GnRH Regulates The Expression Of PGE2 In Astrocytes By MAPK-COX Pathway

Reproductive function is centrally controlled by a group of specialized neurosecretory neurons that produce the neuropeptide gonadotropin-releasing hormone(GnRH). These neurons send their neurosecretory axons to the median eminence of the hypothalamus, where GnRH is released into pituitary portal blood vessels for delivery to the anterior pituitary gland.Within the adenohypophysis, GnRH elicits the secretion of luteinizing hormone(LH) and follicle-stimulating hormone(FSH), which in turn promote gonadal development and support reproductive physiology. Astrocytes are specialized glial cells. They contiguously tile the entire central nervous system(CNS) and exert many essential complex functions in the healthy CNS. In recent years, with the further research of astrocytes, new functions of astrocytes in animals were discovered and confirmed. It had been demonstrated that astrocytes participate in the regulation of animal reproduction by secreting PGE2 to promote the activity and secretion of GnRH neurons. While whether the GnRH can produced a feedback effect in regulated astrocytes to control the reproductive activity have not been reported. In this study, we used the rat hypothalamic astrocytes in vitro to explore the regulation mechanism of GnRH on the secretion of PGE2 in astrocytes. The purpose of this research is to study whether there are GnRHR on astrocytes, to address whether GnRH affects the expression of PGE2 in cultured astrocytes, to detect whether COX and MAPK signaling pathway are involved in the regulation of GnRH on the expression of PGE2 in astrocytes. In order to study the Gn RH on the regulatory mechanism of astrocytes in vitro more effectively, build a reasonable system of astrocytes cultivates in vitro is necessary. These Results were as followed:(1) In this experiment, the primary astrocytes is took from newborn SD rat within 1 to 3 days, 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%. The results inferred that the primary cells could meet the requirement for the research.(2) In the experiment, the astrocytes were selected according to the requirements, by immune-fluorescence, reverse transcription PCR and Western Blot to test whether there are GnRHR on astrocytes, the results shows that astrocytes are capable of producing GnRHR.(3) In order to explore the effect of GnRH on the expression of PGE2 in cultured astrocytes, this experiment with 10-12 mol/L, 10-10 mol/L, 10-8 mol/L, 10-6 mol/L GnRH stimulate cultured astrocytes, respectively 1 h, 6 h, 12 h, 24 h, 48 h to detect the expression of PGE2 in cell culture medium by ELISA, The results showed that the expression of PGE2 was higher in the 10-10 mol/L GnRH group compared with the control, and the difference was significant(P<0.05) in 12 h. The results showed that the expression of PGE2 was less in the 10-8mol/L and 10-6 mol/L GnRH group compared with the control, and the difference was significant(P<0.05).(4) In order to detect whether COX is involved in the regulation of GnRH on the expression of PGE2 in astrocytes. This experiment with 10-10 mol/L Gn RH stimulate cultured astrocytes, respectively 1 h, 6 h, 12 h, 24 h, 48 h to detect the expression of COX-1 and COX-2 in mRNA and protein by real time quantitative PCR, ELISA and Western Blot. The results found that the expression of COX-1 mRNA increased at 12 h, the expression of COX-2 mRNA significantly increased at 6 h and 12 h, and the amount of COX-2 mRNA increase more compared with COX-1 mRNA.In order to verify whether the up-regulation of COX-1 and COX-2 facilitates PGE2 production after GnRH stimulating cultured astrocytes. Cells were treated with the following COX inhibitors: 50 nmol/L COX-1 inhibitor SC-560 and 3μmol/L COX-2 inhibitor NS-398, and given 30 min prior to GnRH. the results show that the astrocytes PGE2 significantly down-regulated which group add in GnRH+SC-560, GnRH+NS-398, GnRH+SC-560+NS-398(P<0.05) than the control, demonstrated that COX-1 and COX-2 facilitates PGE2 production after GnRH stimulating cultured astrocytes, and COX-2 play a major role.(5) In order to detect whether MAPK signaling pathway is involved in the regulation of GnRH on the expression of PGE2 in astrocytes. Cells were treated with the following MAPK inhibitors: 20 μmol/L p38 inhibitor SB203580, 10 μmol/L JNK inhibitor SP600125 and 10 μmol/L ERK inhibitor U0126, and given 30 min prior to GnRH. The results showed that the three inhibitors were able to inhibit the upregulation of PGE2 and the inhibitory effect of U0126 is most obvious, showed that p38, JNK and ERK signal pathway can regulate the secretion of PGE2, and ERK signal pathway is most obvious in the regulative process.Astrocytes were treated with the MAPK inhibitors, to detect the expression of COX-1 and COX-2 by real time quantitative PCR and Western Blot. The results indicated that SB203580 and U0126 reduced COX-1 mRNA and COX-1 protein expression in cultured astrocytes. However, SP600125 had no effect on the expression of COX-1. The results showed that SB203580, SP600125 and U0126 reduced the expression of COX-2 mRNA and COX-2 protein in cultured astrocytes and the inhibitory effect of U0126 is most obvious. The results combined with the expression of PGE2 demonstrated that GnRH induced PGE2 expression in cultured astrocytes by p38/ERK-COX-1 and p38/JNK/ERK-COX-2 signaling pathway, and p38/JNK/ERKCOX-2 signaling way play a major role in PGE2 production.Above all, the experiment can get the following conclusion:(1) The expression of GnRHR in cultured astrocytes was established, which laid a morphological basis for the study of the function of astrocytes.(2) GnRH has a dual role in astrocytes, the higher concentration GnRH(10-8 mol/L, 10-6 mol/L) inhibited PGE2 production and the lower concentration GnRH(10-10 mol/L) increased PGE2 expression.(3) GnRH stimulates the production of PGE2 by up regulation of COX-1 and COX-2 in astrocytes, and COX-2 plays a major role in the process.(4) GnRH can promote the PGE2 expression in cultured astrocytes by p38/ERKCOX-1 and p38/JNK/ERK-COX-2 signaling pathway, and p38/JNK/ERK-COX-2 signaling way play a major role in PGE2 production.