The Protective Effect Of Obeticholic Acid On Intrahepatic Cholestasis Of Pregnancy Induced By DEHP Exposure In Mice

Background: Intrahepatic cholestasis of pregnancy（ICP）is a specific intrahepatic disease during pregnancy that often occurs in the second and third trimesters of pregnancy.The occurrence of ICP will have adverse effects on the mother itself and the fetus,especially the fetus,which can seriously cause premature birth or even death of the fetus.At present,the etiology and pathogenesis of ICP are not well understood.Genetic,environmental and hormonal factors are known to occur in the disease.Previous research by our group found that the occurrence of ICP is directly related to the exposure of phthalates（PAEs）,and dis（2-ethylhexyl）phthalate（DEHP）is currently the most widely used phthalate plasticizer.Therefore,DEHP exposure was selected in the experiment to explore whether DEHP exposure in mice during pregnancy could induce the occurrence of ICP.As a bile acid receptor,farnesoid X receptor（FXR）plays a major regulatory role in bile acid metabolism.Obeticholic acid（OCA）,a synthetic FXR agonist,previous studies in our laboratory have shown that OCA can affect both bile acid metabolism disorders and estradiol（E2）-induced ICP due to bacterial lipopolysaccharide（LPS）exposure during pregnancy and whether OCA has a protective effect on ICP induced by DEHP exposure during pregnancy deserves further investigation.Objective: To investigate whether DEHP exposure in mice during pregnancy can induce the occurrence of ICP and whether later intervention with OCA has a protective effect on ICP.Methods: Experiment 1,24 pregnant ICR mice were randomly divided into 2 groups（n=12）: the control group and the DEHP model group.The control group was given corn oil by gavage,and the model group was given a gavage dose of 200 mg/kg/d.After gavage on GD17,all pregnant mice were fasted for 6 hours,blood and liver tissues were collected,and a biochemical analyser was used to detect total bile acid（TBA）,alanine aminotransferase（ALT）,and aspartate amino acids in the serum of the mother mice.The levels of transferase（AST）and TBA in the liver tissue of the mother mice were observed by HE staining to observe the pathological changes in the liver tissue of the mother mice.Experiment 2: Forty-eight pregnant ICR mice were randomly divided into 4 groups（n=12）and divided into the control group,OCA group,DEHP model group,and DEHP+OCA intervention group.The model group was given an intragastric dose of 200 mg/kg/d,and the gavage time was 9:00 a.m.every day on GD0-GD17,and the OCA gavage time in the OCA group and DEHP+OCA intervention group was 7:00 a.m.every day on GD12-17,and the gavage dose was 5mg/kg/d.After gavage with GD17 DEHP,the rats were fasted for 6 hours.Blood and liver tissues were collected.A biochemical analyser was used to detect the level of TBA in the serum and liver tissue of the mother mice.HE staining was used to observe the pathological changes in the liver tissue of the mother mice.and the bile acid components in liver tissue;Western blot detected the expression levels of FXR nucleoprotein,CYP7A1 and CYP3A11 total protein in the liver tissue of the mother mouse;RT–q PCR detected the SHP,NTCP,BCRP,CYP7A1,CYP8B1,CYP3A11 m RNA expression levels.Results: Experiment 1:The liver mass and liver weight index of dams in the model group were significantly increased after exposure to DEHP during pregnancy.Biochemical results showed that TBA levels in maternal blood and liver were significantly increased after DEHP exposure during pregnancy.The histopathological results of the liver showed that the enlarged portal area,bile duct deformation and bile duct hyperplasia were seen in the maternal liver after DEHP exposure during pregnancy,with a small amount of inflammatory cell infiltration around the bile duct.The results showed that DEHP exposure during pregnancy could lead to ICP,and the modeling was successful.Experiment 2: Compared with the DEHP model group,the liver mass and liver weight index and the TBA level in the serum and liver of the female mice were decreased after the post-treatment with OCA.The histopathological results of the liver showed that the morphology of the liver of the female mice was significantly improved.The results showed that post-treatment with OCA had a significant protective effect on ICP induced by DEHP exposure during pregnancy.The detection results of LC-MS/MS showed that four primary bile acid components CA,CDCA,TCA and GCA in maternal blood and two secondary bile acid components UDCA and TDCA and TCDCA,GCA,CA in maternal liver after exposure to DEHP during pregnancy.,CDCA,TCA five primary bile acid components and TUDCA,UDCA,TDCA three secondary bile acid components were significantly increased,and the bile acid components in maternal blood and liver were significantly decreased after OCA post-treatment;Western blot results showed that during pregnancy DEHP exposure significantly decreased the total protein levels of FXR nucleoprotein and CYP3A11 in the liver tissue of the mother mice and significantly increased the total protein level of CYP7A1 in the liver tissue of the mother mice.At the same time,RT–q PCR results showed that the m RNA levels of FXR,SHP,CYP3A11,NTCP and BCRP in the liver tissue of the mother mice were significantly reduced after exposure to DEHP during pregnancy.The m RNA levels of CYP7A1 and CYP8B1 in the liver tissue of the mother mice were significantly increased.After OCA intervention,the m RNA levels of FXR,SHP,CYP3A11,NTCP,and BCRP in the liver tissue of the mother mice were upregulated,while the m RNA expression levels of CYP7A1 and CYP8B1 in the liver tissue of the mother mice were decreased.Conclusion: DEHP exposure during pregnancy induces ICP by inhibiting the FXR signaling pathway,and the FXR agonist OCA has a significant protective effect on ICP.