基于“肾藏精”理论研究补肾填精法通过孤核受体Nur77调控睾丸分泌睾酮的作用及机制

There is a close relationship between the theory of "kidney storing essence" in Traditional Chinese Medicine and the function of hypothalamic -pituitary-gonads axis. Kidney-reinforcing formulas are usually thought to promote testosterone secretion through the sexual axis, but the mechanism is still unclear. It is recently found that Nur77, an orphan nuclear receptor, is indicated a close involvement in gonads axis function: GnRH can facilitate pituitary gonadotroph cells to secrete LH and FSH; many factors, including LH, all function on Leydig cells through Nur77, promoting testosterone synthesis by up-regulation of StAR, 21-hydroxylase and P450c17. Previously, we demonstrated that kidney reinforcing drug, Yangjing capsule, can promote Leydig cellssecreting testosterone. In order to elucidate the underlying mechanisms, Leydig cells, old SD mouse with hypogonadism and small RNA interference technology would be used in the study. We present a hypothesis that Yangjing capsule could enhance testosterone synthesis in Leydig cells through up-regulation of StAR, P450scc and HSD3B2 induced by Nur77, it also could function on pituitary through Nur77 causing an indirect elevation of testosterone. In conclusion, the study would contribute to understand the working mechanism of kidney reinforcing drugs, to elucidate the nature of "kidney storing essence" in Traditional Chinese Medicine, and to guide the treatment of male with reproductive and sexual disturbance.

"肾藏精"理论与下丘脑-垂体-睾丸性腺轴的关系密切。补肾填精中药可以通过调节性腺轴功能，促进睾酮分泌，但其具体机制不清。近年发现，孤核受体Nur77与性腺轴功能密切相关：GnRH能通过Nur77调控垂体促性腺细胞分泌LH、FSH；许多因素（LH等）均是通过Nur77作用于睾丸间质细胞(Leydig)，影响睾酮合成关键基因（StAR等）的表达，调节睾酮合成。前期的研究表明补肾填精中药养精胶囊可以促进Leydig细胞合成睾酮，本研究拟利用细胞实验、性腺功能减退大鼠及siRNA（小RNA） 干扰技术，进一步研究其促进睾酮分泌的分子机制和作用途径。课题组推测养精胶囊可以通过Nur77诱导StAR等的表达，直接促进睾酮合成；也可以通过Nur77改善垂体分泌功能，间接促进睾酮分泌。这对于理解补肾填精法调控性腺轴、促进睾酮分泌的作用靶点和分子机制，阐释"肾藏精"的科学内涵，均有重要的理论和现实意义。

补肾填精中药可以促进睾酮分泌，但其具体机制不清。孤核受体Nur77是雄激素合成的关键调控分子。前期的研究表明补肾填精中药养精胶囊可以促进Leydig细胞合成睾酮。本项目在前期工作的基础上，进一步研究养精胶囊是通过Nur77调控雄激素合成及睾丸腺的功能。细胞实验表明，养精胶囊可以上调Leydig细胞Nur77表达且成剂量依赖性；进一步分析（荧光素酶报告基因实验）发现，养精胶囊可以增强Nur77基因启动子活性，从而上调Nur77表达；腺病毒干扰实验表明，下调Nur77的表达，Leydig细胞合成雄激素下降，且睾酮合成关键酶StAR、HSD3B2的表达均下降；这提示补肾填精中药是通过Nur77调控雄激素合成。此外，不同发育时期小鼠睾丸Nur77表达研究发现，Nur77 (NR4A1)的mRNA水平和蛋白水平在幼年期小鼠睾丸中低表达，在成年期表达增加，老年期又有一定程度的减退，这种变化模式与体内睾酮的变化趋势相同。这提示Nur77与雄性小鼠的性腺发育呈现同步性，说明Nur77与性腺功能减退及雄激素分泌下降有密切关系。综合上述结果，本研究对于理解补肾填精中药治疗男性性腺功能减退的作用靶点，阐释“肾藏精”的科学内涵，进而指导临床运用中药促进雄激素分泌，改善男性生殖内分泌状况，具有重要的理论和现实意义。项目培养硕士研究生一名，获得硕士学位；资助发表学术论文4篇，其中SCI论文1篇；资助参加国家及省级学术会议多次，并做交流汇报。