| Background Adverse pregnancy outcomes include early pregnancy loss, birth defects,preterm delivery, stillbirth and intra-uterine growth restriction (IUGR). Numerousreports have demonstrated that maternal lipopolysaccharide (LPS) exposure results inadverse pregnancy outcomes, including embryonic resorption, fetal malformation,preterm delivery, fetal death and IUGR in mice. LPS-induced adverse pregnancyoutcomes are associated with excess production of reactive oxygen species (ROS) andrelease of inflammatory cytokines. Folic acid (FA) is a water-soluble B-complexvitamin. FA has anti-inflammatory and antioxidant effects. Nevertheless, it is unknownwhether FA protects against LPS-induced adverse pregnancy outcomes.Objective To explore the protective effects of FA on LPS-induced adverse pregnancyoutcomes and to clarify whether FA could alleviate LPS-induced inflammation andoxidative stress.Methods This study included six experiments.(1) To investigate the effects of FA onLPS-induced adverse pregnancy outcomes during the second trimester, the pregnantmice were divided into six groups randomly. All pregnant mice except controls (eithersaline or FA) were intraperitoneally (i.p.) injected with LPS (20μg/kg) daily from GD8to GD12. FA was administered in three different modes. In LPS+FAig group, pregnantmice were administered with FA (3mg/kg/d) by gavage at1h before LPS injection. In LPS+FAip group, pregnant mice were i.p. injected with FA (3mg/kg/d) at1h beforeLPS injection. In LPS+FAdw, pregnant mice were administered with FA throughdrinking water (15mg/L) from GD7to GD12. All dams were sacrificed on GD18. Foreach litter, the number of resorption sites, dead fetuses and live fetuses were counted.Live fetuses in each litter were weighed and examined for gross morphologicalmalformations. Crown-rump length was measured. All fetuses were subsequentlyevaluated the supraoccipital ossification and skeletal malformations.(2) To investigatethe effects of FA supplementation on LPS-induced oxidative stress and inflammationduring the second trimester, the pregnant mice were divided into four groups randomly.In LPS group, pregnant mice were i.p. injected with LPS (20μg/kg) daily from GD8toGD12. In LPS+FA group, pregnant mice were administered with FA (3mg/kg/d) bygavage at1h before LPS injection. In control group, pregnant mice were i.p. injectedwith NS daily from GD8to GD12. In FA alone group, pregnant mice were administeredwith FA (3mg/kg/d) by gavage before NS injection. All dams were sacrificed on GD12.Maternal serum and amniotic fluid were collected for measurement of inflammatorycytokines (TNF-α, IL-1β and IL-6). Some placentae were collected for real-timeRT-PCR and Western blotting. Maternal liver and other placentae were collected formeasurement of GSH content.(3) To investigate the effects of FA on LPS-inducedpreterm delivery, the pregnant mice were divided randomly into six groups. All pregnantmice except controls (either saline or FA) received an injection of LPS (300μg/kg) onGD15. In LPS+FA groups, the pregnant mice were orally administered with differentdoses of FA (0.6,3,15mg/kg)1h before LPS injection. Pregnant mice were observedclosely for any signs of preterm delivery.(4) To investigate the effects of FA onhigh-dose LPS-induced fetal death, the pregnant mice were divided randomly into sixgroups. All pregnant mice except controls (either saline or FA) received an injection ofLPS (300μg/kg) on GD15. In LPS+FA groups, the pregnant mice were orallyadministered with different doses of FA (0.6,3,15mg/kg)1h before LPS injection. All pregnant mice were sacrificed at14h after LPS injection. It was rapidly determinedwhether the fetus was viable by tactile stimulation.(5) To investigate the effects of FAon LPS-induced IUGR, the pregnant mice were divided randomly into six groups. Allpregnant mice except controls (either saline or FA) received an i.p. injection of LPS (75μg/kg) daily from GD15to GD17. In LPS+FA groups, the pregnant mice were orallyadministered with different doses of FA (0.6,3,15mg/kg/d)1h before LPS injection.All dams were sacrificed on GD18. For each litter, the number of resorption sites, deadfetuses, and live fetuses were counted. Live fetuses in each litter were weighed.Crown-rump length was measured.(6) To investigate the effects of FA on LPS-inducedinflammation during the third trimester, the pregnant mice were divided into four groupsrandomly. All pregnant mice except controls (either saline or FA) received an i.p.injection of LPS (300μg/kg) on GD15. In LPS+FA group, the pregnant mice wereorally administered with3mg/kg of FA1h before LPS injection. Half of the dams weresacrificed2h after LPS injection. Placentae were collected for measurements of nuclearNF-κB p65. The remaining animals were sacrificed at6h after LPS injection. Maternalserum and amniotic fluid was collected for measurement of IL-6, keratinocyte-derivedcytokine (KC) and nitric oxide (NO). Placentae were collected for measurements ofCOX-2.Results A five-day LPS (20μg/kg/d) injection during the second trimester resulted in50%(8/16) of litters with externally malformed fetuses. The incidence of externalmalformed fetuses was significantly increased in fetuses from dams injected with LPSfrom GD8to GD12, in which19.95%of fetuses per litter were externally malformed.Exencephaly and encephalomeningocele were two of the most common malformations.In addition, the incidence of fetus with supraoccipital ossification and skeletalmalformations was significantly increased in LPS-treated mice. FA supplementation bythree ways all attenuated LPS-induced external and skeletal malformations. And thebest protective effect was by orally. Additional experiment showed that FA significantly attenuated LPS-induced expression of MyD88in placenta. Moreover, folic acidinhibited LPS-induced c-Jun NH2-terminal kinase (JNK) phosphorylation, I-κBphosphorylation and NF-κB activation in placenta. Correspondingly, FA significantlyattenuated LPS-induced TNF-α, IL-1β and IL-6in placenta, maternal serum andamniotic fluid. In addition, FA significantly attenuated LPS-induced GSH depletion inmaternal liver and placenta. LPS injection with a high dose of LPS on GD15resulted in100%dams delivered before GD18. FA pretreatment delayed the latency interval ofpreterm delivery and reduced the incidence of preterm delivery. Moreover, FApretreatment significantly reduced the number of dead fetuses per litter in LPS-treatedmice. In addition, FA pretreatment significantly attenuated LPS-induced IUGR in adose-dependent manner. Additional experiment showed that FA pretreatment inhibitedLPS-induced activation of NF-κB in placenta. Correspondingly, FA pretreatmentsignificantly reduced the level of IL-6and KC in amniotic fluid of LPS-treated mice. Inaddition, FA pretreatment significantly attenuated LPS-induced upregulation of placentalCOX-2. However, FA had little effect on LPS-induced release of NO in maternal serumand amniotic fluid.Conclusions (1) FA supplementation during pregnancy efficiently preventsLPS-induced teratogenicity in mice.(2) FA supplementation during pregnancy protectsagainst LPS-induced preterm delivery, fetal death and IUGR in a dose-dependentmanner.(3) The protection of FA against LPS-induced adverse pregnancy outcomesmight be, at least partially, attributed to its anti-inflammatory and antioxidant effects. |
PDF Full Text Request