Research On The Molecular Mechanism Of Postpartum Depression

Postpartum depression(PPD)is a serious medical condition that affects approximately 10% to 20% of mothers within the first 4 weeks after delivery.Within half a year after delivery,symptoms can be relieved spontaneously in 1/3-1/2 patients with PPD.Since the pathological mechanism of PPD remains elusive,effective measures for prevention and treatment are in shortage.The levels of estrogen(E2)and progesterone(P4)during pregnancy increase 50-fold and 10-fold,respectively,compared to menstrual cycle levels.When the placenta is expelled during delivery,levels of E2 and P4 drop off rapidly and remain low(hypogonadal)for a prolonged postpartum period.The “E2 withdrawal” hypothesis of PPD is supported by clinical studies.For example,postpartum E2 treatment can reduce the depressive symptoms in women with PPD.Experimentally-induced E2 withdrawal can precipitate depressive symptoms in women with a history of PPD,but not in women without a PPD history.Ovariectomized rats can be given a dose of E2 and P4 daily with a subsequent single E2 treatment to create a hormone-simulated pregnancy(HSP).Following the E2 withdrawal,all rats show depression-like behavior within 4 days,which is recovered by E2 treatment.Hippocampus is an important center for regulating emotional behavior,and it is one of the major targets of stress hormones and sexual hormones.A large body of evidence demonstrates that E2,through the activation of ER?,induces neuronal nitric oxide synthase(nNOS)expression,releases nitric oxide(NO),which can elicit the phosphorylation of c AMP response element binding protein(CREB)in the hippocampal neurons.nNOS-NO-CREB signaling pathway participates in the regulation of hippocampal affective behavior.Brain-derived steroids are termed neurosteroids.Dehydroepiandrosterone(DHEA)and its sulfate-bound form are the most abundant neurosteroids.DHEA,as an activator of sigma-1 receptors(?1R),affects some aspects of human mood,and modifies some features of human emotion and behaviors.On the other hand,there is evidence that the levels of corticotropin-releasing hormone(CRF),adreno-corticotropic hormone(ACTH)and corticosterone(CORT)are increased significantly during pregnancy and postpartum,suggesting that the activity of the HPA axis is enhanced.The release of CRF in the paraventricular nucleus(PVN)of the hypothalamus is regulated by the glutamate NMDA receptor and GABAA receptors,as well as negative feedback regulation of the glucocorticoid receptor(GR).In PVN,presynaptic activation of NO stimulates GABA release,which can inhibit CRF release.Phosphorylation of GR enhances the GR-mediated negative feedback of the HPA axis.Activation of estrogen membrane receptor GPR30 can increase protein kinase C(PKC)phosphorylation and promote GR activity.Based on the above analysis,we have put forward the following research hypothesis: 1)Whether the withdrawal of E2 influences the regulation of emotion by altering the nNOS-NO-CREB signaling pathway in the hippocampus;2)Whether the withdrawal of E2 induces hyperactivity of the HPA axis by altering the neuro-hormone regulation of CRF.Objective 1.To investigate the influence of E2 withdrawal on nNOS-NO-CREB signaling pathway and affective behavior in hippocampus and the underlying molecular mechanisms.2.To explore the effects of E2 withdrawal on the activity of HPA axis and the underlying molecular mechanisms.The First Part Effect of E2 withdrawal on nNOS-NO-CREB signaling pathway and affective behavior in hippocampus and the underlying molecular mechanisms.Materials and Methods 1.Animal models: According to the protocol of hormone-simulated pregnancy(HSP)reported by Galea Laboratories,mice were firstly either ovariectomized(OVX mice)or sham ovariectomized(control mice)bilaterally under isoflurane anesthesia and given 7 days for recovery.For hormone-simulated pregnancy,the OVX mice were injected subcutaneously(s.c.)with estradiol benzoate(EB,0.5 ?g/day)combined with P4(0.8 mg/day)dissolved in 0.1 ml of sesame oil,and then with EB(10 ?g/day)for 7–11 days.Behavioral examination were performed on days 2-4 of EB withdrawal(mice of early EB withdrawal,abbreviated as e-EW mice)and on days 7-9 of EB withdrawal(mice of late EB withdrawal,abbreviated as l-EW mice).The ?1R knockout mice(?1R-KO mice)were given as a present by Dr.Zorrilla(Neurosciences Graduate Program,University of California).2.Behavioral examination: open field test(OFT)was performed to evaluate the spontaneous activity and exploratory behavior of mice.Tail suspension test(TST)and forced swimming test(FST)were performed to detect the depressive-like behavior.3.Activity of nNOS proteasome was measured by fluorometric assay.4.Level of NO was estimated as a formed nitrite(NO2-)using a ELISA assay.5.Levels of St AR,P450 scc,P450-17? and P450 arom mRNA were detected by real-time quantitative polymerase chain reaction(RT-PCR).6.Protein levels of nNOS,CREB,St AR,P450 scc,and phosphorylation of NR2 B and BDNF was detected by Western blotting(WB).Results 1.In comparison with control mice,early-EW mice showed a significant prolongation of immobility time in the FST or in the TST,while HSP-mice and late-EW mice did not.2.In comparison with controls,the levels of nNOS and NO were increased in HSP-mice,whereas in early-EW mice,nNOS and NO levels significantly decreased and had no difference in the late-EW mice.Similarly,in comparison with control mice,the level of phosphor-CREB was elevated in HSP-mice,followed by a significant decline in early-EW mice,with a subsequent restoration to control levels in late-EW mice.3.The administration of DETA/NONOate in early EW-mice could alleviate the prolongation of immobility time in the FST or in the TST and correct the reduction of phosphor-CREB.In contrast,immobility time in the FST or TST in HSP-mice was increased and phosphor-CREB declined by the treatment with 7-NI or C-PTIO.In addition,the administration of 7-NI or C-PTIO in late-EW mice could significantly increase their immobility time in the FST or TST and reduce phosphor-CREB.4.There was a main effect of the treatment with HSP-EW for the mRNA levels of St AR and P450 scc,but not P450-17? and P450 arom.Consistently,the levels of St AR and P450 scc proteins were affected by the treatment with HSP-EW.Interestingly,late-EW mice showed a significant increase in the levels of St AR and P450 scc mRNA or St AR and P450 scc protein,but early-EW mice and HSP-mice did not.5.The immobility time in the FST and the TST was increased or nNOS,NO and phosphor-CREB were reduced in HSP-mice treated with PHTPP,but not in late-EW mice.6.The administration of DHEA or PRE084 corrected the decline in the levels of nNOS,NO,phosphor-CREB and the increased immobility time in the FST and the TST in early-EW mice.As expected,the levels of nNOS,NO and phosphor-CREB were reduced and immobility time in the FST and the TST was increased in late-EW mice treated by NE100.The prolongation of immobility time in the FST or the TST was observed in late-EW ?1R-KO mice compared to wild-type(WT)mice.Consistently,the levels of nNOS,NO and phosphor-CREB in late-EW ?1R-KO mice were significantly reduced.7.In particular,the reversion of either BDNF and phosphor-NR2 B in the late-EW mice was sensitive to the blockade of ?1R by NE100.Interestingly,the decline of nNOS-NO-CREB signaling in the early-EW mice could be corrected by the administration of NMDAr agonist NMDA.Summary The “pregnancy”-simulated “withdrawal” from E2 leads to the production of depressive behaviors through a reduction of hippocampal nNOS-NO-CREB activities.The Second Part effects of E2 withdrawal on the activity of HPA axis and the underlying molecular mechanisms.Materials and Methods 1.Animal models: The protocol of hormone-simulated pregnancy(HSP)was same as the first part.The activity of HPA axis was measured after 1 days of EB withdrawal(abbreviated as EW-2d mice),2 days of EB withdrawal(abbreviated as EW-3d mice)and 8 days of EB withdrawal(abbreviated as EW-8d mice).The ?1R knockout mice(?1R-KO mice)were given as a present by Dr.Zorrilla(Neurosciences Graduate Program,University of California).2.Level of NO in PVN was estimated as a formed nitrite(NO2-)using a ELISA assay.Serum levels of CORT and ACTH were measured with ELISA assay.3.Levels of CRF mRNA were detected by Real-time quantitative polymerase chain reaction(RT-PCR).4.Coimmunoprecipitation was performed to detect the binding ability of nNOS and PSD-95 in the PVN.5.Protein levels of GR,nNOS,GPR30,PKC,and phosphorylation of GR and PKC was detected by Western blotting(WB).Results 1.Compared with the control mice,the serum levels of CORT and ACTH in the EW-2d and EW-3d mice were significantly increased,and the CRF mRNA levels in PVN of EW-3d mice were also increased.Serum levels of CORT and ACTH in HSP mice were increased after the treatment of E2 receptor blocker ICI.On contrast,E2 treatment could recover the CORT and ACTH levels in EW-2d and EW-3d mice,as well as the increase of CRF mRNA.2.Compared with the control mice,NO levels and the binding capacity of nNOS and PSD-95(nNOS activity)in the PVN region of HSP mice were increased and then significantly decreased in EW-2d mice,while the EW-3d mice did not.E2 treatment could increase NO level and nNOS activity in EW-2d mice.E2 receptor blocker ICI treatment could reduce NO levels and nNOS activity in HSP mice.3.Serum levels of CORT and ACTH in HSP mice were increased with the treatment of NO scavenger C-PTIO.Treatment with NO donor DETA or GABA receptor agonists could restore the serum CORT and ACTH levels in EW-2d mice.4.Compared with the control mice,the inhibitory rate of dexamethasone in EW-3d mice was significantly reduced,and the phosphorylation level of GR in PVN was decreased,while there was no significant changes in EW-2d mice.5.Compared with the control mice,expression levels of estrogen receptor GPR30 and PKC phosphorylation were decreased in EW-3d mice.Treatment of GPR30 agonist G-1 or PKC agonist PMA could increase GR phosphorylation,and reduce the levels of CORT and ACTH and CRF mRNA levels,while ER? agonist DPN had no significant effect.6.Compared with the control mice,the expression level of GPR30 in EW-8d mice was reduced,while the phosphorylation of PKC and GR was not changed.Treatment of the ?1R blocker NE100 caused a decrease in PKC and GR phosphorylation in the EW-8d mice.The reduction of PKC and GR phosphorylation could recover in the EW-3d mice treated with dehydroepiandrosterone(DHEA)or ?1R agonist PRE084.Summary Decrease of nNOS-NO activity prevented the inhibition effect of GABA on CRF releasing in PVN on the second day of postpartum E2 withdrawal.E2 withdrawal decreased the expression of GPR30 on the third day,leading to the inhibition of PKC-GR activity,which could attenuate the negative feedback regulation of HPA axis and increased CRF synthesis(summary diagram).Conclusion and clinical significance: The results of this study suggest that the down-regulation of the nNOS-NO-CREB signaling pathway in the hippocampus or the hyperactivity of the HPA axis is involved in the PPD induced by estrogen withdrawal.The activation of ?1R could alleviate the symptoms of PPD caused by estrogen withdrawal.By exploring the multi-target strategy of hippocampus-hypothalamus interaction,we may provide theoretical basis for the prevention and treatment of PPD with a comprehensive new program.