微粒空氣污染物對疾病動物之心肺毒性研究： 1. 氣懸微粒粒徑分布與季節變異之心肺毒性探討 2. 柴油引擎微粒與微粒內毒素之心肺毒性探討

Abstract

Part I 氣懸微粒粒徑分布與季節變異之心肺毒性探討 流行病學研究指出，當空氣中的懸浮微粒濃度升高時，民眾的總死亡率增加，其中主要是心臟血管和呼吸道疾病的貢獻，不過相關機制仍待釐清，特別是心血管疾病方面資料較少。然而懸浮微粒中的成分相當複雜，且隨著季節的不同會有不同的濃度分布，因而造成不同程度的微粒毒性，因此本研究以新莊超級測站微粒來探討不同季節及粒徑於易感性動物的自發性高血壓大鼠及糖尿病大鼠之心血管疾病相關指標變化，包括系統性發炎反應CRP（C-reactive protein ）、凝血功能指標fibrinogen以及血管內皮細胞功能指標NO（nitric oxide）。 懸浮微粒採集自新莊地區超級測站，我們利用測站質量濃度連續監測儀（Rupprecht & Patashnick 1400a）所使用的TEOM（Tapered-Element Oscillating Microbalance）濾紙上的微粒來進行實驗，其成分及粒徑分佈來自超級測站收集之資料。我們利用氣管灌注（intratracheal instillation）方式來進行暴露。自發性高血壓大鼠（spontaneously hypertensive rat, SHR）及健康WKY（Wistar Kyoto）大鼠和糖尿病大鼠及健康SD（Sprague-Dawley）大鼠各分為PM暴露組與對照組，其中暴露組包括PM10-2.5與PM2.5不同粒徑及2-4月和9-11月不同季節之微粒，劑量均為200 μg/animal。灌注後24小時進行犧牲，然後採集肺部及周邊血液樣本進行CRP、NO、fibrinogen分析，同時採集其肺泡灌洗液（BALF）進行肺部發炎及傷害指標的分析。最後以Wilcoxon Rank Sum test比較實驗組與控制組各項肺部發炎指標及心血管指標，並就不同粒徑及季節進行統計分析。結果發現大氣懸浮微粒在SHR及健康WKY大鼠會產生肺部發炎反應，而SHR還會有周邊血液fibrinogen的增加及內皮細胞放鬆素NO的下降，且相較於9-11月分，2-4月分似乎有較強的心血管效應，但在不同粒徑並無顯著差異，而當我們合併兩種季節的資料後，發現SHR會較健康WKY大鼠有明顯的心肺效應；糖尿病及健康SD大鼠在暴露微粒後亦是有肺部發炎反應及fibrinogen的增加，但糖尿病大鼠並沒有較健康SD大鼠更具易感性，且在不同季節及粒徑之間並無明顯差異。 微粒空氣污染物會造成肺部發炎及心血管指標的改變，且易感性動物SHR有較健康動物明顯的心血管效應，而不同季節有些許不同的效應，可能與周邊懸浮微粒各種不同成分有關。

Part II 柴油引擎微粒與微粒內毒素之心肺毒性探討 柴油引擎微粒（Diesel Exhaust Particles, DEPs）是都會區空氣懸浮微粒的主要成分之一，而研究指出DEPs的暴露會造成肺部的發炎反應，甚至進一步的研究發現，DEPs中的超細微粒與血栓的形成有關，但是心血管相關的研究仍非常缺乏。細菌內毒素（Lipopolysaccharide, LPS）亦是都會區懸浮微粒的一種重要成分，且LPS的暴露會引發肺炎及影響肺部細胞功能，也可能活化TLR（Toll-like receptor）而引起一連串cytokine的釋放並影響內皮功能。上述兩種成分都是都會區空氣懸浮微粒的重要成分，因此我們想釐清這些成分的心血管效應以及交互作用，並利用糖尿病大鼠來探討心血管疾病相關指標變化。 我們將糖尿病大鼠及健康SD大鼠各分為四組，並分別以氣管灌注給予saline、DEPs 2 mg/kg，LPS 2 mg/kg，以及DEPs +LPS。灌注後24小時進行犧牲，然後採集肺部及周邊血液樣本進行系統性發炎反應（CRP）、血管內皮細胞功能（NO）、凝血功能（fibrinogen）分析，同時採集其肺泡灌洗液（BALF）進行肺部發炎及傷害指標的分析。最後以Wilcoxon Rank Sum test比較實驗組與控制組各項肺部發炎指標及心血管指標，並就不同成分進行統計分析。結果發現DEPs會產生肺部傷害和發炎反應以及周邊血液fibrinogen的增加，LPS亦會產生肺部傷害，並且引起fibrinogen的增加及內皮細胞素NO的下降，而DEPs和LPS在心肺各參數上所造成的效應不太一致：DEPs造成較強的肺部發炎反應及fibrinogen的增加，LPS則是造成較多的CRP釋出及NO減少，推測兩者在心肺有不同的作用途徑，使得DEPs和LPS之間沒有明顯的交互作用，導致DEPs+LPS在肺部的效應並沒有比單獨暴露DEPs或LPS要來的強，而在fibrinogen及NO也只有較單獨暴露DEPs或LPS有稍強的效應。我們的研究並無發現糖尿病大鼠有較健康大鼠強的心血管效應。 DEPs和LPS會造成肺部發炎及心血管指標的改變，但是效應並不一致，推測和這兩種成分的作用途徑不太相同有關，使得這兩種成分並沒有明顯的交互作用。

Part I Effects of Particle Size and Season Increased cardiovascular and respiratory mortality have been associated with elevated concentration of ambient particulate matters (PM). However, the underlying mechanism remains unclear. Our study at supersite also revealed that PM might induce cardiovascular toxicity. Thus, we further examined the cardiovascular toxicity of PM of different sizes and seasons in spontaneously hypertensive rat (SHR) and STZ-diabetic rat. The particles were collected on TEOM (Tapered-Element Oscillating Microbalance) at Hsing-Chuang supersite where the data of its composition and size distribution were determined. The SHR and STZ-diabetic rats were intratracheally administered with PM2.5 and PM10-2.5 of different seasons. They were sacrificed 24hrs after exposure and levels of CRP for systemic inflammation, NO for endothelial function and fibrinogen for blood clotting function were determined in peripheral blood. We also collected bronchoalveolar lavage fluid to analyze lung injury. Wilcoxon Rank Sum test was used to compare between the experimental and control groups on lung inflammation and cardiovascular biomarkers.In animals treated with PM collected from supersite, we found that fibrinogen increased in diabetic rats and SHR after PM exposure. NO also decreased in SHR after PM exposure. It appears diseased animals were more susceptible to PM administration. We also found that PM of different seasons might induce different effects, but there was no difference between PM2.5 and PM10-2.5. Ambient PM can cause lung and cardiovascular toxicity, and the the effects are more prominent in diseased animals. The different effects in different seasons may be explained by the components of PM.

Part II Effects of DEPs and LPS Diesel exhausts particles (DEPs) are one of the primary compositions in suspended particulate matter in a metropolitan area. Studies showed that DEPs’ exposure can cause inflammation in lungs. Further research discovered that, in DEPs’ ultrafine particle can result in thrombus formation or blood clots. The bacterium endotoxin (LPS) is one of the main compositions in suspended particulate matter. Exposure to LPS can cause pneumonia and affect functions of the lungs cells, and it may also affect TLR to induce more cytokine release and affect endothelial function. Because these two components are important in ambient particles, therefore we want to clarify these two componetes effects on cardiovascular parameters by using diabetic rats, and try to investigate the interaction between these two components. We respectively divided the diabetic rats and the healthy SD rats into four groups, and each rat was injected by means of intratracheal instillation with saline, DEPs 2 mg/kg, LPS 2 mg/kg, as well as DEPs +LPS. Rats are then sacrificed twenty four hours after the injection, lungs and the peripheral blood samples are then collected to for the analysis of systemic inflammation CRP, endothelial dysfunction NO、blood clotting function fibrinogen, and BALF is also collected simultaneously for the analysis of lung inflammation and injury index. Finally, Wilcoxon Rank Sum test was used to compare the results between experimental group and the controlled group for lung inflammation index and cardiovascular index, statistical analysis is then done on the different compositions. The result showed that DEPs, LPS, and DEPs +LPS can cause lungs inflammation response, increase in peripheral blood fibrinogen and NO drop in the diabetic rats and the healthy SD rats, simultaneous exposure to both DEPs and LPS creates a much stronger effect when compared to individual exposure to DEPs or LPS, additivity response between diabetes and particulate matter on NO can be observed on the diabetic rats. DEPs and LPS can result in lung inflammation and a change in cardiovascular index, susceptibility animals like diabetic rats tends to have a stronger cardiovascular effect, and the speculation is that it is the result of the correlations between the different particles.