RIP1/RIP3信号通路调控纳米TiO2长期暴露致神经元炎症损伤的机制研究

Nano-sized titanium dioxide (Nano-TiO2) are widely used in numerous fields, such as dentistry, owing to their excellent physicochemical properties. Therefore, producers are at high risk of getting exposed to Nano-TiO2, and epidemiologic studies have showed that workplace exposure becomes a health concern to the respiratory system. The central nervous system (CNS) is susceptible to exogenous insults, but no epidemiologic study investigates the relationship between Nano-TiO2 exposure and CNS disorders. However, a great number of in vivo studies have shown that Nano-TiO2 can be detected in animal brains following their exposure. Nano-TiO2 accumulation in the brain may impair neuronal cells, leading to brain disorders. In vitro studies have obtained the similar conclusions. To sum up, the potential neurotoxicity of Nano-TiO2 has been confirmed and Nano-TiO2 become a big threat to the brain. The molecular mechanisms underlying neurotoxicity of Nano-TiO2 mainly include oxidative stress, inflammatory responses, apoptosis, and so on. However, the relationship between inflammation in the brain and Nano-TiO2 exposure remains unclear. Necroptosis is one type of the programmed cell death, which is different from necrosis. It is defined as active cell death and is regulated by certain genes. Necroptosis is reported to be involved in nanotoxicity and inflammation. Necroptosis, which can also be called “programmed necrosis”, is initiated by activating the death receptor with stimuli. Receptor-interacting protein kinase 1 and 3 (RIP1 and RIP3) are frequently involved in necroptosis. However, the relationship between RIP1/RIP3 signaling pathway and neurotoxicity of Nano-TiO2 is unknown. Besides, toll-like receptors 4 (TLR4) is involved in neurotoxicity of Nano-TiO2 and necroptosis. Whether RIP1/RIP3 signaling pathway in the brain could be activated through TLR4 during Nano-TiO2 exposure also remains unknown. In our preliminary experiment, we found that Nec-1 as the specific inhibitor of necroptosis could attenuate neuro-inflammation induced by Nano-TiO2. Meanwhile, the expression of RIP1/RIP3 and TLR4 is up-regulated after Nano-TiO2 exposure. Therefore, we put forward the hypothesis that RIP1/RIP3 signaling pathway might be involved in neurotoxicity of Nano-TiO2, and TLR4 could be the upstream signal of RIP1/RIP3. In this study, we are going to examine the relationship between RIP1/RIP3 signaling pathway and neurotoxicity of Nano-TiO2 firstly. Secondly, the role of TLR4 in neurotoxicity of Nano-TiO2 should be investigated. Thirdly, the role of TLR4 as the upstream signal regulating RIP1/RIP3 signaling pathway in the CNS during Nano-TiO2 exposure should be further explored. This study may help us to further understand the molecular mechanisms underlying neurotoxicity of Nano-TiO2.

纳米TiO2在口腔医学领域的广泛应用，增加了职业暴露风险。除了呼吸系统外，纳米TiO2也能诱导中枢神经系统发生炎症反应，但机制尚不清楚。有报道称，RIP1/RIP3信号通路能够调控炎症反应，TLR4是其上游因子，但其是否参与纳米TiO2的促炎作用，尚未见报道。本课题组通过前期研究发现，纳米TiO2暴露导致中枢神经系统发生炎症损伤，并伴RIP1/RIP3及TLR4的上调；RIP1/RIP3抑制剂的干预，则能够减轻纳米TiO2所导致的炎症损伤作用。据此，本课题组提出如下科学假说：RIP1/RIP3信号通路参与调控纳米TiO2致中枢神经系统的炎症损伤，TLR4可能为其上游调控因子。因此，本研究首先探讨RIP1/RIP3及TLR4在纳米TiO2致中枢神经系统炎症损伤中的作用；其次阐明TLR4对RIP1/RIP3的调控作用。本研究有助于更加深入了解纳米TiO2的中枢神经毒理机制，具有重要的理论价值。

随着纳米二氧化钛（titanium dioxide nanoparticles，TNPs）材料在生活生产领域的广泛应用，TNPs潜在的毒性成为威胁人们健康的一个重要因素。现有的研究表明，TNPs能够造成中枢神经系统的损伤，但其分子机制尚不明确。有研究表明，RIP1/RIP3信号通路参与诸多病理过程，但其是否参与TNPs所致的中枢神经系统毒性机制，目前尚未见相关报道。本课题组采用SH-SY5Y细胞作为神经元细胞系，对RIP1/RIP3是否参与TNPs的神经毒性进行了相关探讨。本项目组首先发现TNPs能够导致SH-SY5Y细胞发生氧化应激损伤及炎症反应，并伴随RIP1、RIP3、TLR4表达的升高。随后采用RIP1抑制剂Nec-1及RIP3抑制剂GSK872对TNPs染毒过程进行干预。结果发现Nec-1及GSK872的干预能够有效抑制TNPs对细胞所造成的毒性效应，并能有效抑制RIP1及RIP3表达的上调。随后课题组采用siRNA技术来进一步进行机制探讨，结果发现，TLR4、RIP1、RIP3表达被沉默后，能够有效抑制TNPs对细胞所造成的毒理效应。最后课题组通过构建TLR4、RIP1、RIP3基因缺陷型细胞，对TRL4、RIP1、RIP3的相互影响进行了探讨。结果提示，TLR4的表达改变能够影响RIP1、RIP3的表达，但RIP1与RIP3的表达改变并不能影响TLR4的表达。通过以上研究，课题组初步证实了TLR4介导的RIP1/RIP3信号通路参与了TNPs对细胞所造成的的氧化应激损伤及炎症反应。通过本课题的完成，为进一步研究RIP1/RIP3信号通路在TNPs中枢神经毒理效应中的作用奠定了基础，进一步丰富了TNPs的毒理机制。