气相色谱-质谱联用结合代谢组学方法研究不同极化状态下小胶质细胞的代谢差异

目的 运用代谢组学方法阐明经典激活型(M1型)、选择活化型(M2型)和静息态小胶质细胞的代谢差异。方法 将体外培养小鼠小胶质细胞系(BV2)细胞,分为M1组、M2组和静息态组,用实时荧光定量聚合酶链反应(qRT-PCR)检测特异性mRNA的表达差异以确定细胞极化状态,采用基于气相色谱-质谱联用(GC-MS)技术的代谢组学方法阐明代谢变化。结果 发现M1型与静息态细胞的差异代谢物15个,M2型与静息态细胞的差异代谢物15个。结论 通过代谢组学方法可以找到小胶质细胞极化的差异代谢物,并解释其可能的极化机制,为神经退行性疾病的防治提供了理论依据。     

肾虚证大鼠尿液的核磁共振谱代谢组学研究

目的：应用1HNMR结合主成分分析(PCA)方法研究氢化可的松诱导的肾虚证大鼠和空白对照大鼠的尿液成分谱差异。方法：使用氢化可的松诱导大鼠建立肾虚证模型,1HNMR分析对照组和模型组大鼠的尿液,使用PCA方法进行模式识别,寻找标记物并做出相关代谢途径的可能解释。结果：PCA方法处理肾虚证和空白对照大鼠尿液数据显示,两组大鼠的数据可以在得分图实现分类,与对照组比较肾虚组大鼠由于氢化可的松诱导,大鼠机体进入一个过消耗后衰弱的状态,相关代谢发生显著变化,乳酸(δ 1.37)代谢发生堆积;二甲胺(δ 2.72)的含量增加,提示肾虚与肾功能异常是密切相关的;天冬氨酸(δ 2.83),牛磺酸(δ 3.44,3.28),马尿酸(δ 7.84,7.56,7.64,3.97),肌氨酸(δ 3.87)等的相对积分面积明显下降,这预示糖皮质激素可能引起了肾上腺皮质分泌功能的损害;琥珀酸(δ 2.41)和柠檬酸(δ 2.53,2.68)是三羧酸循环能量代谢及糖酵解的中间产物,其含量的降低,通常是由于线粒体功能紊乱所引起。结论：1HNMR结合PCA模式识别的代谢组学方法具有研究复杂条件下机体病理生理变化的优势,本研究为理解中医的肾虚证和为该类疾病临床诊治的研究提供了证据。     

基于1H-NMR的当归四逆汤抗凝血作用的代谢组学研究

采用¹H-NMR代谢组学技术研究当归四逆汤抗凝血作用机制以及对小鼠血浆内源性代谢产物的影响。当归四逆汤灌胃给药1周后,观察当归四逆汤对小鼠凝血酶时间的影响,并采用¹H-NMR技术分析小鼠血浆代谢指纹图谱,采用主成分分析(principal component analysis,PCA)和正交偏最小二乘法判别分析(orthogonal partial least squares-discrimination analysis,OPLS-DA),研究给药组与正常组之间的代谢物组差异,筛选与抗凝血作用相关的潜在生物标志物。连续当归四逆汤灌胃给药1周后,能显著延长小鼠凝血酶时间(P<0.05)。代谢组学结果显示空白组和复方组能明显区分,与空白组比较,复方组内源性代谢产物肉碱、苯丙氨酸和异丁酸显著上调(P<0.01),赖氨酸、组氨酸和胆固醇显著下调(P<0.05)。肉碱、苯丙氨酸、组氨酸和胆固醇为当归四逆汤抗凝过程中潜在代谢标志物,当归四逆汤可能通过调节脂质代谢、能量代谢和氨基酸代谢影响血小板聚集功能和组织因子、纤维蛋白酶的表达来发挥抗凝作用。     

Comparison of the specificity and sensitivity of traditional methods for assessment of nephrotoxicity in the rat with metabonomic and proteomic methodologies

There is currently a great deal of scientific interest and debate concerning the possible advantages that proteomic and metabonomic technologies might have over traditional biomarkers of toxicity (blood and urine chemistry, histopathology). Numerous papers have been published that make impressive claims concerning potential applications for these novel technologies, however there appears to be little hard evidence in the literature of their advantages over the traditional techniques for assessing toxicity. The aim of this review was to evaluate the relative sensitivity and specificity of proteomic and metabonomic techniques, compared with traditional techniques, for assessing xenobiotic-induced nephrotoxicity. A review of studies was performed where both one of the novel methods as well as traditional techniques were used for assessment of xenobiotic-induced nephrotoxicity. There was no consistent evidence from the literature that the novel methodologies were any more sensitive than the traditional methods for assessing nephrotoxicity. This could be due to the relatively small number of studies available for review (n = 13), the fact that generally these studies were not aimed at determining relative sensitivity or specificity and may not be the case with other target organs, such as the liver. However, it was clear that the novel methodologies were able to discriminate between the effects caused by different toxicants. There was evidence both that this discrimination was on the basis of different mechanisms of toxicity and on the basis of different locations of nephrotoxic lesion. A great deal of validation work is necessary before these techniques could gain full acceptance by regulatory authorities, and it is unclear whether their use in anything other than non-regulatory, mechanistic studies is likely to become widespread.     

(1) H-nuclear magnetic resonance-based metabonomic analysis of brain in rhesus monkeys with morphine treatment and withdrawal intervention

Comprehensive cerebral metabolites involved in morphine dependence have not been well explored. To gain a better understanding of morphine dependence and withdrawal therapy in a model highly related to humans, metabolic changes in brain hippocampus and prefrontal cortex (PFC) of rhesus monkeys were measured by ¹H‐nuclear magnetic resonance spectroscopy, coupled with partial least squares and orthogonal signal correction analysis. The results showed that concentrations of myoinositol (M‐Ins) and taurine were significantly reduced, whereas lactic acid was increased in hippocampus and PFC of morphine‐dependent monkeys. Phosphocholine and creatine increased in PFC but decreased in hippocampus after chronic treatment of morphine. Moreover, N‐acetyl aspartate (NAA), γ‐aminobutyric acid, glutamate, glutathione, methionine, and homocysteic acid also changed in these brain regions. These results suggest that chronic morphine exposure causes profound disturbances of neurotransmitters, membrane, and energy metabolism in the brain. Notably, morphine‐induced dysregulations in NAA, creatine, lactic acid, taurine, M‐Ins, and phosphocholine were clearly reversed after intervention with methadone or clonidine. Our study highlights the potential of metabolic profiling to enhance our understanding of metabolite alteration and neurobiological actions associated with morphine addiction and withdrawal therapy in primates. © 2012 Wiley Periodicals, Inc.     

Metabonomic analysis of fatty acids in seminal plasma between healthy and asthenozoospermic men based on gas chromatography mass spectrometry

The mechanism of asthenozoospermia remains unclear. The knowledge of the metabolism of fatty acids in seminal plasma is important and meaningful for the pathological study of asthenozoospermia. We present an optimised assay of extraction and derivatisation followed by GC/MS to analyse metabolites, especially fatty acids, in seminal plasma from healthy and asthenozoospermic men. Eighty‐nine peaks including 17 kinds of fatty acids were analysed and identified in the chromatogram. The GC/MS data were analysed using t test, fold change and partial least squares discriminant analysis to explore the potential biomarkers of asthenozoospermia. Seven metabolites in asthenozoospermic group were found to be significantly different from those in the normal group (with p < .05, fold change >1.2 and variable importance for projection >1). Of which, high levels of oleic acid and palmitic acid in seminal plasma from asthenozoospermic men may indicate a membrane metabolism disorder in spermatozoa and the lack of valine in the asthenozoospermic group may contribute to poor sperm motility. The results may facilitate the understanding of the role of fatty acids and amino acids in asthenozoospermia and provide solid foundation for further pathological study of asthenozoospermia.     

基于生物标志物探索系统性红斑狼疮中医药治疗机制的研究进展

系统性红斑狼疮（SLE）是一种多器官受累的自身免疫性疾病,目前基于糖皮质激素和免疫抑制剂的治疗,仍然存在很多局限性和个体差异。近年来,越来越多的研究表明,联合中医药治疗SLE具有疗效好,不良反应低和安全性高等优点。但是,中医药治疗SLE的确切调节机制和作用环节尚不明确,本文从代谢组学、免疫细胞、淋巴细胞因子和细胞凋亡等,对中医药治疗SLE机制的研究进行综述,为利用现代化方法探索祖国传统医学治疗SLE的机制研究提供思路。     

A Metabonomic Approach to a Unique Detoxification Effect of Co‐use of Euphorbia kansui and Zizyphus jujuba

Euphorbia kansui (EK) has been widely used in traditional Chinese medicine (TCM); however, it possesses toxic effects. The fruits of Zizyphus jujuba (ZJ) are frequently co‐used with EK to reduce EK's toxicity. The present study is to clarify the toxicity of water extract of EK and explore the detox effect of ZJ using ¹H NMR‐based metabonomic approach. The water extracts of ZJ, EK and the co‐use of EK and ZJ (CEZ) were orally administered to SD rats at designed doses for 1 week, respectively, and one more week observation was further conducted. Histopathological studies of liver samples from all groups showed no negative impacts. In metabonomic analyses of urines, ZJ showed no toxicity, while significant changes of metabolites indicating liver damages, kidney lesions and imbalance of gut microbes were clearly observed during the second week in EK‐treated rats. Very meaningfully, CEZ clearly indicated that the toxicities appeared at the first week and became weaker, and furthermore, was recovered during the second week. These results clearly demonstrated the rationality of traditional co‐use of EK together with ZJ, and the metabonomic approach should be a promising tool to research the toxicity of TCM. Copyright © 2012 John Wiley & Sons, Ltd.     

三七总皂苷对顺铂肾损害大鼠的保护作用

目的:应用血清代谢组学方法探讨三七总皂苷(PNS)对早期的顺铂肾损害大鼠的保护作用。方法:顺铂组大鼠单剂量腹腔注射顺铂,PNS干预组大鼠注射顺铂后再腹腔注射PNS,每天1次。24 h后,评价大鼠肾功能,苏木精-伊红染色(HE)观察肾脏组织病理学的改变,并使用核磁共振代谢组学技术进行血清代谢组学的检查。结果:PNS可降低血清肌酐(SCr),血尿素氮(BUN)的浓度和改善肾组织病理损伤。另外,血清标本的主成分分析(PCA),偏最小二乘法判别分析(PLSDA)和正交偏最小二乘法判别(OPLS-DA)分析显示,顺铂模型组,PNS干预组均与正常组有显著的区分,而顺铂模型组与PNS干预组之间有部分重叠。与正常组比较,顺铂模型组和PNS干预组的丙酮,n-乙酰糖蛋白信号,丙酮酸,脂质和极低密度脂蛋白(VLDL)表达降低,而葡萄糖、缬氨酸和酪氨酸的表达升高。而且,顺铂模型组的乳酸表达较PNS干预组高。另外,正常组和PNS干预组大鼠苯丙氨酸的表达较顺铂模型组高,而酪氨酸的表达较顺铂模型组低。差异代谢物涉及的代谢通路主要有戊糖磷酸途径、糖酵解/糖异生途径、丙酮酸途径等与能量代谢相关的通路。结论:PNS对顺铂肾毒性有保护作用,其保护机制可能和逆转顺铂肾损害引起的代谢紊乱尤其是能量代谢障碍有关。