以FD-LC-MS/MS分析茶酚對小鼠腎間質纖維化模型之蛋白質體影響

Proteome analysis of the effect of (+)-Catechin toward tubular interstitial fibrosis by fluorogenic derivatization-liquid chromatography-tandem mass spectrometry method

黃國展(Kuo-Chan Hwang)

指導教授 ： 林美香

共同指導教授 ： 李仁愛(Jen-Ai Lee)

臺北醫學大學/藥學院/藥學研究所/碩士(2012年)

摘要

馬兜鈴酸腎病變 (aristolochic acid nephropathy, AAN) 是特指因誤用含有馬兜鈴酸的生藥而導致近曲小管有明顯細胞浸潤、腎小管萎縮，以及間質纖維化等情形，目前尚無有效的治療用藥。而本研究所用之治療藥物(+)- Catechin(CAT)，在過去研究顯示，其為強力的抗氧化劑，能夠有效地清除多種自由基，並有螯合過渡金屬離子、抑制脂質過氧化、抗突變、抗發炎等作用。本研究旨在以蛋白質體分析方法，比較腎臟組織在馬兜鈴酸腎病變後以及給予治療後表現量改變的蛋白質，以評估 CAT對AAN的改善效果。
實驗所用之組織，來源於前人已建立之小鼠腎病變模型；將組織均質後，利用螢光衍生化試劑 DAABD-Cl 進行衍生化反應， 再以螢光高效能液相層析儀 (FD-HPLC) 分離並定量，收集表現量有差異的蛋白質以液相層析串聯質譜儀 (LC-MS/MS) 與 Mascot 數據庫檢索系統定性。
在先前實驗結果顯示，誘導八週並投與CAT 50 mg/kg治療兩週後，CAT組別的血糖值、血中尿素氮、尿蛋白及尿中 N-acetyl-β-D-glucos- aminidase (NAG) 均有下降，而MMP-9呈現上升；組織鏡檢中，發現腎小管萎縮、間質細胞浸潤及纖維化等情形均較為改善。
本實驗中，延續先前顯示的生化值檢測結果，將投與CAT 50 mg/kg，與僅投與蒸餾水之對照組小鼠進行蛋白質體分析比較後，分別有30個蛋白質有顯著的差異 ( p < 0.05)，這些蛋白質經鑑定分別與抗氧化、細胞外基質蛋白質與其相關訊息路徑、發炎、細胞凋亡及 ATP 合成有關。本研究結果將有助於確立(+)-Catechin對馬兜鈴酸腎病變療效的發展，並了解(+)-Catechin於治療馬兜鈴酸腎病變的機轉中影響的路徑與環節，以利於後續研究的拓展以及臨床上相應產品的開發。

關鍵字

馬兜鈴酸腎病變 ； 蛋白質體學 ； (+)-Catechin ； DAABD-Cl ； 乙型轉型生長因子 ； 基質金屬蛋白分解酶甲型腫瘤壞死因子 ； 螢光衍生化-液相層析串聯質譜法

並列摘要

Aristolochic acid nephropathy (AAN), a unique type of nephropathy caused by mistaken the herb which contains aristolochic acid (AA), is a progressive interstitial nephritis which rapidly leading to end-stage renal disease. Features of AAN were characterized by extensive cell infiltration, tubular atrophy, and interstitial fibrosis. So far, there was no efficacy pharmacotherapy to relief or reverse this progression. In this research, we used (+)-Catechin (CAT), one of ingredients in tea, as our therapeutic drug; in past studies, there were many functions of CAT had been reported, such as antioxidation, anti-inflammation, anti-mutation, chelation with peroxidative metal ions and inhibition of lipid-peroxidation. Therefore, this study was designed to establish a chronic interstitial fibrosis model of AAN in inbred mice and investigated the efficacy of (+)-Catechin (CAT) on AAN by using a new method of proteomics to identify the differential expression of proteins in those two groups.
The pathologic kidney tissue which we used came from the model build by others before; after homogenation of those kidneys, we took the homo- genate of the kidney to derivatize with DAABD-Cl. The dirivatized proteins were separated and quantified by high performance liquid chromatography with fluorescence detection (FD-HPLC).The differential proteins were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with MASCOT database searching system.
The past results showed that, after 8 weeks of AA administration and treated with CAT 50 mg/kg, the CAT group were showed improvements of renal injury bydecreasing blood glucose, blood urea nitrogen (BUN), urine protein and N-acetyl-β-D-glucosaminidase (NAG). In addition, decreased amount of tubular atrophy, interstitial infiltration and fibrosis were found in histopathology.
In our results, the significantly altered expression between the normal and AA group now had 30 proteins. Those proteins were identified as anti-oxidant, extracellular matrix related, inflammatory, apoptotic and in ATP synthesis. These results can be used as proof for experiment before.
The conclusion of our study is that AAN mice treated with CAT 50 mg/kg had improved the renal function, and maybe the results can assist us to clarify which is the role (+)-Catechin played in AAN mechanism, and then, be benefit to research foe clinical therapy in the future.

並列關鍵字

Aristolochic acid nephropathy (AAN) ； proteomics ； fluorogenic derivatization-liquid chromatography-tandem mass spectrometry ； DAABD-Cl ； TGF-β ； MMP-9 ； TNF-α

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