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Metabolomics Study of Aristolochic Acid Nephrotoxicity in Rodents
|Advisor:||曾宇鳳(Yufeng J Tseng)|
Aristolochic Acid,Aristolochia contorta,Madouling,Bu-Fei-A-Jiau-Tang,1H NMR Spectroscopy,Metabolomics,Principal Component Analysis,Nephrotoxicity,
|Publication Year :||2009|
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。
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.
|Appears in Collections:||生醫電子與資訊學研究所|
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