中成药六味地黄丸对急性肾损伤保护作用的研究

利用代谢组学方法研究了六味地黄丸治疗庆大霉素诱导的急性肾损伤的作用机制,并将超高效液相色谱-质谱联用(UPLC-MS)技术与主成分分析(PCA)、正交偏最小二乘判别分析(OPLS-DA)相结合来研究正常组、模型组、给药组间生物标记物的变化情况。在PCA与OPLS-DA模型中,正常组、模型组、给药组均可明显区分。共鉴定出17个正常组与模型组间的生物标记物,包括多种磷脂酰胆碱、鞘氨醇、植物鞘氨醇、鞘磷脂(d18∶0/16∶1(9z))、花生四烯酸。与正常组相比,鞘氨醇、植物鞘氨醇、鞘磷脂(d18∶0/16∶1(9z))、花生四烯酸的含量升高,而给药组中这4种化合物的含量下降,因此六味地黄丸可能通过调节这4种生物标记物的含量从而达到治疗庆大霉素诱导的急性肾损伤的目的。该研究为临床上六味地黄丸治疗肾损伤提供了理论依据。     

丹参多酚酸盐改善扩张性心肌病心肌功能的作用机制

将代谢组学与分子生物学方法相结合,研究了丹参多酚酸盐治疗扩张性心肌病的作用机制.采用主成分分析(PCA)法,分析了健康组、模型组及丹参多酚酸盐给药组大鼠血清代谢轮廓,采用正交校正的偏最小二乘判别分析(OPLS-DA)法寻找潜在的生物标记物,共鉴定得到磷酯酰丝氨酸[16∶0/18∶1(9Z)]、溶血磷脂(16∶0)、溶血磷脂[20∶4(5Z,8Z,11Z,14Z)]、溶血磷脂[22∶6(4Z,7Z,10Z,13Z,16Z,19Z)]、胆固醇硫酸酯、胆汁酸、γ-亚麻酸、二十二碳五烯酸和9'-羧基-γ-生育酚9种潜在的生物标记物.其中,γ-亚麻酸、二十二碳五烯酸和9'-羧基-γ-生育酚的含量在模型组中下降,经丹参多酚酸盐治疗后含量上升.通过Western bloting法和酶联免疫吸附法证实丹参多酚酸盐通过影响体内与γ-亚麻酸和9'-羧基-γ-生育酚相关的超氧化物岐化酶(SOD)、丙二醛(MDA)及其下游Bcl-2和Bax蛋白分子表达量,从而减少氧化应激所致的心肌细胞凋亡数量,达到治疗阿霉素所致扩张型心肌病的目的.     

采用柱切换色谱-离子阱质谱联用法研究复方丹参滴丸中丹参素及其代谢产物

采用柱切换高效液相色谱技术对人血清中的丹参素及其代谢产物进行富集和分离,利用对电喷雾离子阱多级质谱负离子模式进行结构分析。一级流动相为甲醇-水(30：70,V/V),流速0．8mL／min;二级流动相为甲醇-水-甲酸铵(15：85：0．5,V/V/V),流速0．2mL／min;在人血清中检测到丹参素及其代谢产物,并探讨了主要代谢产物乙酰化的丹参素(m/z 240．2)。建立一种复方丹参滴丸中丹参素及其代谢产物在人体内代谢过程的高效分析方法。该方法可用于复方丹参滴丸中丹参素及与其结构类似的其它药物的体内代谢研究。     

中药通心络对抑郁-动脉粥样硬化大鼠干预作用的血浆代谢组学研究

采用快速液相色谱串联离子阱飞行时间质谱(UFLC/MS-IT-TOF)技术对通心络干预下的抑郁-动脉粥样硬化大鼠的血浆代谢谱进行研究.结果表明,抑郁-动脉粥样硬化模型大鼠体内的氨基酸代谢、脂肪酸的β-氧化、胆固醇代谢及磷脂代谢发生了异常.中药通心络通过调节模型大鼠体内的色氨酸、苯丙氨酸代谢及某些胆汁酸的代谢,抑制大鼠抑郁-动脉粥样硬化的形成.该研究结果表明代谢组学在中药整体性药效评价、作用机理的阐明等方面具有很好的应用前景.     

基于脂质代谢组学研究褐藻糖胶对黄颡鱼幼鱼的影响

采用超高效液相色谱-四极杆-飞行时间质谱 (UPLC-Q-TOF-MS) 技术,结合多元变量数据分析方法,研究0.1%褐藻糖胶喂养黄颡鱼幼鱼1~8周的脂质代谢组学.通过不同喂养时间的黄颡鱼脂质成分的重要性(VIP)值和差异性(P)值,筛选出98个差异性代谢物,其中鉴定出与褐藻糖胶对黄颡鱼随喂养时间变化的影响有重要作用的11种脂质生物标志物,包括Lyso-PC 16∶0、PC 22∶6/16∶0、PE 22∶6/16∶0、PI 18∶0/22∶6、DAG 16∶0/16∶0、DAG 16∶0/18∶1、DAG 18∶1/18∶1、TAG 20∶5/16∶0/18∶3、TAG 20∶4/16∶1/18∶2、TAG 16∶0/18∶2/20∶5 和TAG 18∶2/14∶0/18∶1.研究表明,经0.1%褐藻糖胶喂养不同时期的黄颡鱼中Lyso-PC、PC、PE和PI的含量在第8周时达到最大值;DAG的含量呈现出先减少后增加的变化规律,而TAG的变化不定,TAG 18∶2/14∶0/18∶1、TAG 20∶5/16∶0/18∶3和TAG 20∶4/16∶1/18∶2的含量随着喂养褐藻糖胶时间的延长而增加,而TAG 16∶0/18∶2/20∶5呈减少的趋势.根据上述实验结果,推测褐藻糖胶可能通过调节脂质成分的含量来增加黄颡鱼幼鱼的免疫力.本研究结果为褐藻糖胶对黄颡鱼脂质代谢响应机理的影响的研究提供理论依据.     

黄芪黄酮干预脾虚水湿不化大鼠血浆代谢组学研究

采用高效液相色谱结合飞行时间质谱检测正常大鼠、脾虚水湿不化证大鼠及黄芪黄酮组分干预后大鼠血浆内源性代谢物变化,获取大鼠血浆代谢图谱,研究黄芪黄酮组分干预脾虚水湿不化证的作用机制.采用高脂低蛋白饮食加负重游泳力竭建立脾虚水湿不化证大鼠模型,选择Halo C18色谱柱,流动相为0.05％甲酸-水与0.05％甲酸-乙腈,梯度洗脱,利用液相色谱-串联质谱分析测定大鼠血浆样品.利用主成分分析法对造模前后大鼠血浆样本进行代谢轮廓分析,结合变量投影重要性及显著性分析等筛选对分组贡献最大的差异标志物及相关通路,阐明药物的作用机制.结果表明, 黄芪黄酮组代谢轮廓异于模型组,而接近于正常组,共鉴定出了包含甘油磷脂类、鞘酯类、氨基酸类等在内的11种潜在生物标志物,其代谢通路包括三羧酸循环、甘油磷脂代谢、脂肪酸代谢及氨基酸代谢等,主要涉及体内能量代谢和脂肪代谢.黄芪黄酮干预脾虚水湿不化证大鼠后, 宏观指标(如体重、自主活动)和微观指标(如代谢标志物、血脂)均明显回调,表明黄芪黄酮可能主要通过调节能量代谢、脂肪代谢等途径而发挥健脾利水作用.     

基于UPLC/Q-TOFMS的大鼠血浆磷脂成分的快速分析

采用超高效液相色谱-四极杆飞行时间高分辨质谱(UPLC/Q-TOF MS)联用技术,对大鼠血浆磷脂提取液进行分析,建立了一种快速、简便、可靠的血浆磷脂鉴别方法。以经典的氯仿甲醇法为磷脂的提取方法,以10 mmol/L甲酸铵水溶液(A)-含10 mmol/L甲酸铵的乙腈-异丙醇(1∶1)混合溶液(B)为流动相,采用ESI源,分别在正、负离子模式下进行数据采集。根据实验结果,结合二级质谱裂解数据、元素组成、数据库匹配,鉴定出81个成分,其中包括7个PE、7个PI和1个SM,其余均为PC。所建立的液相色谱-质谱联用分析方法,可为大鼠血浆中磷脂成分的鉴别提供一种快速、简便、可靠的分析手段,以及为血浆磷脂代谢组学的研究奠定基础。     

尿液代谢组学方法研究人参总皂苷治疗糖尿病心肌病大鼠作用机制

利用基于质谱的代谢组学方法考察了人参总皂苷(TG)治疗糖尿病心肌病(DCM)大鼠的效应机制;建立了糖尿病心肌病大鼠模型,并连续12周口服人参总皂苷,采用快速高分辨液相色谱/四级杆-飞行时间/质谱(RRLC/Q-TOF/MS)技术对糖尿病心肌病模型组(DCM组)和人参总皂苷治疗组(TG组)大鼠尿样的尿液代谢物进行分析,采用主成分分析(PCA)对两组代谢物进行分类,并寻找潜在生物标记物,同时检测心肌病理超微结构、血液生化指标和心肌氧化应激水平。RRLC/Q-TOF/MS检测结果表明,DCM组和TG组大鼠的尿液代谢物谱能得到很好的区分,发现并鉴定了3种生物标记物。TG降低了DCM大鼠心肌超微结构损伤并改善其血脂、血糖及心肌氧化应激水平,代谢组学研究结果表明:作用机制可能是TG对柠檬酸循环、脂肪酸代谢和氧化应激水平的调节作用。     

基因芯片用于组分中药新双龙方的配伍机制研究

采用基因芯片技术研究了中药新双龙方及其有效组分人参总皂苷和丹参总酚酸治疗急性心肌梗塞(AMI)大鼠的作用机制. 以结扎大鼠冠脉左前降支的方法制备急性心梗模型, 采用大鼠全基因组表达谱芯片分别筛选出AMI大鼠给药前及新双龙方与组分给药后的差异表达基因(Ratio>2或<0.5), 通过对4组差异基因的综合分析, 比较了新双龙方与组分作用方式的异同, 从分子调控水平上探讨复方配伍后的增效作用, 同时对与药效作用机理直接相关的钙离子转运相关的Cacna1d基因和血管修复相关的Flnb基因进行了实时荧光逆转录聚合酶链式反应(RT-PCR)定量. 结果表明, 新双龙方与2种有效组分对心梗大鼠基因表达的影响不同. 对差异基因的聚类分析以及药物的调控作用比较结果均表明复方比组分的疗效更好; 通路分析结果表明, 新双龙方与组分均主要通过调控钙信号转导通路、 丝裂原活化蛋白激酶信号转导通路(MAPK)信号等通路发挥药效作用, 且复方涉及更多通路; 筛选出了复方与组分共同调控的靶点基因, 其在3组中具有不完全相同的表达趋势, 但复方具有更积极的调控作用. 本研究结果从分子调控角度证实了复方新双龙方的配伍优势.     

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。     

基于气相色谱-飞行时间质谱技术的佐剂性关节炎大鼠尿液代谢组学的研究

采用弗氏完全佐剂(FCA)诱导佐剂性关节炎(AA)大鼠模型,观察大鼠足趾肿胀度和踝关节组织的病理学形态变化。应用气相色谱-飞行时间质谱(GC-TOF MS)技术检测AA大鼠尿液代谢物谱,并对数据进行主成分分析(PCA)、偏最小二乘法-判别分析(PLS-DA)及正交偏最小二乘法-判别分析(OPLS-DA),探讨可能的发病机制。通过变量重要性投影值(VIP>1)和P值(<0.05),筛选出尿液中的差异代谢物。在模型组大鼠的尿液中共发现异柠檬酸、α-酮戊二酸、柠康酸、肌酸、3-羟基丁酸等20种差异代谢物。推断AA代谢组学的发病机制可能与能量代谢、氨基酸代谢、脂肪酸代谢途径有关。     

Plasma metabonomics study on toxicity biomarker in rats treated with Euphorbia fischeriana based on LC–MS

Lang‐du (LD) has been traditionally used to treat human diseases in China. Plasma metabolic profiling was applied in this study based on LC–MS to elucidate the toxicity in rats induced by injected ethanol extract of LD. LD injection was given by intraperitoneal injection at doses of 0.1, 0.05, 0.025 and 0 g kg^?1 body weight per day to rats. The blood biochemical levels of alanine aminotransferase, direct bilirubin, creatinine, serum β2‐microglobulin and low‐density lipoprotein increased in LD‐injected rats, and the levels of total protein and albumin decreased in these groups. The metabolic profiles of the samples were analyzed by multivariate statistics analysis, including principal component analysis, partial least squares discriminant analysis and orthogonal projection to latent structures discriminate analysis (OPLS‐DA). The metabolic characters in rats injected with LD were perturbed in a dose‐dependent manner. By OPLS‐DA, 18 metabolites were served as the potential toxicity biomarkers. Moreover, LD treatment resulted in an increase in the p‐cresol, p‐cresol sulfate, lysophosphatidylethanolamine (LPE) (18:0), LPE (16:0), lysophosphatidylcholine (16:0) and 12‐HETE concentrations, and a decrease in hippuric acid, cholic acid and N‐acetyl‐l ‐phenylalanine. These results suggested that chronic exposure to LD could cause a disturbance in lipids metabolism and amino acids metabolism, etc. Therefore, an analysis of the metabolic profiles can contribute to a better understanding of the adverse effects of LD. Copyright © 2016 John Wiley & Sons, Ltd.     

Metabonomics study of atherosclerosis rats by ultra fast liquid chromatography coupled with ion trap-time of flight mass spectrometry

An ultra fast liquid chromatography coupled with IT-TOF mass spectrometry (UFLC/MS-IT-TOF) metabonomic approach was employed to study the plasma and urine metabolic profiling of atherosclerosis rats. Acquired data were subjected to principal component analysis (PCA) for differentiating the atherosclerosis and the control groups. Potential biomarkers were screened by using S-plot and were identified by the accurate mass and MSⁿ fragments information obtained from UFLC/MS-IT-TOF analysis. 12 metabolites in rat plasma and 8 metabolites in urine were identified as potential biomarkers. Concentrations of leucine, phenylalanine, tryptophan, acetylcarnitine, butyrylcarnitine, propionylcarnitine and spermine in plasma and 3-O-methyl-dopa, ethyl N2-acetyl-l-argininate, leucylproline, glucuronate, t6A N(6)-(N-threonylcarbonyl)-adenosine and methyl-hippuric acid in urine decreased in atherosclerosis rats. Ursodeoxycholic acid, chenodeoxycholic acid, LPC (C16:0), LPC (C18:0) and LPC (C18:1) in plasma and hippuric acid in urine were in higher levels in atherosclerosis rats. The alterated metabolites demonstrated abnormal metabolism of phenylalanine, tryptophan, bile acids and amino acids. This research proved that metabonomics is a promising tool for disease research.     

High-throughput serum metabolomics analysis of gouty arthritis rat treated by total saponins of Rhizoma Dioscoreae Makino by UPLC–Q/TOF–MS

Rhizoma Dioscoreae Makino (RDM) is effective in treating gouty arthritis (GA) and hyperuricacidemia, especially in promoting uric acid excretion and reducing the inflammatory reaction. Bioactive constituents in RDM are mainly steroidal saponins such as dioscin, trillin, protodioscin and protogracillin. However, the mechanism of its anti-GA action is still unclear, owing to the complex pathological and physiological characteristics of GA, and integration of RDM with multiple components, multiple targets and multiple pathways. Herein, a GA rat model was induced with monosodium urate (MSU), and RDM reduced inflammation of rat synovium tissue. Through metabolomics analysis, 35 potential biomarkers with significant changes involved in the pathogenesis of GA induced by MSU were identified, and perturbations were restored after RDM treatment. The most correlated pathways involved in d -galactose, d -mannose, d -glucose, myoinositol, Phosphatidylcholine (PC) (16:0/16:0), LysoPC (15:0), phosphatidic acid (PA) [18:1(9Z)/18:1(11Z)] and glutathione induced by MSU were galactose metabolism, inositol phosphate metabolism, glycerophospholipid metabolism and glutathione metabolism, and the derivations of all those biomarkers could be regulated by RDM treatment. RDM has a therapeutic effect on GA by intervening in changes in endogenous metabolisms and the related metabolic pathways.     

基于超高效液相色谱-四极杆飞行时间质谱联用技术的血瘀模型大鼠血浆代谢组学分析

采用盐酸肾上腺素加冰水浴建立急性血瘀大鼠模型，使用超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF/MS）检测空白对照组与血瘀模型组中血浆代谢物，用主成分分析（PCA）、有监督偏最小二乘法判别分析（PLS-DA）及正交偏最小二乘法判别分析（OPLS-DA）对代谢组学数据进行多维统计分析，筛选潜在生物标志物。与对照组相比，在血瘀模型组大鼠血浆中检测出46个差异代谢物，血瘀模型组中乙酰胆碱、N6，N6，N6-三甲基-L-赖氨酸、胞嘧啶、乙酰肉碱等21个代谢物显著上调，吲哚丙酸、LysoPC（14：0）等25个代谢物显著下调，可能与脂质代谢、半乳糖代谢、亚油酸代谢、不饱和脂肪酸生物合成、糖酵解、花生四烯酸代谢等通路有关。代谢产物可作为血瘀证研究中的重要标记物，该研究结果有助于揭示血瘀证的发病机制，可为临床血瘀疾病的诊断及选用药物治疗提供思路，为后续治疗手段提供参考依据。     

Nootkatone,a Dietary Fragrant Bioactive Compound,Attenuates Dyslipidemia and Intramyocardial Lipid Accumulation and Favorably Alters Lipid Metabolism in a Rat Model of Myocardial Injury: An In Vivo and In Vitro Study

In the present study, we assessed whether nootkatone (NKT), a sesquiterpene in edible plants, can provide protection against dyslipidemia, intramyocardial lipid accumulation, and altered lipid metabolism in a rat model of myocardial infarction (MI) induced by subcutaneous injections of isoproterenol (ISO, 85 mg/kg) on days 9 and 10. The rats were pre- and co-treated with NKT (10 mg/kg, p.o.) administered daily for 11 days. A significant reduction in the activities of myocardial creatine kinase and lactate dehydrogenase, as well as non-enzymatic antioxidants, and alterations in lipids and lipoproteins, along with a rise in plasma lipid peroxidation and intramyocardial lipid accumulation, were observed in ISO-treated rats. ISO administration induced alterations in the activities of enzymes/expressions that played a significant role in altering lipid metabolism. However, NKT treatment favorably modulated all biochemical and molecular parameters altered by ISO and showed protective effects against oxidative stress, dyslipidemia, and altered lipid metabolism, attributed to its free-radical-scavenging and antihyperlipidemic activities in rats with ISO-induced MI. Additionally, NKT decreased the accumulation of lipids in the myocardium as evidenced from Oil red O staining. Furthermore, the in vitro observations demonstrate the potent antioxidant property of NKT. The present study findings are suggestive of the protective effects of NKT on dyslipidemia and the underlying mechanisms. Based on our findings, it can be suggested that NKT or plants rich in NKT can be promising for use as a phytopharmaceutical or nutraceutical in protecting the heart and correcting lipid abnormalities and dyslipidemia, which are risk factors for ischemic heart diseases.