Vitamin D3 Pretreatment Protects Against Lipopolysaccharide-induced Early Embryo Loss Through Its Anti-inflammatory Effects

Objective We aimed to investigate the effects of vitamin D3(Vit D3)on LPS-induced early embryo loss in mice.Methods Females were checked by 7:00 am the next morning,and the presence of a vaginal plug was designated as gestational day(GD)0.Pregnant mice were divided into four groups.In this study,pregnantmice were divided into four groups.In the Vit D3+LPS group,pregnant mice were pretreated with Vit D3(25 ?g/kg,dissolved in corn oil)daily by gavage beginning at 2 days before LPS injection(150?g/kg,dissolved in normal saline,ip)on GD7.In the LPS-alone group,pregnant mice were pretreated with corn oil daily by gavage beginning at 2 days before LPS injection on GD7.In the Vit D3-alone group,pregnant mice were pretreated with Vit D3(25 ?g/kg,dissolved in corn oil)daily by gavage beginning at 2 days before normal saline injection on GD7.In the control group,pregnant mice were pretreated with corn oil daily by gavage beginning at 2 days before normal saline injection on GD7.The doses of LPS used in this study referred to others.To investigate the effects of Vit D3 on LPS-induced early embryo loss,dams were killed on GD8.The uteri were examined macroscopically to count the number of gross implantation sites and viable embryos.Early embryo loss was assessed by observing the contents of uterus.As most of pregnant mice only lost one or a few embryos at 24 hours after LPS injection,pregnant mice with one or more embryos loss were considered abortion pregnancy.The ratio of abortion to normal pregnancy was calculated according to others.Mice without gross implantation sites or tissue debris in the uterus were considered not pregnant and excluded from the experiment.To investigate the effects of Vit D3 on LPS-induced placental inflammatory signaling,some pregnant mice(six each group)were killed at 2 hours after NS or LPS injection.The other pregnant mice(six each group)were killed at 6 hours after NS or LPS injection.Maternal sera were collected for biochemical analysis.Some uteri were collected for immunohistochemistry.Results LPS caused 62.5% pregnant mice with early embryo loss.Interestingly,the rate of abortion dropped to 14.3% when pregnant mice were pretreated with Vit D3.Additional experiment showed that Vit D3 significantly attenuated LPS-evoked elevation on TNF-?,IFN-?,MIP-2,and nitrate plus nitrite in maternal serum.In addition,Vit D3 alleviated LPS-induced COX-2 expression in the decidua and attenuated the elevation of PGF2? in maternal serum.Although Vit D3 had no effect on IL-10 in maternal serum,it induced further elevation of serum IL-10 level in LPS-treated mice.Further analysis showed that Vit D3 activated VDR signaling,simultaneously inhibited LPS-induced nuclear translocation of NF-?B p65 subunits in the decidua.Conclusions Vit D3 protects mice from LPS-induced early embryo loss at least partially through its anti-inflammatory effects.