基于快速高分辨液相色谱-质谱的中波紫外线辐射诱导大鼠急性光损伤的代谢组学研究

Urinary Metabonomic Investigation into Acute Photo Damage of Rats Induced by Ultraviolet B Irradiation using Rapid Resolution Liquid Chromatography-Mass Spectrometry

利用快速高分辨液相色谱-四极杆-飞行时间质谱（RRLC-Q-TOF-MS）联用技术结合多元统计分析方法,考察在中波紫外线（Ultraviolet B,UVB）辐射前后,大鼠尿液中内源性代谢物谱的变化,研究UVB辐射导致急性光损伤的生理机制。急性光损伤大鼠模型由窄谱中波紫外线光源（TL-01,峰值312 nm）照射,采用离心沉降后四倍稀释法处理尿液样本,Supelco Ascentis Express C18色谱柱,水（含0.1%甲酸）与乙腈为流动相梯度洗脱,液相色谱-串联质谱分析测定。利用主成分分析（PCA）法、聚类分析（CA）法等对辐射前后的大鼠尿液样本进行代谢轮廓分析,寻找对分组贡献大的差异代谢物及通路,并阐明其作用机制;运用偏最小二乘判别分析（PLS-DA）法建立预测模型,考察此模型在UVB致光损伤模型诊断上的预测能力。多元统计分析结果显示,空白对照组与UVB模型组能够获得很好地区分,通过将差异代谢物与数据库、串联质谱数据及标准品比对,发现并鉴定出11种潜在生物标记物,表明UVB辐射可影响正常大鼠的鞘脂类代谢、核酸代谢、亚油酸代谢、氨基酸代谢等通路,这些差异代谢物对UVB辐射致光损伤类疾病的诊断具有较好的预判能力。

A rapid resolution liquid chromatography quadruple time-of-flight mass spectrometric（ RRLC-QTOF / MS） method combined with multivariate statistical analysis was applied to investigate the changes of endogenous metabolites in murine urine before and after ultraviolet B（ UVB） irradiation for the purpose of discussing the physiological mechanism of acute injury caused by UVB radiation. A narrow-band UVB（ NBUVB）（ TL-01,peak value 312nm） was used to establish the acute light damage model. The urine samples were centrifuged before four times dilution treatment,subsequently the diluted urine samples were separated on a Supelco Ascentis Express C18 column using water（ 0.1% formic acid） and acetonitrile as mobile phase by gradient elution. The differences metabolites with major contribution for grouping were found out based on the metabolic profiling analysis of principal component analysis（ PCA） and cluster analysis（ CA）,which could illustrate their possible mechanism of actions by means of relevant pathways. A prediction model was built to investigate the forecasting ability of the acute photo damage induced by UVB irradiation through the partial least square discriminant analysis（ PLS-DA）. The results of multivariate statistical analysis showed that the blank control group was separated from UVB model group quite well,11 endogenous metabolites were identified as the potential biomarkers through comparison with the database,tandem mass spectrum data and standard substance,which indicated the UVB radiation may affect the sphingolipid metabolism,nucleic acid metabolism,linoleic acid metabolism and amino acid metabolism pathways. These different metabolites could be helpful for diagnosing the light damage induced by UVB radiation