Obeticholic Acid Protects Against Gestational Cholestasis-induced Fetal Intrauterine Growth Restriction In Mice

Background Intrahepatic cholestasis of pregnancy(ICP)is the most common liver disease in the third trimester of pregnancy,there are still no effective treatments.Many epidemiological studies and animal experiments have shown that ICP is associated with adverse pregnant outcomes.Estradiol treatment during the late pregnancy was usually used to induce cholestasis in animals.This method was used to establish the ICP model in our research.Obeticholic acid(OCA)is a pharmaceutical currently in clinical trials to treat nonalcoholic steatohepatitis and is also a synthetic agonist of FXR.However,the effects of OCA on E217? cholestasis induced fetal intrauterine growth restriction(IUGR)have not been proved.Objective This study was to investigate whether OCA pretreatment protects the IUGR during gestational cholestasis.Finally clarify its mechanism.Method ICR mice were divided into four groups: control,E2 treatment group,OCA treatment group and OCA+E2 group on gestation day(GD)12.Control was treatment with solvent of plant oil at 8 am from GD12 to GD17 by gavage.In the E2 alone and the OCA+E2 groups,pregnant mice were subcutaneous injected with E2(0.625 mg/kg)daily from GD13 to GD17.In OCA alone and OCA+E2 groups,pregnant mice were orally administered with OCA(5 mg/kg)daily from GD12 to GD17.All dams were sacrificed on GD18 and gravid uterine weights were recorded.For each litter,the number of live fetuses,dead fetuses,and resorption sites were counted.Live fetuses and placentas were weighed.Maternal serum total bile acid(TBA),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were detected by using an automated bio analyzer.Calculate the incidence of IUGR.The livers and placentas of pregnant and fetal mice were weighted and collected for the measure of the glutathione(GSH)and malondialdehyde(MDA).Used in IHC and real-time RT-PCR to detect placenta function,development and the occurrence of oxidative stress.Results Exposure with E2 during late pregnancy induced cholestasis,whereas OCA alleviated E2-induced cholestasis.According to the serum of biochemical outcomes,the levels of serum TBA,ALT and AST were significantly higher in E2 group than those in control group,indicating successful modeling.OCA almost completely suppressed E2-induced elevation of serum TBA,ALT and AST levels.Maternal liver weight and liver coefficient were significantly increased in E2-treated mice.Additionally,OCA markedly attenuated E2-induced necrosis of hepatocytes,irregular arrangement of hepatic cords and cytoplasm rarefaction.Fetal weight and crown-rump length were significantly reduced in E2-treated mice.OCA significantly alleviated E2-induced reduction of fetal weight and crown-rump length.After treatment of E2,the incidence of IUGR was increased could be found through observing fetal outcome,while OCA significantly alleviated E2-treatment induced IUGR but did not the death.Besides,OCA significantly alleviated E2-induced reduction of fetal weight and crown-rump length.The results of real-time PCR,western blot have shown that OCA could active FXR and its downstream target genes in the liver,placenta,and fetal liver.IHC have shown that OCA promoted nuclear translocation of FXR in maternal hepatocytes and mononuclear sinusoidal trophoblast giant cells of the placental labyrinth zone.OCA pretreatment up-regulated the gene expressions of Bsep,Mdr2 and Mrp2,three target genes of FXR,in maternal liver,placenta and fetal liver.By contrast,OCA pretreatment reduced the gene expressions of Cyp7a1 and Cyp8b1,two suppressive target genes of FXR,in maternal liver,placenta and fetal liver.Through analysis of the placental development and function,it was found that the blood sinusoid area in placental labyrinth layer was reduced in E2-treated mice,and OCA completely alleviated it.The results of real-rime PCR and IHC have shown that the m RNA and protein levels of the placental sodium-dependent neutral amino acid transporter 2 Snat2 were significantly down-regulated after treatment with E2,this were alleviated after OCA treatment.IHC have shown that a strong SNAT2 immunoreactivity was observed in the placental labyrinth cells.In addition,Through analysis of oxidative stress and placental peroxidation of mice,we found the GSH level of mice placenta tissue after E2 treatment was significantly lower than that of control,while the level of lipid peroxidation was significantly higher than control.Western blot and IHC have shown that the placental 3-NT residue,a marker of protein nitration,was highly increased in E2-treated mice.OCA could reduce E2-induced GSH depletion and lipid peroxidation in placenta and fetal liver,and significantly reduce E2-induced placental protein nitrification.Moreover,ICP could up-regulate the placental NADPH oxidase 4(NOX-4)protein level and down-regulate the expression of two antioxidant genes peroxiredoxin(prdx)1 and Prdx3;OCA significantly inhibits E2-induced increase in placental NOX-4 protein and down-regulation of Prdx1 gene expression.In addition,various experimental results have shown that OCA could activate the Nrf2-Keap1 antioxidant signaling pathway in mice placenta.The nuclear protein level of placental Nrf2 was significantly increased in OCA-treated and OCA+E2-treated mice.IHC have shown that a strong nuclear Nrf2 immunoreactivity in mononuclear sinusoidal TGCs of the placental labyrinth zone in OCA-treated and OCA+E2-treated mice.Moreover,Placental keap1,an inhibitory protein of Nrf2,was significantly increased in E2-treated mice,whereas it was significantly decreased in OCA+E2-treated mice.The protein level of placental HO-1 was markedly downregulated in E2-treated mice,whereas it was significantly up-regulated in OCA-treated and OCA+E2-treated mice.Conclusion The present study found that gestational cholestasis induced by E2 resulted in fetal death and IUGR in mice.OCA pretreatment activated placental and hepatic FXR signaling.Additionally,OCA pretreatment protected against gestational cholestasis-induced placental dysfunction and fetal IUGR through suppressing placental oxidative stress and maintaining bile acid homeostasis.