琼玉膏抗衰老与下丘脑nf-kb炎症通路及GnRH1关系的相关性研究

The research process in connection of anti-aging with Qiong Yugao in nf-kb inflammation signaling pathway of hypothalamic and GnRH1

目的探索琼玉膏抗衰老以及下丘脑nf-kb炎症通路的激活与GnRH1基因甲基化相关性的可能分子机制。方法(1)观察比较6只24月龄自然衰老SD大鼠和6只2月龄青年SD大鼠下丘脑衰老相关蛋白及其基因mRNA的表达水平:Western Blot检测GnRH1、Sirt1、DNMT1、DNMT3a的蛋白表达水平;Real-Time PCR检测GnRH1、Sirt1、DNMT1、DNMT3a基因mRNA的表达水平;使用SPSS19.0统计软件做统计学分析。MSP法检测老年鼠和青年鼠GnRH1基因甲基化情况。(2)通过分离新生SD大鼠下丘脑神经元干细胞,建立D-半乳糖诱导的下丘脑神经元干细胞衰老模型,比较正常神经元干细胞组、D-半乳糖衰老组以及不同浓度琼玉膏干预组之间炎性因子及NF-κB表达的差异。结果 (1)GnRH1、Sirt1、DNMT1的蛋白及其基因mRNA表达水平在老年鼠中明显降低,与青年鼠比较有显著性差异(P<0.05);DNMT3a的蛋白及基因mRNA表达水平在老年鼠中无明显变化,与青年鼠比较无显著性差异(P>0.05);老年鼠中GnRH1基因出现部分甲基化,青年鼠中GnRH1基因未发生甲基化。(2)D-半乳糖诱导建立的神经元干细胞衰老模型呈现炎性指标高表达以及NF-κB信号通路激活;琼玉膏可抑制炎性因子、nuclear P65的表达水平以及NF-κB的转录活性;并且,随着琼玉膏浓度从45μg/ml增加到90μg/ml,其提前干预D-半乳糖诱导的神经元干细胞衰老的作用也相应增强。结论琼玉膏抗衰老的可能分子机制在于降低下丘脑神经元干细胞炎性因子表达和NF-κB转录活性,进而能够逆转衰老进程中GnRH1基因甲基化及其表达水平下降。

Objective To investigate the mechanism of anti-aging with Qiong Yugao in nf-kb inflammation signaling pathway of hypothalamic and methylation of GnRH1 Methods 1. We observed and compared the expression levels of aging-related proteins and mRNA in hypothalamic of 6 24 month aged SD rats and 6 2 month adult SD rats： the protein expression of GnRH1,Sirt1,DNMT1,DNMT3 a were detected by Western blot; the mRNA expression of the proteins above were detected by Real-Time PCR; did statistics analysis with spss19. 0 software. The methylation status of GnRH1 gene as examined by means of methylation specific PCR. 2. By separating the neural stem cells in hypothalamic of neonatal rats,building the aging model of neural stem cells in hypothalamic caused by D-galactose,To compare the expression of inflammatory cytokines and NF-κB between the normal neuron stem cell group,D-galactose induced aging group and the intervention group of different concentration of Qiong Yugao.Results We established aging model of neural stem cell successfully,it appeared typical inflammatory changes： the expression levels of mRNA and protein such as TNF-α and IL-1β were increased significantly,NF-κB signaling pathway was activated and the expression levels of nuclear P65 was increased. Conclusion By inhibiting the expression of inflammatory factors,nuclear P65 and the transcriptional activity of NF-κB,Qiong Yugao played a role in reversing GnRH1 mRNA reduction and delaying aging.