| Objective:Prenatal caffeine exposure(PCE)can cause the hypothalamus-pituitary-adrenal(HPA)axis low basic activity and high stress sensitivity in intrauterine growth retardation(IUGR)offspring rats.The change of HPA axis activity was associated with the dysplasia of hippocampus,which is the regulating center of HPA axis.Based on our previous study,we reconstructed the model of caffeine exposure during pregnancy to explore the intrauterine origin mechanism of the hippocampal excitability damage.Methods:Animal experiments:Pregnant Wistar rats were randomly divided into control and different doses of caffeine groups.From gestational day(GD)9 to GD20,rats in the caffeine groups were administered 30 and 120 mg/kg-d caffeine by gavage.Rats in control group were given equal volume of distilled water by gavage.On GD20,pregnant rats were sacrificed.Then fetal rats were obtained for further hippocampus separation and blood collection.Some brain tissues were used to make frozen sections and electron microscopic sections.The mRNA expression of proliferation(CDK2,CyclinA),apoptosis(Bax,Bcl-2,caspase-3),synaptic plasticity(NR1,NR2A,NR2B,Syn1,SNAP25)and brain-derived neurotropic factor pathway(CREB,BDNF,TrkB)related genes were detected by realtime-PCR.The concentration of cyclic adenosine monophosphate(cAMP)and protein kinase A(PKA)in hippocampus were detected by ELISA kit.The protein expressions of BDNF and glutamate decarboxylase(GAD67)in hippocampus were detected by Western bloting.The number of glutamatergic neurons and y-mminobutyrate(GABA)ergic neurons were detected by immunofluorescence stain.Cell experiments:Fetal rat hippocampal neuronal cells(H19-7/IGF1R)were provided by American Type Culture Collection(ATCC).H19-7 cells were treated with different concentration(1?M,10 ?M and 100 ?M)caffeine for different days(1 day,2 days and 3 days),and treated with 2 ?M adenosine A2A receptor(A2AR)agonist(CGS-21680,Abcam,USA)or 4 ?M Adenylate cyclase agonist(Forskolin,Abcam,USA)plus 100 ?M caffeine for 3 days.The culture medium was renewed every other day.At the end of this treatment,the cells and culture mediums were harvested for further analysis.Detected the mRNA expression of cell proliferation,synaptic plasticity and brain-derived neurotropic factor pathway related genes related genes and glutamic acid decarboxylase 67(GAD67)by real time PCR.The concentration of glutamate(Glu),GABA,cAMP and PKA of cells were detected by ELISA kit.The protein expression of BDNF and GAD67 were detected by Western bloting.Results:Fetal rat hippocampus:(1)Scanning electron microcopy images showed that neurons of fetal rats in caffeine group suffered from decrease in number of organelles,dilation of endoplasmic reticulum and mitochondrial swelling.(2)The changes of proliferation,apoptosis and synaptic plasticity in hippocampus:Compared with the control group,the mRNA expression of CDK2 was decreased significantly(P<0.05,P<0.01).The mRNA expression of CDK was unchanged.The mRNA expression of Bax and caspase-3 was increased significantly(P<0.05,P<0.01).The mRNA expression of Bcl-2 was decreased significantly(P<0.01)in female fetal rats.The expression ratio of Bax/Bcl-2 was increased.The mRNA expression of NR1,NR2B,Synl and SNAP25 were decreased significantly(P<0.05,P<0.01).The mRNA expression of NR2A was increased significantly(P<0.01)and the expression ratio of NR2A/NR2B was increased.(3)Immunofluorescence showed that the number of glutamatergic neuron was decreased and the number of GABAergic neuron was increased(P<0.05,P<0.01)in the high dose of caffeine group.The mRNA and protein expression of GAD67 were increased significantly(P<0.01).(4)The mRNA expression of AC,A2AR,CREB,BDNF and TrkB were decreased significantly(P<0.05,P<0.01).The concentration of cAMP and PKA were decreased significantly(P<0.05,P<0.01).The protein expression of BDNF was decreased significantly in high dose of caffeine group.H19-7/IGF1R cells:(1)Compared with the control group,the mRNA expression of CDK2 was decreased significantly(P<0.05,P<0.01)with treatment of 100 ?M caffeine for different days(1,2 and 3 days),and the mRNA expression of CyclinA was decreased significantly(P<0.05,P<0.01)in 2 and 3 days.The mRNA expression of CDK2 was decreased significantly(P<0.05,P<0.01)with treatment of 1,10 and 100 ?M caffeine for 3 days,and the CyclinA was decreased significantly(P<0.05)in 100 ?M caffeine croup.(2)Compared with the control group,the mRNA expression of Bax and Bcl-2 were decreased significantly(P<0.01)with treatment of 100 ?M caffeine for different days,but the expression ratio of Bax/Bcl-2 was increased,the mRNA expression of caspase-3 was increased(P<0.01)significantly.Cells flow showed that the ratio of apoptotic cells was increased significantly(P<0.05,P<0.01)with treatment of 100 ?M caffeine for different days or 1,10 and 100 ?M caffeine for 3 days.(3)Compared with the control group,the mRNA expression of NR1,NR2B,Synl and SNAP25 were decreased significantly(P<0.05,P<0.01)with treatment of 100 ?M caffeine for different days.The mRNA expression of NR2A was increased significantly(P<0.01)and the expression ratio of NR2A/NR2B was increased with treatment of 100 ?M caffeine for different days.The mRNA expression of NR1,NR2B,Synl and SNAP25 were decreased significantly(P<0.01)with treatment of 1,10 and 100 ?M caffeine for 3 days.The mRNA expression of NR2A was increased significantly(P<0.01)and the expression ratio of NR2A/NR2B was increased with treatment of 1,10 and 100 ?M caffeine for 3 days.(4)The concentration of glutamate and GABA were increased significantly(P<0.01)with treatment of 1,10 and 100?M caffeine for 3 days.The mRNA and protein expression of GAD67 were increased significantly(P<0.05,P<0.01)with treatment of 1,10 and 100 ?M caffeine for 3 days.(5)The mRNA expression of AC was decreased significantly(P<0.01)with treatment of 100 ?M caffeine for 3 days.The mRNA expression of A2AR,CREB,BDNF and TrkB were decreased significantly(P<0.05,P<0.01)with 100 ?M caffeine for dffrent days.The mRNA expression of AC,CREB,BDNF and TrkB were decreased significantly(P<0.05,P<0.01)with treatment of 1,10 and 100 ?M caffeine for 3 days.The concentration of cAMP and PKA were decreased significantly(P<0.05,P<0.01)with treatment of 1,10 and 100 ?M caffeine for 3 days.The protein expression of BDNF was decreased with treatment of 1,10 and 100?M caffeine for 3 days.(6)The treatment of CGS21680 and Forskolin can reverse the inhibition of caffeine on the BDNF signaling pathway(P<0.01).Conclusion:PCE can down-regulate fetal hippocampus BDNF signaling pathway and its downstream gene expression by inhibiting cAMP signaling pathway,and eventually lead to the hippocampal excitability damage of fetal rats.The compensatory increased of GAD67 can translate the overloaded glutamate into GABA.The changes induce the neurotransmitter imbalance and the damage of hippocampal negative feedback regulation to hypothalamus PVN,eventually mediated the HPA axis high stress sensitivity in offspring. |
PDF Full Text Request