叶酸对细菌脂多糖诱发小鼠不良妊娠结局的保护作用
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摘要
背景：不良妊娠结局主要包括自然流产、出生缺陷、早产、死胎和低出生体重等。大量研究表明，孕鼠接触细菌脂多糖(LPS)引起胚胎吸收、流产、胚胎和胎儿死亡、畸胎、宫内生长发育迟缓和早产等不良妊娠结局。LPS引起的这些不良妊娠结局与Toll样受体信号下游活性氧和炎性细胞因子密切相关。叶酸(FA)属B族水溶性维生素，具有抗炎和抗氧化作用，但叶酸是否能有效预防LPS引起的不良妊娠结局目前尚不清楚。
目的：研究孕鼠补充适量叶酸对孕中期和孕晚期LPS暴露所致不良妊娠结局的保护效应，阐明叶酸对孕期接触LPS致炎和氧化应激的拮抗效应及分子机理。
方法：本研究由六个独立的实验组成。实验一，孕鼠被随机分成6组，即生理盐水对照组、叶酸对照组、LPS组、LPS+叶酸灌胃组、LPS+叶酸注射组和LPS+叶酸饮用组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于孕第8-12天(GD8-GD12)经腹腔注射LPS (20μg/kg/d)；LPS+叶酸灌胃组孕鼠于注射LPS前1h经口灌胃补充叶酸(3mg/kg/d)；LPS+叶酸注射组孕鼠于注射LPS前1h经腹腔注射补充叶酸(3mg/kg/d)；LPS+叶酸饮用组孕鼠于GD7-GD12通过饮水补充叶酸(15mg/L，根据孕鼠饮水量换算相当于口服叶酸3mg/kg/d)。所有孕鼠于GD18剖杀，记录吸收胎数、死胎数和活胎数，检查并记录活胎外观畸形，称量活胎体重和测量活胎身长，评价活胎鼠骨骼畸形及发育情况。实验二，孕鼠被随机分为4组，即生理盐水对照组、叶酸对照组、LPS组和LPS+叶酸组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于GD8-GD12经腹腔注射LPS (20μg/kg/d)；LPS+叶酸组孕鼠于注射LPS前1h经口灌胃补充叶酸(3mg/kg/d)。孕鼠于末次LPS处理后2h剖杀取材，用Western blotting检测胎盘TLR4/NF-κB信号通路相关蛋白水平，用实时定量RT-PCR检测胎盘tnf-α、il-1β和il-6mRNA水平，用ELISA检测孕鼠血清和羊水TNF-α、IL-1β和IL-6水平，用Beutler改良法测定孕鼠肝脏和胎盘还原型谷胱甘肽(GSH)含量。实验三，孕鼠被随机分成6组，即生理盐水对照组、叶酸对照组、LPS组、LPS+低剂量叶酸组、LPS+中剂量叶酸组和LPS+高剂量叶酸组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于GD15经腹腔注射LPS (300μg/kg)；三个叶酸补充组于LPS注射前1h分别经口灌胃补充不同剂量叶酸(0.6、3、15mg/kg)。LPS注射后，密切观察并记录孕鼠早产和早产潜伏时间。实验四，孕鼠被随机分成6组，即生理盐水对照组、叶酸对照组、LPS组、LPS+低剂量叶酸组、LPS+中剂量叶酸组和LPS+高剂量叶酸组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于GD15经腹腔注射LPS (300μg/kg)；三个叶酸补充组于LPS注射前1h分别经口灌胃补充不同剂量叶酸(0.6、3、15mg/kg)。所有孕鼠于LPS注射后14h剖杀，判断并记录活胎和死胎数。实验五，孕鼠被随机分为6组，即生理盐水对照组、叶酸对照组、LPS组、LPS+低剂量叶酸组、LPS+中剂量叶酸组和LPS+高剂量叶酸组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于GD15-GD17经腹腔注射LPS (75μg/kg/d)；三个叶酸补充组于LPS注射前1h分别经口灌胃补充不同剂量叶酸(0.6、3、15mg/kg/d)。GD18剖杀孕鼠，记录吸收胎数、死胎和活胎数，称量活胎胎盘重量和胎仔体重，测量活胎胎仔身长。实验六，孕鼠随机分为4组，即生理盐水对照组、叶酸对照组、LPS组和LPS+叶酸组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于GD15经腹腔注射LPS(300μg/kg)；LPS+叶酸组于LPS注射前1h经口灌胃补充叶酸(3mg/kg)。LPS处理后2h剖杀半数孕鼠并收集胎盘标本，用Western blotting检测胎盘核蛋白NF-κBp65水平，用免疫组化方法对胎盘NF-κB p65进行细胞定位。剩余孕鼠于LPS注射后6h剖杀，收集孕鼠血清、羊水和胎盘，用Western blotting检测胎盘COX-2蛋白水平，用ELISA检测孕鼠血清和羊水IL-6和角化细胞源性细胞因子(KC)水平，用硝酸还原酶法检测孕鼠血清和羊水NO水平。
结果：主要包括以下两个方面：(1)补充叶酸对孕中期LPS暴露所致不良妊娠结局的保护作用。结果发现，孕中期暴露低剂量LPS主要引起胎鼠外观和骨骼畸形，最常见的外观畸形为露脑和脑膜膨出，最常见的骨骼畸形为胸骨畸形和肋骨畸形。经腹腔注射、经口灌胃和饮用等三种方式补充叶酸均能明显降低胎鼠外观和骨骼畸形发生率，其中以经口灌胃方式效果最明显，其平均每窝外观畸形率从19.95%降至0.96%，胸骨畸形率从19.10%降至2.95%，肋骨畸形率从26.86%降至11.62%。为探讨叶酸抑制LPS致畸的机理，进一步研究了叶酸的抗炎和抗氧化效应，结果发现，补充叶酸抑制孕中期LPS暴露诱导的胎盘MyD88上调、JNK和I-κB磷酸化及NF-κB激活；补充叶酸对抗孕中期LPS暴露引起的胎盘tnf-α、il-1β和il-6mRNA上调，降低LPS处理组孕鼠血清和羊水TNF-α、IL-1β和IL-6水平；补充叶酸减轻孕中期LPS暴露引起的孕鼠肝脏和胎盘GSH耗竭。(2)叶酸补充对孕晚期LPS暴露所致不良妊娠结局的保护效应。结果显示，孕晚期暴露高剂量LPS引起100%孕鼠发生早产，平均早产潜伏时间为14h；孕晚期暴露高剂量LPS引起89.3%胎仔死亡。孕晚期暴露低剂量LPS导致29.3%胎仔死亡；孕晚期暴露低剂量LPS引起活胎平均体重明显下降、身长降低。补充叶酸显著减轻孕晚期LPS暴露引起的早产，并呈剂量-效应关系；补充叶酸明显减少孕晚期LPS暴露引起的胎仔死亡；补充叶酸明显对抗孕晚期LPS暴露引起的宫内生长迟缓。进一步研究结果显示，补充叶酸抑制孕晚期LPS暴露引起的胎盘NF-κB信号激活；补充叶酸抑制孕晚期LPS暴露引起的胎盘迷路层滋养巨细胞NF-κB p65核转位；补充叶酸抑制孕晚期LPS暴露引起的胎盘COX-2上调；补充叶酸显著降低LPS处理组孕鼠羊水IL-6和KC水平。叶酸对LPS上调孕鼠血清和羊水NO水平无明显影响。
结论本研究得出如下结论：(1)叶酸补充可有效预防孕中期LPS暴露引起的胎鼠外观和骨骼畸形，其中经口灌胃方式效果最佳；(2)叶酸补充可减轻小鼠孕晚期LPS暴露引起的早产、死胎和宫内生长发育迟缓；(3)叶酸补充对LPS诱发小鼠不良妊娠结局的保护与其抗炎和抗氧化性能有关。
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.
目的：研究孕鼠补充适量叶酸对孕中期和孕晚期LPS暴露所致不良妊娠结局的保护效应，阐明叶酸对孕期接触LPS致炎和氧化应激的拮抗效应及分子机理。
方法：本研究由六个独立的实验组成。实验一，孕鼠被随机分成6组，即生理盐水对照组、叶酸对照组、LPS组、LPS+叶酸灌胃组、LPS+叶酸注射组和LPS+叶酸饮用组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于孕第8-12天(GD8-GD12)经腹腔注射LPS (20μg/kg/d)；LPS+叶酸灌胃组孕鼠于注射LPS前1h经口灌胃补充叶酸(3mg/kg/d)；LPS+叶酸注射组孕鼠于注射LPS前1h经腹腔注射补充叶酸(3mg/kg/d)；LPS+叶酸饮用组孕鼠于GD7-GD12通过饮水补充叶酸(15mg/L，根据孕鼠饮水量换算相当于口服叶酸3mg/kg/d)。所有孕鼠于GD18剖杀，记录吸收胎数、死胎数和活胎数，检查并记录活胎外观畸形，称量活胎体重和测量活胎身长，评价活胎鼠骨骼畸形及发育情况。实验二，孕鼠被随机分为4组，即生理盐水对照组、叶酸对照组、LPS组和LPS+叶酸组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于GD8-GD12经腹腔注射LPS (20μg/kg/d)；LPS+叶酸组孕鼠于注射LPS前1h经口灌胃补充叶酸(3mg/kg/d)。孕鼠于末次LPS处理后2h剖杀取材，用Western blotting检测胎盘TLR4/NF-κB信号通路相关蛋白水平，用实时定量RT-PCR检测胎盘tnf-α、il-1β和il-6mRNA水平，用ELISA检测孕鼠血清和羊水TNF-α、IL-1β和IL-6水平，用Beutler改良法测定孕鼠肝脏和胎盘还原型谷胱甘肽(GSH)含量。实验三，孕鼠被随机分成6组，即生理盐水对照组、叶酸对照组、LPS组、LPS+低剂量叶酸组、LPS+中剂量叶酸组和LPS+高剂量叶酸组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于GD15经腹腔注射LPS (300μg/kg)；三个叶酸补充组于LPS注射前1h分别经口灌胃补充不同剂量叶酸(0.6、3、15mg/kg)。LPS注射后，密切观察并记录孕鼠早产和早产潜伏时间。实验四，孕鼠被随机分成6组，即生理盐水对照组、叶酸对照组、LPS组、LPS+低剂量叶酸组、LPS+中剂量叶酸组和LPS+高剂量叶酸组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于GD15经腹腔注射LPS (300μg/kg)；三个叶酸补充组于LPS注射前1h分别经口灌胃补充不同剂量叶酸(0.6、3、15mg/kg)。所有孕鼠于LPS注射后14h剖杀，判断并记录活胎和死胎数。实验五，孕鼠被随机分为6组，即生理盐水对照组、叶酸对照组、LPS组、LPS+低剂量叶酸组、LPS+中剂量叶酸组和LPS+高剂量叶酸组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于GD15-GD17经腹腔注射LPS (75μg/kg/d)；三个叶酸补充组于LPS注射前1h分别经口灌胃补充不同剂量叶酸(0.6、3、15mg/kg/d)。GD18剖杀孕鼠，记录吸收胎数、死胎和活胎数，称量活胎胎盘重量和胎仔体重，测量活胎胎仔身长。实验六，孕鼠随机分为4组，即生理盐水对照组、叶酸对照组、LPS组和LPS+叶酸组。对照组孕鼠分别给予生理盐水或叶酸；LPS组孕鼠于GD15经腹腔注射LPS(300μg/kg)；LPS+叶酸组于LPS注射前1h经口灌胃补充叶酸(3mg/kg)。LPS处理后2h剖杀半数孕鼠并收集胎盘标本，用Western blotting检测胎盘核蛋白NF-κBp65水平，用免疫组化方法对胎盘NF-κB p65进行细胞定位。剩余孕鼠于LPS注射后6h剖杀，收集孕鼠血清、羊水和胎盘，用Western blotting检测胎盘COX-2蛋白水平，用ELISA检测孕鼠血清和羊水IL-6和角化细胞源性细胞因子(KC)水平，用硝酸还原酶法检测孕鼠血清和羊水NO水平。
结果：主要包括以下两个方面：(1)补充叶酸对孕中期LPS暴露所致不良妊娠结局的保护作用。结果发现，孕中期暴露低剂量LPS主要引起胎鼠外观和骨骼畸形，最常见的外观畸形为露脑和脑膜膨出，最常见的骨骼畸形为胸骨畸形和肋骨畸形。经腹腔注射、经口灌胃和饮用等三种方式补充叶酸均能明显降低胎鼠外观和骨骼畸形发生率，其中以经口灌胃方式效果最明显，其平均每窝外观畸形率从19.95%降至0.96%，胸骨畸形率从19.10%降至2.95%，肋骨畸形率从26.86%降至11.62%。为探讨叶酸抑制LPS致畸的机理，进一步研究了叶酸的抗炎和抗氧化效应，结果发现，补充叶酸抑制孕中期LPS暴露诱导的胎盘MyD88上调、JNK和I-κB磷酸化及NF-κB激活；补充叶酸对抗孕中期LPS暴露引起的胎盘tnf-α、il-1β和il-6mRNA上调，降低LPS处理组孕鼠血清和羊水TNF-α、IL-1β和IL-6水平；补充叶酸减轻孕中期LPS暴露引起的孕鼠肝脏和胎盘GSH耗竭。(2)叶酸补充对孕晚期LPS暴露所致不良妊娠结局的保护效应。结果显示，孕晚期暴露高剂量LPS引起100%孕鼠发生早产，平均早产潜伏时间为14h；孕晚期暴露高剂量LPS引起89.3%胎仔死亡。孕晚期暴露低剂量LPS导致29.3%胎仔死亡；孕晚期暴露低剂量LPS引起活胎平均体重明显下降、身长降低。补充叶酸显著减轻孕晚期LPS暴露引起的早产，并呈剂量-效应关系；补充叶酸明显减少孕晚期LPS暴露引起的胎仔死亡；补充叶酸明显对抗孕晚期LPS暴露引起的宫内生长迟缓。进一步研究结果显示，补充叶酸抑制孕晚期LPS暴露引起的胎盘NF-κB信号激活；补充叶酸抑制孕晚期LPS暴露引起的胎盘迷路层滋养巨细胞NF-κB p65核转位；补充叶酸抑制孕晚期LPS暴露引起的胎盘COX-2上调；补充叶酸显著降低LPS处理组孕鼠羊水IL-6和KC水平。叶酸对LPS上调孕鼠血清和羊水NO水平无明显影响。
结论本研究得出如下结论：(1)叶酸补充可有效预防孕中期LPS暴露引起的胎鼠外观和骨骼畸形，其中经口灌胃方式效果最佳；(2)叶酸补充可减轻小鼠孕晚期LPS暴露引起的早产、死胎和宫内生长发育迟缓；(3)叶酸补充对LPS诱发小鼠不良妊娠结局的保护与其抗炎和抗氧化性能有关。
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.
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