The Regulatory Mechanism Of Glucocorticoid In Astrocyte Secreting The GnRH

Glucocorticoid (GC) is a stress hormones synthesized and secreted by the adrenal cortex-zona fasciculate, and regulated by the hypothalamic-pituitary-adrenal (HPA axis). A normal concentrations is important to maintain the body’s normal growth and the stable of internal environment under physiological and stress state, meanwhile, it is also very strong immune inhibitors which participates in regulating various metabolism and the central nervous system function in body. Studies has confirmed that when stress reaction, the adrenal GC secretes excessively is closely related to the reproductive endocrine disease, GC can disturb the Gonadotropin-releasing hormone (GnRH) neurons’s normal activity in the hypothalamus levels, thereby cause the inhibition of the reproductive endocrine function. The GC to affect GnRH neurons activities may through the Glucocorticoid receptor (GR) mediated directly effects on GnRH neurons, there is GR in hypothalamus GnRH neurons, at the same time, Glucocorticoid under stress state will have an impact on astrocyte. There are data to show that the GC can regulate astrocyte’s proliferation, differentiation process and the secretion of cytokines, it is the key hormone to affect the metabolism of astrocytes in neurodegenerative diseases. Astrocytes are the main part of glial cells in the nervous system, in recent years, the studies found that the astrocytes in addition to have nutrition and protection to neurons, the characteristics of neural stem cells and involve in signaling and pain regulation mechanism, it also plays an important role in the regulation of reproduction. Studies have shown that the hypothalamus astrocytes can secrete active factor like Prostaglandin E2 (PGE2), Transforming growth factor-beta (TGFβ) etc, unite with corresponding receptors on GnRH neurons, promote the synthesis and secretion of GnRH, and control the reproductive activities. In addition, the hypothalamus astrocytes can unite with the GnRH neurons through adhesion molecules to complete signal transduction, accelerate the GnRH neurons secrete GnRH, thus regulate the reproductive activity. While whether the astrocytes can directly secrete GnRH to control the reproductive activity have not been reported.In order to study the glucocorticoid on the immunological mechanism of astrocytes in vitro more effectively, build a reasonable system of astrocytes cultivates in vitro is necessary. In this experiment, the primary astrocytes is took from newborn mice of SD rat within 1 to 3 days age, and then through the primary cell culture separately, to get the required cells by repeatedly purification and extend, the cells purification rate reach above 99%. Which provide required astrocytes to studies in vitro.This experiment through the required astrocytes cultured in vitro, by immune-fluorescence, fluorescence quantitative PCR, GnRH ELISA kit to test whether the astrocyte secrete GnRH, the results shows that astrocytes can secrete GnRH.In order to test the influence of GnRH in AST by GC. This experiment with 10-6mol/L, 10-7mol/L,10-8mol/L GC culture AST 10min、20min、30min、40min, choose the best time and concentration. The results shows that the expression quantity of GnRH compared with the control group was significantly increased by using AST which stimulated and cultured by GC of 10-8mol/L at 30min (P<0.05), the experimental concentration and time is the best experimental concentration and time.In order to test the influence of GR in AST by GC, This experiment with 10-6mol/L, 10-7mol/L,10-8mol/L GC culture AST 10min 20min、30min、40min, check the level of AST GR at 10min、20min、30min、40min, The results found every time and concentration has no significant influence on AST GR (P>0.05).In order to verify the GC stimulate AST secrete GnRH is through non-genomic mechanism instead of the classical genomic mechanism. This experiment using 10-8mol/L bovine serum albumin coupling with Glucocorticoid (BSA-GC), glucocorticoid receptor antagonist RU486 10-6mol/L,10-8mol/L GC stimulate AST, check the level of GnRH at 30min, the results show that the AST GnRH significantly increased which group add in RU486+GC,GC, GC+BSA-GC (P<0.05) than in the control group, demonstrated that GC stimulated AST secrete GnRH is mainly through the non-genomic mechanism.In order to understand the signal transduction pathway which GC act on, cultured AST use in the experiments, each add 10μM p38 of inhibitor SB203580, 10μM ERK-1/-2 of inhibitor U0126, 10μM JNK of inhibitor SP600125, the control group add 0μM of SB203580、U0126 and SP600125, continue to culture for 30min, each add in 10-6mol/L、10"7mol/L、10-8mol/L of GC, check the level of AST GnRH at 10min、 20min、30min、40min. The results found that AST GnRH levels in which group of 10μM SB203580 and 10μM U0126 were decreased than that in the control group (P<0.05), the AST GnRH level 10μM SP600125 changes of is not obvious. Prove that the changes of GC can adjust the AST GnRH through the p38MAPK, ERK-1/-2MAPK transduction pathways.In order to further explore the MAPK signal transduction pathway which GC act on is whether the PKC-MAPK signal transduction pathway or not, cultured AST use in the experiments, add 1μM pkc of Inhibitor staurosporine, continue to culture for 30min, each add in 10-6 mol/L、10-7 mol/L、10-8 mol/L of GC, check the phosphoric acid situation of p38、ERK-1/-2 at 10min,20min,30min in 40min, determine the level of AST GnRH in the 10min、20min、30min、40min. The results show that the staurosporine group AST GnRH is significantly decreased than the control group (P<0.05), which prove that the upstream signaling pathway of MAPK signal transduction pathway of GC is related to PKC signal transduction pathways.Above all, the experiment can get the following conclusion:1、Astrocytes cultured in vitro can expression glucocorticoid receptor, lay the foundation of study for glucocorticoid regulate the function of astrocytes.2、For the first time found that astrocytes can secrete GnRH, it provides an important physiological basis on astrocutes regulation of reproductive.3、Glucocorticoid can though the PKC-MAPK(p38) reglation GnRH mRNA expression, though the PKC-MAPK(p38、ERK-1/-2) reglation its secretion. For the glucocorticoid regulate reproductive mechanism provides a new theoretical support, to understand astrocytes in the role of the hypothalamus-pituitary-ovarian axis has important physiological significance.