The Effects Of P38 MAPK And GCR On GnRH Secretion Stimulated By Glucocorticoid In Astrocytes

Glucocorticoid(GC) is controlled by the Hypothalamic- Pituitary- Adrenocoyical axis(HPA), which regulates and maintain the steady of endocrine and reproductive function, and plays an important role in immune function. Gonadotropin-Relesaing Horme, Gn RH is a 10 peptides hormone, which is the most important regulating reproduction hormone, and it is influnenced by the hypothalamic-pituitary-gonadal axis(HPG). In case of stress, HPG axis is influenced by HPA axis mainly through GC secretion, female reproductive ability will be decreased by this. Astrocytes play an important role in the central nervous system(CNS), especially in reproduction, immune, stress. According to the completed work, it has been proved that Astrocytes can secretate Gn RH in vitro. But the mechanism of the regulation is still unknown. GC-GCR is the most improtant genomic effect system of glucocorticoid, and P38 MAPK is the most important signaling pathway related to immune,but it is unknown how these influence the Gn RH secretion in Astrocytes. Hered with, we focus on the mechanism of the regulation on Gn RH secretion in Astrocytes by GCR and P38 MAPK signaling pathway.Culturing the rat hypothalamus area astrocytes was used to build the vitro model in this experiment, to explore the effects of P38 MAPK and GCR on Gn RH secretion stimulated by slucocorticoid in astrocytes.The purpose of this research is to study: the effects of different dose GC on Gn RH secretion; the effects of GCR on Gn RH secretion; the effects of P38 MAPK on Gn RH secretion and the effects of NF-κB on Gn RH secretion.The original astrocytes culture was collected from the 1 ~ 3 days age female SD newborn rats. To test the purity rate of Astrocytes by GFAP signed immunofluorescence staining after the original culture, purification and subculture. The purity rate result is 99% of astrocytes, and it can be used to the experiment with this rate.To explore the effects of different dose GC on Gn RH secretion, this experiment with 10-4 mol/L、10-5 mol/L、10-6 mol/L、10-7 mol/L、10-8 mol/L DEX(GC treatment) stimulating cultured astrocytes, respectively 1h, 12 h, 24 h, 48 h to detect the expression of Gn RH secretion and Gn RH m RNA by ELISA and Real time quantitative PCR, the the expression of GCR by Western Blot, The results showed that the expression of Gn RH secretion and Gn RH m RNA was higher in all the GC treatment group compared with the control group, and the difference was significant(P<0.05) in these time. The 10-8 mol/L GC group was higher than control group, and the difference was was significant(P<0.05). The results showed that 10-8 mol/L GC is the best dose above.To test the effects of GCR on Gn RH secretion this experiment with 10-8 mol/L DEX-BSA(GC-BSA group) and 10-8 mol/L DEX(GC group) stimulating cultured astrocytes, respectively 1h, 12 h, 24 h, 48 h to detect the expression of Gn RH secretion, Gn RH m RNA by ELISA, Real time quantitative PCR and the GCR transportation into caryon stained by immunofluorescence, The results showed that the expression of Gn RH secretion was higher in the GC treatment group compared with the control, and the difference was was significant(P<0.05) at 1h, 24h; the expression of Gn RH m RNA was higher in the GC treatment group compared with the control, and the difference was was significant(P<0.05) at 12 h, 24h; and There was no significant difference between the GC-BSA group and the control group; the more GCR transportation into caryon can be ovserved in the GC group, yet neither the GC-BSA group nor control group. We certified that the GCR is the key pathways of Gn RH secretion.To verify the effects of P38 MAPK on Gn RH secretion, these experimental groups treated by the 10 μmol/L P38 inhibitor SB203580, the 10-8 mol/L GC and both the P38 inhibitor and GC treatment were setted. Respectively 1h, 12 h, 24 h, 48 h to to detect the expression of Gn RH secretion and Gn RH m RNA by ELISA and Real time quantitative PCR, the GCR transportation into caryon stained by immunofluorescence. The results showed that the expression of Gn RH secretion and m RNA was higher in the GC treatment group and P38 inhibitor + GC group compared with the control, and the difference was significant(P<0.05), the more the level of m RNA in P38 inhibitor + GC group was higher than GC group with significant(P<0.05) at 1h, 24h; the expression of Gn RH secretion in P38 inhibitor + GC group was higher than control with significant(P<0.05) at 48 h. Mean that the P38 MAPK can reduce the level of Gn RH secretion stimulated by GC.To verify the effects of P38 MAPK on NF-κB(P65) phosphory expression, these experimental groups treated by the 10 μmol/L P38 inhibitor SB203580, the 10-8 mol/L GC and both the P38 inhibitor and GC treatment were setted. Respectively 1h, 12 h, 24 h, 48 h to to detect the expression of NF-κB phosphory by Western Blot. The results showed that the expression of NF-κB(P65) phosphorylation in GC group was higher than the control with significant(P<0.01);Both the expression in P38 inhibitor group and P38 inhibitor + GC group were lower than control with significant(P<0.01) at 1h to 24 h. this mean that P38 MAPK was the key to active the phosphory expression of NF-κB(P65).To verify the effects of NF-κB on Gn RH secretion, these experimental groups treated by the 10 μmol/L NF-κB inhibitor BAY 11-7082, the 10-8 mol/L GC and both the NF-κB inhibitor and GC treatment were setted. Respectively 1h, 12 h, 24 h, 48 h to to detect the expression of Gn RH secretion and Gn RH m RNA by ELISA and Real time quantitative PCR, the GCR transportation into caryon stained by immunofluorescence. The results showed that the expression of Gn RH secretion was higher in the GC treatment group and NF-κB inhibitor + GC group compared with the control, and the difference was significant(P<0.05) at 12 h, 24h;the level of m RNA in NF-κB inhibitor + GC group was higher than GC group and control with significant(P<0.01) at at 1h, 12 h, 24 h, 48 h. Mean that the NF-κB can reduce the level of Gn RH secretion stimulated by GC.We get these conclusions:1. GC can promote the Gn RH secretion in astrocytes and the higher dose the more secretion between the 10-8 mol/Lto10-4 mol/L.2. The GCR is the key pathway of Gn RH secretion in astrocytes.3. P38 MAPK-NF-κB signaling pathway is one of the relugation mechanisms of Gn RH secretion by GC in astrocytes.