Kiss1/Gpr54系统和肠道微生物组的交互作用调控GnRH节律性分泌的机制研究

Kiss1/Gpr54 systems in the ARC are key components of the HPG axis, have been demonstrated as targets of the most important regulators of the generation of pulsatile GnRH secretion. However, most of the abnormal puberty onset cannot be accounted for the genetic and transcriptional abnormalities of Kiss1/Gpr54 genes. Kisspeptin neurons Kiss1 action in ARC is dependent on energy status and unmasking metabolic factors responsible for modulating ARC Kiss1/Gpr54 neurons remains one of neuroendocrinology’s most fundamental problems. One possible factor is glucagon-like peptide 1 (GLP-1), which can promote kiss1 expression in vivo an in vitro. Our previous researches and preliminary studies have revealed that besides transcription factors such as EAP1 and CUX1, Kiss1/Gpr54 activation was also regulated by nutritional metabolism. We also observed significant metabolomics and microbiomics changes in the girls with precocious puberty, which suggests that the development of sex may involve an alteration in symbiotic gut microbial composition. While, besides regulation of sex development, Kiss1/Gpr54 also plays an important role in the regulation of glucose metabolism. So we hypothesize that via regulating carbohydrate and energy metabolism, through GLP-1/GLP-1r pathways the interaction between gut microbiome and Kiss1/Gpr54 system plays a major role in maintaining hypothalamic pituitary gonadal axis homeostasis. To establish the hypothesis, we will investigate the interaction between microbes and Kiss1/Gpr54 system by comparing germfree, gonadectomy and hormone replaced mice. To investigate the effects of microbes on the signaling pathways that may be involved in GnRH secretion from the hypothalamus neurons, the GT1–7 cell line, an in vitro model for GnRH-secreting neurons being used, we will examine signal transduction pathways affected by microbes associated carbohydrate metabolism. These results will provide new evidence for understanding of the mechanism of the interplay between Kiss1/GPR54 system and gut microbes in the mechanism of puberty onset and hold great promise for future novel pathways of intervention in sexual development disease.

Kiss1/Gpr54触发下丘脑GnRH节律性分泌是性发育启动研究的重大发现，但Kiss1/Gpr54基因表达及转录异常并不能解释大部分性发育启动异常。除调节性发育外，Kiss14/Gpr5还与糖代谢及体重控制相关。前期及预实验发现：除转录因子外，Kiss1表达还受营养影响；性早熟女童代谢产物、肠道菌群及能量代谢平衡均存在异常；GLP-1可以促进下丘脑GnRH神经元Kiss1表达。据此提出假说：肠道菌群可能通过GLP-1信号影响糖代谢，实现与Kiss1/Gpr54的交互作用，进而改变GnRH神经元的发育、分化以及功能稳态参与性发育进程的调控。本项目拟通过性腺摘除及菌群移植小鼠研究肠道菌群与Kiss1/Gpr54系统的交互作用；以GLP-1、利拉鲁肽及菌群代谢产物刺激GT1-7细胞，研究肠道微生物对GnRH神经元功能影响的分子机制。研究可为从肠道微生态及代谢紊乱角度干预及调控性发育提供基础。

青春期是人类发育过程中最重要的阶段之一，下丘脑GnRH神经元的适时激活决定个体青春期性发育的启动。肠道菌群可以通过与植物神经和中枢神经系统建立交互作用体系，参与机体众多神经内分泌功能的调控。随着“脑-肠轴”研究的深入，发现由肠道-神经系统-肠道微生物共同组成人体的“第二大脑”，而肠道微生物在物质能量代谢与神经内分泌之间起着重要的桥梁作用。前期研究发现性早熟（CPP）女童粪菌代谢产物、肠道菌群及能量代谢平衡均存在异常。Kiss1/GPR54可能参与肠道菌群的相互作用，通过GLP1影响下丘脑GnRH神经元的发育、分化以及功能稳态，参与调控性发育进程。本项目执行期间我们采用高通量测序分析CPP女童肠道菌群及代谢组学特征谱，筛选差异菌属和代谢物并对其进行菌群代谢功能预测和代谢物相关性分析结果发现：CPP女童肠道微生物的多样性比对照组显著减少，筛选代谢物发现肠道内L-色氨酸含量在CPP组显著减少，而CPP女童接受GnRHa治疗后色氨酸有明显恢复；在动物水平通过筛选青春期不同发育阶段小鼠粪菌代谢物以及将CPP女童的粪菌移植到小鼠体内，观察发现CPP组粪菌移植后明显导致青春期启动提前；进一步探究色氨酸经脑肠轴影响GnRH的合成与分泌发现，肠道内色氨酸含量的减少造成下丘脑5-HT能神经元代谢产生的5-HT降低，减弱了青春发育期前期对GnRH神经元的抑制作用，从而使GnRH提前大量合成，诱发性早熟的发生。本研究从色氨酸-5HT代谢途径探究肠道菌群多样性变化对青春期性发育的影响，可以从肠道微生态及代谢紊乱角度为性发育异常疾病的干预提供理论基础。