- 學位論文
馬兜鈴酸腎毒性在囓齒類動物的代謝體學研究
Metabolomics Study of Aristolochic Acid Nephrotoxicity in Rodents
若您是本文的作者，可授權文章由華藝線上圖書館中協助推廣。
摘要
馬兜鈴酸具腎毒性，過去傳統中藥裏常使用的馬兜鈴酸屬的植物含有此物質。本實驗研究馬兜鈴酸標準品、馬兜鈴草果實及含馬兜鈴草的中藥複方補肺阿膠湯3種物質在囓齒類動物上的腎毒性，並以代謝體學方法分析尿液核磁共振氫譜在馬兜鈴酸腎病變中的變化。實驗目的在初步探討如何以容易取得之尿液來建立腎毒性之診斷模式。實驗共分4部份進行，一為大鼠以胃灌食馬兜鈴酸標準品測試。二為小鼠以胃灌食馬兜鈴酸標準品測試。三為小鼠以胃灌食馬兜鈴草果粉末測試。四為小鼠以胃灌食補肺阿膠湯粉末測試。實驗收集每日尿液樣本並冷凍保存以備分析，實驗結束後犧牲鼠隻取其肝腎做病理分析。 結果：大鼠馬兜鈴酸標準品給藥5劑後腎病理檢查顯示高劑量組有1-3度的急性腎小管間質病變。小鼠馬兜鈴酸標準品給藥10劑後，高低劑量組皆有3-4度的急性腎小管間質病變。小鼠馬兜鈴草給藥21劑後，低及中劑量組有1-2度而高劑量組有3-4度之急性腎小管間質病變。小鼠補肺阿膠湯給藥20劑後，低劑量組有2度而高劑量組有3-4度之急性腎小管間質病變。尿液核磁共振氫譜以主成份分析法分析各實驗中各個組別在同一天之分數圖，結果發現大鼠馬兜鈴酸標準品給藥後2天3組皆能分群。小鼠馬兜鈴酸標準品給藥後8天高劑量組能與其他2組分群，10天時給藥的2組能與對照組分群。小鼠馬兜鈴草給藥後8天高劑量組能與其他3組分群，10天時高劑量與中劑量組能與對照及低劑量組分群。小鼠補肺阿膠湯各組則至給藥後16天仍未見分群。以t檢定比對給藥組與對照組尿液中內生代謝物隨時間相對濃度變化發現大鼠馬兜鈴酸標準品實驗早期時甘氨酸、琥珀酸、氧化三甲胺及2-酮戊二酸有下降的現象，而到晚期時糖類、尿囊素、肌酐、二甲基甘胺酸、2-酮戊二酸及氧化三甲胺有上升現象(p < 0.05)。小鼠馬兜鈴酸酸標準品實驗之代謝物濃度有明顯變化者相對較少，但在實驗後期可見給藥組有丙氨酸、乳酸及甲酸平均濃度相對增加之現象。 結論：馬兜鈴酸標準品及含有此物質的中草藥馬兜鈴和其複方補肺阿膠湯在鼠類是有腎毒性的。以主成份分析尿液的核磁共振氫譜可以分辨出有發生腎病變的組別，其代謝體的濃度變化也可進一步分析找出相關的生物標誌。但在含低劑量馬兜鈴酸的複方補肺阿膠湯在本實驗中則未能以主成份分析法從尿液分析出小鼠發生的腎病變，這有待進一步的代謝體學分析研究。
並列摘要
Aristolochic acid (AA) is a potent nephrotoxic agent that can be found in several herbs of the genus Aristolochia. It was once commonly used in traditional Chinese medicine remedy. In this study, we applied our metabolomics platform of the 1H NMR spectroscopy on urine with principal component analysis (PCA) to detect and further dissect the nephrotoxicity in rodent of four AA containing materials, AA standard, Madouling, an AA containing herb Aristolochia contorta and Bu-Fei-A-Jiau-Tang (BFAJT), a herb compound containing Aristolochia contorta. The aim was to use the easy available urine samples to establish a model for early diagnosis of nephrotoxicity. The experiment was divided into four parts. The first was rat AA experiment. The second was mouse AA experiment. The third was mouse Madouling experiment. The fourth was mouse BFAJT experiment. Urine samples were collected daily and freeze-dried for storage. All animals were euthanized after experiment and their kidneys and livers procured for pathological studies. Results: In the rat AA experiment, pathology showed grade (gr.) 1-3 acute renal tubulointerstitial necrotic change (ATIN) after 5 doses in the high dose group. In the mouse AA experiment, 10 doses were given. Both high and low dose group showed gr. 3-4 ATIN. In the mouse Madouling experiment, 21 doses were given. It was gr. 1-2 for low and moderate groups, and gr. 3-4 ATIN for the high dose group. In the BFAJT experiment, 20 doses were given. It was gr. 2 ATIN in the low dose group and gr. 3-4 in the high dose group. PCA scoring plots for the urine NMR spectral chemical shifts variables showed early cluster at 2 day and later for each group in the rat AA experiment. In the mouse AA experiment, the high dose group was clustered from the other 2 groups at day 8. It was at day 10 that the 2 dose groups were clustered from the control group. In the mouse Madouling experiment, the high dose group clustered from all other 3 groups. It was at day 10 that the high dose and the moderate dose groups clustered together from the other 2 groups. In the BFAJT experiment, PCA scoring plots failed to classify across control and dose groups even at day 16. The endogenous metabolites assigned from NMR spectroscopy and their integral concentration among dose and control groups were tested by t-test. In the rat AA experiment glycine, succinate, 2-oxoglutarate and trimethylamine-N-oxide (TMAO) were decreased in the dose groups in earlier days of the experiment. At later days, sugar, allantoin, creatine, dimethylglycine, 2-oxoglutarate and TMAO were increased in the dose groups (p < 0.05). In the mouse AA experiment no significant metabolite difference was noted, only alanine, lactate and formate had more than two fold change at day 10. In conclusion, AA standard, Madouling and BFAJT were all nephrotoxic. PCA scoring plots for the urine NMR spectroscopy collected from living animals showed the ability to classify the dose and control groups days before confirming the renal pathology. But toxicity classifying failed in the mouse BFAJT experiment by PCA scoring plot. Further metabolomics study is expected to resolve this issue.
參考文獻
1. Zhang X, Wu H, Liao P, Li X, Ni J, Pei F: NMR-based metabonomic study on the subacute toxicity of aristolochic acid in rats. Food Chem Toxicol 2006, 44:1006-1014.
2. Sieber M, Hoffmann D, Adler M, Vaidya VS, Clement M, Bonventre JV, Zidek N, Rached E, Amberg A, Callanan JJ, et al: Comparative Analysis of Novel Noninvasive Renal Biomarkers and Metabonomic Changes in a Rat Model of Gentamicin Nephrotoxicity. Toxicol Sci 2009, 109:336-349.
3. Li X, Wang H: Aristolochic acid nephropathy: what we know and what we have to do. Nephrology (Carlton) 2004, 9:109-111.
4. Debelle FD, Vanherweghem J-L, Nortier JL: Aristolochic acid nephropathy: A worldwide problem. Kidney Int 2008, 74:158-169.
5. Feigin VL: Herbal medicine in stroke: does it have a future? Stroke 2007, 38:1734-1736.