The Effect Of Prenatal Exposure To Lipopolysaccharide On Intra-uterine Fetal Growth Development And Skeletal Development In Mice

Lipopolysaccharide (LPS) or endotoxin (ET) is a toxic component of cell walls of gram-negative bacteria and is widely present in the digestive tracts of humans and animals. Gastrointestinal distress and alcohol drinking often increase permeability of LPS from gastrointestinal tract into blood. LPS has been associated with adverse developmental outcome, including embryonic resorption, abortion and preterm labor in animals. However, the exact mechanism of LPS-induced developmental toxicity remains unclear. In this study, we investigated the effect of prenatal exposure to lipopolysaccharide on intra-uterine fetal growth development and skeletal development in mice. We also explored the roles of reactive oxygen species (ROS), tumor necrosis factor-α(TNF-α) and nitric oxide (NO) on LPS-induced IUFD, IUGR and skeletal development retardation in mice, and the effect of N-acetylcysteine (NAC), ascorbic acid (AA) and melatonin (N-acetyl-5-methoxytryptamine, MT) on LPS induced IUFD, IUGR and skeletal development retardation.1. The effect of prenatal exposure to lipopolysaccharide on intra-uterine fetal growth development and skeletal development in miceTo investigate the effects of LPS on IUFD, IUGR and skeletal development retardation. The timed pregnant mice were injected with different doses of LPS (25~75μg/kg, ip) on gestational day (gd) 15-17. The number of live fetuses, dead fetuses and resorption sites was counted on gd 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were examined and skeletal development was evaluated. Results showed that maternally administered LPS significantly increased fetal mortality in a dose-dependent manner. In addition, maternally administered LPS significantly reduced fetal weight and crown-rump and tail lengths of live fetuses and retarded skeletal ossification in caudal vertebrae, anterior and posterior phalanges, and supraoccipital bone in a dose-dependent manner. These results indicate that maternal LPS exposure at late gestational stages results in IUFD, IUGR and skeletal development retardation in mice.2. The role of TNF-αin LPS-induced IUFD, IUGR and skeletal development retardation in miceIn this study, we investigated the role of TNF-αon LPS-induced IUFD, IUGR and skeletal development retardation in mice. All pregnant mice except controls accepted an intraperitoneal injection of LPS (75μg/kg, ip) daily on gestational late stage. In LPS+PTX group, the pregnant mice were injected with pentoxifylline (PTX, 100 mg/kg, ip) at 0.5 h before LPS treatment. The saline- and PTX- treated pregnant mice served as controls. Some pregnant mice were sacrificed on the day of injection, maternal liver and fetal liver and placenta were dissected for total RNA extraction, maternal serum and amniotic fluid were collected for measurement of TNF-αconcentration, and the left were injected the drug continuously and killed on gd 18. The number of live fetuses, dead fetuses and resorption sites were counted in each litter. Live fetuses were weighted, Crown-rump and tail lengths were measured, and the skeleton of live fetuses were evaluated in each litter. Results showed that pretreatment with PTX, an inhibitor of TNF-αsynthesis, was used to inhibit LPS-evoked TNF-αproduction, As expected, almost blocked LPS-induced IUFD. In addition, PTX significantly alleviated LPS-induced decreases in crown-rump and tail lengths and decrease in fetal weight, and reversed LPS-induced developmental retardation in Posterior phalanx, Caudal vertebrae and breastbone. PTX significantly attenuated LPS-induced lipid peroxidation in maternal liver, fetal liver and placenta. However, PTX had not effect on LPS-induced GSH depletion in maternal liver. Furthermore, PTX evidently mitigated LPS-induced increase in TNF-αconcentration in amniotic fluid and maternal serum. These results indicate that TNF-αis, at least in part, mediated in LPS-induced IUFD, IUGR and skeletal development retardation in mice.3. The roles of ROS and NO in LPS-induced IUFD, IUGR and skeletal development retardation in miceTo investigated the role of Reactive oxygen species (ROS) and nitric oxide (NO) on LPS-induced IUFD, IUGR and skeletal development retardation in mice. In LPS-treated group, the pregnant mice were injected with LPS (75μg/kg, ip) daily on gestational late stage. In the LPS+PBN group, the pregnant mice were injected with 100 mg/kg of Alpha-phenyl-N-t-butylnitrone (PBN, ip) at 0.5 h before LPS (75μg/kg, ip) treatment, followed by additional dose (50 mg/kg) of PBN at 3 h after LPS treatment. In the LPS+AG group, the pregnant mice were injected with 50 mg/kg of aminoguanidine (AG, ip) at 0.5 h before LPS (75μg/kg, ip) treatment, followed by additional dose (25 mg/kg) of AG at 3 h after LPS treatment. the saline- and AG- treated pregnant mice served as controls. Some pregnant mice were sacrificed on the day of injection, maternal liver, fetal liver and placenta were dissected for measurement of MDA concentration and so on. The left were injected the drug continuously and killed on gd 18, For each litter, the number of live fetuses, dead fetuses and resorption sites were counted. Live fetuses in each litter were weighted. Crown-rump and tail lengths were measured. And the skeleton of all live fetuses in each litter were evaluated. Results showed that PBN, a free radical spin-trapping agent, almost blocked LPS-induced IUFD and revered LPS-induced intra-uterine skeletal development retardation. And abolished LPS-induced decrease in fetal weight and crown-rump and tail lengths. However, AG, an inhibitor of inducible nitric oxide synthase (iNOS) activity, which was used to inhibit LPS-evoked NO production, had no effect on LPS-induced IUFD, IUGR and skeletal development retardation in mice. Furthermore, PBN significantly attenuated LPS-induced lipid peroxidation in maternal liver, fetal liver and placenta, and GSH depletion in maternal liver and fetal liver. In addition, NO contents in serum and amniotic fluid were statistically indistinguishable between LPS-treated groups and control (data not shown). These results suggest that oxidative stress is, at least in part, mediated in LPS-induced IUFD, IUGR and skeletal development retardation; and LPS-induced IUFD, IUGR and skeletal development retardation in mice are independent of NO production.4. The effect of NAC, AA and MT on LPS-induced IUFD, IUGR and skeletal development retardation in miceNAC, AA and MT are potent antioxidants. To investigate the role of NAC, AA and MT on LPS-induced IUFD, IUGR and skeletal development retardation in ICR mice. The present study included two separate experiments. In experiment one, All pregnant mice except controls (either saline or NAC, AA, MT) received an intraperitoneal injection of LPS (75μg/kg, ip) on gd 15-17. In LPS+NAC, LPS+AA and LPS+MT groups, the pregnant mice were treated with NAC, AA or MT at before and / or after LPS. The number of live fetuses, dead fetuses and resorption sites were counted on gd 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were examined and skeletal development was evaluated. In experiment two, All pregnant mice except controls (either saline or NAC, AA, MT) received an intraperitoneal (75μg/kg) injection of LPS on gd 15. In LPS+NAC, LPS+AA and LPS+MT groups, the pregnant mice were treated with NAC, AA or MT at before and / or after LPS. All dams were sacrificed at 1.5 h or 6 h after LPS treatment. Maternal liver, fetal liver and placenta were dissected for GSH and MDA measurements, maternal serum and amniotic fluid were collected for TNF-αanalyses. Results showed that pretreatment with NAC, AA, and pre- plus post- treatments and post-treatment with MT significantly attenuated LPS-induced IUFD. However, post-treatment with NAC, both post- and pre- plus post- treatments with AA had less effect on LPS-induced IUFD. In addition, pre- and post- treatment with NAC, AA- and MT- treatments significantly alleviated LPS-induced IUGR, and reversed LPS-induced skeletal developmental retardation. Furthermore, pretreatment with NAC significantly attenuated LPS-induced elevation in TNF-a concentration in maternal serum and amniotic fluid and lipid peroxidation in maternal liver, fetal livers and placenta. By contrast to pretreatment, post-treatment with NAC had no effect on LPS-induced TNF-a production and lipid peroxidation; Pre- and post- treatmentwith AA significantly attenuated LPS-induced lipid peroxidation; pre- plus post- treatments with melatonin significantly attenuated LPS-induced lipid peroxidation in maternal liver and placenta. However, melatonin had no effect on LPS-induced GSH depletion. These results indicate that NAC had a dual effect on LPS-induced IUFD, IUGR and skeletal development. Pretreatment with NAC improves fetal survival and reverses LPS-induced IUGR and skeletal development retardation, whereas post-treatment with NAC aggravates LPS induced preterm labor; Pretreatment with AA protected against LPS-induced IUFD and reversed LPS-induced IUGR and skeletal development retardation, whereas post-treatment and pre- + post-treatment had less effect on LPS-induced IUFD. MT protects against LPS-induced IURD, IUGR and skeletal development retardation via counteracting LPS-induced oxidative stress.In summary, the present results allow us to reach the following conclusions. First, the timed pregnant mice were injected low dose of LPS on gestational day (gd) 15-17 resulted in intra-uterine fetal death (IUFD), intra-uterine growth retardation (IUGR) and skeletal development retardation in mice; second, TNF-αpartially contributes to LPS-induced IUFD, IUGR and skeletal development retardation in mice; third, LPS-induced IUFD, IUGR and skeletal development retardation in mice is mediated, at least in part, by reactive oxygen species; fourth, LPS-induced IURD, IUGR and skeletal development retardation in mice is independent of NO production; and fifth, NAC had a dual effect on LPS-induced IUFD, IUGR and skeletal development retardation, pretreatment with NAC improves fetal survival and reverses LPS-induced IUGR and skeletal development retardation, whereas post-treatment with NAC aggravates LPS induced preterm labor; Pretreatment with AA protected against LPS-induced IUFD and reversed LPS-induced IUGR and skeletal development retardation, whereas post-treatment and pre- + post-treatment had less effect on LPS-induced IUFD; and MT protects against LPS-induced IURD, IUGR and skeletal development retardation in mice.