| 摘要: | 健大黴素(gentamicin)是一種可以有效治療革蘭氏陰性菌所引起之各種感染症的抗生素，但是在臨床上的應用往往受限於腎臟毒性。先前已有研究證實metformin對於gentamicin引起之腎臟損傷具有預防與保護的效果。本篇研究利用蛋白質體學的方法，比較健康的大鼠、以gentamicin誘導腎臟損傷的大鼠和投與metformin的大鼠腎臟組織之蛋白質表現量變化，希望能釐清gentamicin造成腎臟毒性以及metformin具有腎臟保護效果的詳細原因，以提升gentamicin在臨床上使用的安全性。
在連續經腹腔施打六天的gentamicin (150 mg/kg/day)的七天前先經口投與metformin (100 mg/kg/day)作為預防藥物，並持續投與至大鼠犧牲的當天；誘導腎損傷組則是經口給與蒸餾水並連續施打六天的gentamicin；健康組除了經口給與蒸餾水也會施打六天的生理食鹽水0.1mL作為對照。注射完之後將大鼠犧牲，接著取出腎臟組織均質化並且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(FD-HPLC)分析，最後用液相層析-串聯質譜(LC-MS/MS)定性表現量有顯著差異的蛋白質，並以MASCOT資料庫進行比對。
研究結果顯示健康對照組、誘導腎損傷組以及給與藥物預防組之間共有32個蛋白質的表現量有顯著差異(p＜0.05)。實驗中以質譜定性出來的蛋白質可依照功能分為下面幾類：抗氧化酵素、ATP合成酶、細胞凋亡相關蛋白、運輸蛋白、糖解酵素以及細胞骨架蛋白等。本實驗證實了metformin能夠恢復抗氧化酵素的活性，防止細胞失去合成ATP的能力而凋亡，也能調控葡萄糖代謝酵素的活性。
綜合本研究的結果，藉由分析各組間不同蛋白質表現量的變化，可以對gentamicin的腎毒性以及metformin的腎臟保護效果有更進一步的了解，對於未來開發更靈敏的腎臟損傷生物標記以及讓gentamicin更安全地被使用都將有莫大的幫助。
Gentamicin is an antibiotic which can effectively treat various infections caused by gram-negative bacteria. However, the clinical utility of gentamicin is limited by its nephrotoxicity. Previous study has shown that metformin has protective effects on gentamicin-induced renal injury. To understand the detailed mechanisms of metformin on gentamicin-induced nephritis, proteomic methods were used to identify the altered proteins between healthy control group, gentamicin-induced nephritis group and metformin prevention group.
Before injection of gentamicin for 6 days (i.p. 150 mg/kg/day on day 14 to day 20), Wistar Albino rats (7 week, male) were administered orally with metformin dissolved in ddwater for 7 days (100 mg/kg/day on day 7 to day 14) after 1 week accumulation. The homogenate of kidney cortex tissue was derivatized with 4-[2-(dimethylamino)ethyl aminosulfonyl]-7- chloro-2,1,3-benzoxadiazole (DAABD-Cl) and analyzed by Fluorogenic Derivatization-High-Performance Liquid Chromatography (FD-HPLC). Finally, altered proteins were identified by LC-MS/MS with MASCOT database searching system.
Results showed that 32 proteins had significant differences in expressions (p＜0.05) between healthy control, renal injury and metformin prevention group. The proteins identified by mass spectrum in this study could be separated into several categories: anti-oxidant, ATP synthesis, apoptotic, transporter, glycolysis and cell skeleton. This study proved that metformin could restore the activity of anti-oxidant enzyme, preventing cell apoptosis by keeping its ability to produce ATP, moreover, regulating activity of glucose-metabolic enzymes.
In conclusion, the results provided useful information about gentamicin nephrotoxicity and renal protective effect of metformin. It would be helpful in finding new biomarkers to improve early detection and ameliorate clinical safety of gentamicin. |
