五味子治疗大鼠糖尿病肾病作用机制的血清代谢组学研究

采用基于超高效液相色谱与串联四级杆飞行时间质谱仪(Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,UPLC/Q-TOF-MS)联用技术的代谢组学方法,通过分析大鼠血清内源性代谢物的变化,研究五味子治疗糖尿病肾病的作用机制。利用高脂高糖饲料喂养并腹腔注射链脲佐菌素(STZ)建立糖尿病大鼠模型。给药12周后,采用试剂盒方法测定尿蛋白、尿肌酐的含量,结果表明五味子水提取物可以显著降低模型动物的尿蛋白含量(p<0.05),对糖尿病大鼠肾病并发症具有一定的改善作用。采用UPLC/Q-TOF-MS方法分析了五味子对糖尿病肾病大鼠的血清代谢轮廓,分析了健康组、模型组和五味子给药组的大鼠血清,采用偏最小二乘法-判别分析(Partial least squares discriminant analysis,PLS-DA)进行数据分析。PLS-DA得分图显示健康组、模型组和五味子组的代谢轮廓有显著差别,根据正交偏最小二乘法-判别分析(Orthogonal partial least squares discriminant analysis,OPLS-DA)载荷图筛选,将对各组分离贡献大的化合物的串联质谱分析数据,经Human Metabolome Database(HMDB)等数据库检索,进行质谱信息匹配,鉴定出黄尿酸、油酰胺、棕榈酰胺、尿酸、5-羟基己酸、硫酸对甲酚、对甲酚葡萄糖苷酸7种内源性代谢物为生物标记物。研究结果表明五味子通过影响色氨酸代谢、嘌呤代谢、肠内菌代谢、脂肪酸代谢等通路对糖尿病肾病发挥治疗作用,其中嘌呤代谢、肠内菌代谢通路可能是五味子发挥治疗作用的重要途径。     

黄芩-栀子配伍治疗糖尿病肾病大鼠的尿液代谢组学研究

采用基于超高效液相色谱与串联四极杆飞行时间质谱仪(Ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,UHPLC-Q-TOF MS)联用的代谢组学方法,通过分析大鼠尿液内源性代谢物的变化,研究黄芩-栀子药对治疗糖尿病肾病的作用机制。利用高脂高糖饲料喂养并腹腔注射链脲佐菌素(STZ)建立糖尿病大鼠模型。给药12周后,采用UHPLC-Q-TOF MS对正常对照组、模型组、黄芩-栀子药对治疗组的尿液样品进行代谢轮廓分析。利用正交偏最小二乘判别分析(Orthogonal partial least squares discriminant analysis,OPLS-DA)对实验数据进行分析,挑选出对各组分分离贡献较大(VIP>1,p<0.05)的化合物在Human Metabolome Database(HMDB)等数据库进行质谱信息匹配,共鉴定出糖尿病肾病潜在生物标记物31个,给药后回调生物标记物16个。研究结果表明,黄芩-栀子药对通过影响胆汁酸的合成与代谢、尿毒素代谢、能量代谢、色氨酸代谢、苯丙氨酸代谢、酪氨酸代谢等通路对糖尿病肾病起到治疗作用,其中胆汁酸的合成与代谢、尿毒素代谢和能量代谢可能是黄芩-栀子药对发挥治疗作用的主要途径。与单味药相比,配伍后的药对具有了新的药理作用。     

基于代谢组学技术的辛伐他汀对磷脂类物质作用的初探

用超高效液相色谱和高分辨飞行时间质谱（UPLC／Q-TOF MS）联用技术,跟踪小鼠给予药物辛伐他汀前后血清中的内源性代谢物的变化,开展基于代谢组学技术的辛伐他汀对磷脂类物质作用的研究．研究结果显示：辛伐他汀药物对血清中磷脂类物质有较大影响,本文对其中6种具有显著性差异的标记物进行了结构确认,并对这些物质与降脂作用的关系进行了讨论．研究结果表明,将代谢组学技术应用于药物的作用效果研究,对新药研发和合理临床用药具有重要的指导意义．     

糖肾方对自发性Ⅱ型糖尿病鼠磷脂代谢的影响

利用正相液相色谱飞行时间质谱建立了大鼠血浆磷脂的轮廓谱。通过对磷脂轮廓谱的PLS-DA判别分析及14种化合物的定量,考察了中药糖肾方对自发性II型糖尿病鼠磷脂代谢的影响,并对比了糖肾方与西药蒙诺在糖尿病发展过程中的不同作用。结果表明：与正常对照组相比,糖尿病鼠的血浆磷脂代谢发生异常。糖肾方能够调节糖尿病鼠的磷脂代谢紊乱,蒙诺则加剧了病鼠的磷脂代谢异常。此外,8种磷脂化合物可能成为糖尿病发展过程中的潜在生物标志物。     

阿卡波糖对Ⅱ型糖尿病大鼠尿液代谢轮廓的影响

采用超高效液相色谱-质谱联用(UPLC-MS/MS)方法研究了阿卡波糖对Ⅱ型糖尿病大鼠代谢轮廓的影响, 分析了健康组、 糖尿病模型组和糖尿病给予阿卡波糖组的大鼠尿样, 采用主成分分析法(PCA)和偏最小二乘法-判别分析(PLS-DA)对数据进行分析. PCA得分图表明, 健康组、 糖尿病组和阿卡波糖组的代谢轮廓有显著差别, 根据PLS-DA载荷图筛选, 将对各组分离贡献大的化合物的串联质谱分析数据经Human Metabolome Database(HMDB)和Mass Bank.jp等数据库检索, 进行质谱信息匹配, 鉴定出苯乙酰甘氨酸、 肌酐及葡萄糖酸等8种内源性代谢物为潜在生物标记物.     

基于UPLC-Q/TOF MS技术探讨竹节参总皂苷对HepG2细胞脂质代谢的改善作用

基于细胞代谢组学技术研究竹节参总皂苷改善HepG2细胞脂质代谢的机制。应用棕榈酸和油酸诱导HepG2细胞脂质沉积模型，竹节参总皂苷干预24 h后进行油红O染色和甘油三酯分析。采用改良的Bligh-Dyer法将细胞内源性代谢物分为极性和非极性部位，运用超高效液相色谱-四极杆飞行时间质谱（UPLC-Q/TOF MS）技术检测代谢物，结合多元统计分析和火山图筛选差异代谢物，并富集代谢通路。结果显示，竹节参总皂苷可显著改善HepG2细胞脂质沉积模型的脂质代谢。从极性部位鉴定了34个差异代谢物，主要为脂肪酸和氨基酸类成分；从非极性部位鉴定了28个差异代谢物，主要为磷脂酰胆碱、磷脂酰乙醇胺、神经酰胺类成分。差异代谢物在竹节参总皂苷干预后均有恢复至正常水平的趋势。该研究提示竹节参总皂苷改善HepG2细胞脂质沉积模型的脂质代谢与调控鞘脂代谢、甘油磷脂代谢、脂肪酸代谢、炎症反应密切相关。     

基于修饰组和探针分子的重要途径代谢物筛选和注释新方法北大核心CSCD

植物次生代谢物在抵御生物/非生物胁迫、生物间互作以及信息传递等方面发挥重要作用,次生代谢途径解析对植物分子育种、天然产物合成等方面具有重要意义。液相色谱-高分辨串联质谱(LC-HRMS/MS)为次生代谢物鉴定及途径表征提供了技术手段。非靶向LC-HRMS/MS方法可获得丰富的质谱信号,包括一级质谱和二级质谱(MS,MS/MS),但受质谱数据库规模以及次生代谢物复杂性的制约,次生代谢物注释十分困难。该研究以玉米叶片中苯丙烷途径代谢物为例,发展用于非靶向代谢组数据中重要途径代谢物的高效筛选和注释新方法。首先,利用公共代谢途径数据库及文献获取参与苯丙烷代谢途径的61种修饰反应类型,进而从非靶向实验数据中筛选出修饰代谢组。其次,获取开源串联质谱数据中的苯丙烷类化合物作为探针分子,构建探针分子质谱数据库。将探针分子与修饰代谢组共建分子网络,锁定目标途径代谢物并注释结构。该方法在正、负离子模式下分别筛选出玉米叶片中392个和417个苯丙烷途径候选代谢物,去冗余后共注释出129个代谢物,涉及苯丙烷代谢的主要分支途径,如黄酮途径的8个类黄酮、19个氧苷类黄酮和32个碳苷类黄酮,31个羟基肉桂酸途径代谢物以及22个木脂素途径代谢物;其中26个在PubChem和SciFinder数据库中未见收录。该研究利用探针分子结合修饰组可快速锁定途径代谢物,且有助于快速、准确的网络传播注释,可显著提高目标途径代谢物筛选与注释效率,为植物次生代谢途径的深入解析提供分析手段。     

基于代谢组学研究橙黄决明素对高脂血症大鼠的调脂作用

本文运用代谢组学的研究方法,考察橙黄决明素对高脂血症大鼠血液内源性代谢物的影响,并寻找相关生物标志物。采用高脂饲料喂养的方法建立高脂血症大鼠模型,给予橙黄决明素灌胃治疗。利用气相色谱-质谱(GC-MS)联用技术分析高脂血症大鼠血浆中的代谢物,并使用主成分分析、偏最小二乘-判别分析和随机森林算法,研究大鼠样本在造模前后及橙黄决明素治疗后血浆内源性代谢物的变化情况,结果得到丙氨酸、甘氨酸、L-缬氨酸、异亮氨酸、富马酸、丝氨酸、1,5-脱水山梨醇、亚油酸、硬脂酸和胆固醇10种潜在的生物标志物。研究表明橙黄决明素主要通过影响机体氨基酸及脂肪酸的代谢,从而产生较理想的调脂作用。     

超高效液相色谱/飞行时间质谱用于人参皂甙Rg3作用后大鼠尿液代谢物指纹图谱分析及标记物的鉴定

超高效液相色谱技术(UPLC)采用1.7 μm的色谱柱填料,有更好的分离效率、峰容量以及灵敏度;高分辨的时间飞行质谱(TOF-MS)能够测定化合物准确的分子质量并具有MS/MS功能。 两者的联用适合于复杂体系的分离分析和未知物的结构鉴定。 因此建立了一种基于UPLC/TOF-MS测定人参皂甙Rg3给药后大鼠尿液代谢物变化的方法,对其中2种发生显著变化的代谢物分别通过准确的质量测定得到其元素组成,通过MS/MS技术得到其结构信息,并通过检索数据库最终分别鉴定为4,8-二羟喹啉甲酸和4-羟基-2-喹啉酸。     

基于气相气谱-质谱的代谢组学方法研究四氯化碳致小鼠急性肝损伤

建立了一种基于气相色谱-质谱(GC-MS)技术的代谢组学分析方法。以CCl4致小鼠肝损伤模型为研究对象,检测了染毒小鼠肝组织和血浆中内源性代谢物随时间的变化情况,并运用主成分分析方法对这一变化过程进行模式识别。研究发现,随着染毒时间的推移,小鼠的整体代谢物谱存在着一定的变化规律。肝组织中苹果酸和部分游离脂肪酸含量升高;血浆中柠檬酸和部分氨基酸含量有所上升。这些代谢物的变化都与肝损程度密切相关,且变化趋势与肝组织病理检查结果一致。研究结果表明:GC-MS技术在代谢物谱的获取和代谢物鉴定方面具有优势,可作为核磁共振(NMR)技术的补充工具用于代谢组学研究。     

Study of plasma metabolic profiling and biomarkers of chronic unpredictable mild stress rats based on gas chromatography/mass spectrometry

A metabolomic investigation of chronic unpredictable mild stress (CUMS) rats was carried out. Plasma obtained from Sprague-Dawley (SD) rats treated by CUMS was analyzed using gas chromatography/mass spectrometry. Thirty-seven metabolites were identified among the detected compounds. Subsequent data analysis using the t test and principal component analysis (PCA) revealed significant metabolic changes in the rats' plasma after CUMS treatment. Clear separation between the model and control group was achieved, and the level of twelve metabolites, including amino acids, sugar, organic acids and fatty acids, were significantly different between plasma samples from the controls and CUMS group. These observations suggested that the depressed state may be associated with perturbation of amino acid metabolism, energy metabolism and glycometabolism. The study suggested that the metabolomics approach could be used as a potential powerful tool to investigate the biochemical change in certain physiopathological conditions, such as depression, as an early diagnostic means.     

Integrated metabolomics analysis of the effect of PPARδ agonist GW501516 on catabolism of BCAAs and carboxylic acids in diabetic mice

The peroxisome proliferator-activated receptor(PPARδ) agonists are reported to improve insulin sensitivity,reduce glucose levels,and alleviate dysfunctional lipid metabolism in animal models of type 2 diabetes mellitus.However,the underlying mechanisms remain incompletely understood.Metabolism plays an essential role in the biological system.Monitoring of metabolic changes in response to disease conditions or drug treatment is critical for better understanding of the pathophysiological mechanisms.In this study,metabolic profiling analysis by gas chromatography-mass spectrometry integrated with targeted analysis by liquid chro matography-mass spectrometry was carried out in plasma samples of db/db diabetic mice after six-week treatment of PPARδ agonist GW501516.GW501516 treatment significantly altered levels of metabolites,such as branched-chain amino acids(BCAAs),BCAA metabolites(3-hydroxyisobutyric acid and 3-hydroxyisovaleric acid),long-chain fatty acids,uric acid and ketone bodies(3-hydroxybutyric acid and 2-hydroxybutyric acid) which are all associated with the impaired systemic insulin sensitivity.The pre sent results indicate the beneficial effect of PPARδ agonist in alleviating insulin resistance of diabetic mice by favorably modulating metabolic profile,thus providing valuable information in understanding the therapeutic potential of PPARδ agonists in correcting metabolic dysfunction in diabetes.     

基于液相色谱-质谱联用系统的系统性红斑狼疮患者血浆代谢组学分析

应用快速分离液相色谱-串联四极杆飞行时间质谱(RRLC-Q-TOF/MS)系统对系统性红斑狼疮(SLE)患者血浆样本进行代谢指纹图谱的分析。分别应用监督模式识别方法正交信号校正结合偏最小二乘法-判别分析(OSC-PLS-DA)对代谢组数据进行处理,结果显示SLE患者与健康人群对照组的代谢指纹存在明显差异;进一步从SLE患者血清代谢图谱中筛选出10个对分类有显著贡献的离子,定性鉴定出7种代谢标志物,发现SLE患者存在异常的氨基酸、磷脂和卟啉的代谢状态。本研究可为SLE的监测和诊断以及SLE发病的分子基础研究提供科学依据。     

基于液相色谱-质谱代谢组学方法研究中药定喘汤对呼吸道合胞病毒感染的疗效

呼吸道合胞病毒(RSV)容易引发下呼吸道感染,尤其是小儿毛细支气管炎.中药定喘汤用于RSV感染的治疗在中国有着广泛的临床实践基础.由于中药体系的复杂性,其治疗机制和主要药效成分还不够明确.采用代谢组学方法分析中药药效,可以为传统中药治疗提供现代科学论证.该研究对大鼠各干预组分别采用中药定喘汤全方,宣、降、清分解剂以及利...     

Metabonomic analysis of serum reveals antifatigue effects of Yi Guan Jian on fatigue mice using gas chromatography coupled to mass spectrometry

Yi Guan Jian (YGJ), one of the most commonly used traditional Chinese medicines, has been reported to possess significant antifatigue effects. However, the mechanisms underlying its antifatigue effects remain largely unresolved. In this study, a metabonomics approach, involving gas chromatography coupled to mass spectrometry and a multivariate statistical technique, was developed to estimate the extent to which YGJ alleviated the exhausting swimming‐induced fatigue of mice. High‐dose treatment with YGJ significantly extended the swimming time of fatigued mice. Significant alterations of metabolites involving amino acids, organic acids and carbohydrates were observed in the serum of fatigued mice, which were reversed by YGJ treatment while biochemical indexes returned to normal. These metabolic changes suggest that the antifatigue effect of YGJ is associated with the impairement of amino acid, organic acids and carbohydrates. It also appears that YGJ can induce significant metabolic alterations independent of the exhausting swimming‐induced metabolic changes. The significantly altered metabolites induced by YGJ intervention include l‐ 2‐amino‐acetoacetate, taurine, fumaric acid, malic acid, oxoadipic acid and l‐ aspartate, all of which are associated with antifatigue properties. This suggests that YGJ exerts chemopreventive effects via antifatigue mechanisms.     

A computational strategy for metabolic network construction based on the overlapping ratio: Study of patients’ metabolic responses to different dialysis patterns

BackgroundUremia is a worldwide epidemic disease and poses a serious threat to human health. Both maintenance hemodialysis (HD) and maintenance high flux hemodialysis (HFD) are common treatments for uremia and are generally used in clinical applications. In-depth exploration of patients’ metabolic responses to different dialysis patterns can facilitate the understanding of pathological alterations associated with uremia and the effects of different dialysis methods on uremia, which may be used for future personalized therapy. However, due to variations of multiple factors (i.e., genetic, epigenetic and environment) in the process of disease treatments, identification of the similarities and differences in plasma metabolite changes in uremic patients in response to HD and HFD remains challenging.MethodsIn this study, a computational strategy for metabolic network construction based on the overlapping ratio (MNC-OR) was proposed for disease treatment effect research. In MNC-OR, the overlapping ratio was introduced to measure metabolic reactions and to construct metabolic networks for analysis of different treatment options. Then, MNC-OR was employed to analyze HD-pattern-dependent changes in plasma metabolites to explore the pathological alterations associated with uremia and the effectiveness of different dialysis patterns (i.e., HD and HFD) on uremia. Based on the networks constructed by MNC-OR, two network analysis techniques, namely, similarity analysis and difference analysis of network topology, were used to find the similarity and differences in metabolic signals in patients under treatment with either HD or HFD, which can facilitate the understanding of pathological alterations associated with uremia and provide the guidance for personalized dialysis therapy.ResultsSimilarity analysis of network topology suggested that abnormal energy metabolism, gut metabolism and pyrimidine metabolism might occur in uremic patients, and maintenance of both HFD and HD therapies have beneficial effects on uremia. Then, difference analysis of network topology was employed to extract the crucial information related to HD-pattern-dependent changes in plasma metabolites. Experimental results indicated that the amino acid metabolism was closer to the normal status in HFD-treated patients; however, in HD-treated patients, the ability of antioxidation showed greater reduction, and the protein O-GlcNAcylation level was higher. Our findings demonstrate the potential of MNC-OR for explaining the metabolic similarities and differences of patients in response to different dialysis methods, thereby contributing to the guidance of personalized dialysis therapy.     

Plasma esterified and non-esterified fatty acids metabolic profiling using gas chromatography-mass spectrometry and its application in the study of diabetic mellitus and diabetic nephropathy

Using gas chromatography–mass spectrometry (GC–MS), a new metabolic profiling method was established to assess the levels of non-esterified fatty acids (NEFAs) and esterified fatty acids (EFAs) in plasma. The extraction method was simple and robust without removing protein process. With this method 25 fatty acids (FAs), both EFAs and NEFAs, can be recognized simultaneously with only 10 μL plasma. 15 of the 25 can be precisely quantified. The method was validated and then applied into clinical metabonomics research. Five clinical groups including 150 cases were involved. The relationship between FA levels and diabetic mellitus (DM) as well as diabetic nephropathy (DN) pathology was speculated. Furthermore, the possible pathological causes and effects were discussed in detail. Potential biomarkers (p value <0.01) were screened with Student's t-test. With the application of partial least squares-discriminant analysis (PLS-DA), different stages were distinguished. The result may be useful for the pathology study of metabolic syndromes, and may also be helpful for monitoring the progression of DM and DN.     

血浆游离脂肪酸代谢轮廓柱前衍生定量方法在糖尿病患者中医虚证分型中的应用

以α-溴代苯乙酮为衍生化试剂,十七酸为内标物,建立了糖尿病患者血浆中游离脂肪酸(FFA)代谢谱分析法,实现了6种主要FFA及6种微量FFA的定量分析。用此方法分析了75位临床糖尿病患者的血浆FFA代谢谱,并通过线性判别分析(LDA)对气虚和气阴两虚两种中医虚证进行了关联分析,正判率为94.3%。逐步判别分析结果表明,花生四烯酸(C20:4)和油酸(C18:1)承载了这两种虚证的重要信息,可作为潜在的标志物。利用代谢组学技术研究血浆FFA代谢谱与中医虚证的相关关系对规范证候临床诊断,提高中医药诊疗体系的可信度与可重复性具有重要意义。     

气相色谱-质谱和液相色谱-质谱联用方法用于口腔癌代谢组学分析

口腔癌的发病率占全身恶性肿瘤的第6位,正确区分正常状态与良性和恶性口腔肿瘤,是恰当选择治疗方案的关键所在。本研究中,首先利用液相色谱-质谱和气相色谱-质谱联用方法分别得到健康人、良性口腔肿瘤患者和恶性口腔肿瘤患者血浆、尿液和唾液的代谢轮廓,然后应用正交信号校正的偏最小二乘法进行多变量统计分析。结果表明健康人、良性肿瘤患者和恶性肿瘤患者在血浆、尿液和唾液等3种体液代谢中都可以被区分开,而且找到和鉴定出19个重要差异代谢物。相关代谢通路分析显示,与健康人相比,良性和恶性口腔肿瘤患者都存在能量代谢紊乱和脂类代谢失衡的现象,但恶性口腔肿瘤患者还表现出三羧酸循环和肌醇代谢异常,这为临床诊断及治疗提供了重要信息。