基于1H-核磁共振代谢组学研究黄药子乙醇提取物致肝损伤的潜在生物标志物

Metabonomic study on early biomarkers of hepatic injury induced by ethanolic extract from Rhizoma Dioscoreae Bulbiferae in rats based on ~1H-NMR

目的采用基于1H-核磁共振（NMR）的代谢组学方法,分析黄药子乙醇提取物（RDB）致肝损伤大鼠血清和尿液中代谢产物随时间的动态变化,探寻RDB肝毒性早期的潜在生物标志物。方法大鼠ig给予RDB5 g·kg-1,连续给药28 d,于给药后3,7,14和28 d及停药后7 d（恢复期）取6只大鼠采血,进行常规血液生化分析;取肝组织,计算肝指数,并用HE染色进行肝组织病理观察。收集给药后0,3,7,14和28 d及停药后7 d血清和尿液,进行1H-NMR的代谢组学研究,采用主成分分析和正交偏最小二乘法分析对早期潜在生物标志物进行筛选和鉴别。结果与正常对照组相比,RDB组血清总胆红素（TB）和总胆固醇（TC）于给药后3~28 d显著升高（P〈0.05）,总胆汁酸（TBA）于7~28 d显著升高（P〈0.05）,TB,TC和TBA于停药后7 d恢复正常,谷丙转氨酶和谷草转氨酶活性以及葡萄糖含量均无显著变化。RDB组给药后肝指数显著升高（P〈0.01）,停药后7 d恢复正常;肝组织于给药后7 d开始出现单细胞坏死和肝细胞肿大,随给药时间延长,病变程度加重;停药7 d后有所恢复。代谢组学检测显示,RDB给药早期血清脂质〔低密度脂蛋白（LDL）/极低密度脂蛋白（VLDL）〕、谷氨酸、磷酸胆碱和甘油磷酸胆碱等升高（P〈0.05）,尿液丙酮酸盐和N-乙酰谷氨酸等降低（P〈0.05）;上述代谢产物在停药7 d后均恢复正常。结论早期肝损伤与糖脂代谢、能量代谢和胆汁淤积有关。随着RDB给药的持续,产生了组织过氧化损伤。线粒体损伤导致体内能量代谢不足。血清脂质（LDL/VLDL）、谷氨酸、磷酸胆碱和甘油磷酸胆碱,及尿液丙酮酸盐和N-乙酰谷氨酸均可作为RDB致肝损伤的早期潜在生物标志物。

OBJECTIVE Dynamics of serum and urine metabolites in hepatic injury rats induced by ethanolic extract from Rhizoma Dioscoreae Bulbiferae（RDB）was investigated by1H-NMR-based metabonomic methods in order to discover early biomarkers of liver toxicity induced by RDB. METHODS Rats were ig adminisetred with RDB at a dose of 5 g·kg-1for 28 d. Rats were sacrificed 3,7,14 and 28 d after RDB administration,as well as after a recovery period,respectively. Blood was taken for routine biochemical analysis by an automatic biochemical analyzer. Liver/body mass indexes were calculated,and liver pathological changes were observed with hematoxylin-eosin staining. Urine samples were collected before and 3,7,14 and 28 d after RDB administration,respectively,as wel as after withdrawal. Metabonomic analysis was carried out for serum and urine samples. Principal component analysis and orthogonal partial least squares- discriminant analysis were used for screening and identifiying early biomarkers.RESULTS Compared with the control group,total bilirubin（TB）and total cholesterol（TC）values were increased in 3-28 d in RDB group（P〈0.05,P〈0.01）. Total bile acid（TBA）was elevated in 7-28 d（P〈0.05,P〈0.01）. TB,TC and TBA became normal after discontinuation with RDB. There was no significant difference between RBD- treated group and control group in the activity of glutamic- pyruvic transaminase and glutamic-oxaloacetic transaminase,and the content of glucose also was not different between the two groups. The ratio of liver/body mass was elevated at 3-28 d（P〈0.01）but returned to normal after withdraval of RDB. The enlargement and necrosis of hepatocytes were observed 7 d after RDB administration,and lesion degree was aggravated with the extension of RDB delivery time. Metabonomic analysis showed that the serum lipids（low density lipoprotein/very low density lipoprotein（LDL/VLDL）,glutamic acid,choline phosphate and glycerolphosphatecholine were increased in the early stage. Pyruvate and N- acetylglutamate were decreased in urine. These metabolites became normal 7 d after discontinuation with RDB. CONCLUSION The serum lipids（LDL/VLDL）,glutamic acid,glycerol phosphate choline,as well as urine pyruvic acid salt and N-acetyl glutamate may be used as the early biomarkers for liver toxicity induced by RDB.