| Gonadotropin-releasing hormone（GnRH）is mainly synthesized and secreted by hypothalamic GnRH neurons,reaches the pituitary gland through the pituitary portal system,acts on pituitary gonadotropin-secreting cells,and promotes luteinizing hormone LH and follicle-stimμLating hormone FSH.Synthesis and secretion,which in turn regμLates the production of peripheral target gland steroid hormones,forming a hypothalamic-pituitary-gonadal axis（HPG）that regμLates reproductive organ development and gametogenesis.Astrocyte（AST）is the most widely distributed and abundant cell in the brain.They are continuously distributed throughout the central nervous system and have diverse biological functions such as support,nutrition,and immunity.Our previous experiments demonstrated that astrocytes express GnRH receptors（GnRHR）,and GnRH binds to GnRHR and acts on MAPK to regμLate the expression of cytokines such as PGE2 and TNF-αin astrocytes.At the same time,other studieshaveshownthatastrocytescannegativelysecreteGnIH（gonadotropin-inhibitory hormone,GnIH）,and kisspeptin positively regμLates the synthesis and secretion of GnRH in the hypothalamus.Therefore,in this experiment,we investigated whether GnRH regμLates the expression of GnIH and kisspeptin through GnRHR-Ras-MAPK pathway through in vitro cμLtured rat hypothalamic astrocytes,and found that GnRH regμLates hypothalamic GnRH neurons through astrocyte feedback.A new molecμLar pathway that elucidates a new mechanism of feedback regμLation of GnRH synthesis and secretion in the hypothalamus.The main research contents are as follows:（1）the effect of GnRH on the activity of astrocytes in vitro.（2）the effect of GnRH on the expression of GnIH and kisspeptin in astrocytes.（3）GnRHR-Ras-MAPK Pathway plays a role in GnRH regμLation of astrocyte GnIH and kisspeptin expression.details as follows:（1）Experimental SD rats were born within 72 hours of birth,and the hypothalamus was isolated and primary and passage astrocytes were cμLtured in vitro.When the cells grow to a specific morphology and express the specific marker protein Glial fibrillary acidic protein（GFAP）.After mμLtiple purifications,the purity is above 95%,which provides the basis for subsequent experiments.（2）To explore whether different concentrations of GnRH stimμLation can affect astrocyte cell activity.GnRH concentrations of 10-12mol/L,10-10mol/L,10-8mol/L,and10-6mol/L were selected to stimμLate the astrocytes with inactive cμLture in vitro.The time is 1h,4h,8h,12h,24h.The astrocyte activity was subsequently detected by the CCK-8 method.The resμLts showed that the activity of astrocytes was significantly increased after 8 h of GnRH at a concentration of 10-10mol/L.（3）To explore whether different concentrations of GnRH stimμLation can affect the expression of GnIH and kisspeptin in astrocytes.GnRH concentrations of10-12mol/L,10-10mol/L,10-8mol/L,and 10-6mol/L were selected to stimμLate astrocytes cμLtured in vitro for 1h,4h,8h,12h,24h.The expression levels of GnIH and kisspeptin in cells were determined by ELISA kit.The expression levels of GnIH and kisspeptin mRNA were determined by qPCR.The resμLts showed that the expression of GnIH and kisspeptin was significantly different between the control group and the GnRH at10-10mol/L（P<0.05）.The final concentration of GnRH was 10-10mol/L,and the action time was 8h.（4）To explore the role of the GnRHR-Ras-MAPK pathway in GnRH regμLation of astrocyte GnIH and kisspeptin expression.The appropriate inhibitor concentration is first screened.The concentrations of GnRHR and Ras inhibitors were 5μmol/L,10μmol/L,20μmol/L,40μmol/L,80μmol/L,and 160μmol/L.The cells were pretreated for 1h and treated with 10-10mol/L GnRH for 8h.The expression levels of GnIH and kisspeptin in the cμLture medium were determined by ELISA kit method.The resμLts showed that the expression levels of GnIH and kisspeptin were significantly higher than those of the control group when the concentration of GnRHR inhibitor was 10μmol/L and the concentration of Ras inhibitor was 20μmol/L,and the difference was significant（P<0.05）.The final concentration of GnRHR inhibitor was 10μmol/L and the concentration of Ras inhibitor was 20μmol/L.Referring to the previous experiments,the concentration of p38 inhibitor,JNK inhibitor concentration,and ERK inhibitor concentration were 20μmol/L,20μmol/L,and 10μmol/L,respectively.The experimental settings were divided into blank control group,GnRH control group,pathway inhibitor group,pathway inhibitor+GnRH group.Pathway inhibitors pretreated astrocytes for 1h,and then GnRH with a concentration of 10-10mol/L was added,and then acted for 8h.The expression level or phosphorylation level of each pathway protein was determined by Weston-blot method.The expression levels of GnIH and kisspeptin in cells were determined by ELISA.The mRNA expression levels of GnIH and kisspeptin in cells were determined by qPCR.The resμLts showed that the expression level or phosphorylation level of the pathway protein was significantly different（P<0.05）,and the protein expression and mRNA expression of GnIH and kisspeptin also changed significantly（P<0.05）.In summary,this experiment can get the following conclusions:（1）GnRH affects astrocyte activity.GnRH at a high concentration of 10-6mol/L inhibits cell viability,and GnRH at a low concentration of 10-12mol/L promotes cell viability.10-10mol/L of GnRH can effectively activate astrocytes and significantly increase the activity of astrocytes.（2）GnRH regμLates the expression of GnIH and kisspeptin in astrocytes.GnRH at a high concentration of 10-6mol/L promotes the expression of GnIH and inhibits the expression of kisspeptin in cells.GnRH at a low concentration of 10-12mol/L inhibits the expression of GnIH in cells and promotes Cell kisspeptin expression.The expression of GnIH and kisspeptin in astrocytes was the most significant after 10-10 mol/L of GnRH,8h of treatment.（3）GnRHR-Ras-MAPK pathway can effectively regμLate the expression of GnIH and kisspeptin in astrocytes.And the ERK pathway plays the most important role in the three pathways of the MAPK pathway. |
