| Intrauterine growth restriction (IUGR) is a common birth defect that is often considered as an essential cause for a series of perinatal diseases including fetal distress, neonatal asphyxia and perinatal death. Besides, the adverse effects of IUGR can be prolonged to adulthood and result in physical and mental stunting as well as increased susceptibility to adult metabolic syndrome. Maternal exposure to smoking during pregnancy is a definite inducement of IUGR. The lipophilic nicotine, a major alkaloid component in cigarette, readily passes through the placenta and is largely accumulated in fetus, which impedes the fetal development. Prenatal nicotine exposure always causes IUGR as well. The increasing evidences suggest that programmed alteration of the hypothalamic-pituitary-adrenal (HPA) axis is most likely to be involved in the mechanism underlying the IUGR-originated adult metabolic syndrome. Adrenal is the terminal organ of HPA axis that is responsible for the steroidogenesis, which is of great significance for maintaining normal gestation, promoting fetal growth and nervous system maturation. Low birth weight induced dysfunction of adrenal glucocorticoid (corticosterone in rodents and cortisol in human) synthesis increases the risk of postnatal metabolic syndrome. Therefore, the programmed alteration of adrenal steroidogenesis may play an important role in IUGR originated adult metabolic syndrome.We have previously demonstrated that prenatal nicotine exposure inhibits the key/rate-limiting enzyme-steroidogenic acute regulation protein (StAR) and cytochrome P450cholesterol side chain cleavage (P450scc) involved in fetal adrenal steroidogenesis thereby reducing corticosterone synthesis and leading to IUGR. Meanwhile, nicotine could directly reduce StAR/P450scc expression and cortisol synthesis in human fetal adrenal cortex cells in vitro. These results indicate that fetal adrenal might be the target organ for nicotine, and nicotine might have direct toxic effect on adrenal. However, the detail in mechanism of nicotine’s effects on adrenal corticosterone synthesis of IUGR fetus has not been clarified yet, and no relevant reports about the functional changes of enzymes regulating adrenal steroidogenesis in different postnatal stages of prenatal nicotine-exposed IUGR offspring were seen.Steroidogenic factor-1(SF-1) plays a key role in adrenal cortex morphogenesis and transcriptional regulation of steroidogenic genes. All the steroidogenic genes’promoter region have recognition sequence for SF-1. It has been demonstrated that nicotine exposure results in abnormal expression genes via changing the pattern of DNA methylation and/or histone acetylation. The inhibitor of DNA methylation or histone acetylation could reduce the SF-1expression and affect the secretion of steroid. In addition, insulin-like growth factor1(IGF-1) and its receptor IGF-1R are widely expressed in adrenal cortex.1GF-1and its receptor could modulate the expression of SF-1as well as relevant steroidogenic enzyme system and enhance the synthesis of steroid by phosphorylating P13K/Akt signaling pathway. All of the above suggest that prenatal nicotine exposure might change the expression of the steroidogenic enzyme via affecting SF-1DNA methylation/histone acetylation and/or1GF-1signaling pathway, and consequently program the adrenal corticosterone synthesis and secretion.In this study, on the1UGR rat model induced by prenatal nicotine exposure, we systematically investigate the expressions of fetal adrenal steroidogenic enzyme system and SF-1, IGF-1signaling pathway, and adrenal corticosterone level alteration under different conditions (i.e. prenatal stage and postnatal stage with normal or high-fat diet). Moreover, from the perspective of DNA methylation and histone acetylation modification of SF-1gene promoter, we aimed to confirm the epigenetic mechanism underlying the change of nicotine-induced fetal adrenal steroidogenic function. This study is of great theoretical and realistic significance for comprehensive understanding of nicotine’s developmental toxicities, controlling of IUGR and corresponding susceptibility to adult metabolic syndrome, and improvement of population quality.PART ONEPrenatal nicotine exposure inhibited the function of adrenal corticosterone synthesis in fetal ratsObjective:To investigate the effects of prenatal nicotine exposure on fetal adrenal steroidogenic function, IGF-1signaling pathway and the gene expression of relevant steroidogenic enzymes in vivo, and to address the regulatory mechanism from the perspective of DNA methylation and histone modification. Methods:From GD11to GD20, the pregnant rats were subcutaneously administered1.0mg/kg of nicotine twice per day to establish a stable IUGR rat model. The control group received the same volume of the vehicle. Gas chromatography coupled with mass spectrometry (GC-MS) was employed to measure the content of nicotine in maternal and fetal blood; The adrenal histopathological and cytopathological examination was based on hematoxylin-eosin staining (H&E) and transmission electron microscope (TEM); enzyme-linked immuno sorbent assay (ELISA) was used to detect the level of adrenal corticosterone and blood IGF-1; real-time quantitative PCR was performed to measure mRNA expression of fetal adrenal genes, including StAR, P450scc, 3β-hydroxysteroid dehydrogenase (3(3HSD), steroid11(3-hydroxylase (P450c11), steroid21-hydroxylase (P450c21), SF-1, IGF-1signaling pathway (IGF-I, IGF-1R. AKT1and MC2R), DNA Methyltransferase (Dnmts), histone deacetylases (HDACs); immunohistochemistry was employed to detect the protein expression of fetal adrenal SF-1; the methylation of CpG sites on SF-1promoter was determined by bisulfite sequence PCR (BSP); the histone acetylation of SF-1promoter was detected by chromatin immunoprecipitation (ChIP). Results:①Body weight and IUGR rate:When being compared with control, the fetal bodyweight was decreased to87.5%of that of control (P<0.01), IUGR rate reached to85.8%(P<0.01).②Blood nicotine level:5.39±0.27μM in maternal blood and3.7H0.15μM in fetal blood (P<0.05), the average level of nicotine in fetus is68.8%of that in dam.③Pathological changes:In nicotine group, the adrenal cells were swelling and the cytoplasm were under-stained; the results from the TEM showed that the inner tube structure in mitochondrial became arrangement disorder, quantity reduction, membrane structure damage, content overflow and widen nuclear weeks gap in nicotine-treated fetal adrenals.④The functional change of fetal adrenal corticosterone synthesis:When being compared with control, the level of fetal adrenal corticosterone were decreased (P<0.05) and the expressions of fetal adrenal StAR,30-HSD and P450c11were decreased (P<0.05), both mRNA and protein expression of SF-1were decreased (P<0.05).⑤Fetal blood IGF-1level and adrenal IGF-1signaling pathway:when being compared with control, blood IGF-1level was decreased, especially in female fetal rat (P<0.05), the expression of IGF-1R and AK.T1was increased in male of nicotine group (P<0.01) but decreased in female(P<0.01); however, the expression of IGF-1and MC2R had no significant change.⑥Epigenetic modification of SF-1promoter:Compared with control, the fetal adrenal Dnmt1and Dnmt3a mRNA expression were respectively increased by67.3%and42.9%(P=0.07), and HDAC2mRNA expression was also increased (P<0.05); ChIP technique was employed to detect the histone modification of H3K9and H3K14within-832～-653and-334～-172region, and the result showed that the histone acetylation of H3K9in nicotine group were dereased to54.58%and58.83%of the control (P<0.05), however, the histone acetylation of H3K14presented no obvious alteration; the total methylation ratio of SF-1promoter (-260-+60bp) and the individual site methylation ratio were all unchanged. Conclusion:Prenatal nicotine exposure could inhibit fetal adrenal corticosterone synthesis; the mechanism is associated with decreased histone acetylation and expression of SF-1, and/or IGF-1signaling pathway caused by lower IGF-1level in fetal serum. PART TWONicotine exposure induced inhibition of steroidogenic function in human fetal adrenal cellsObjective:To investigate the effects of nicotine on steroidogenic function in fetal adrenal NCI-H295A cells in vitro, and to elucidate the potential mechanism from the perspective of SF-1histone acetylation modification. Methods:The human fetal adrenal cortex cells were treated with nicotine with different concentrations (0,25,50μM) for5days, or with50μM of nicotine for different times (0,3,5days). Cytotoxicity was evaluated by MTT assay; the levels of cortisol and dehydroepiandrosterone-sulfate (DHEAS) in media were measured by ELISA. Real-time PCR was to determine the mRNA expressions of steroidogenic enzymes (StAR, P450scc and P450c17), histone deacetylases (HDAC1, HDAC2, HDAC3and HDAC7A) and SF-1, Western blot and/or immunohistochemistry were used to detect protein expression of SF-1. After100ng/ml of trichostatin A (TSA) and/or50μM nicotine treatment for5days, the mRNA expression of SF-1, the histone acetylation modification of SF-1promoter was determined by ChIP. Results:①Cytotoxicity:Nicotine (0,25,50μM) treatment for5days had no significant effect on cell viability.②Cortisol and DHEA content: Compared with control, the concentration of cortisol and DHEA in media were dose-and time-dependently decreased in nicotine groups, respectively (P<0.05, P<0.01).③Steroidogenic enzymes and SF-1expression:Compared with control, the mRNA expression of StAR, P450scc, P450c17and SF-1were dose-dependently decreased by nicotine (P<0.05, P<0.01); Western Blotting and immunohistochemistry showed that the SF-1protein expression was dose-dependently decreased (P<0.05).④Histone deacetylase expression:The mRNA expression of HDAC1, HDAC2and HDAC7A were dose-dependently increased (P<0.05, P<0.01) while HDAC3mRNA expression had no change.⑤Histone acetylation modification in SF-1promoter:Compared with control, the acetylation of H3K9and H3K14within SF-1promoter region were decreased to37.7%(P<0.01) and22.9%(P<0.01) of control, respectively.⑥TSA inhibition test:TSA increased the SF-1mRNA expression by1.5folds (P<0.01) of control, and2.2folds of nicotine treatment (P<0.01). Conclusion: Nicotine could directly inhibit steroidogenic enzyme system and function in fetal adrenal cells and the mechanism is associated with altered deacetylation of SF-1.PART THREEThe functional change of adrenal corticosterone synthesis in nicotine-induced IUGR offspring fed with normal diet Objective:To observe changes of HPA axis activity, adrenal steroidogenic enzyme system and IGF-1signaling pathway in prenatal nicotine exposure-induced IUGR offspring fed by normal diet after birth as well as the potential relationship among them, and to clarify the programmed alteration of adrenal corticosterone synthesis function in IUGR offspring induced by prenatal caffeine exposure. Methods:The first batch of animals:IUGR rat model was established by prenatal nicotine (2mg/kg-d) exposure, the weights of the offspring were recorded at different periods after birth. The offspring fed by normal diet were divided into four groups according to the time to maturity, including postnatal day (PD)1, PD7, PD35, and PD125. Specifically,4,3and2littermates from each maternal rat were decapitated at PD1, PD7and PD35, respectively, and the serum and adrenal samples of littermates were pooled to one independent sample. The second batch of animals:IUGR rat model was established by prenatal nicotine exposure. The pregnant rats were allowed to deliver spontaneously at term, and the offspring were fed by normal diet until PD150and then sacrificed to collect blood and adrenal tissue. ELISA was used to measure the level of adreno-cortico-tropic-hormone (ACTH), corticosterone and IGF-1, Real-time PCR was to determine the mRNA expressions of steroidogenic enzymes (SF-1, StAR, P450scc,3βHSD, P450c21and P450c11), histone deacetylases (HDAC1, HDAC2, HDAC3and HDAC7A), IGF-1signaling pathway (IGF-1, IGF-1R, AKT1and MC2R) and SF-1. Results:①Body weight:The mean bodyweight of nicotine groups was lower than that of control from PD1to PD150(P<0.05, P<0.01).②HPA axis activity:When being compared with control, the blood ACTH and CORT level in nicotine group was increased in the early postnatal stage (PD1, PD7and PD35, P<0.05, P<0.01), but fallen from PD60to PD100(P<0.01) and presented no significant change in PD150.③Adrenal steroidogenic enzyme system expression:When being compared with control, the mRNA expression of StAR, P450scc, P450c11and SF-1were significantly decreased in nicotine group on PD100(P<0.05,P<0.01). The expression of3βHSD (P<0.01) and P450c21(P<0.01) on PD150were increased in male but decreased in female, and SF-1expression were all increased.④Blood IGF-1and adrenal IGF-1signaling pathway expression:When being compared with control, the mRNA expression of IGF-1, IGF-1R and AKT1of nicotine group were increased in male on PD150(P<0.01), however, the expression of IGF-1, Akt and MCR2mRNA were decreased in female on PD150(P<0.05,P<0.01).Conclusion:The alteration of adrenal SF-1and steroidogenesis in IUGR offspring induced by prenatal nicotine exposure could be maintained after birth and even lasted to adulthood. When fed by normal diet, the enhanced IGF-1signaling pathway expression in adrenal of male could partially compensate the impaired adrenal corticosterone synthesis; however, the expression of IGF-1signaling pathway had no significant change in female.PART FOURThe altered adrenal corticosterone synthesis function of nicotine-induced IUGR offspring fed by high-fat diet after birthObjective:To mimic the high-fat diet in human, and further observe the adrenal corticosterone synthesis function and mechanism of nicotine-induced IUGR offspring fed by high-fat diet after birth. Methods:IUGR rat model was established by prenatal nicotine (2mg/kg-d) exposure, the weights of the offspring were recorded at different periods after birth. The offspring were fed by high-fat diet containing89.5%of corn flour,10%of lard and0.5%of cholesterol from the day after weaning to PD150. On PD120, the blood samples were collected via the vena caudalis every4hours in a day to determine the diurnal rhythm of ACTH. The animals were anesthetized with isoflurane and decapitated to collect blood and adrenal samples. Radioimmunoassay was employed to detect serum ACTH, ELISA for serum CORT and1GF-1level, real-time PCR was to determine the mRNA expressions of steroidogenic enzymes system (SF-1, StAR, P450scc,3P-HSD, P450c21and P450c11), IGF-1signaling pathway (IGF-1, IGF-1R, AKT1and MC2R). Result:①Body weight:The male and female pup’s body weight of nicotine group were lower than high-fat control on postnatal week1(P<0.01). The offspring were fed by high-fat diet after weaning, the body weight of male and female offspring in nicotine group were approximate to or higher than high-fat diet.②ACTH diurnal rhythm and HPA activity:When being compared with control, the ACTH level in male of nicotine group was increased (P<0.05) while the female only had the increasing tendency; the CORT level in male of nicotine group exhibited a decreased tendency but the female remained unchanged; the diurnal rhythm of ACTH in high-fat diet control and nicotine group all presented low level at night but high at day; the area under curve (AUC) of ACTH was decreased (P<0.01) in male but increased in female (P<0.01).③Adrenal steroidogenic enzyme system and SF-1expression:when being compared with high-fat diet, the mRNAd expression of adrenal StAR, P450scc,3βHSD. P450c11were all decreased in male of nicotine group (P<0.05. P<0.01) while P450c21expression had no obvious change; for the female of nicotine group, the mRNA expression of3βHSD and P450c11were decreased (P<0.05) while that of P450c21was increased (P<0.05), the StAR P450scc expression showed no alteration. The SF-1mRNA expression was not changed in both male and female of nicotine group.④Blood IGF-1and adrenal IGF-1signaling pathway: When being compared with high-fat diet control, the level of blood IGF-1in nicotine group was increased, especially for the female (P<0.05); the mRNA expression of IGF-1and IGF-1R in male of nicotine group were increased while that of Aktl and MC2R remained unchanged. The mRNA expression of IGF1, IGF1R, AKT1and MC2R in female of nicotine group were all increased (P<0.05. P<0.01). Conclusion:The inhibition of adrenal corticosterone synthesis in IUGR offspring induced by prenatal nicotine exposure could be maintained after birth and even lasted to adulthood, especially for male. The enhanced adrenal IGF-1signaling pathway expression could partially compensate the suppressed adrenal corticosterone synthesis in female. The results indicated that postnatal high-fat diet could aggravate the disorder of HPA axis function induced by prenatal nicotine exposure. |
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