慢性暴露大氣細懸浮微粒後大鼠不同器官與血清的脂質變化

Abstract

細懸浮微粒又稱PM2.5，它進入人體呼吸道後能在肺泡穿越氣血屏障，進而抵達血液循環系統然後傳遞至其他器官。過去文獻指出PM2.5誘導的體內氧化壓力上升可能造成脂質的擾動，而含有磷酸膽鹼的脂質(phosphorylcholine-containing lipids)是人體構成細胞膜的最主要成分，在面對氧化攻擊時細胞膜是體內的第一道防線。本實驗室先前的研究使用脂質體學的方法探討在慢性暴露PM2.5後大鼠肺部含有磷酸膽鹼脂質的變化，發現磷脂醯膽鹼(phosphatidylcholines, PCs)的顯著改變可能與肺部表面活性劑功能的損傷有關。然而除了肺臟外，PM2.5對人體其他器官的脂質效應目前仍不清楚。因此，在本研究中我們利用脂質體學的方法透過大鼠長期吸入PM2.5的動物模型，去探討慢性暴露PM2.5後不同器官與血液的脂質效應。 十隻六週大的雄性SD大鼠，五隻全身持續性地暴露於未經濃縮的含有PM2.5的外來一般空氣，其他五隻則吸入通過懸浮微粒過濾器的空氣。整個實驗在臺北市臺大公衛大樓進行長達八個月，實驗結束後暴露組與控制組所測得的平均PM2.5濃度分別為16.7 ± 10.1 μg/m3與0.70 ± 0.46 μg/m3。隨後採集動物的血液樣本及其各式器官，包括心臟、肝臟、胰臟、腎臟、脾臟、睪丸及副睪丸，接著將脂質從每個器官組織及血清中萃取出來進行極致液相層析串聯式質譜儀(UPLC-MS/MS)分析，得到的圖譜經過數據前處理後，再利用偏最小平方判別分析(partial least squares discriminant analysis)搭配無母數統計方法Wilcoxon rank sum tests去檢驗暴露組與控制組間的脂質變異。 本研究結果指出慢性暴露PM2.5確實會在肺以外的器官造成脂質擾動的情形。偏最小平方判別分析顯示含磷酸膽鹼脂質在大鼠睪丸、胰臟、心臟、肝臟及腎臟的兩組別中有顯著差異，而無母數統計分析顯示在大鼠睪丸中發現最大量的含磷酸膽鹼脂質變化，包括多種磷脂醯膽鹼(lyso-PCs, diacyl-PCs, ether-linked PCs)及神經磷脂(sphingomyelins)，此改變推測可能與維持精子細胞膜完整性、抗氧化、抗發炎及輕微生精功能障礙有關。此外，在血液中的脂質調查發現與睪丸有一致的特定脂質PC(16:0/18:1)下降趨勢，但其是否為PM2.5毒性的潛在生物指標物需要更進一步的研究證實。總結，脂質體學是一種有效不偏頗且靈敏的方法去探究在PM2.5造成嚴重損傷前體內的分子變化，同時幫助於潛在生物指標物的開發。

Fine particulate matter (PM2.5) is able to pass the respiratory barrier and further enter the circulatory system, and consequently spread to the whole body. PM2.5-induced toxicity has been correlated with oxidative stress, which may lead to lipid perturbation. Our previous studies have applied a lipidomic platform to investigate the chronic effects of PM2.5 exposure on the pulmonary lipids in rats inhaled ambient air, and found that significantly altered levels of phosphorylcholine-containing lipids, which might impair pulmonary surfactant functions. However, the effects of PM2.5 on phosphorylcholine-containing lipids in other organs have not been fully elucidated yet. In this study, we examined the lipid effects of chronic PM2.5 exposure on various organs and serum using a rat inhalation model. Five male Sprague-Dawley rats were continually whole-body exposed to non-filtered and non-concentrated ambient air containing PM2.5 from the outside of the Public Health building in Taipei city for 8 months, while five rats were inhaled filtered air. The mean concentrations of PM2.5 in the exposure and control group were 16.7 ± 10.1 μg/m3 and 0.70 ± 0.46 μg/m3, respectively. Blood samples and various tissues, including heart, liver, kidney, pancreas, spleen, testis and epididymis were collected. Then lipids from each organ and serum were extracted for further ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) analysis. Subsequently, the partial least squares discriminant analysis (PLS-DA) and Wilcoxon rank sum tests were used to examine the variations of phosphorylcholine‐containing lipids among samples. Our results demonstrated that after the chronic and low-dose PM2.5 exposure, the lipidome were significantly different in the certain organs. In the PLS-DA models, the patterns of phosphorylcholine-containing lipids were altered in the testis, pancreas, heart, liver and kidney of rats exposed to PM2.5. After statistical analyses, most of significantly changed phosphorylcholine-containing lipids were discovered in the rat testis after chronic PM2.5 exposure. The changed lipids include decreased lyso-phosphatidylcholines (PCs), increased unsaturated diacyl-PCs, a decreased ether-linked PC and increased sphingomyelins, which may be related to maintain membrane integrity of spermatozoa, play anti-oxidants and anti-inflammatory roles, and dysfunction of spermatogenesis. Additionally, our results showed decreased PC(16:0/18:1) was both observed in the serum and testis. Further studies to verify potential biomarkers for PM2.5-induced toxicity are needed. We concluded lipidomics is a powerful, unbiased, and sensitive approach to study biological molecular effects in different organs after long-term and low concentration PM2.5 exposure. Our study suggested target organs of PM2.5 exposure and revealed the underlying possible mechanisms of PM-induced toxicity and potential biomarkers.