Wen Yu-jie, Su Tao, Tang Jia-wei, Zhang Cui-ying, Wang Xuang, Cai Shao-qing, Li Xiao-mei
Renal Division, First Hospital, Peking University, Beijing, China.
Nephron Exp Nephrol. 2006;103(3):e95-e102. doi: 10.1159/000092194. Epub 2006 Mar 22.
BACKGROUND/AIMS: Aristolochic acid nephropathy, a progressive tubulointerstitial renal disease, is predominantly a result of aristolochic acid I (AA-I) intoxication. However, other unidentified phytotoxins have indeed been postulated as the cause of this unique interstitial nephropathy. The purpose of this study was to investigate the cytotoxicity of other phenanthrene derivatives extracted from Aristolochia contorta in the human proximal tubular epithelial cell line HK-2.
After HK-2 cells were incubated with an indicated concentration of test compounds for 24 h, cell viability was assessed by lactate dehydrogenase (LDH) leakage assay (cell membrane damage) in combination with MTT assay (metabolic capability). Cellular morphologic assessments were performed with a phase-contrast inverted microscope and transmission electron microscope.
In all test compounds at 5 microg/ml, AA-I, 7-methoxy-aristololactam IV and aristololactam IVa showed cytotoxic activity in HK-2 cells in both MTT assay and LDH leakage assay (p < 0.01). At high concentration (5-80 microg/ml), these three compounds caused a dose-dependent decrease in MTT reduction and a dose-dependent increase in LDH leakage compared to non-treated cells (p <0.01). In LDH leakage assay, 40 mug/ml 7-methoxy-aristololactam IV induced a 1.58-fold LDH leakage compared to AA-I at the same concentration (p < 0.01). Moreover, the IC50 of these three compounds were 16.675 microg/ml for AA-I, 4.535 microg/ml for 7-methoxy-aristololactam IV, and 30.244 microg/ml for aristololactam IVa in MTT assay. The cellular morphologic assessments suggest interactions with cell membrane and intracellular structures such as lysosome and mitochondria are likely to be involved in cell injury induced by these three compounds.
The potency of cytotoxic activity of aristololactam IVa and 7-methoxy-aristololactam IV extracted from A. contorta is similar to or even stronger than that of AA-I.
背景/目的：马兜铃酸肾病是一种进行性肾小管间质性肾病，主要是马兜铃酸I（AA-I）中毒的结果。然而，其他未明确的植物毒素确实被认为是这种独特的间质性肾病的病因。本研究的目的是调查从北马兜铃中提取的其他菲衍生物对人近端肾小管上皮细胞系HK-2的细胞毒性。
将HK-2细胞与指定浓度的受试化合物孵育24小时后，通过乳酸脱氢酶（LDH）泄漏试验（细胞膜损伤）结合MTT试验（代谢能力）评估细胞活力。用相差倒置显微镜和透射电子显微镜进行细胞形态学评估。
在所有受试化合物浓度为5微克/毫升时，AA-I、7-甲氧基马兜铃内酰胺IV和马兜铃内酰胺IVa在MTT试验和LDH泄漏试验中均对HK-2细胞显示出细胞毒性活性（p<0.01）。在高浓度（5-80微克/毫升）时，与未处理细胞相比，这三种化合物导致MTT还原呈剂量依赖性降低，LDH泄漏呈剂量依赖性增加（p<0.01）。在LDH泄漏试验中，40微克/毫升的7-甲氧基马兜铃内酰胺IV在相同浓度下比AA-I诱导的LDH泄漏高1.58倍（p<0.01）。此外，在MTT试验中，这三种化合物的半数抑制浓度（IC50）分别为：AA-I为16.675微克/毫升，7-甲氧基马兜铃内酰胺IV为4.535微克/毫升，马兜铃内酰胺IVa为30.244微克/毫升。细胞形态学评估表明，与细胞膜以及溶酶体和线粒体等细胞内结构的相互作用可能参与了这三种化合物诱导的细胞损伤。
从北马兜铃中提取的马兜铃内酰胺IVa和7-甲氧基马兜铃内酰胺IV的细胞毒性活性效力与AA-I相似，甚至更强。
