Molecular Mechanisms Of Cadmium-induced Inflammatorycytokines In Placental Trophoblast Cells

Objective The present study was to investigate the effect of cadmium on the expression of inflammatory cytokines in mouse placenta and human placental trophoblast cells,and to explore the role of PI3K/Akt signaling activation in cadmium-upregulated the expression of inflammatory cytokines in placental trophoblast cells.Methods The present study includes experiments both in vitro and in vivo.The following four experiments were main contents of in vitro.Experiment 1,human JEG-3cells were incubated with distinct concentrations of CdCl₂（0,3.125,6.25,12.5,25,and50μΜ）for different time points（0,2,6,12,and 24 h）,and then cell viability and the level of Akt and pAkt were measured.Experient 2,human JEG-3 cells were treated with CdCl₂（25.0μM）for 2,12 and 24 h,the expressions of cytokines in JEG-3 cells were detected.Experient 3,human JEG-3 cells were pre-incubated with LY294002（50.0μM）for 30 min,and subsequently incubated with CdCl₂（25μM）for 6 h and 12 h.The mRNA expression of IL-8 and the level of Akt and pAkt were further detected.Experient 4,human JEG-3 cells were pre-treated with NAC（4.0 mM）for 30 min,and then incubated with CdCl₂（25.0μM）for 6 h.The level of Akt and pAkt were finally measured.Next,the present study consists of two in vivo experiments.Experient 1,the pregnant mice of GD15 were respectively administered with a single dose of CdCl₂（3.0mg/kg,i.p.）at two different time points,all dams were euthanized on GD16.The expression of cytokines,the level of Akt and pAkt,as well as the KC levels in maternal serum and amniotic fluid were measured.Experient 2,pregnant mice were i.p.injected with two doses of NAC（150 mg/kg）from GD15 to GD16,one 30 min before CdCl₂injection,the other 4 h after CdCl₂ injection.All dams were euthanized on GD16.Maternal serum and amniotic fluid were collected for detection of KC,and then the level of Akt and pAkt from mouse placentas were also determined.Results In vitro results showed that the level of TNF-α,IL-8 and IL-6 mRNAs was elevated in CdCl₂-treated JEG-3 cells.In vivo results demonstrated that the expression of Tnf-α、Il-1β、Kc and Mip-2 mRNAs were up-regulated in CdCl₂-treated placentas of mice.The level of keratinocyte chemokine（KC）was increased in maternal serum and amniotic fluid from CdCl₂-exposed mice.Gestational Cd exposure activated Akt signaling in mouse placenta.In addition,co-culture with CdCl₂ elevated the pAkt level in JEG-3 cells in concentration-dependent manners.LY294002,a specific inhibitor of PI3K,blocked CdCl₂-evoked Akt phosphorylation in human JEG-3 cells.Concomitantly,LY294002 inhibited CdCl₂-induced the expression of IL-8 mRNA in human JEG-3 cells.N-acetylcysteine（NAC）,an antioxidant,blocked CdCl₂-evoked Akt phosphorylation in mouse placenta and human trophoblast cells.Additionally,NAC attenuated CdCl₂-induced the elevation of KC level in amniotic fluid.Conclusion In the present study,CdCl₂ significantly upregulated the expression of inflammatory cytokines in placental trophoblast cells.The activation of PI3k/Akt signaling controlled by ROS that plays an important role in CdCl₂-induced upregulation of inflammatory cytokines in placental trophoblast cells.