口咽吸入柴油引擎微粒在阿茲海默症三基因轉殖小鼠中對中樞神經系統毒性的影響

Effects of oropharyngeal aspiration to diesel exhaust particles on central nervous system toxicity in 3xTg-AD mice

傅晨歡(Chen-Huan Fu)

指導教授 ： 鄭尊仁

國立臺灣大學/公共衛生學院/職業醫學與工業衛生研究所/碩士(2019年)

https://doi.org/10.6342/NTU201901961

摘要

許多空氣污染的流行病學研究已經證實大氣微粒會對人類造成健康危害，越來越多動物研究的結果也支持暴露微粒會引起中樞神經系統毒性，包括神經發炎、氧化壓力，進而引起神經退化性疾病，如阿茲海默症(Alzheimer’s disease, AD)。而都市中最主要的污染來源是交通相關的空氣污染物，其中由柴油引擎燃燒所排放的柴油引擎微粒(diesel exhaust particles, DEPs)更是微粒的主要來源。先前我們實驗室已利用C57BL/6小鼠急性暴露柴油引擎微粒，發現腦中促發炎反應因子、脂質過氧化及Tau蛋白均有顯著地增加，但其中微粒的所引起的中樞神經毒理機制仍不清楚，且使用一般品系小鼠的結果並不一致，故本研究選用阿茲海默症模式動物—三基因轉殖小鼠(3xTg-AD mice)來進行實驗，這種小鼠是一種能夠同時發展出兩種阿茲海默症特定病理變化的小鼠，包括由乙型類澱粉蛋白(amyloid-beta, Aβ)累積所形成的類澱粉蛋白斑塊(amyloid plaque)，以及由過度磷酸化Tau蛋白(phosphorylated Tau protein)聚集而成的神經纖維纏結(Neurofibrillary Tangles, NFTs)。因此本研究利用3xTg-AD 小鼠來探討暴露柴油引擎微粒是否會引起中樞神經系統毒性，包括氧化壓力、微膠細胞活化、自噬作用失調、阿茲海默症病理變化等現象。
本研究使用6-8週大的34隻3xTg-AD母鼠，以口咽吸入的方式在三週內重複暴露柴油引擎微粒6次，每次暴露50μg的微粒，每隻小鼠的總劑量為300μg，並於最後一次暴露後24小時犧牲。柴油引擎微粒是由美國國家標準暨技術研究院購買的標準品，型號為SRM1650b，代表重型柴油引擎燃燒所排放出的微粒。動物犧牲後會進行生化指標分析及組織病理分析，生化指標分析的部分會利用液相層析串聯質譜法(Liquid chromatography-tandem mass spectrometry, LC-MS/MS)以及西方點墨法(Western Blot)分別測量脂質過氧化(malondialdehyde, MDA)、微膠細胞(Iba-1)、自噬作用(LC3b)、阿茲海默症特定病理變化(Aβ42, t-Tau)等指標，另外組織病理分析則是將灌流固定後的腦、肺部進行石蠟包埋，並以H&E染色觀察組織病理。
實驗結果顯示，在大腦皮質、小腦、海馬迴的脂質過氧化指標MDA，暴露組均比控制組高，但僅在海馬迴中達顯著差異(p<0.05)，其餘中樞神經系統毒性指標在腦部均無顯著差異；在暴露組的肺部病理切片中觀察到輕微的發炎反應、碳粒沉積，部分控制組則是呈現最小程度的發炎反應，而暴露組和控制組的腦部病沒有顯著的病理變化。
根據本次的實驗結果發現，急性暴露柴油引擎微粒會誘導腦部的脂質過氧化，其中掌管記憶與學習的海馬迴是最易感的腦區，但其餘指標則是沒有顯著的變化，對於柴油引擎微粒是否會引起微膠細胞活化、自噬作用失調，甚至是阿茲海默症病徵的生成並不清楚。我們認為未來可以依照本次的研究設計進行修正，包括實驗動物的年齡、暴露後的實驗終點等，仰賴更近一步的研究以釐清柴油引擎微粒如何引起中樞神經系統毒性。

關鍵字

柴油引擎微粒 ； 口咽吸入 ； 中樞神經系統毒性 ； 微膠細胞 ； 氧化壓力 ； 自噬作用

並列摘要

Air pollution has been known as a major health threat for decades. Many epidemiological and toxicological studies of air pollution have confirmed that ambient particulate matter can cause adverse effects on the central nervous system (CNS), including neuroinflammation, oxidative stress, autophagy dysfunction and contribute to neurodegenerative diseases, such as Alzheimer’s disease (AD).
In the modern city, the most important source of pollution is traffic-related air pollutants. Diesel exhaust particles (DEPs) emitted by diesel engine combustion is the most important component of near-road and urban air pollution. In our previous study, C57BL/6 mice were acutely exposed to DEPs and found that pro-inflammatory cytokines, lipid peroxidation, and tau tauopathies increased significantly in the brain. However, the mechanisms are still unclear. Therefore, the objective of this study was to investigate the central nervous system toxicity caused by DEPs in Alzheimer’s disease mouse model.
6 to 8-week-old 3xTg-AD female mice were exposed to 300μg of DEPs SRM 1650b by oropharyngeal aspiration (50 μg/time, 2 times/week and for 3 weeks). SRM 1650b was purchased from National Institute of Standards and Technology, and it represents particulate from the combustion of heavy-duty diesel engines. After the last exposure, all animals were sacrificed at 24 hours, and the brain tissue, including the cerebral cortex, hippocampus and cerebellum regions, were collected. Brain region-specific malondialdehyde (MDA) level was measured by LC-MS/MS to assess lipid peroxidation. LC3b, Iba-1, Aβ42, and total tau protein were also evaluated using western blot. Brain and lung tissue were stained by hematoxylin and eosin (H&E) for histopathological examination.
The results showed that MDA concentration significantly increased in the hippocampus (p<0.05), but not in cerebral cortex and cerebellum. Expression of LC3b, Iba-1, Aβ42, and total tau protein was not significant between the control and the exposure group in 3 brain region. The DEPs exposure did not cause histopathological changes in the brain, but there was slight inflammation in the lungs.
In all, the data support that the hippocampus may be more sensitive to the oxidative stress from the exposure to DEPs. However, further studies should include the aged animals for prolonged period of time to elucidate the underlying mechanisms of DEPs induced neurotoxicity.

並列關鍵字

diesel exhaust particulates ； oropharyngeal aspiration ； central nervous system toxicity ； microglia activation ； oxidative stress ； autophagy

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