纳米氧化钛暴露致小鼠睾丸炎过程中TAM受体/TLR3信号通路的调控机制

Although numerous studies demonstrated that titanium dioxide nanoparticles (TiO2 NPs) have been demonstrated to accumulate in organs resulting in toxicity, there is currently only limited data regarding reproductive toxicity by TiO2 NPs. Our previous studies indicated that TiO2 NPs exposure caused significant decreases in sperm numbers and sperm motility, and increases in abnormal sperm numbers in mice, which are closely associated with testicular inflammation. However, how the orchitis caused by TiO2 NPs occur and develop, whether the orchitis is associated with activation of TLR3-mediated signal pathway regulated by TAM receptor are still unclear. Therefore, we will investigate several scientific problems in vivo and in vitro and coupling with dose-effect and time-effect using various techniques of molecular biology, including: (1) effect of TiO2 NPs on expression of Tyro3, Axl and Mer and TLR3 in mouse testis and Sertoli cell; (2) whether TiO2 NPs continue to induce high TLR3 expression when expression of TAM receptors is increased by lentiviral vector carrying TAM receptor genes infecting mouse testis or Sertoli cells; (3) whether testicular inflammation is associated with over activation of TLR3 signal pathway-related cytokines by decreasing TLR3 expression with SOCS inhibitors injecting testis and treating Sertoli cells. The studies would help to understand reproductive toxic mechanisms, and provide theory evidences for reproductive system protection under TiO2 NP-induced reproductive toxicity.

关于纳米TiO2毒理研究较多，但生殖毒性报道较少。前期工作已证实纳米TiO2可引起小鼠精子数量和活力下降及畸形率增加，并发现这些现象与睾丸炎有关。但这种炎症是如何发生发展、是否与TAM受体参与调控的TLR3信号通路的作用有关尚不明确。本课题将结合体内外实验及其剂量和时间效应，利用多种分子生物学技术研究纳米TiO2暴露小鼠和Sertoli细胞后的以下问题: 1)对睾丸组织和Sertoli细胞中Tyro3、Axl、Mer和TLR3表达影响；2) 通过TAM受体慢病毒过表达重组载体转染睾丸和Sertoli细胞以增强TAM受体的表达，研究纳米TiO2是否会继续诱导TLR3受体高表达；3) 通过SOCS抑制剂注射小鼠睾丸和处理Sertoli细胞以抑制TLR3表达，研究纳米TiO2致睾丸炎是否因TLR3信号通路相关因子的过度活化造成，为阐明纳米TiO2的生殖毒性机制提供实验数据。

已证实纳米TiO2暴露可引起小鼠睾丸炎，但这种炎症是否与TAM受体参与调控的TLR3信号通路的作用有关尚不明确。本研究发现纳米TiO2长期暴露可导致雄性小鼠睾丸炎症发生，且引起睾丸组织中Tyro3、Axl、Mer、IB、SOCS1和SOCS3的基因和蛋白表达的下调，而TLR–3、TLR–4、NF–kB、IL–lb、IL–6、TNF–α、IFN–α和IFN–r的基因和蛋白表达上调，显示睾丸炎发生与其免疫功能的异常有密切关系，并认为与TAM受体表达被抑制及TLR3介导的信号通路相关因子过度活化有关。体外纳米TiO2染毒对原代培养小鼠睾丸Sertoli细胞的毒性作用研究结果表明，纳米TiO2染毒后Sertoli细胞活性下降、LDH大量释放、细胞凋亡或坏死、ROS大量积累、SOD、CAT、GSH-PX活性显著下降，线粒体膜电位明显降低，Cytc、caspase-3、Bax、caspase-12、GRP7和CHOP的蛋白表达都显著上调，而bcl-2蛋白表达下调，这显示纳米TiO2导致的Sertoli细胞凋亡与线粒体介导的信号通路和内质网介导的信号通路有关；纳米TiO2可进入原代培养Sertoli细胞，诱导TLR3、NF-kB、IL-lb、IL-6、TNF-α、IFN-α和IFN-r蛋白表达，抑制Tyro3、Axl、Mer和I-kB蛋白表达，进一步证实睾丸免疫功能的破坏与TAM/TLR3信号通路的调控紊乱相关；利用染毒原代培养的大鼠睾丸Sertoli细胞研究了纳米TiO2致睾丸免疫功能破坏是否还涉及Ca2+/PKC/p38 MAPK/NF-κB通路，结果表明纳米TiO2可导致Ca2+-ATP酶和Ca2+/Mg2+-ATP酶活性下降和细胞内Ca2+水平显著上升，NF-kB、TNF-α、IL-1b、p-PKC和p-p38MAPK蛋白表达上调，而I-kB蛋白表达下调，证实纳米TiO2也可通过Ca2+/PKC/p38 MAPK/NF-κB通路失调导致睾丸免疫功能破坏。. 纳米TiO2可进入小鼠睾丸和附睾中积累，引起精子数量和活力下降及畸形率增加，诱导睾丸特异基因如Cdc2、Cyclin B1、Dmcl、TERT、Tesmin、TESP-1、XPD和XRCCI的基因和蛋白表达的上调，而Gsk3-β和PGAM4表达水平下调；纳米TiO2导致小鼠精子发生的抑制也与睾丸生化功能破坏有关。