右旋兒茶素作用於順氯氨鉑引發腎毒性小鼠之蛋白質體分析

Proteome Analysis of Cisplatin Nephrotoxicity in Mice with (+)-Catechin Treatment

巫蕙宜(Hui-Yi Wu)

指導教授 ： 李仁愛

臺北醫學大學/藥學院/藥學研究所/碩士(2011年)

摘要

順氯氨鉑(cis-diamminedichloroplatinum (II), CDDP)是目前廣泛應用於治療固體癌的化學治療藥物之一，然而在臨床上的應用常受限於其腎毒性。先前的研究已證實(+)-catechin對於CDDP造成的腎毒性具有保護的效果。本研究利用蛋白質體學的研究方法，比較以CDDP誘導腎毒性小鼠與服用抗氧化藥物(+)-catechin小鼠腎臟組織蛋白質表現差異，希望能釐清(+)-catechin對於腎臟的保護機轉，也期望從中找到臨床上可使用的生物標記來早期診斷腎損傷的發生。
在施打CDDP前5天開始經口投予(+)-catechin作為預防藥物，持續投藥直至動物犧牲前，對照組經口投予蒸餾水 0.1 ml/mice。在CDDP 5 mg/kg/d連續3天及5天腹腔投予BALB/c小鼠後，將小鼠予以犧牲。取出小鼠腎臟將之均質後，加入螢光衍生化試劑4-[2-(dimethylamino)ethylaminosulfonyl]-7- chloro-2,1,3-benzoxadiazole (DAABD-Cl)，利用螢光高效能液相層析法分析。之後，利用LC-MS/MS定性表現量產生變化的蛋白質，並以MASCOT資料庫比對蛋白質。
實驗結果顯示，連續給予CDDP 3天的小鼠相較於給予預防藥物的小鼠之腎臟，共有44個蛋白質表現量有顯著差異(P < 0.05)；連續給予CDDP 5天的小鼠相較於給予預防藥物的小鼠之腎臟，則有9個蛋白質表現量具有顯著差異(P < 0.05)。實驗中定性之蛋白質可依功能分為數個種類，包括：運輸蛋白、抗氧化酵素、 ATP 合成相關蛋白、抗發炎蛋白 、細胞凋亡相關蛋白、細胞骨架及 DNA結合蛋白等。
本研究證實(+)-catechin可藉由增加體內抗氧化酵素活性，抑制發炎反應、抑制MAPK pathway等機轉，達到腎臟保護的效果。另一方面也發現，細胞在遭受外來壓力時，會啟動自我保護機制，減少細胞毒性物質進入細胞中(OCT2表現量下降)，並增加細胞毒性物質輸出量(ABC transporter表現量上升)以調適外來的傷害。
總結本研究的結果，可更加了解腎臟組織受損與修復之間蛋白質表現量的變化，對於日後CDDP腎毒性機轉之研究以及保護腎臟藥物之開發具有很大的意義。

關鍵字

蛋白質體學 ； 順氯氨鉑腎毒性 ； (+)-兒茶素 ； 螢光高效能液相層析 ； 液相層析-串聯質譜

並列摘要

Cisplatin (CDDP) has emerged as a principal chemotherapeutic agent in the treatment of various solid tumors. However, the full clinical utility of CDDP is limited due to its dose-related nephrotoxicity. In our previously study, (+)-catechin (CAT) acted as a protective agent against CDDP nephrotoxicity. To understand the protection mechanism of CAT in CDDP-induced nephritis, the aim of this study was to identify the altered proteins between CDDP-induced nephritis group and with CAT prevention group. Moreover, to find out some biomarkers to predict kidney injury effectively.
Before administration of CDDP (i.p. 5 mg/kg/d on day 1 to day 5), BALB/c mice (6 week, female) were administered orally with CAT 50 mg/kg once daily for 10 days (on day -5 to day 5, i.e. treat for 5 days) and 8 days (on day -5 to day 3, i.e. treat for 3 days). The homogenate of the kidney was derivatized with 4-[2-(dimethylamino)ethylaminosulfonyl]-7- chloro-2,1,3-benzoxadiazole (DAABD-Cl), and subjected to proteome analysis by Fluorogenic Derivatization-High-Performance Liquid Chromatography (FD-HPLC). Finally, the altered proteins were identified by LC-MS/MS with MASCOT database searching system.
The results showed that proteins changed significantly in both 3 days and 5 days samples. Between the CDDP and CAT tissues of the 3-day and 5-days treatment group, 44 proteins and 9 proteins had significantly difference in expressions (P < 0.05), respectively. The proteins found in this study can be separated into several categories: transporter, antioxidase, ATP-synthesis, anti-inflammatory, apoptotic, cell skeleton, DNA binding protein.
Our finding demonstrated that CAT can attenuate CDDP-induced nephrotoxicity by increasing antioxidase level, suppressing inflammation, and inhibition of MAPK signaling pathway. On the other hand, transporter associated with CDDP transport into cells was downregulated, and transporter associated with CDDP transport out of cells was upregulated, suggesting a defense response against subsequent exposure and renal uptake of CDDP.
In conclusion, this study may serve to find new biomarkers to improve early detection and new drug development in CDDP-induced nephrotoxicity.

並列關鍵字

proteomics ； cisplatin nephrotoxicity ； (+)-catechin ； ROS ； FD-HPLC ； LC-MS/MS

參考文獻

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