-HSD2 protein expression in mouse placentae and human placental trophoblast cells.Further studies showed that environmental Cd exposure significantly up-regulated the expression of PERK and p-e IF2α proteins in placental trophoblast cells.PERK si RNA pretreatment significantly alleviated the down-regulation effect of Cd on 11β-HSD2 protein expression in placental trophoblast cells.In addition,the antioxidant N-acetylcysteine antagonized the activation of PERK/p-e IF2α signaling and reversed Cd down-regulated the expression of 11β-HSD2 protein in mouse placentae and human placental trophoblast cells.Conclusion: Maternal environmental cadmium exposure during pregnancy impairs placental angiogenesis via activating endoplasmic reticulum PERK-regulated GC/GR signaling.