Direct Effects And Mechanism Of Corticotropin Releasing Factor On Structure Of Rat Hippocampal Neurons

Abstract:Stress is defined as various physiological changes in biology, including imbalance of inner environment and activation of pituitary adrenal axis. CRF is released by hypothalamic under stress. In normal circumstances, negative feedback loops can suppress the synthesis and release of CRF by activating HPA axis. Dysregulation of HPA breaks negative feedback function.Too much glucocorticoid is synthesized and released, and then it gets into the central nervous system wich can causes damage to neurons. Therefore exploring the damage effect of CRF on hippocampal neurons and its possible mechanism can provide new theoretical and experimental basis for the molecular mechanism of central neurons stress injury.Objective:In order to reveal the pathophysiological mechanisms of mental diseases associated with chronic stress, systemic study of molecular mechanism of CRF and CRFR1signaling was performed. The experiment provides new theoretical and experimental basis for the molecular mechanism of central neurons stress injury.Method:1. Analysis of the effect of CRF on structure of hippocampal neurons. On the fifth day, treated primary cultured hippocampal neurons with CRF (0.02μM,0.2μM,2μM), continue to culture to tenth days. Observed the cell structure changes of hippocampal neurons under the microscope. Then labled hippocampal neuronal dendrites by MAP2, observed the changes of hippocampal neuronal dendrites by immunofluorescence. Treated primary cultured hippocampal neurons of rats with DMP696which is CRFR1specific antagonists. Observed the cell structure changes of hippocampal neurons of control group, CRF treatment group, DMP696treatment group under the microscope, then labled hippocampal dendrites by MAP2, observed the changes of hippocampal neuronal dendrites by immunofluorescence.2. Analysis of the effect of CRF on viability of hippocampal neuron cells by SRB. Cultured primary rat hippocampal neurons for five days, Treated primary cultured hippocampal neurons with different concentrations of CRF, the experiment was divided into:blank group (no cells), control group (no adding drug group), CRF(0.02μM,0.2μM,2μM) treatment group. Detected the effect of CRF on viability of rat hippocampal neuron cells by SRB.3. Analyse protein levels of key molecule which is closely associated with the growth of hippocampal dendrites:Cultured primary rat hippocampal neurons for five days. Treated primary cultured hippocampal neurons by CRF (0.02μM,0.2μM,2μM). Continue to culture to tenth days. Detected phosphorylation levels of CREB and Tau in primary cultured hippocampal neurons, and analysed protein levels of MAP2and PSD95. Treated primary cultured rat hippocampal neuron with CRF and H89, Calphostisc, PD98059,2-APB, U-73122which is specific inhibitor of PKA, PKC, MAPK, IP3, PLC. Detected phosphorylation levels of CREB and Tau in primary cultured hippocampal neurons, and analysed protein levels of MAP2and PSD95. Analysed the signaling pathway involved in this change.4. Analyse mRNA expression of key molecule which is closely associated with the growth of neurons. Cultured primary rat hippocampal neurons for five days, treated primary cultured hippocampal neurons with CRF and DMP696. Continue to culture to tenth days. Analysed mRNA expression of CREB, Tau, MAP2, PSD95.5. Detection of cAMP content changes in primary cultured hippocampal neurons by LANCE cAMP assay:Cultured primary rat hippocampal neurons to tenth days, detected cAMP content changes in primary cultured hippocampal neurons which were treatmented with CRF, and then analysed the effects of DMP696on cAMP content changes in primary cultured hippocampal neurons which was treatmented with CRF.Result:1. Dendritic density of primary cultured hippocampal neurons was reduced by2μM CRF, and this phenomenon was suppressed by DMP696.2. Cell viability of CRF(0.2μM,2μM) treatment group:71.6±3.1%,72.6±3.5%(n=3,***P<0.001vs control).3. CRF reduced protein levels of MAP2and P-CREB, raised protein levels of PSD95, P-Tau. This phenomenon was suppressed by DMP696.4. The change of mRNA expression of PSD95and MAP2was consistent with protein levels.5. CRF regulated the variation of cAMP content in hippocampal neuron cells(EC50=(3.157±0.133)×10-9M, n=3). And the release of cAMP can be suppressed by DMP696(n=3,***P<0.001,*P<0.05VS-5group).Conclusion:High concentration of CRF induced injury on the structure of cultured hippocampal neurons by combining with CRFR1. The change of protein levels of PSD95, P-Tau, MAP2, P-CREB was regulated through a combination of CRF and CRFR1. The PKA pathway may regulate the change of protein levels of P-Tau and MAP2. The release of cAMP in hippocampal neuron cells may through the combination of CRF and CRFR1. It suggested that CRFR1may couple to Gs, and then activate adenylate cyclase, stimulates the release of cAMP.