miR-299a-5p/NF-κB调控环路促进睾丸Leydig细胞衰老的分子机制研究

In addition to affecting the health of the reproductive system, testosterone deficiency can also cause metabolic abnormalities and cognitive decline in the male body, which seriously affects the physical health and quality of life of men. The main cause of testosterone decline in middle-aged and elderly men is the aging of testosterone-producing cells (Leydig cells), but the mechanism is unknown. Previous results showed that the inflammation-related signaling pathway in aging Leydig cells was abnormally active and the expression of miR-299a-5p was down-regulated. Further studies showed that silencing of miR-299a-5p expression inhibits the production of testosterone in Leydig cells and promotes the synthesis of senescence-associated secretory phenotype (SASP) factor. Based on the observation that miR-299a-5p targets NF-κB1, we propose the hypothesis that inflammatory factors mediate the aging of Leydig cells by regulating the expression of miR-299a-5p. We will use co-immunoprecipitation, CHIP-Seq, single-cell sequencing and bio-informatics and other methods to systematically elucidate the molecular mechanism by which extracellular inflammatory microenvironment inducesLeydig cell senescence in the testis. This study will provide an important theoretical basis and experimental evidences for revealing the epigenetic regulation of male gonad aging and the diagnosis and treatment of hypogonadism.

睾酮缺乏除影响生殖系统健康外，还会造成男性机体代谢异常和认知功能下降，严重影响男性的身体健康和生活质量。中老年男性睾酮下降的主要原因是睾酮分泌细胞（Leydig细胞）的老化，但机理未明。前期结果显示，老化Leydig细胞内炎症相关信号通路异常活跃，且miR-299a-5p表达下调。进一步研究表明，沉默miR-299a-5p的表达可抑制Leydig细胞雄激素的产生并促进其衰老分泌相关表型（SASP）因子的合成。由于miR-299a-5p靶向NF-κB1，因此，本项目提出炎症因子通过调控miR-299a-5p的表达介导Leydig细胞衰老的假设，并拟采用免疫共沉淀、CHIP-Seq、单细胞测序及生物信息学等方法，系统阐明细胞外炎症微环境诱发睾丸Leydig细胞衰老的分子机制。本研究将为揭示男性性腺组织衰老的表观遗传调控及性腺机能低下疾病的诊断治疗提供重要的理论基础和实验依据。

男性随着年龄增长，血液睾酮水平会逐步下降。雄激素缺乏不但影响中老年男性生殖系统的健康，也会导致2 型糖尿病、肥胖、骨质疏松、记忆力下降等多种疾病的发生。血清睾酮主要由睾丸间质细胞（Leydig cell，LC）合成，因此，LC功能衰退是中老年男性体内睾酮水平降低关键因素。本项目研究了炎症因素在LC老化过程中的作用。对睾丸进行单细胞测序，发现老化睾丸免疫微环境处于低度的慢性炎症状态，且衰老相关分泌表型（SASP）因子含量及其受体表达增加。转录组分析进一步表明，睾丸间质免疫细胞及其它体细胞随年龄变化基因大多富集在各种炎症信号通路，尤其是IL-17、TNF及NF-ĸB/MAPK信号通路。另外，LC的IL17R、TNFR及CXCR7等免疫因子受体表达也随老化而上升，表明老化增加了LC对免疫因子反应的敏感度。同时，睾丸免疫微环境的改变诱发LC老化表型，降低其异质性，同时减弱了睾丸先天性免疫功能。在众多随年龄上升的炎症因子中，TNFa及IL17均可以剂量依赖性地抑制LC产生睾酮，该效应主要通过MAPK及NF-ĸB通路的激活并随LC老化程度而增加。LC衰老过程中NF-ĸB通路的激活与miR-299a-5p表达量的变化也有密切关系。miR-299a-5p的靶标分子为Nf-kb1。抑制miR-299a-5p的表达或者过表达Nf-kb1都可以激活NF-ĸB通路并引起LC中SASP因子的表达，其机制是通过上调胞质中IkB-α及p65蛋白的磷酸化水平。睾丸间质微环境中炎症因子激活LC中NF-ĸB通路后同样可以下调miR-299a-5p的表达，而抑制该信号通路则增加miR-299a-5p的表达，两者间形成负反馈调控环路。此外，miR-299a-5p介导LC表达的多种SASP，可以通过旁分泌的方式作用LC，抑制其合成雄激素并促其表达更多SASP，造成睾丸微环境持续恶化，加速LC功能性老化。本研究结果阐明了炎症是促进LC功能性老化的关键因素，为日后该类疾病的诊疗提供实验基础和科学依据。