Supplementation With Vitamin D3 During Pregnancy Protects Against Lipopolysaccharide-Induced Neural Tube Defects

Objective Several reports demonstrated that maternal LPS exposure at middle gestational stage caused neural tube defects(NTDs). The present study investigated the effects of supplementation with vitamin D3(Vit D3) during pregnancy on LPS-induced NTDs.Methods The animals were fed ad libitum with free access to food and water and were housed in a room with controlled lighting(12-h light/12-h dark cycle), temperature(20–25°C) and humidity(50%±5%) for 2 weeks before use. After acclimatization, four female mice were mated with two males in a cage at about 9:30 PM. Females were checked at 7:30 AM the next morning, and the presence of a vaginal plug was designated as gestational day(GD) 0. To investigate the effects of vitamin D3 on LPS-induced NTDs, pregnant mice were randomly divided into four groups. In the LPS group, pregnant mice were intraperitoneally(i.p.) injected with LPS(25 μg/kg) daily from GD8 to GD12. In the LPS+Vit D3 group, pregnant mice were pretreated with vitamin D3(25 μg/kg) by gavage daily 2 days before LPS treatment. In the Vit D3 group, pregnant mice were administered with vitamin D3(25 μg/kg, i.g.) by gavage daily from GD6 to GD12.The normal saline-treated pregnant mice served as controls. All dams were sacrificed on GD18. Numbers of live fetuses, dead fetuses and resorption sites were counted in every litter. Live fetuses were weighed and examined for external malformations. All fetuses were then stored in ethanol a minimum of two weeks for subsequent skeletal evaluation. To investigate the effects of vitamin D3 on the transport of folate through placentas into the embryos, pregnant mice were randomly divided into four groups. In the LPS group, pregnant mice were injected with LPS(25 μg/kg, i.p.)daily from GD8 to GD12. In the LPS+Vit D3 group, pregnant mice were pretreated with vitamin D3(25 μg/kg) by gavage daily 2 days before LPS injection. In the Vit D3 group, pregnant mice were administered with vitamin D3(25 μg/kg) by gavage daily from GD6 to GD12. In the control group, pregnant mice were i.p. injected with NS daily from GD8 to GD12. All pregnant mice were sacrificed 12 h after the last LPS injection. Maternal sera and embryos were collected for folate measurements. To investigate the effects of vitamin D3 on the expression of placental folate transportors, pregnant mice were randomly divided into four groups. In the LPS group, pregnant mice were injected with a single dose of LPS(25 μg/kg, i.p.) on GD10. In the LPS+Vit D3 group, pregnant mice were pretreated with vitamin D3(25 μg/kg) by gavage daily 2 days before LPS injection. In the Vit D3 group, pregnant mice were administered with vitamin D3(25 μg/kg) by gavage daily from GD8 to GD10. In the control group, pregnant mice were i.p. injected with NS on GD10. All pregnant mice were sacrificed 12 h after LPS injection. Placentas were collected for real-time RT-PCR. To investigate the effects of vitamin D3 on LPS-induced inflammatory cytokines, pregnant mice were randomly divided into four groups. In the LPS group, pregnant mice were i.p. injected with a single dose of LPS(25 μg/kg) on GD10. In the LPS+Vit D3 group, pregnant mice were administered with vitamin D3(25 μg/kg) by gavage daily 2 days before LPS injection. In the Vit D3 group, pregnant mice were administered with vitamin D3(25 μg/kg) by gavage daily from GD8 to GD10. In the control group, pregnant mice were i.p. injected with NS on GD10. All pregnant mice were sacrificed 2 h after LPS injection. Maternal sera and amniotic fluid were collected for measurement of inflammatory cytokines. Placentas were collected for real-time RT-PCR.Result A five-day LPS injection resulted in 62.5%(10/16) of litters and 20.3% of fetuses with NTDs, and the average weights of fetuses and placenta were reduced in LPS groups, LPS injection resulted in skeletal development retardation in fetuses.Additional experiment showed that a five-day LPS injection down-regulated placental proton-coupled folate transporter(pcft) and reduced folate carrier 1(rfc1), two major folate transporters in placentas. Consistent with down-regulation of placental folate transporters, the level of folate acid in fetuses of LPS groups was lower than controls, but there was no significant difference between each group of folate contents in maternal sera, indicating that folate transport from maternal circulation into embryos was disturbed in LPS-treated mice. LPS induced the release of TNF-α and IL-6 in maternal sera and amniotic fluid and up-regulated the expression of placental inflammatory cytokines and chemokines. Interestingly, Vit D3 not only inhibited placental inflammation but also attenuated LPS-induced down-regulation of placental folate transporters. Correspondingly, Vit D3 markedly improved folate transport from maternal circulation into the embryos. Importantly, supplementation with Vit D3 during pregnancy protected mice from LPS-induced NTDs.Conclusion Supplementation with Vit D3 during pregnancy prevents LPS-induced NTDs through inhibiting placental inflammation and improving folate transport from maternal circulation into the embryos.