The Protective Role Of Obeticholic Acid On Lipopolysaccharide-Induced Disorder Of Maternal Bile Acid Metabolism During Pregnancy

Background: Microbial infection is a common disease in pregnant women,which severe patients can cause pregnancy complications and adverse birth outcomes.The developmental toxicity caused by microbial infection has been widely concerned.The infection model in mice during pregnancy was successfully established by our group using bacterial lipopolysaccharide(LPS).Our series of studies found that LPS exposure during pregnancy caused abortion,embryo and fetal death,malformation,intrauterine growth restriction,premature delivery,impaired neurodevelopment and metabolic abnormalities after birth.The previous studies have paid more attention to the developmental toxicity and its mechanism of LPS on offspring,but less attention to the maternal damage effect.The disorder of bile acid metabolism is a common disease during pregnancy,which can not only lead to adverse birth outcomes in offspring,but also induce maternal death and pregnancy complications,and increase the risk of postpartum liver disease and metabolic changes in pregnant women.It has been found that LPS disorder the metabolism of bile acids in adult mice,suggesting that LPS may induce the disorder of bile acid metabolism during pregnancy.Bile acids can be divided into two types according to their chemical structure: free bile acids and conjugated bile acids.Different bile acids have significant differences in toxicity to tissues and cells due to their hydrophobicity and hydrophilicity.Therefore,it is important to determine the effect of LPS on bile acid profile during pregnancy.Bile acid can activate the farnesoid X receptor(FXR)as a signaling molecule to,which is involved in the regulation of glucose,lipid,amino acid,energy,endocrine and inflammation,and has attracted more and more attention.FXR as bile acid receptor plays an important role in regulating the synthesis,transport and metabolism of bile acids.Tight junctions of hepatocytes also play an important role in the development of bile acid disorders.The bile acid homeostasis in the body is also regulated by circadian rhythm,the circadian rhythm disorder can induce cholestasis,and inflammation can also impairs the expression of the genes with circadian rhythm and metabolic homeostasis in liver.In addition,LPS induces endoplasmic reticulum stress in liver,and endoplasmic reticulum stress also inhibits bile acid synthesis by down-regulating bile acid synthase Cyp7a1.Endoplasmic reticulum stress sensor inositol-requiring enzyme 1α(IRE1α)autophosphorylates and activates its endoribonuclease activity,which cleaves mRNA and mi RNA containing specific base sequences,and plays a role in hepatic metabolism and diseases.A study found that obeticholic acid(OCA),a FXR agonist,attenuates liver injury by inducing IRE1 signaling.Therefore,it needs to be further studied that is the mechanism of OCA in LPS-induced the disorder of bile acid metabolism during pregnancy.Whether the genes of circadian rhythm in liver are involved in the regulation of OCA on LPS-induced the disorder of bile acid metabolism,and the role of IRE1α endoribonuclease in OCA affecting LPS-induced the disorder of bile acid metabolism also needs to be further investigated.Aims:For determining the effect of LPS on maternal bile acid metabolism during pregnancy,the study will observe the effect of maternal bile acid profile in a single of dose LPS treated pregnant mice.For determining the protective role of OCA on LPS-induced disorder of maternal bile acid metabolism during pregnancy,the study will observe the effect of OCA on maternal bile acid profile in a single of dose LPS treated pregnant mice.Then,it will also be explored that the role of FXR signaling in LPS-induced disorder of bile acid metabolism during pregnancy;and the association between the gene of circadian rhythm in liver and the regulation of OCA on LPS-induced disorder of bile acid metabolism.To explore the role of IRE1 signaling in OCA on LPS-induced disorder of bile acid,it will be analysed that the effect of OCA on LPS activating IRE1 signal in maternal liver in vivo,and observed that the effect of selective inhibition of IRE1α endoribonuclease on the activation of IRE1 signaling and Cyp7a1,a key enzyme of bile acid synthesis,in vitro.Methods:The study included the experiments in vivo and in vitro.In order to observe the effect of LPS on bile acid profile during pregnancy,pregnant ICR mice were randomly divided into control group and LPS exposure group.The pregnant mice of LPS exposure group were treated with a single of dose LPS(200 μg/kg)in GD17 at 9:00 AM,and the pregnant mice of control group were treated with saline.In order to observe the role of FXR in the disorder of LPS-induced bile acid metabolism during pregnancy,pregnant ICR mice were randomly divided into 6 groups.The pregnant ICR mice of LPS 0h,1h and 6h exposure groups were treated with a single of dose LPS(200 μg/kg)in GD17,and OCA(5 mg/kg)was pretreated by GD15-17 at 8:00 AM every morning in the OCA pretreatment group,which is followed by a single of dose LPS treatment.All mice were fasted at 9:00 AM at GD17,and sacrificed at 15:00 PM after 6 hours to collect maternal blood and liver.The experiments in vitro:(1)in order to confirm the activation of OCA on LPS inhibiting FXR signal,the AML12 cells were divided into 6 groups.In the LPS 0h,1h and 6h groups,OCA+LPS 0h,1h and 6h groups.The AML12 hepatocytes were treated with 2 μg/ml LPS,and pretreated with 10 μm OCA at 1h before LPS treatment.All AML12 hepatocytes are collected at the same time.(2)In order to explore the effect of IRE1α endoribonuclease on bile acid synthase CYP7A1,the effect of IRE1α endoribonuclease selective inhibitor STF-083010(STF)pretreatment to inhibite the activation of IRE1α endoribonuclease on Cyp7a1 mRNA was observed.AML12 hepatocytes were divided into 4 groups: control group,STF pretreatment group,tunicamycin(TM)treatment group and STF+TM co-treatment group.AML12 hepatocytes were treated with 1.5 μg/ml TM and pretreated with 60 μM STF 1 h before LPS treatment.All AML12 hepatocytes were collected at 6 h after LPS treatment.The level of total bile acid(TBA)and bile acid profiles in maternal serum and liver were detected by LC-MS/MS;the levels of FXR in nuclear protein,and the levels of Cyp7a1,Cyp3 a,and p IRE1α / IRE1α in total protein from maternal liver were detected by Western blotting,the levels of Fxr、Shp、Cyp7a1、Cyp8b1、Cyp3a11、Bsep、Bcrp、Ntcp、Mrp2、Mrp3、Mdr1a、Mdr1b、Mdr3、Bmal1、Clock、Cry1、Cry2、Per1、Per2、Rev-erbα、Rorα、Dbp、Tjp1、Tjp2、E-cadherin、Occludinand Xbp-1s mRNA from maternal liver were detected by real time RT-PCR.Results:(1)This study found that LPS exposure can induce the disorder of bile acid metabolism through investigating the effects of LPS on maternal bile acid profile in a single of dose LPS treated pregnant mice,and LPS increased the levels of CA,TCA,DCA,CDCA,GCA,TCDCA,GCA and TUDCA in maternal serum,and DCA and CA in maternal liver,and changed the bile acid profile of maternal serum and liver.(2)This study found that LPS exposure in the third trimester of pregnancy can induce the disorder of bile acid metabolism through investigating the dynamic effects of FXR agonist OCA on maternal bile acid metabolism and bile acid profile in a single of dose LPS treated pregnant mice.OCA pretreatment protected LPS-induced the disorder of bile acid metabolism with obviously reducing the levels of CA,TCA,DCA,TCDCA,CDCA,GCA and TDCA in maternal serum and DCA,TCA,TDCA,TUDCA,CDCA and TCDCA in maternal liver.(3)In this study,we also observed the effect of FXR agonist OCA on the bile acid metabolism of maternal liver in a single of dose LPS treated pregnant mice.It was found that LPS reduced the bile acid metabolism enzyme Cyp3 a and bile acid synthase Cyp7a1 and Cyp8b1,obviously decreased the mRNA levels of the bile acid transporters,such as Bsep,Ntcp,Mrp2,Mrp3,Mdr1 a and Mdr3,and the key genes of tight junction such as Tjp1,E-cadherin and Occludin in the maternal liver.OCA pretreatment obviously down-regulated the mRNA level of Cyp7a1 and up-regulated the mRNA level of Cyp3 a and transporter Mdr1 a and Mdr1 b.These results suggest that LPS exposure induced disorder of bile acid metabolism by down-regulating bile acid synthesis,transport,metabolism and tight junctional during pregnancy,and OCA pretreatment can protect LPS induces disorder of bile acid metabolism through reducing synthesis and enhancing metabolism and transport of bile acid.(4)In this study,we observed the dynamic effect of FXR agonist OCA on the genes of circadian rhythm from maternal liver in a single of dose LPS treated pregnant mice during the third trimester of pregnancy.It was found that LPS obviously increased the mRNA level of the positive regulation factor Bmal1 in the maternal liver,but obviously decreased the mRNA level of negative regulation factor Dbp,Per2 and Rev-erbα,especially the decrease of DBP was the most significant,which showed a significant time-effect relationship.OCA pretreatment obviously increased the more mRNA level of the positive regulation factor Bmal1 in the maternal liver than LPS treatment.The mRNA levels of Dbp,Per1 and Per2 also were obviously reduced by OCA pretreatment.The mRNA level of DBP was the most significant decrease by OCA pretreatment.These results indicate the association between circadian rhythm outout gene DBP and LPS-induced disorder of bile acid metabolism or the protection of OCA on LPS-induced the disorder of bile acid metabolism during the third trimester of pregnancy.(5)This study observed the effect of OCA on LPS-induced IRE1-XBP1 signaling in maternal liver,and the effect of selective inhibition of IRE1α endoribonuclease on the activation of IRE1 signaling and Cyp7a1 in vitro.The results showed that LPS activated IRE1α-XBP1 signaling from maternal liver,and tunicamycin treatment significantly reduced the mRNA level of bile acid synthetase Cyp7a1 in AML12 hepatocyte,and STF pretreatment completely blocked the decrease of tunicamycin-induced the mRNA level of Cyp7a1 in AML12 hepatocyte,which indicate that the activation IRE1α endoribonuclease plays a protective role through its down-regulation of the mRNA level of Cyp7a1 in LPS-idisordered bile acid metabolism in the third trimester of pregnancy.Conclusions: The present study demonstrates that LPS disorders maternal bile acid metabolism and changes bile acid profile by decreasing synthesis,transport and metabolism of bile acid,and hepatic tight junctions during pregnancy.OCA protects LPS-induced the disorder of bile acid metabolism by decreasing synthesis of bile acid,enhancing bile acid metabolism and transport.The circadian rhythm outout gene DBP is associated with OCA protected LPS-induced disorder of bile acid metabolism during pregnancy.IRE1α endoribonuclease protects LPS-induced the disorder of bile acid metabolism by reducing bile acid synthetase.The findings of this study will provide new targets and theoretical evidence for the prevention and therapy of bile acid disorder during pregnancy.