探討應用於牙科醫療之奈米二氧化鈦粒子長期暴露對於帕金森氏症的影響及其作用機轉

Abstract

研究目的 奈米二氧化鈦 (titanium dioxide nanoparticles, TiO2-NPs) 被廣泛應用於牙科材料中，具有良好的生物相容性以及抗菌能力。儘管TiO2-NPs在生醫領域已有許多研究，但TiO2-NPs對生物體之毒性效應並未明確，值得注意的是應用於牙科醫療的TiO2-NPs 可能會增加人體暴露於TiO2-NPs的風險。TiO2-NPs能成功穿過血腦屏障進入腦部，卻鮮少有研究探討TiO2-NPs與帕金森氏症之間的關係。本研究的目的在於探討TiO2-NPs造成帕金森氏症的可能風險。 研究程序及方法 利用基因轉殖之秀麗隱桿線蟲 (Caenorhabditis elegans, C. elegans) NL5901以及 BZ555評估長期暴露於TiO2-NPs 是否會使前突觸蛋白累積、多巴胺神經受損。透過分析C. elegans體內氧化壓力 (reactive oxidative stress, ROS) 以及多巴胺相關行為模式分析評估TiO2-NPs是否對C. elegans產生不良影響，並透過即時定量聚合酶連鎖反應 (qRT-PCR) 分析轉錄因子SKN-1的表達量。最後透過RNA干擾 (RNA interference, RNAi) 的技術了解TiO2-NPs、SKN-1以及帕金森氏症的交互作用以及其作用機轉。 結果 研究結果指出，長期暴露於低濃度1 µg/mL TiO2-NPs會顯著提升NL5901體內前突觸蛋白濃度，並使BZ555的多巴胺神經不正常百分比達16%，顯示長期暴露TiO2-NPs會對多巴胺神經造成損傷。體內氧化壓力分析中發現，長期暴露低濃度1 µg/mL TiO2-NPs會顯著提升BZ555體內ROS水平1.3倍。進一步的多巴胺相關行為模式分析發現，暴露於10 µg/mL TiO2-NPs其每20秒身體擺動次數相較於控制組會顯著減少30%，此結果代表暴露於TiO2-NPs影響多巴胺神經正常功能，影響C. elegans行為模式。在氧化壓力相關的轉錄因子SKN-1的RT-qPCR分析中，結果發現在1 µg/mL TiO2-NPs的濃度下，SKN-1以及其下游基因GST-38， GCS-1表現量顯著提升約2倍。在RNAi的實驗中發現，1 µg/mL TiO2-NPs的暴露濃度抑制SKN-1轉錄因子且會更顯著增加13% C. elegans多巴胺神經不正常比例，表示SKN-1在對抗TiO2-NPs產生的氧化壓力扮演重要角色。 結論 本研究結果顯示，在模式生物秀麗隱桿線蟲中，長期暴露於1 µg/mL TiO2-NPs會透過前突觸蛋白以及細胞內ROS累積，造成多巴胺神經損傷影響到其正常功能，進而可能造成帕金森氏症。TiO2-NPs的毒性效應和SKN-1 pathway相關，因此可以推斷SKN-1在保護TiO2-NPs所引起之帕金森氏症病徵中扮演重要角色。

Objective Titanium dioxide nanoparticles (TiO2-NPs) are widely used in nowadays dental ma-terial with good biocompatibility and anti-bacteria effects. Despite many studies for TiO2-NPs in biomedical fields, the toxicity of TiO2-NPs to organisms was still unclear. In particular, the TiO2-NPs used in dental materials might potentially increase potential risk to human. TiO2-NPs could penetrate through the blood-brain-barrier (BBB), however, studies on the relationship between TiO2-NPs and Parkinson’s disease (PD) are limited. Herein, the purpose of this study was to evaluate the possible risk of PD by TiO2-NPs and its possible underlying mechanisms Materials and methods The transgenic strains of Caenorhabditis elegans (C. elegans) NL5901 and BZ555 were used to evaluate whether long-term exposure to TiO2-NPs results in α-synuclein accumulation, dopamine neuron damage or not. The effects of TiO2-NPs were further explored on C. elegans through intracellular ROS analysis and dopamine-related behavior analysis. In addition, qRT-PCR was performed to examine the mRNA levels of SKN-1 and its downstream genes. Finally, RNA interference technique was used to explore the association between SKN-1 and PD. Results The results showed that long-term exposure to TiO2-NPs (1 µg/mL) significantly increased α-synuclein level in NL5901 up to 10%, and enhanced dopamine neuron ab-normality in BZ555 around 16%. In addition, long-term exposure to 1 µg/mL TiO2-NPs significantly elevated the ROS level by 1.3 folds comparing to the unexposed control. Further dopamine neuron-related behavior analysis found that under 10 µg/mL of TiO2-NPs exposure, the body bending of C. elegans significantly reduced by 30%, com-pared with the unexposed control. This suggests that TiO2-NPs exposure affects the nor-mal function of dopamine neuron, leading to the behavior change of C. elegans. Moreover, TiO2-NPs significantly increased the mRNA levels of SKN-1 and its downstream gene, GST-38 and GCS-1. Furthermore, under TiO2-NPs (1 µg/mL) exposure, the dopamine neuron abnormality was further significantly increased 13% compared with the vector control after skn-1 knockdown, suggesting the vital role of SKN-1 on PD upon long-term TiO2-NPs exposure. Conclusion Results of this study showed that long-term exposure to 1 µg/mL TiO2-NPs in-creased accumulation of α-synuclein, and intracellular ROS, and caused dopamine neu-rons damage. The damaged dopamine neurons affected the normal function of dopamine neurons, leading to Parkinson's symptoms in C. elegans model. This study showed that the toxicity of TiO2-NPs was associated with the SKN-1 pathway, indicating that SKN-1 played an important role in protecting dopamine neurons against ROS caused by TiO2-NPs.