應用質譜儀為基礎的脂質體學探討萘對小鼠多重器官的劑量效應關係

Abstract

萘為環境中常見的空氣汙染物之一，主要的來源來自於不完全燃燒、交通排放或是含萘製品(如：萘丸)，過去有許多動物實驗探討萘的致毒機制，小鼠在經過不同劑量的萘暴露之後，其呼吸道上非纖毛細胞─克氏細胞(Clara cell)為主要的攻擊標靶，且細胞型態上的改變和暴露劑量的多寡有劑量效應性，在100 mg/kg的劑量暴露24小時後，看到克氏細胞膜上出現腫脹以及囊泡化現象，而200 mg/kg的暴露則出現細胞脫落和壞死，表示萘暴露可能會改變細胞膜的成分，脂質為細胞膜上最主要構成的物質，因此本研究利用脂質體學的研究方法，針對細胞膜中重要的脂質-磷脂醯膽鹼(Phosphatidylcholines)來探討不同劑量的萘對小鼠多重器官暴露造成的影響。 本研究動物實驗將小鼠分成三組，其中兩組分別經腹腔注射注入100、200 mg/kg萘，剩下一組只施打橄欖油載體做為實驗控制組，24小時後犧牲， 取下肺臟、肝臟以及腎臟樣本進行樣本前處理和脂質萃取，再利用極致液相層析儀搭配串聯式質譜儀(UPLC-MS/MS)進行後續脂質分析，得到的數據經過前處理後，再利用多變量分析─偏最小平方判別分析(Partial least squared discriminate analysis)以看出不同組別之間的脂質代謝物是否有明顯分群，另外利用無母數單變量分析─Kruskal-Wallis以及Dunnett’s test的事後檢定方法，可以找出主要改變的特定脂質，方便建立完整的代謝途徑擾動。 本研究發現不同萘劑量暴露會造成脂質體顯著差異。在萘的標靶器官–肺臟中，偏最小平方判別分析結果顯示低劑量組與高劑量組的脂質體分群是不同方向，在低劑量時，多種磷脂醯膽鹼(如:diacyl-phosphatidylcholines, plasmanylcholines和plasmenylcholines)有上升趨勢，推測可以增加細胞膜流動性，並產生抗氧化劑應付萘在體內代謝出的氧化壓力(oxidative stress)，在高劑量萘暴露下，這些磷脂醯膽鹼則出現下降趨勢。我們推測小鼠肺臟暴露在低劑量萘時，生物會採取保護性修復機制，然而在高劑量暴露下，克氏細胞可能無法逆轉萘產生的壓力以至於發現有明顯細胞膜破裂。另外，在 肝臟及腎臟中，低劑量萘暴露下，無法觀察到有脂質擾動，但高劑量萘暴露下，卻可以看到磷脂醯膽鹼有顯著改變，過去文獻中並未看到肝臟和腎臟經過萘暴露之後，有出現明顯的型態學上的改變，而利用脂質體學的方法，可以觀察到脂質體的確有出現變化。 本研究顯示在不同劑量的萘暴露下，小鼠不同器官的磷脂醯膽鹼會呈現不同改變趨勢，研究結果可以透過比較肺臟與肝臟、腎臟之間的差異，找到有潛力的脂質，來進一步解釋萘暴露後造成不同傷害的原因。除此之外，利用脂質體學研究法，可以進一步應用在疾病預防、藥物開發和公共衛生上。

Naphthalene, the most common polycyclic aromatic hydrocarbons, is widely spread in the environment. Previous studies have demonstrated that naphthalene caused dose-dependent damage to nonciliated bronchiolar cell, Clara cell, and alteration of lipidome in mice. After 100 mg/kg naphthalene treatment, the histopathological results showed that cell membrane was swollen and vacuolated; after 200 naphthalene treatment, the cell membrane was exfoliated. This result indicated naphthalene-induced oxidative stress could change the component of cell membrane. In this study, main lipid components of cell membrane, phosphorylcholine-containing lipids including phosphatidylcholine and sphingomyelin were profiled in various organs of mice treated with naphthalene. In this study, seven-week male ICR mice were treated with naphthalene (0, 100 or 200 mg/kg, ip.). After 24h, the liver, lung and kidney were collected and extracted. Phosphorylcholine-containing lipids were analyzed by using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) following partial least squares discriminant analysis (PLS-DA). After PLS-DA, the scores plots provide similarity of lipidome among groups. Additionally, statistical analysis, Kruskal-Wallis test and Dunnett’s test, were used to compare variance of selected lipid species between two groups. Our results demonstrated that the lipidome after the naphthalene treatment were significantly different. In the lung, which is a naphthalene target organ, PLS-DA model showed that control group could be clearly separated from the low and high dose groups. Moreover, the lipid responses of the low-dose and the high-dose groups towards different direction. After low-dose naphthalene treatment, up-regulated diacyl-phosphatidylcholines, plasmanylcholines, and plasmenylcholines were observed which might strengthen the fluidity of membrane and be used as antioxidants to alleviate naphthalene-induced oxidative stress. On the other hand, high-dose naphthalene treatment caused down-regulated of those phosphatidylcholines which might indicate that cell fail to alleviate the higher naphthalene-induced assaults. Besides, dose-respond trends of phosphorylcholine-containing lipids were found in the PLS-DA models of liver and kidney results. We conclude that MS-based lipidomic approaches are an effective and powerful tool to understand changes of metabolic pathways and their possible biological impacts after toxicant exposure. Furthermore, the platform can be applied to disease prevention, drug development, and public health.