Melatonin protects against lipopolysaccharide-induced intra-uterine fetal death and growth retardation in mice.

Lipopolysaccharide (LPS) has been associated with adverse developmental outcomes, including intra-uterine fetal death (IUFD) and intra-uterine growth retardation (IUGR). However, the exact mechanism for LPS-induced IUFD and IURD remains unclear. LPS stimulates macrophages to generate reactive oxygen species (ROS). Therefore, we hypothesize that ROS may be involved in LPS-induced IUFD and IURD. Melatonin is a powerful endogenous antioxidant. In this study, we investigated the protective effects of melatonin on LPS-induced IUFD and IURD in ICR mice. All pregnant mice except controls received an intraperitoneal (75 microg/kg, i.p.) injection of LPS on gestational day (gd) 15-17. The experiment was carried out in two different modes. In mode A, the pregnant mice received two doses of melatonin within 24 hr, one (5 or 10 mg/kg) injected immediately after LPS and the other (5 or 10 mg/kg) injected at 3 hr after LPS. In mode B, the pregnant mice were pretreated with 10 mg/kg of melatonin 18 hr before LPS and then received two doses of melatonin in 24 hr, one (10 mg/kg) injected immediately after LPS and the other (10 mg/kg) injected 3 hr 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. Results showed that post-treatments with melatonin significantly attenuated LPS-induced IUFD in a dose-dependent manner. Surprisingly, pre- plus post-treatments with melatonin almost blocked LPS-induced IUFD. In addition, both post-treatments and pre- plus post-treatments with melatonin significantly alleviated LPS-induced decreases in crown-rump and tail lengths and reversed LPS-induced skeletal developmental retardation. However, melatonin had little effect on LPS-induced decrease in fetal weight. Furthermore, pre- plus post-treatments with melatonin significantly attenuated LPS-induced lipid peroxidation in maternal liver. These results indicate that melatonin protects against LPS-induced IURD and IUGR via counteracting LPS-induced oxidative stress.

Perinatal lipopolysaccharide exposure downregulates pregnane X receptor and Cyp3a11 expression in fetal mouse liver.

The pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates cytochrome P450 3A (CYP3A) gene transcription in a ligand-dependent manner. Lipopolysaccharide (LPS)-induced downregulation on PXR and cyp3a11 in adult mouse liver has been well characterized. In this study, we investigated the effects of maternal LPS exposure on PXR and cyp3a11 expression in fetal mouse liver. Pregnant ICR mice were injected intraperitoneally with different doses of LPS (0.1 approximately 0.5 mg/kg) on gestational day (GD) 17. PXR and cyp3a11 mRNA levels were determined using RT-PCR. Erythromycin N-demethylase (ERND) activity was used as an indicator of CYP3A expression in this study. Results showed that LPS significantly downregulated PXR and cyp3a11 mRNA levels and ERND activity in fetal liver in a dose-dependent manner. LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity was attenuated after pregnant mice were pretreated with alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin trapping agent. Additional experiment revealed that LPS significantly increased lipid peroxidation in fetal liver, which was also attenuated by PBN pretreatment. Furthermore, LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity was prevented by maternal pretreatment with N-acetylcysteine (NAC). Maternal pretreatment with NAC also inhibited LPS-initiated lipid peroxidation and GSH depletion in fetal liver. However, maternal LPS treatment did not affect nitrite plus nitrate concentration in fetal liver. Correspondingly, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), has no effect on LPS-induced downregulation of PXR and cyp3a11 expression and ERND activity in fetal liver. These results indicated that maternal LPS exposure downregulates PXR and cyp3a11 in fetal mouse liver. Reactive oxygen species (ROS) may be involved in LPS-induced downregulation of PXR and cyp3a11 in fetal mouse liver.

Lipopolysaccharide treatment downregulates the expression of the pregnane X receptor, cyp3a11 and mdr1a genes in mouse placenta.

The cytochrome P450 3A (CYP3A) is a member of the cytochrome P450 monooxygenase superfamily. The multidrug resistance 1 (MDR1) gene belongs to the ATP-binding cassette (ABC) family. Pregnane X receptor (PXR) is a nuclear receptor that regulates its target gene transcription in a ligand-dependent manner. Lipopolysaccharide (LPS)-induced downregulation of PXR, CYP3A and MDR1 in liver has been demonstrated in a series of studies. However, it is not clear whether LPS represses the expression of PXR, CYP3A and MDR1 in placenta. In the present study, we investigated the effects of LPS on the expression of PXR, cyp3a11 and mdr1a in mouse placenta. Pregnant ICR mice were injected intraperitoneally with different doses of LPS (0.1-0.5 mg/kg) on gestational day (gd) 17. Placental PXR, cyp3a11 and mdr1a mRNA levels were determined at 12 h after LPS treatment using RT-PCR. Results showed that LPS significantly downregulated PXR, cyp3a11 and mdr1a mRNA levels in a dose-dependent manner. LPS-induced downregulation of PXR, cyp3a11 and mdr1a mRNA in placenta was significantly attenuated after pregnant mice were pre- and post-treated with alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin trapping agent. Additional experiments revealed that LPS increased lipid peroxidation and proinflammatory cytokine expressions in mouse placenta, all of which were also attenuated by PBN. Furthermore, LPS-induced downregulation of PXR, cyp3a11 and mdr1a mRNA in mouse placenta was prevented by N-acetylcysteine (NAC). NAC also inhibited LPS-initiated lipid peroxidation, GSH depletion and proinflammatory cytokine expressions in mouse placenta. These results indicated that LPS downregulates placental PXR, cyp3a11 and mdr1a mRNA expressions. Reactive oxygen species (ROS) may be involved in LPS-induced downregulation of PXR, cyp3a11 and mdr1a in mouse placenta.

Melatonin attenuates lipopolysaccharide-induced down-regulation of pregnane X receptor and its target gene CYP3A in mouse liver.

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates target gene transcription in a ligand-dependent manner. Our earlier study indicated that reactive oxygen species contribute to lipopolysaccharide (LPS)-induced down-regulation of PXR and its target gene CYP3A in mouse liver. Melatonin is a powerful endogenous antioxidants. In this study, we investigated the effects of melatonin on LPS-induced down-regulation of PXR and CYP3A in mouse liver. Mice were intraperitoneally administrated different doses of melatonin before and/or after LPS treatment. PXR and CYP3A11 mRNA levels were measured using RT-PCR. Erythromycin N-demethylase (ERND) was used as an indicator of CYP3A catalytic activity. Results indicated that melatonin significantly attenuated LPS-induced down-regulation of PXR and CYP3A11 mRNA levels in a dose-dependent manner. Repeated doses of melatonin (10 mg/kg) treatments also significantly attenuated LPS-induced down-regulation of dexamethasone-inducible CYP3A11 mRNA level and ERND activity in mouse liver. In addition, the present study also shows that melatonin significantly increased hepatic superoxide dismutase, Se-dependent glutathione peroxidase, glutathione reductase and catalase activities and glutathione levels in LPS-treated mice. These findings suggest that melatonin may exert its protective effects on LPS-induced down-regulation of PXR and CYP3A via counteracting LPS-induced oxidative stress in mouse liver.

Kupffer cells and reactive oxygen species partially mediate lipopolysaccharide-induced downregulation of nuclear receptor pregnane x receptor and its target gene CYP3a in mouse liver.

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates target gene transcription in a ligand-dependent manner. The in vivo effects of lipopolysaccharide (LPS) on expression of PXR and its target gene cytochrome P450 3A (CYP3A) in mouse liver were investigated in this study. Mice were injected intraperitoneally with different doses of LPS (0.1-5.0 mg/kg). PXR and CYP3A11 mRNA levels were measured using reverse transcription polymerase chain reaction. Results indicate that LPS significantly inhibits the expression of PXR mRNA in a dose-dependent manner, followed by suppression of CYP3A11 mRNA in mouse liver. LPS also represses the upregulation of CYP3A11 mRNA levels and erythromycin N-demethylase (ERND) catalytic activity in mice pretreated with PXR ligands dexamethasone, rifampicin, mifepristone, and phenobarbital. LPS-induced downregulation of PXR and CYP3A11 mRNA in liver was significantly attenuated in mice pretreated with gadolinium chloride, a selective Kupffer cell toxicant. Pretreatment with a single dose of gadolinium chloride (10 mg/kg) also significantly attenuated LPS-induced downregulation of dexamethasone-, rifampicim-, mifepristone-, and phenobarbital-inducible, CYP3A11 mRNA expression and ERND activity in mouse liver. Furthermore, LPS-induced downregulation of PXR and CYP3A11 mRNA was significantly attenuated in mice pretreated with allopurinol, an inhibitor of xanthine oxidase, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. Allopurinol and diphenyleneiodonium chloride pretreatment also attenuated the repressive effects of LPS on dexamethasone-, rifampicin-, mifepristone-, and phenobarbital-inducible CYP3A11 mRNA expression and ERND catalytic activity in mouse liver. However, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, has no effect on LPS-induced downregulation of PXR and CYP3A11 mRNA. Finally, LPS-induced downregulation of PXR and CYP3A11 mRNA was prevented in mice pretreated with either N-acetylcysteine or ascorbic acid. These antioxidants also prevented the repressive effects of LPS on dexamethasone-, rifampicin-, mifepristone-, and phenobarbital-inducible CYP3A11 mRNA expression and ERND catalytic activity in mouse liver. These results indicate that Kupffer cells contribute to LPS-induced downregulation of PXR and CYP3A in mouse liver. Reactive oxygen species, produced possibly by NADPH oxidase and perhaps by xanthine oxidase, are involved in LPS-induced downregulation of nuclear receptor PXR and its target gene CYP3A in mouse liver.