NMR-based metabonomic approach on the toxicological effects of a Cimicifuga triterpenoid

Cimicifugae Rhizoma, a well‐known botanical dietary supplement, has been the subject of intense interest due to its potential application for alleviating menopausal symptom. Although there are clinic data that the Cimicifuga extract should have hepatotoxicity, no evidence on the main chemical components has been reported. Cimicidol‐3‐O‐β ‐d‐ xyloside (CX) is one of the main triterpenoids of the rhizome. This work studies the toxicological effects of CX after oral administration (50 mg kg^?1 per day) over a 7‐day period in female SD rats using metabonomic analyses of ¹H NMR spectra of urine, serum and liver tissue extracts. Histopathological studies of liver and analyses of blood biochemical parameter, such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, blood urea nitrogen and creatinine revealed that CX had no negative impacts on liver and kidney. However, the metabolic signature of ¹H NMR‐based urinalysis of daily samples displayed an increment in the levels of taurine, trimethylamine‐N‐oxide (TMAO), betaine and acetate. Elevated serum levels of creatinine, glucose, alanine, TMAO and betaine and lower levels of lactate were observed. Metabolic profiling on aqueous soluble extracts of liver showed simultaneously increases in succinate, glycogen, choline, glycerophosphorylcholine, TMAO and betaine levels and reduction in valine, glucose and lactate levels. Nevertheless, no changes in any metabonomic level were found in lipid‐soluble extracts of liver. These findings indicate that CX has a slight toxicity in liver and kidney via disturbance of the metabolisms of energy and amino acids. The present study provides a reasonable explanation for the clinical hepatotoxicity of Cimicifuga extract. Copyright © 2011 John Wiley & Sons, Ltd.     

基于1H-NMR的代谢组学方法对醋制甘遂解毒作用的研究

甘遂为有毒中药,临床上常经醋制后入药以降低其毒性。该文采用代谢组学方法比较生甘遂和醋甘遂对正常大鼠的损伤情况,研究醋制对甘遂毒性的缓解作用。连续7 d以生甘遂（EK）、醋甘遂（VEK）的水提取物对大鼠灌胃给药,药量均为9 g·kg^-1·d^-1（按生药量计）,对照组给予生理盐水。停止灌胃后继续观察7 d,收集14 d白天尿液,用于¹H-NMR检测;于第8天将每组大鼠处死一半,第15天处死另一半,采集肝脏,一部分用于¹H-NMR检测,另一部分用于病理学切片检测。病理学切片检测结果显示在本实验剂量下甘遂及醋制甘遂对大鼠肝脏没有造成损伤,但代谢组学方法分析发现在实验的第2周甘遂造成大鼠肝、肾及消化系统内部内源性代谢产物谱的紊乱;同时发现醋甘遂的毒性要比生甘遂低得多,传统醋制可以降低甘遂的毒性。该研究显示代谢组学方法有助于评价甘遂的毒性及醋制对甘遂的解毒作用。     

清热降压胶囊干预SHR海马区组织代谢组学研究

选取30只SHR大鼠随机分为3组模型组,卡托普利组,清热降压胶囊组,wistar大鼠10只作为正常对照组,连续给药14 d后取海马组织,采用核磁共振（¹H-NMR）代谢组学方法探讨高血压大鼠的海马组织损伤以及清热降压胶囊的保护作用。研究结果发现卡托普利组海马区代谢轮廓明显异于其余3组,另3组大鼠样本分类的趋势非常明显,提示清热降压胶囊能显著改善大鼠海马组织的代谢,鉴定4种代谢产物为二甲基甘氨酸、甘油磷酸胆碱、醛固酮和去甲肾上腺素。高血压大鼠机体代谢异常主要表现在酪氨酸代谢、血管平滑肌收缩以及醛固酮控制的钠盐再吸收失常,清热降压胶囊在有效降压的同时通过影响这些代谢产物来改善高血压的海马区损伤。     

Global metabolic profiling for the study of Rhizoma Paridis saponins‐induced hepatotoxicity in rats

Rhizoma Paridis saponins (RPS) is a traditional Chinese medicine (TCM) from the plant Paris polyphylla var. yunnanensis (Fr.) Hand.‐Mazz. Despite its potentially clinical utility such as anticancer and anti‐inflammation, it has slight side effects and toxicity as previous report. In this work, 90‐day administration of RPS induced liver injury. ¹H‐NMR‐ and GC/MS‐based metabonomic analyses in conjunction with histopathological examinations, blood biochemistry and hepatic phase I and II enzymes assays were performed to evaluate the toxic mechanisms of RPS induced in rats. As a result, oral administration of RPS possessed certain liver toxicity in SD rats. ¹H‐NMR and GC/MS data indicated that RPS inhibited the oxidation of fatty acids, glycolysis, and TCA cycle pathway, and disturbed glycine, serine, and threonine metabolism. Low expression of TG, T‐CHO, and LDL‐C and high levels of ALT and AST indicated that chronic exposure to RPS caused hepatocyte damage, synthesis dysfunction, and transportation failure of lipoproteins. In addition, RPS downregulated the mRNA levels of CYP1A2, CYP2E1, and UGTs. In conclusion, we used metabonomics approach to study the toxicity of RPS for the first time. This research demonstrated that metabonomics method was a promising tool to study and diagnose TCM‐induced toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 99–108, 2017.     

大鼠溃疡性结肠炎的代谢组学研究

目的应用代谢组学研究技术探讨大鼠溃疡性结肠炎（Ulcerative colitis,UC）相关的小分子标志物。方法 Wistar大鼠随机分为溃疡性结肠炎模型组（21只）和正常对照组（7只）,并采用2,4-二硝基氯苯（2,4-dinitrochlorobenzene,DNCB）复合乙酸法建立溃疡性结肠炎大鼠模型,收集正常对照组和模型组大鼠血浆,进行1H-NMR谱测定和主成分分析。结果与正常对照组大鼠相比,模型组大鼠血浆中亮氨酸、异亮氨酸、缬氨酸等支链氨基酸、苯丙氨酸、丙酮酸、β-羟丁酸、肌酸等小分子化合物含量明显降低,而VLDL含量升高,差异有统计学意义（P〈0.05）。结论 UC大鼠血浆的代谢谱明显偏离正常大鼠,亮氨酸、异亮氨酸、缬氨酸、苯丙氨酸、丙酮酸有可能作为溃疡性结肠炎早期诊断的分子标志物。     

抗结核药物利福平肝毒性时效关系的代谢组学

目的研究利福平(RFP)灌胃后不同时段大鼠尿液的代谢表型改变及其与组织病理学和血液生化指标的相关性。方法将36只雄性Wistar大鼠随机分为对照组、50和100mg/kgRFP3组,每组12只。每日灌胃给药1次,分别于给药第3、7、14天次日每组处死4只大鼠,采集血液标本和肝标本,进行血液生化指标测定和肝脏组织病理学检查。收集给药前24h及给药期间每天24小时的尿液,测定质子核磁共振(1H-NMR)谱,并运用主成分分析方法进行模式识别。结果在给药7d后,100mg/kgRFP组的血清总胆红素水平显著高于对照组(P<0.05)。肝脏组织病理显示RFP仅在较高剂量(100mg/kg)、较长给药时间(14d)时表现出轻度的肝毒性。各组大鼠尿液的代谢谱各不相同,随着给药时间的推移,大鼠尿液1HNMR谱发生一定改变。与对照组比较,给药组尿样1HNMR谱葡萄糖和牛磺酸显著增加,2-酮戊二酸和柠檬酸显著降低。结论尿液代谢谱的改变与常规毒性评价指标的改变相符合且更灵敏。大鼠尿液1H-NMR代谢轨迹与RFP作用时间密切相关,RFP引起的肝毒性与三羧酸循环中能量代谢异常及葡萄糖代谢紊乱有关。     

A comparative study of intratracheal and aerosolization instillations of bleomycin inducing experimental lung fibrosis in rat

We aimed to investigate in the present work, using metabonomics approaches, the scalability of lung fibrosis-biomarkers, in bleomycin (BLM) model of pulmonary fibrosis in rats. Sixty male Wistar rats, weighing 250?±?10?g, were randomly divided into three groups: a negative control group receiving normal saline treatment (G₁), an intratracheal BLM instilled group (G₂), and an aerosol BLM instilled group (G₃). Rats were investigated at various times after BLM instillation. Metabolic changes observed in different biofluids have been integrated into the results of the histological examination (increase in inflammation, fibrosis score, and TGF-β immunostaining) which provide a novel pathway of biomarkers in pulmonary fibrosis. These two BLM-models showed an efficacy in the production of pulmonary fibrosis in rats, accompanied by an oxidative stress in lung tissue as assessed by the increase of lipid peroxidation and the depletion in the level of antioxidant enzymes such as superoxide dismutase and catalase. The aerosol model was more advantageous showing fibrotic foci occupying the majority of the lung in contrast to intratracheal instillation characterized by a non-homogeneous distribution of the fibroblastic foci.     

Establishing 1H nuclear magnetic resonance based metabonomics fingerprinting profile for spinal cord injury: a pilot study

Background Spinal cord injury （SCI） is a complex trauma that consists of multiple pathological mechanisms involving cytotoxic, oxidation stress and immune-endocrine. This study aimed to establish plasma metabonomics fingerprinting atlas for SCI using 1H nuclear magnetic resonance （NMR） based metabonomics methodology and principal component analysis techniques.Methods Nine Sprague-Dawley （SD） male rats were randomly divided into SCI, normal and sham-operation control groups. Plasma samples were collected for 1H NMR spectroscopy 3 days after operation. The NMR data were analyzed using principal component analysis technique with Matlab software.Results Metabonomics analysis was able to distinguish the three groups （SCI, normal control, sham-operation）. The fingerprinting atlas indicated that, compared with those without SCI, the SCI group demonstrated the following characteristics with regard to second principal component： it is made up of fatty acids, myc-inositol, arginine, very low-density lipoprotein （VLDL）, low-density lipoprotein （LDL）, triglyceride （TG）, glucose, and 3-methyl-histamine.Conclusions The data indicated that SCI results in several significant changes in plasma metabolism early on and that a metabonomics approach based on 1H NMR spectroscopy can provide a metabolic profile comprising several metabolite classes and allow for relative quantification of such changes. The results also provided support for further development and application of metabonomics technologies for studying SCI and for the utilization of multivariate models for classifying the extent of trauma within an individual.     

Metabolic profiling of bile in cholangiocarcinoma using in vitro magnetic resonance spectroscopy

Objectives:

Cholangiocarcinoma (CCA) has a poor prognosis and its aetiology is inadequately understood. Magnetic resonance spectroscopy (MRS) of bile may provide insights into the pathogenesis of CCA and help identify novel diagnostic biomarkers. The aim of this study was to compare the chemical composition of bile from patients with CCA with that of bile from patients with benign biliary disease.

Methods:

Magnetic resonance spectra were acquired from the bile of five CCA patients and compared with MRS of control bile from patients with benign biliary disease (seven with gallstones, eight with sphincter of Oddi dysfunction [SOD], five with primary sclerosing cholangitis [PSC]). Metabolic profiles were compared using both univariate and multivariate pattern-recognition analysis.

Results:

Univariate analysis showed that levels of glycine-conjugated bile acids were significantly increased in patients with CCA, compared with the benign disease groups (P= 0.002). 7β primary bile acids were significantly increased (P= 0.030) and biliary phosphatidylcholine (PtC) levels were reduced (P= 0.010) in bile from patients with CCA compared with bile from gallstone patients. These compounds were also of primary importance in the multivariate analysis: the cohorts were differentiated by partial least squares discriminant analysis (PLS-DA).

Conclusions:

These preliminary data suggest that altered bile acid and PtC metabolism play an important role in CCA aetiopathogenesis and that specific metabolites may have potential as future biomarkers.     

Systems biochemical responses of rats to Kansui and vinegar-processed Kansui exposure by integrated metabonomics

Ethnopharmacological relevance

The dried root of Kansui (Euphorbia kansui L.) is an effective and commonly used traditional Chinese medicine. Even so, Kansui cannot be satisfactorily applied clinically because of toxic side effects. In China, the most common Kansui-processing method uses vinegar to reduce its toxicity. The present study was designed to investigate the toxic effects caused by Kansui and evaluate detoxification of Kansui by vinegar processing of Kansui.

Materials and method

Thirty male Sprague Dawley (SD) rats were randomly assigned to five groups of six rats. Two experimental groups were oral gavaged with 7.875 and 15.75 g Kansui/kg body weight, two treated with 7.875 and 15.75 g VP-Kansui/kg body weight for 14 d, and the control group concurrently subjected to oral gavage with only distilled water. On day 14, plasma, liver and kidney tissues were collected from all rats for biochemistry assessments, histopathological examination, and NMR analyses.

Results

The metabonome of rats treated with Kansui and vinegar-processed (VP-) Kansui was found to differ from that of controls. In liver extracts, the variational metabolites included elevated concentrations of isoleucine, leucine, valine, glutamate, and phenylalanine, with decreased taurine, glucose, and glycogen. However, changes in lysine, methionine, choline, phosphorylcholine, and tyrosine were only observed in Kansui-treated rats. In kidney extracts, prominent changes included elevations in isoleucine, leucine, valine, methionine, creatine/creatinine, and phenylalanine as well as decreased glutamine. Only Kansui treatment induced variations in alanine, lysine, acetate, choline, and phosphorylcholine.

Conclusion

Perturbations in endogenous metabolites induced by Kansui correlated with disturbances in glycolysis and amino acid and lipid metabolism, while biochemical pathway disorders caused by VP-Kansui only involved glycolysis and amino acid metabolism. All results were confirmed by histopathological examination of liver and kidney tissues and clinical biochemistry analyses.