三氯生暴露通过miR-17家族介导线粒体自噬和凋亡致雄性生殖细胞损伤的机制研究

Triclosan (TCS) is a commonly used chemical which could induce multi-system toxicity. However, few studies pay attention to the detrimental effects of TCS on male reproductive system. In the preliminary study, we found that after TCS exposure, the structures of testis and sperm cells were disarranged. The swollen mitochondria, autophagic vacuoles and apoptotic bodies were also observed in spermatogenic cells. Gene microarray results showed that the expressions of ten mitochondrial autophagy genes were declined. Moreover, the software prediction and preliminary verification suggested that the miR-17 family might play an important role in regulating the expressions of these genes. Therefore, we propose that TCS exposure leads to the changes of miR-17 family, followed by the downregulated mitochondrial autophagy and upregulated mitochondrial apoptosis, leading to the damage of spermatogenic cells and sperm cells. Three aspects (population, animal and cell models) are included in this project. mtDNA sequence analysis, Adeno-associated virus vector construction, Co-immunoprecipitation, confocal microscopic imaging and optical in vivo imaging technology are applied to verify our hypotheses. This study aims to provide scientific insights into TCS-induced male reproductive toxicity, and develop new strategies and potential targets for the prevention and treatment of male infertility.

三氯生（Triclosan，TCS）是一种常用化学物，暴露广泛且可致多系统毒性。然而，TCS对雄性生殖系统的影响鲜有报道。本项目发现，TCS可造成睾丸组织结构紊乱、精子线粒体肿胀、生精细胞出现自噬泡和凋亡小体。芯片结果显示，TCS显著下调10种线粒体自噬相关基因表达。经软件预测和初步验证发现，miR-17家族可能在调控自噬相关基因表达中发挥作用。据此，本项目提出以下假设：TCS暴露通过改变miR-17家族表达，进而下调线粒体自噬靶基因，激活线粒体凋亡通路，导致雄性生殖细胞损伤。本项目拟从人群、动物和细胞三个层面开展研究，采用mtDNA测序、腺相关病毒载体构建、免疫共沉淀、激光共聚焦及活体成像等技术手段，验证以上假设。本研究旨在为TCS暴露致雄性生殖毒性提供科学依据，同时为男性不育的防治提供新的策略和潜在靶点。

三氯生（Triclosan, TCS）作为广谱抗菌剂，在男性精液和尿液生物样本中检出率高，提示TCS对男性精液质量可能产生影响。然而，TCS对男性精液质量影响方式和机制尚不明确。本项目从人群、细胞和动物三个方面，探索了TCS暴露对雄性生殖细胞和精子的影响。.人群研究方面，1.选择低精子质量病例和对照精子样本，通过自噬相关功能基因微阵列分析差异表达基因（DE基因）。然后使用较大人群验证DE基因的表达。结果表明ESR1的表达显著降低，揭示了自噬受损在精子质量低下的机制中可能发生作用。2.人群尿液样本TCS浓度检测。收集男性尿液样品191份，检测尿液中TCS浓度。结果显示，TCS浓度为38.3±248.09 ng/ml；浓度范围为0-3184.4 ng/ml，总检出率为91.10%，不同地区尿液样本TCS浓度差异无统计学意义。3.通过对200例人群精液样本进行TCS浓度检测和与精子质量相关性研究，TCS浓度对精液质量的影响有统计学意义，人体内TCS浓度与精液质量呈负相关。.细胞研究方面，通过不同浓度TCS进行干预后，检测生精细胞GC2和睾丸支持细胞TM4中miRNA及自噬和凋亡功能基因表达改变。研究表明，TCS暴露可能通过影响miR-20a-5p表达，从而调控PTEN表达，出现了LC3介导的自噬反应，最终导致了细胞自噬功能异常。.雄性SD大鼠干预实验表明，miR-20a-5p过表达，ATP浓度显著升高，睾丸线粒体染色体突变发生最多，miR-20a-5p过表达导致线粒体自噬显著下调。未来仍需开展进一步研究，揭示TCS暴露对男性生殖细胞的影响和调控机制。