| Gonadotropin-releasing hormone?GnRH?is synthesized and secreted by GnRH neurons in the hypothalamus,acts on gonadotropin cells via the hypothalamic-pituitary-portal system,stimulates synthesis and secretion of gonadotropins,and regulates animal reproductive activity.Studies have found that GnRH exerts an important immunomodulatory effect on immune cells in the cellular activity,proliferation,immune medium release and expression.Astrocytes?AST?are specialized immune cells in the brain,have many complex physiological functions,secrete many kinds of cytokines and have antigen presentation function,and participates in the immune response of the central nervous system.Previous experiments have demonstrated that GnRH receptors are present on astrocytes,suggesting that GnRH maybe act on astrocytes to exert an immunomodulatory function.In this experiment,astrocytes from SD-rat hypothalamus were cultured in vitro to reveal the molecular mechanism of GnRH regulating astrocyte immunity.The main objectives are:?1?Select suitable concentrations of lipopolysaccharide?LPS?activate astrocytes under in vitro conditions and verify its effect on the expression of TNF-?,IL-1?and NO in the inflammatory mediators of astrocytes;?2?GnRH regulates the expression of inflammatory mediators TNF-?,IL-1?,and NO in activated astrocytes under in vitro conditions;?3?To investigate the role of GnRH receptors in GnRH-regulated expression of inflammatory mediators TNF-?,IL-1?,and NO in activated astrocytes;?4?The role of MAPK signaling pathways in the regulation of astrocyte immunity by GnRH.The specific study is as follows:?1?Experiments Select healthy 1–3 day old newborn SD rats and isolated the hypothalamus for primary culture,purification,and subcultures.To obtain astrocytes with a purification efficiency of 95%or more,which have typical astrocyte morphology and expression the astrocytes specific marker protein Glial fibrillary acidic protein?GFAP?.Used as a follow-up study.?2?In order to select suitable concentrations of LPS to activate astrocytes,1?g/mL,10?g/mL,and 100?g/mL LPS were used to stimulate cultured astrocytes in vitro for 10h,detected the cell activity by CCK-8 Kit.The results showed that 10?g/mL LPS could activate astrocytes and significantly increase the astrocytic cell activity?P<0.05?.To verify the effect of LPS on the expression of inflammatory mediators in astrocytes,astrocytes were stimulated with 10?g/mL LPS for 1 h,4 h,8 h,12 h,and 24 h,detected the expression of TNF-?,IL-1?,and NO in cell culture medium by enzyme linked immunosorbent assay?ELISA?and Nitric Oxide Colorimetric Assay Kit?NO Kit?,and detected the expression levels of TNF-?,IL-1?,and iNOS mRNA by real-time fluorescence quantitative PCR?RT-qPCR?.The results showed that 10?g/mL of LPS can promote the expression of TNF-?,NO,and IL-1?.Therefore,10?g/mL of LPS was used to activate astrocytes in the latter experiment.?3?In order to explore the role of GnRH in the regulation of the expression of activated astrocyte inflammatory mediators,in the experiment,selected 10-12 mol/L,10-1010 mol/L,10-8 mol/L,and 10-66 mol/L GnRH stimulates activated astrocytes at 1 h,4 h,8h,12 h,24 h,detected the cell activity by CCK-8 Kit,detected the expression of TNF-?,IL-1?,and NO in cell culture medium by ELISA and NO Kit,and detected the expression levels of TNF-?,IL-1?,and iNOS mRNA by RT-qPCR.The results showed that 10-12mol/L GnRH increased the astroglial cell activity?P<0.05?,while 10-6 mol/L GnRH significantly inhibited the astroglial cell activity?P<0.05?.10-10 mol/L GnRH promotes the expression of inflammatory mediators in astrocytes,while 10-6 mol/L and 10-8 mol/L GnRH inhibit the expression of inflammatory mediators in astrocytes?P<0.05?.Therefore,the experiment selected 10-8 mol/L GnRH to stimulate astrocytes.?4?In order to investigate the role of GnRH receptors in GnRH-regulated expression of inflammatory mediators TNF-?,IL-1?,and NO in activated astrocytes,after activation of astrocytes with LPS,10-12 mol/L,10-10 mol/L,10-8 mol/L,and 10-6 mol/L GnRH inhibitor Cetrorelix Acetate?CET?were added respectively,detected the cell activity by CCK-8 Kit.The results showed that the above concentrations of Cetrorelix Acetate had no significant effect on astrocyte activity?P>0.05?.After LPS activated astrocytes,they were pretreated respectively with 10-12 mol/L,10-10 mol/L,10-8 mol/L,and 10-6 mol/L Cetrorelix Acetate for 1 h,then adding 10-8 mol/L GnRH for 8 h,detected the expression of TNF-?,IL-1?,and NO in cell culture medium by ELISA and NO Kit,and detected the expression levels of TNF-?,IL-1?,and iNOS mRNA by RT-qPCR.The results showed that different concentrations of Cetrorelix Acetate have inhibitory effects on the changes of inflammatory mediators induced by GnRH stimulation,indicating that the changes of activated astrocyte inflammatory mediators is caused by GnRH stimulation.?5?In order to study the role of JNK,ERK,and p38 MAPK signaling pathways in GnRH-regulated expression of inflammatory mediators in activated astrocytes,we set up a control group,LPS group,LPS+CET group,LPS+GnRH group and LPS+CET+GnRH group,the expression changes of ERK,JNK,p38 and their phosphorylation proteins in each group was detected by Western Blot.The results showed that ERK,JNK,and p38 signaling pathways play a role in GnRH-regulated expression of inflammatory mediators in activated astrocytes,with the ERK signaling pathway changing most significantly.To further verify the role of ERK,JNK,and p38 signaling pathways,10?mol/L ERK inhibitor U0126,10?mol/L JNK inhibitor SP600125,and 20?mol/L p38 inhibitor SB203580 were used to pretreat LPS-activated astrocytes,then the cells were treated with10-8 mol/L GnRH for 8 h,detected the expression of TNF-?,IL-1?,and NO in cell culture medium by ELISA and NO Kit,and detected the expression levels of TNF-?,IL-1?,and iNOS mRNA by RT-qPCR.The results showed that the three inhibitors inhibited the down-regulation of GnRH on the expression of inflammatory mediators in activated astrocytes on varying degrees,indicating once again that ERK,JNK,and p38 signaling pathways play a role in GnRH-regulated expression of inflammatory mediators in activated astrocytes,and the work of ERK signaling pathway is the most obvious.In summary,the following conclusions can be drawn from this experiment:?1?10?g/mL LPS treatment for 10 h can activate astrocytes in vitro,increase the astrocyte cell viability,and induce the up-regulation of astrocyte inflammatory mediators.?2?GnRH has a dual effect on LPS-activated astrocyte activity.High concentration of GnRH(10-6 mol/L)inhibits cell activity,while low concentration of GnRH(10-12mol/L)promotes cell active.?3?GnRH has a dual role in the expression of LPS-activated astrocyte inflammatory mediators through binding to GnRH receptors.High concentrations of GnRH(10-6 mol/L and 10-8 mol/L)inhibit inflammation,while low concentration of GnRH(10-10 mol/L)promotes the expression of inflammatory mediators.?4?The three ERK,JNK,and p38 MAPK signaling pathways all play a role in GnRH's regulation of LPS-activated astrocyte inflammatory mediators,of which the ERK signaling pathway plays the most prominent role. |
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