利用自發性高血壓大鼠探討亞慢性呼吸暴露大氣細懸浮微粒對大腦脂質的影響

Abstract

空氣汙染所造成的健康危害日趨嚴重，過去的流行病學研究和動物研究皆已經證實大氣細懸浮微粒(PM2.5)與呼吸系統和心血管系統的不良健康效應有關。而近幾年流行病學研究發現長期暴露大氣細懸浮微粒可能造成中樞神經系統的不良影響，如認知功能下降、大腦結構改變以及加速大腦老化速度，甚至與神經退化性疾病有關。由於大腦為脂質含量非常豐富之器官，我們假設暴露大氣懸浮微離會造成腦脂質組成改變，此改變可能造成大腦功能障礙或腦部傷害。我們希望利用質譜儀為基礎的脂質體學，針對大腦中重要的脂質－磷脂醯膽鹼(Phosphatidylcholines)和神經磷脂(Sphingomyelins)，以探討亞慢性暴露大氣細懸浮微粒對大腦所造成的影響，進一步釐清可能的毒理機制。 本實驗利用將八週齡自發性高血壓(spontaneously hypertensive rat)公鼠，隨機分為暴露組及控制組各五隻，其中暴露組全身暴露於大氣微粒暴露系統，控制組暴露經HEPA過濾懸浮微粒之空氣。暴露時間為期三個月，犧牲後取下右腦並分為五個腦區：嗅球、小腦、大腦髓質、海馬迴以及大腦皮質，進行樣本前處理和脂質萃取後，再利用極致液相層析儀搭配串聯式質譜儀(UPLC-MS/MS)進行脂質體分析。經數據前處理後，利用多變量分析－偏最小平方判別分析(PLS-DA)觀察兩個組別的脂質體是否有分群的結果。另外，利用無母數單變量分析－Wilcoxon rank sum test找出兩組間顯著差異的phosphatidylcholines和sphingomyelins。 PLS-DA結果顯示五個腦區的脂質體在暴露組和控制組之間皆呈現分群，其中，PM2.5對海馬迴的脂質造成最大影響，且顯著改變的脂質體在海馬迴及小腦有相同的變化趨勢。在海馬迴中多元不飽和diacyl-phosphatidylcholines、plasmanylcholines以及plasmenylcholines皆有顯著上升的趨勢，而有些sphingomyelins在海馬迴及小腦中也呈現顯著上升，透過代謝途徑的推測，這些脂質可能在海馬迴及小腦中扮演著神經保護性機制，以防禦PM2.5可能導致之氧化壓力及細胞損傷；在大腦髓質中發現部份diacyl-phosphatidylcholines顯著下降且lyso-phosphatidylcholines顯著上升，另外在大腦皮質中少數的飽和plasmanylcholines、飽和plasmenylcholines及sphingomyelins呈現顯著下降，此結果顯示PM2.5暴露可能造成大腦皮質和大腦髓質中的脂質產生擾動，導致細胞膜不穩定、發炎反應、及抗氧化能力下降；而在嗅球則未發現顯著的脂質改變。我們利用脂質體學的方法觀察到海馬迴及小腦的脂質體改變趨勢與其他腦區有差異。然而總顯著改變的脂質不多，我們推測可能與低濃度及亞慢性暴露有關。 本研究證實亞慢性暴露大氣PM2.5會造成自發性高血壓大鼠的海馬迴、小腦、大腦皮質、大腦髓質以及嗅球之phosphatidylcholines和sphingomyelins產生擾動，在不同腦區觀察到脂質有不同的改變趨勢，可能與特定腦區對PM2.5的易感受性和傷害不同有關。此外，本研究可支持以質譜為基礎的脂質體學為一個有效的方法以探討暴露與可能之健康效應的相關性，並幫助建議未來在公共衛生研究上的方向。

A large number of experimental and epidemiological studies have demonstrated the association between ambient fine particulate matter (PM2.5) with adverse effects of pulmonary and cardiovascular systems. However, recent epidemiological studies indi-cated that long term PM2.5 exposure could cause brain damage, such as cognitive de-cline, cerebral structure change and brain aging acceleration, and also associated with the risk of neurodegenerative diseases. Due to the brain is rich in lipids, we hypothe-size that PM2.5 may cause lipid alteration in brain, which may lead to brain dysfunction. We intend to identify possible critical lipids associated with PM2.5 exposure by MS-based lipidomic approach and further associate those changes with biological function. In this study, five male spontaneously hypertensive rats were whole-body exposed to ambient air from outside of the building for 3 months, while the control (n=5) in-haled HEPA filtered air. After animals were sacrificed, five brain regions including ol-factory bulb, cerebellum, cerebral medulla, hippocampus and cerebral cortex were taken. Phosphorylcholine-containing lipids including phosphatidylcholines and sphin-gomyelins were extracted from each brain region, and profiled by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). MS spectra from the analysis of lipids from exposure and control animals were analyzed by partial least squares discriminant analysis (PLS-DA). Moreover, Wilcoxon rank sum tests were used to examine the significant changes between the two groups. The results showed that the phosphorylcholine-containing lipid profiles of the ex-posure group were different from those of the control group in the PLS-DA models of each brain regions. The greatest lipid changes are in hippocampus. Moreover, the pat-tern of lipid changes in the hippocampus and cerebellum were similar. In the hippo-campus, increased polyunsaturated diacyl-phosphatidylcholines, plasmanylcholines, plasmenylcholines and sphingomyelins may play roles to strengthen membrane integ-rity and protect against PM2.5-induced oxidative stress. The increase of sphingomyelins in the hippocampus and cerebellum may attempt to protect against PM2.5-induced neu-ron death and degeneration. The hippocampus and cerebellum were likely to have neuroprotective effects. On the other hand, decrease of some di-acyl-phosphatidylcholines as well as increase of some lyso-phosphatidylcholines in the cerebral medulla, and decrease of saturated ether-linked phosphatidylcholines and sphingomyelins in the cerebral cortex indicated that membrane lipid perturbation may disrupt membrane raft integrity, regulate inflammatory responses, and decrease defense under PM2.5-induced stress. There were no significant changes of lipids in the olfactory bulb. Our result also indicated that PM2.5-induced lipid alteration was region-specific. However, although lipids are abundant in the brain, the numbers of changed lipids are few after ambient PM2.5 exposure. We suggested it may be due to low concentration and sub-chronic exposure. In conclusion, our results demonstrated that sub-chronic exposure to relatively low levels of PM2.5 lead to the alteration of lipids in the brain tissue. Moreover, the changes of lipids in different brain regions may be associated with the susceptibility and impairment of brain regions to PM2.5. This study supported that MS-based lip-idomic approach is a powerful platform to examine the brain lipid perturbation by am-bient PM2.5, and also able to link the changes with possible adverse effects and provide information for further study.