| Intrauterine growth retardation(IUGR)refers to the limitation of growth and development of embryos or fetuses during adverse pregnancy,which is a common type of birth defects and often manifests as low birth weight.In addition to congenital genetic factors,IUGR is largely caused by poor intrauterine environment(including maternal and exogenous environmental factors).Researchers have conducted a lot of research on the relationship between prenatal adverse environment,IUGR and the susceptibility to various chronic diseases in adulthood,and concluded that the adult disease resulting from early life – “Development origin of health and disease(DOHa D)”.Studies have shown that programming changes of hypothalamic-pituitary-adrenal(HPA)axis are the core mechanisms for the increased risk of metabolic syndrome in adult IUGR progeny.As the terminal organ of HPA axis,the development of adrenal gland is the earliest and fastest in the fetal HPA axis,which synthesises many important steroid hormones.Studies have shown that IUGR progeny adrenal dysplasia caused by poor intrauterine exposure during pregnancy,resulting in abnormal secretion of glucocorticoid(GC),which leads to increased risk of metabolic syndrome in offspring adult.Therefore,the development of adrenal function and the level of GC in intrauterine period are the core factors and determine the development of fetal organs and the fate of their offspring.Our previous studies suggested that prenatal caffeine exposure(PCE)can cause maternal GC overexposure in IUGR fetal rats,causing inhibition of HPA axis function,which was a low basal activity and high sensitivity after stress after birth,while the changes of glucose and lipid metabolism phenotype were GC-dependent,the expression of steroid hormone synthesis genes in fetal adrenal were inhibited.Based on these,we proposed that maternal GC over-exposure induced by PCE inhibited the function of fetal adrenal may be the core mechanism of the increased susceptibility of metabolic syndrome and related diseases.11?-hydroxysteroid dehydrogenase type 2(11b-HSD2)is a GC-metabolizing enzyme,responsible for the conversion of active glucocorticoids(cortisol in human,corticosterone In rodents)to inactive glucocorticoids(cortisone,11-dehydrocorticosterone),constituting the GC “barrier” that control the GC enter into the tissue.11b-HSD2 is expressed in the adrenal,which indicates that there may be a GC “barrier” in the adrenal.This study confirmed that PCE induced abnormal expression of 11?-HSD2 in rat adrenal gland,which mediated the functional susceptibility change of adrenal after PCE progeny,and to confirm the relationship between adrenal 11?-HSD2 expression or corticosterone level and the maximum crosssectional area and maximum diameter of adrenal.The human fetal adrenal cell line H295 R was adopted to explore the specific molecular mechanism of decreased adrenal 11?-HSD2expression induced by PCE,and the effect of caffeine with cortisol on the expression of 11?-HSD2 was explored in vitro.This study has important theoretical value for analyzing the developmental toxicity of adrenal gland,realizing the intrauterine origin of fetal disease induced by PCE,and exploring the potential mechanism.PART ONE Characteristics of serum metabolic profiles of pre-and post-natal rats in rats induced by caffeine exposure during pregnancyObjective: Epidemiological investigations have confirmed that prenatal caffeine intake can cause increased risk of IUGR,cognitive dysfunction and obesity in offspring.Our previous studies have confirmed that PCE can induce IUGR in progeny rats,accompanying by multiple organ developmental toxicity and multiple disease susceptibility after birth,and suggest that maternal glucocorticoid overexposure in utero mediated intrauterine neuroendocrine metabolic programming mechanism of IUGR progeny disease susceptibility.In this study,the mature established models by our group through PCE was used to analyze serum metabolomics of pre-and post-natal in male and female offspring rats based on liquid chromatography-mass spectrometry(LC-MS),and to discuss the short-term and long-term toxicity of IUGR individuals,metabolic changes and gender differences.Methods: Wistar rats were given caffeine(120 mg/kg.d)by gavage on gestation day(GD)9 to 20.Some animals were anesthetized and sacrificed on GD20 for serum.The other parts were raised to the postnatal period.The body weight was weighed once every two weeks,and serum was obtained after the anesthesia at postnatal 12 week(PW12),detecting the indicators of liver and kidney function.Then,based on LC-MS,the serum metabolic profiles of male and female offspring were compared.Results: The weight of PCE female and male progeny rats were decreased in utero and catchup growth after birth,and the function of liver and kidney were abnormal.The differential metabolites between PCE female and male fetal rats with the respective control groups were mainly manifested in: amino acid and lipid metabolism were disorder,and multiple metabolic pathways were abnormal,and there were certain gender differences.In adulthood,the number of differential metabolites in female and male offspring of PCE was significantly lower than that in the intrauterine period,especially in females.The types of differential metabolites were also not consistent with intrauterine,and there was more obvious gender differences.Among them,differential metabolites such as phospholipids,platelet activating factor,arachidonic acid,bile acids,sphingosine-1-phosphate,indoxyl-sulfate and 11-deoxycorticosterone may be involved in developmental toxicity of multiple organs in PCE progeny(such as hippocampus,liver,kidney and adrenal)and the main pathophysiological processes of diseases susceptibility in adult.Conclusion: PCE affected the metabolism of three major substances such as glucose,amino and lipid,which was closely related to the developmental toxicity of multiple organs and diseases susceptibility in adult.At the same time,this study demonstrated the short-term and long-term developmental toxicity and gender differences of caffeine comprehensively and systematically,and provided a new idea for exploring the early warning and drug intervention targets of multiple disease susceptibility in adult offspring of IUGR.PART TWO Intrauterine programming mechanism of changes in adrenal function susceptibility in offspring of rats induced by caffeine exposure during pregnancyObjective: To observe the changes of serum corticosterone(CORT),adrenal CORT level and adrenal 11?-HSD2 expression induced by PCE.Further,to explore the relationship between adrenal 11?-HSD2 expression and CORT level and adrenal development,and potential mechanisms which the change of 11?-HSD2 expression involved in this process.Methods: Wistar pregnant rats were randomly divided into control group and PCE group,and were given saline or caffeine(30,120 mg.kg/day)from GD9-20.Some pregnant rats were sacrificed by isoflurane anesthesia on GD20,and the other pregnant rat were delivered naturally and fed to PW6 or 28 for obtaining serum and adrenal.Enzyme linked immunosorbent assay(ELISA)detected fetal serum CORT,adult serum and adrenal CORT;hematoxylin-eosin(HE)stain observed histopathological change;immunofluorescence assay detected the protein levels of 11?-HSD2;chromatin immunoprecipitation assay(Ch IP)detected the level of histone 3 lysine 9 acetylation(H3K9ac)and H3K14 ac,H3K27ac in the promoter region of 11?-HSD2.Real-time quantitative PCR(RT-q PCR)was used to detect m RNA expression of multiple genes,including: 11?-HSD2,GR,HDAC1-11,orphan nuclear receptor subfamily 4,group A,member 1(NR4A1),steroidogenic acute regulatory protein(St AR),cytochrome P450 cholesterol side chain cleavage(P450scc),steroid 21-hydroxylase(P450c21),3?-hydroxysteroid dehydrogenase(3?-HSD).Results:(1)By HE staining and quantitative analysis of fetal adrenal tissue,compared with control,the maximum cross-sectional area and maximum diameter of adrenal in female and male rats of PCE group were decreased(P<0.05,P<0.01).It indicated that PCE could cause adrenal dysplasia in female and male rats.It is known that the main function of adrenal is synthesizing CORT,which plays an important role in the development of the body.So we examined some key genes in the synthesis of fetal CORT,and found that PCE can significantly reduce the genes expression of CORT synthesis in female and male fetal rats,such as NR4A1,St AR,P450 scc,3?-HSD,P450c21 and P450c11 expression(P<0.05,P<0.01).(2)Compared with the control group,the fetal serum CORT of the female and male rats in PCE group were increased(P<0.05,P<0.01),and the expression of 11?-HSD2 in fetal adrenal of female and male were decreased(P<0.05,P<0.01).There was a negative correlation between fetal serum CORT in the female and male and the expression of 11?-HSD2 in adrenal.The expression of 11?-HSD2 in the fetal adrenal was positively correlated with the maximum cross-sectional area and maximum diameter of the adrenal,and the female was more prominent than the male.(3)At PW6 and 28,compared with the control group,the maximum cross-sectional area and maximum diameter of the adrenal of the female progeny in PCE group were significantly decreased(P<0.05,P<0.01);there was no significant change in the maximum cross-sectional area and maximum diameter of the adrenal in PCE group.In detecting the genes of adrenal steroid hormone synthesis,at PW6,the expression of female NR4A1,St AR,P450 scc and P450c21 in PCE group were decreased(P<0.05,P<0.01),while 3?-HSD and P450c11 were not changed significantly,the expression of male P450 scc in PCE group was decreased(P<0.01),and other genes were not significantly changed;at PW28,the expression of female NR4A1,St AR,P450 scc,P450c21 and P450c11 in PCE group were decreased(P<0.05,P<0.01),while 3?-HSD was not changed;male steroid hormone synthesis marker genes are not changed.(4)Compared with the control group,the levels of PCE group female fetal adrenal 11?-HSD2 promoter H3K9 ac,H3K14ac and H3K27 ac were decreased(P<0.05,P<0.01);while the levels of PCE group male H3K14 ac and H3K27 ac levels were decreased(P<0.05,P<0.01)and H3K9 ac level was not changed.At PW6,compared with the control group,the levels of PCE group female adrenal 11?-HSD2 promoter region H3K14 ac and H3K27 ac levels were decreased(P<0.05,P<0.01),H3K9 ac was not changed;while the levels of PCE group male H3K27 ac level was decreased(P<0.05),H3K9 ac and H3K14 ac levels were not changed.At PW28,compared with the control group,the levels of PCE group female adrenal promoter region H3K14 ac level was decreased(P<0.05),H3K9 ac and H3K27 ac levels were not changed;while the levels of PCE group male adrenal 11?-HSD2 promoter region H3K9 ac,H3K14ac and H3K27 ac levels were unchanged.Compared with the control group,GR expression was increased and Sp1 expression was decreased in PCE female and male fetal adrenal(P<0.05,P<0.01).When the 11?-HSD2 promoter region was analyzed by bioinformatics,we found that the 11?-HSD2 promoter region exists GR binding sites,and PCE can significantly increase the binding of GR to the 11?-HSD2 promoter region compared with the control group(P<0.01).The expression of HDAC4 in the female adrenal of PCE group was higher than that of control group(P<0.01),and there was no significant changes in other subtypes.Conclusion: PCE can cause fetal adrenal dysplasia and may be related to the inhibition of 11?-HSD2 expression in the adrenal under PCE.On one hand,the inactivation of adrenal GC was weakened,which was exposed to high level of GC,leading to the development abnormalities and steroidal synthesis;on the other hand,the reduction of the adrenal GC barrier caused by the decreased 11?-HSD2 in utero continue to the postnatal,affecting development of adrenal in PCE progeny.The inhibition of 11?-HSD2 expression induced by PCE may be related to the abnormal histone modification of the 11?-HSD2 promoter region induced by GC activating GR/HDAC4/11?-HSD2 regulatory axis.PART THREE Cortisol inhibits the expression of 11?-HSD2 and the potential molecular mechanism in human fetal adrenal cellObjective: To investigate the effects of cortisol on the expression of GR,HDAC4 and 11?-HSD2 in human fetal adrenal H295 R cell,confirming the mechanism of cortisol affecting the expression of 11?-HSD2,and to explore the effect of cortisol on H295 cell function and the mediation of 11?-HSD2 expression.Methods: Human fetal adrenal H295 R cell was cultured and treated with different concentrations of cortisol(0,300,600,1200 n M)for 5 days.H295 R cell were further treated with cortisol(600 n M)in combination with GR antagonist RU486(10 ?M),HDAC4 antagonist LMK-235(10 ?M)and pc DNA3.1-11?-HSD2(2.5 ?g).The MTS was used to detect cell viability,the m RNA levels of GR,HDAC4,Sp1 and 11?-HSD2 were detected by RT-q PCR,colocalization of HDAC4 and 11?-HSD2 was detected by immunofluorescence,and the protein level of GR and 11?-HSD2 were detected by Western blotting,Ch IP technology detect the binding of GR and 11?-HSD2 gene promoter region and H3K14 ac level in the promoter region of 11?-HSD2 gene,and co-immunoprecipitation(Co-IP)detects the coactions of GR and HDAC4.Results:(1)Cellular activity: there was no significant change in cell viability after treatment of cells with different concentrations of cortisol for 5 days compared with the control group.(2)The expression of 11?-HSD2: compared with the control group,cortisol concentrationdependence decreased the m RNA and protein expression of 11?-HSD2(P<0.05,P<0.01).(3)The changes of GR/HDAC4/Sp-1: compared with the control group,cortisol concentrationdependence increased m RNA expression of GR and HDAC4(P<0.05,P<0.01),but had no significant effect on Sp1 expression,cortisol increased the HDAC4 protein level compared with the control group(P<0.05,P<0.01).Compared with the control group,the protein expression of GR in the cytoplasm was not changed and in the nucleus increased after 600 n M cortisol treatment.Immunofluorescence result showed that GR and HDAC4 were colocalized in cell.Co-IP results showed that the interaction between GR and HDAC4 were increased after 600 n M cortisol treatment.(4)GR mediates cortisol-induced the change of HDAC4 and 11?-HSD2 expression: compared with the control group,600 n M cortisol up-regulated HDAC4 expression and down-regulated 11?-HSD2 expression(P<0.05,P<0.01),while GR antagonist RU486 reversed the effect of cortisol on HDAC4 and 11?-HSD2 expression(P<0.05,P<0.01).In addition,Ch IP results showed that 600 n M cortisol treatment increased the binding of GR to the promoter region of 11?-HSD2 gene and reduced the H3K14 ac level in the promoter region of 11?-HSD2 gene,whereas RU486 reversed this phenomenon(P<0.05,P<0.01).(5)HDAC4 mediates cortisol-induced the change of 11?-HSD2 expression: compared with the control group,LMK-235 treatment had no significant effect on GR expression,but increased 11?-HSD2 expression(P<0.05,P<0.01),while reversed the decreased 11?-HSD2 expression induced by cortilsol treatment(P<0.05,P<0.01).In addition,Ch IP assay showed that 600 n M cortisol treatment reduced the H3K14 ac level in 11?-HSD2 gene promoter region,while LMK-235 reversed this phenomenon(P<0.05,P<0.01).(6)11?-HSD2 mediates cortisol-induced the change of steroidal synthesis in H295 R cells: cortisol inhibited the m RNA expression of NR4A1,St AR,P450 scc,P450c21 and 3?-HSD in concentration-dependence compared with the control group(P<0.05,P<0.01),these effect of inhibitions were reversed by 11?-HSD2 overexpression(P<0.05,P<0.01),while cortisol had no significant effect on P450c11 m RNA expression.Conclusion: High concentration of cortisol can inhibit the synthesis of steroid hormones in human adrenal H295 cell,and may be mediated by the decreased 11?-HSD2 expression in cell induced by high concentrations of cortisol.The potential mechanism of the reduced 11?-HSD2 expression may be: on one hand,cortisol activates GR,and GR translocates into the nucleus and interacts with HDAC4,causing the decreased H3K14 ac level in the 11?-HSD2 promoter region,thereby reducing the expression of 11?-HSD2;on the other hand,the activated GR increased the binding between GR and the 11?-HSD2 promoter region,and inhibits its transcription and reduces its expression.PART FOUR Caffeine inhibits the expression of 11?-HSD2 and the potential molecular mechanism in human fetal adrenocortical cellObjective: To investigate the effects of caffeine on A2AR/c AMP/PKA/Sp1 signaling and 11?-HSD2 expression in human fetal adrenal H295 R cell,and to confirm that A2AR/c AMP/PKA/Sp1 mediates that caffeine influences on the expression of 11?-HSD2.The effect combining with caffeine and cortisol influences on the expression of 11?-HSD2 were initially explored.Methods: Human fetal adrenal H295 R cell were treated with different concentrations of caffeine(0,0.1,1,10 and 100 ?M)for 5 days.H295 R cell were further treated with caffeine(10 ?M)combining with A2 AR agonist CGS21680(2 ?M)and adenylate cyclase agonist Forskolin(20 ?M).Finally,H295 R cell were treated with 600 n M cortisol combining with different concentrations of caffeine(0,0.1,1,10,100 ?M)for 5 days.The cell viability was detected by MTS.The m RNA levels of A2 AR,Sp1 and 11?-HSD2 were detected by RTq PCR.The intracellular c AMP/PKA content were detected by ELISA,the protein level of 11?-HSD2 was detected by Western blotting,and Ch IP detected the level of H3K14 ac in 11?-HSD2 gene promoter region.Results:(1)Cell proliferation: Compared with the control group,there was no significant change in cell viability after treatment with different concentrations of caffeine for 5 days.(2)11?-HSD2 expression in cell: compared with the control group,caffeine decreased the m RNA and protein levels of 11?-HSD2 in concentration-dependence(P<0.05,P<0.01),and did not effect on H3K14 ac levels in 11?-HSD2 promoter region.(3)cell signal change of A2AR/c AMP/PKA/Sp1: compared with the control group,caffeine concentration-dependence decreased A2AR/c AMP/PKA/Sp1 signal(P<0.05,P<0.01).(4)A2AR mediated caffeineinduced c AMP/PKA/Sp1 signaling and 11?-HSD2 expression changes: compared with the control group,10 ?M caffeine inhibited c AMP/PKA/Sp1 signaling(P<0.05,P<0.01).However,these effects were reversed by A2 AR agonist CGS21680(P<0.05).In addition,compared with the control group,the m RNA and protein expression of 11?-HSD2 was decreased after treatment with 10 ?M caffeine(P<0.01),the effect was reversed by the A2 AR agonist CGS21680(P<0.05).(5)c AMP/PKA mediates the change of 11?-HSD2 expression induced by caffeine: compared with the control group,10 ?M caffeine can inhibit the signal of c AMP/PKA/Sp1(P<0.05,P<0.01),these effects were reversed by the adenylate cyclase agonist Forskolin(P<0.05);in addition,the m RNA and protein expression of 11?-HSD2 was decreased after treatment with 10 ?M caffeine compared with the control group(P<0.01),these effects can be reversed by the adenylate cyclase agonist Forskolin(P<0.05).(6)The effect of cortisol and caffeine on 11?-HSD2 expression: different concentrations of caffeine combining with cortisol can amplify the inhibition of cortisol on 11?-HSD2 expression(P<0.05,P<0.01).Conclusion: Caffeine inhibits the expression of 11?-HSD2 in human fetal adrenal H295 R cell.The mechanism may be that caffeine inhibits c AMP/PKA by A2 AR,thereby down-regulating the expression of Sp1,leading to the attenuation of 11?-HSD2 transcriptional activation,resulting in 11?-HSD2 expression inhibition.At the same time,caffeine can amplify the inhibitory effect of cortisol on 11?-HSD2 expression.Summary: In this study,we used firstly metabolomics to detect adrenal dysfunction in PCE offspring rats.Then we explored the role of 11?-HSD2 in the inhibition of adrenal function in offspring rats induced by PCE.Both of GC and caffeine were discovered to play a key role.On the one hand,GC may induce epigenetic and expression changes of adrenal 11?-HSD2 by activating GR,translocating it into the nucleus,and interacting with HDAC4,which may mediate changes in adrenal functional susceptibility after birth.On the other hand,caffeine inhibits the expression of 11?-HSD2 by suppressing c AMP/PKA/Sp1 signaling pathway through antagonism A2 AR.At the same time,caffeine can enhance the inhibitory effect of GC on the expression and epigenetic inheritance of 11?-HSD2.This study comprehensively and systematically demonstrated the short-term and long-term developmental toxicity and gender differences of caffeine,and provided new ideas for exploring the early warning and drug intervention targets for the adult multi-disease susceptibility of the offspring of IUGR. |
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