Mechanism And Prevention Targets Of Cholestasis Liver Injury In Offspring Rats Induced By Prenatal Dexamethasone Exposure

A large number of studies suggest that adult diseases have fetal origins.Fetal-originated disease refer to the continuous changes in the physiological development of important tissues and organs caused by adverse environmental stimuli in the intrauterine period.In the near term,they can be manifested as abnormal perinatal fetal structure and function or even death,and in the long term,it can be manifested as susceptibility to a variety of chronic diseases after birth.Dexamethasone is a synthetic glucocorticoid that can promote fetal lung maturation,reduce the occurrence of neonatal respiratory distress syndrome and reduce perinatal mortality,so it is widely used in the treatment of preterm birth and related pregnancy diseases.Clinical studies have found that mothers receiving dexamethasone before delivery can increase the rate of fetal bile acid synthesis.It is suggested that prenatal dexamethasone exposure may cause abnormal bile acid metabolism in the offspring.However,its long-term effect on the bile acid metabolism in the offspring has not been reported.Bile acid is the main component of bile and the end product of cholesterol metabolism,which plays an important role in lipid digestion,absorption and transport.In recent years,the important role of bile acids in endocrine regulation,energy metabolism,inflammation and other physiological processes has been gradually discovered.Cholestatic liver injury refers to liver injury induced by the accumulation of bile in the liver and blood,and is a common feature of many liver diseases.Our laboratory recently used non-targeted metabolic profiling to find that prenatal dexamethasone exposure(PDE)can cause some bile acid components in the serum of progeny to increase,and abnormal liver function indexes in adults.The liver mainly uses cholesterol as a raw material to synthesize bile acids through the sterol-7α-hydroxylase(CYP7A1)and sterol-27 hydroxylase(CYP27A1)pathways.There is a difference in the bile acid metabolism function between the fetus and adult liver,and the fetal liver in the intrauterine period mainly functions through CYP27A1.However,the role and regulatory mechanism of CYP27A1 in the occurrence of fetal cholestasis-induced liver injury has not been reported so far.Intrauterine programming refers to the process of permanent changes in fetal tissue morphology and function caused by intrauterine injury.Earlier studies in our laboratory found that PDE can cause persistent changes which eventually leads to diseases such as glomerulosclerosis,testicular and ovarian dysplasia in the offspring.This study intends to observe PDE induced cholestatic liver injury and its mechanism at the animal level,and further establishes human Wharton’s jelly mesenchymal stem cells(WJ-MSCs)hepatoid like cell differentiation model,confirming the effect of dexamethasone on the expression of CYP27A1 in hepatocytes and its intrauterine origin mechanism,and finally at the animal level through early postnatal intervention experiments to determine drug targets and prevention methods.This study has important theoretical and practical significance for analyzing the intrauterine programming mechanism of PDE-induced adult cholestasis liver injury,exploring the drug prevention targets and early prevention strategies for fetal cholestasis liver injury.PART ONE Cholestasis liver injury in adult offspring rats induced by prenatal dexamethasone exposureObjective:We observe the effects of PDE on cholestasis serological and histological indicators of and liver function in adult offspring rats,to confirm that PDE can cause cholestasis liver injury in adult offspring rats through using animal experiments,which intends to confirm that PDE can cause cholestasis liver injury in adult offspring rats and explore its possible occurrence mechanism.Methods:Pregnant Wistar rats were given dexamethasone(0.2 mg/kg·d)subcutaneously for 9 to 20 days of gestation day(GD).Animals were fed normally after birth and sacrificed to obtain serum and liver in postal week 28(PW28).Biochemical methods are used to detect serum liver function-related indicators,including:aspartate aminotransferase(AST),alanine aminotransferase(ALT),glutamyl transpeptidase(GGT).Cholestasis related indicators:alkaline phosphatase(ALP),total bilirubin(TBI),direct bilirubin(DBI),and total bile acid(TBA);Rhodanning staining detects liver bile accumulation;real-time quantitative PCR(RTqPCR)technology detects mRNA expression of multiple genes in the liver,including:glucose regulated protein 78(GRP78),C/EBP homologous protein(CHOP),cholesterol 7αhydroxylase(CYP7A1)and cholesterol 27-hydroxylase(CYP27A1),bile salt output pump(BSEP)and multidrug resistance-associated proteins 2(MRP2)and sodium ion-dependent taurocholate transporter(NTCP);The protein expression levels of GRP78 and CHOP in liver were detected by western blotting.Results:① Liver function index:compared with the control group,serum AST levels increased in adult PDE female offspring(P<0.05),serum TBI and DBI levels increased significantly(P<0.01),and serum DBI levels increased(P<0.05).However,only serum AST levels increased in adult PDE male offspring(P<0.05,P<0.01).②Cholestasis index:it was further found that the serum TBA level increased in adult PDE female offspring(P<0.05)but no significant change in males.Rhodanning staining showed that there was bile accumulation in the liver of PDE female offspring(P<0.05).③Liver endoplasmic reticulum stress index:GRP78 mRNA expression was significantly increased(P<0.05),the mRNA expression of CHOP also had a certain increase trend,and GRP78 and CHOP protein expression were significantly increased(P<0.05).However,the mRNA and protein expressions of GRP78 and CHOP in the liver of PDE male adult offspring rats were not significantly changed.④ Bile acid metabolism function:the mRNA expression of bile acid synthase CYP27A1 increased in the liver of PDE female adult offspring rats(P<0.05),while the mRNA expression of CYP7A1 decreased(P<0.05),the mRNA expression of BSEP decreased(P<0.01).The mRNA expression of MRP2 was not significantly changed,and the mRNA expression of NTCP decreased(P<0.01).However,none of the above-mentioned related genes changed significantly in PDE male adult offspring rats.Conclusion:PDE can cause cholestatic liver injury in adult offspring rats,and can also cause abnormal liver metabolic function,and there are obvious sex differences,mainly in females.The occurrence of cholestasis-induced liver injury in adult offspring caused by PDE is related to the increased stress of endoplasmic reticulum in the liver.PART TWO Intrauterine programming mechanism of abnormal bile acid metabolism in adult offspring caused by prenatal dexamethasone exposureObjective:In order to observe the changes of serum and liver bile acid metabolism profiles in prenatal and postnatal period of PDE offspring at the animal level.At the same time,the changes of liver bile acid metabolism genes were detected,and the intrauterine programming mechanism of abnormal liver bile acid metabolism caused by PDE in adult rats was clarified.Methods:Pregnant Wistar rats were given dexamethasone(0.2 and 0.8 mg/kg·d)subcutaneously fromGD 9 to 20.At GD20,some pregnant rats were randomly selected and anesthetized to collect fetal blood and liver.Part of the control group and PDE group(0.2 mg/kg.d)were selected and fed normally to PW12 after natural delivery.They were anesthetized for collecting blood and liver.LC-MS was used to detect changes in serum and bile acid metabolism profiles of PDE offspring in prenatal and postnatal period.Highthroughput sequencing was used to detect changes in mRNA and miRNA transcriptomes of fetal liver;RT-qPCR was used to detect mRNA expression levels of liver bile acid metabolism genes,the protein expression levels of CYP27A1 and CYP7A1 were detected by western blotting.Results:① Bile acid metabolism profile after birth:at PW12,the serum TBA levels of was increased in PDE female offspring rats(P<0.05),the liver TBA content did not change significantly.Serum cholic acid(CA)and CDCA levels increased,and taurochenodeoxycholic acid(TCDCA)levels decreased(P<0.01,P<0.05),but the CA level in liver tissue increased(P<0.05).②Intrauterine bile acid metabolism profile:compared with the control group,in GD20,lower serum TBA levels PDE female fetal rats(P<0.05),the TBA levels in fetal liver tissues did not change significantly but serum chenodeoxycholic acid(CDCA)and muricholic acid(MCA)levels increased(P<0.05,P<0.01),,while tauro-cholicacid(TCA)levels in liver tissues increased(P<0.05).③Fetal liver mRNA genome sequencing:the result showed that 58 genes were up-regulated and 19 genes were down-regulated in the PDE group.The main enrichment pathways include metabolic pathways,peroxisome proliferators-activated receptors(PPARs)signaling pathways,and primary bile acid synthesis pathways,and the primary bile acid synthesis pathways are most enriched.④Bile acid metabolism function of liver in prenatal and postnatal period:the basic expression value of CYP27A1 in fetal liver tissue was significantly higher than that of CYP7A1(P<0.05).It was found that at GD20,the mRNA expression bile acid synthase of CYP27A1,CYP7A1 and cholesterol 25-hydroxylase(CH25H)were increased(P<0.05),while at PW12,CYP27A1 expression continued to increase but CYP7A1 expression level decreased(P<0.05).And the protein expression changes of CYP27A1 and CYP7A1 are consistent with the changes of mRNA expression(P<0.05).AtGD20,the liver bile acid output functional genes BSEP and MRP2 mRNA expression levels increased(P<0.05),the intake of functional gene NTCP mRNA expression levels increased(P<0.05).While at PW12,the mRNA expression levels of MRP2 was still increased(P<0.05)but there was no significant change in NTCP.Conclusion:PDE can cause changes in the serum and liver bile acid metabolism profiles in female offspring before and after birth,mainly including free bile acid increases,such as CA and CDCA.The mechanism of intrauterine programming of PDE-induced abnormal bile acid metabolism in adult female rats may be related to the continuous increase of CYP27A1 expression levels in liver before and after birthPART THREE Regulation mechanism of dexamethasone-induced epigenetic and expression changes of CYP27A1 in hepatocytesObjective:To investigate the effects of dexamethasone on GR/miR-450b-3p/SIRT1 signaling and CYP27A1 epigenetic expression in human WJ-MSCs hepatoid differentiated cells and HepG2 cell lines.Further,confirm the molecular mechanism of dexamethasone affecting the epigenetic and expression changes of CYP27A1.Methods:ChIP technology to detect changes in the CYP27A1 gene promoter region histone 3 Lysine 9 acetylation(H3K9ac),H3K14ac and H3K27ac;WJ-MSCs hepatoid differentiated cells were cultured and treated with different dexamethasone(0,100,500,2500 nM)for 3 days.HepG2 cells were further treated with dexamethasone(2500 nM)combined with GR siRNA,miR-450b-3p antagonist and pcDNA3.1-SIRT1(2.5 μg).Biochemical methods were used to detect the level of bile acid in cells.RT-qPCR was used to detect the expression levels of CYP27A1,miR-450b-3p and SIRT1.Western blotting was used to detect the GR nuclear translocation,protein levels of SIRT1 and CYP27A1.ChIP experiment was used to detect the combination of GR and CYP27A1 gene promoter region and the change of CYP27A1 gene promoter region H3K14ac.Dual luciferase reporter gene experiments verified that miR-450b-3p targeted SIRT1.Results:①Epigenetic modification of CYP27A1 gene:compared with the control group,the levels of H3K14ac in the CYP27A1 promoter region of the liver of PDE female rats increased significantly in prenatal and postnatal period.(P<0.05),while the H3K9ac and H3K27ac levels did not change.②Screening of epigenetic enzymes:at the same time,we screened the subtypes of histone acetylase in fetal liver and found that the mRNA and protein expression of sirtuinl(SIRT1)was significantly reduced in PDE female rats(P<0.05).③Related expression of fetal liver miRNA:fetal liver miRNA sequencing showed that the expression level of miR-450b-3p increased in PDE female fetal rat liver.We used bioinformatics analysis to find that miR-450b-3p can target SIRT1.The expression of miR450b-3p increased in PDE female fetal rats(P<0.05),and the mRNA expression levels of liver GR and its downstream target gene SGK1 were increased(P<0.05).④Effect of dexamethasone on WJ-MSCs hepatoid like differentiated cells:it was found that bile acid levels and CYP27A1 expression were significantly increased after 2500 nM dexamethasone treatment for 3 days(P<0.05).After treatment with different concentrations of dexamethasone for 3 days,the TBA levels in the cell supernatant were increased in a concentration-dependent manner(P<0.05,P<0.01),while miR450b-3p expression increased(P<0.05)and SIRT1 protein expression decreased(P<0.01),the H3K14ac level on CYP27A1 promoter region increased(P<0.05,P<0.01),the mRNA and protein expression of CYP27A1increased(P<0.01).⑤SIRT1 mediated the alteration of CYP27A1 expression induced by dexamethasone:after 2500 nM dexamethasone treatment of HepG2 cells,the SIRT1 protein expression level was significantly reduced(P<0.01),and the SIRT1 protein expression increased 8-fold(P<0.01)after transfection with pcDNA-SIRT1 plasmid.Dexamethasone can increase the bile acid level in HepG2 cells,while overexpression of SIRT1 can reverse the increase of bile acid level in the cell supernatant caused by dexamethasone(P<0.01),H3K14ac level in the CYP27A1 promoter region,and mRNA/protein level of CYP27A1 increase(P<0.05).⑥ miR-450b-3p mediated the alteration of CYP27A1 expression induced by dexamethasone:dexamethasone can increase the expression level of miR-450b-3p in HepG2 cells(P<0.05),while administration of miR450b-3p inhibitor can reverse the decrease of SIRT1 expression(P<0.05)and mRNA/protein levels of CYP27A1 caused by dexamethasone(P<0.05,P<0.01).⑦miR-450b-3p combined with SIRT1:under the stimulation of miR-450b-3p mimics,the ratio of firefly luciferin to renilla luciferin in the SIRT1 unmutated vector transfection group decreased significantly(P<0.05),while the SIRT1 mutant vector transfection group under the stimulation of miR-450b3p mimics the ratio of firefly luciferin to renilla luciferin did not change significantly.⑧GR mediated the alteration of CYP27A1 expression induced by dexamethasone:after HepG2 cells were treated with 2500 nM dexamethasone,the expression of nuclear protein GR increased while the expression of cytoplasmic protein GR decreased(P<0.05),and it was found that dexamethasone can promote GR combination with CYP27A1 promoter region(P<0.01).After GR siRNA treatment of HepG2 cells,GR protein expression was significantly reduced(P<0.01),while GR siRNA treatment can reverse the increase of TBAlevel(P<0.05),the increase of miR450b-3p expression(P<0.05),the decrease of SIRT1 protein expression(P<0.01),the increase of of CYP27A1 mRNA/protein expression caused by dexamethasone.Conclusion:Dexamethasone can affect the miR-450b-3p/SIRT1 signal of human WJMSCs hepatoid differentiated cells,increase CYP27A1 expression and TBA production.The miR-450b-3p and SIRT1 3’UTR have binding sites.Dexamethasone can directly increase the expression of CYP27A1 by activating GR,and can also enhance the expression of CYP27A1 acetylation by miR-450b-3p/SIRT1.PART FOUR Effect of nilvadipine on cholestatic liver injury in adult offspring caused by prenatal dexamethasone exposureObjective:Nivardipine,the active inhibitor of CYP27A1,was administered at the overall animal level to verify that intervention of CYP27A1 can be used as a target for the prevention and treatment of cholestasis liver injury in PDE offspring.Methods:Pregnant Wistar rats were given dexamethasone(0.2 mg/kg·d)subcutaneously fromGD 9 to 20 and were fed normally after birth.PW8 to PW12 were given nilvadipine(1.4 mg/kg·d),Rats were sacrificed at PW12 and PW28 to obtain serum and liver.Biochemical methods were used to detect serum liver function and cholestasis-related indicators,ELISA was used to detect liver 27-hydroxycholesterol content,and Western blotting was used to detect liver GRP78 and CHOP protein expression levels.Results:① Index of cholestatic liver injury in PW12 rats:compared with the control group,serum TBA levels was increased in the PDE group(P<0.05),meanwhile serum ALT,AST,GGT,ALP and TBI levels were increased(P<0.05).Compared with the PDE group,the serum TBA level was decreased in nilvadipine group(P<0.05),while the serum AST,GGT and ALP levels were decreased(P<0.05,P<0.01).②Liver CYP27A1 metaboliteshe:compared with the control group,27-hydroxycholesterol content were increased(P<0.05),compared with the PDE group,the 27-hydroxycholesterol content was decreased in nilvadipine group(P<0.05).③Liver endoplasmic reticulum stress index in PW12 rats:compared with the control group,the protein expression of GRP78 and CHOP were increased(P<0.05),compared with the PDE group,the protein expression of GRP78 and CHOP were decreased(P<0.05).④ Index of cholestatic liver injury in PW28 rats,the serum TBA levels in PDE offspring rats was increased(P<0.05),while the serum ALT,AST,GGT,ALP,TBI and DBI levels were increased(P<0.05),compared with the PDE group,the serum TBA level decreased in the nilvadipine group(P<0.05),while the serum AST,GGT,ALP and TBI levels were decreased(P<0.05).⑤Liver endoplasmic reticulum stress index in PW28 rats:compared with the control group,the protein expression of GRP78 and CHOP were increased(P<0.05),compared with the PDE group,the protein expression of GRP78 and CHOP were decreased(P<0.05).Conclusion:Nivardipine early administration after birth can effectively reverse the occurrence of cholestasis liver injury in female offspring of PDE and has long-term efficacy.CYP27A1 can be used as a drug prevention and treatment target for cholestasis liver injury in adult offspring of PDE.Full paper summary1.This study confirmed that PDE can cause cholestatic liver injury in adult offspring rats,and its mechanism may be related to the increased endoplasmic reticulum stress caused by cholestatic.2.It is confirmed that under PDE,dexamethasone directly increases the expression of the CYP27A1 promoter region by activating the liver GR,and on the other hand increases the level of H3K14ac in the CYP27A1 promoter region by miR-450b-3p/SIRT1 signal.expression.The continuous high expression of CYP27A1 mediated by this miR-450b-3p/SIRT1 pathway is the main cause of the occurrence of cholestatic liver injury in adult offspring of PDE.3.It has been confirmed that Nilvadipine,which inhibits the activity inhibitor of CYP27A1,can alleviate cholestasis liver injury of PDE adult offspring.CYP27A1 can be used as a prevention and treatment target for cholestasis liver injury of PDE adult offspring.Early administration after birth can effectively reverse the occurrence of cholestatic liver injury in female offspring of PDE and has long-term efficacy.