大鼠肝郁脾虚证的代谢组学研究

目的:研究慢性束缚应激大鼠(肝郁脾虚模型)的血浆代谢表型改变,开展中医证候学的研究,试图通过对慢性束缚应激大鼠代谢物组的共性分析和生物标记物的发现,寻找肝郁脾虚证候的生物学本质,探讨代谢组学在中医证候学研究中的应用。方法:选用实验用二级雄性SD大鼠24只,随机分为4组:7 d正常对照组(A组)、21 d正常对照组(B组)、7 d模型组(C组)和21 d模型组(D组),每组6只。以慢性束缚方法制作应激大鼠模型。分别于第8天和第22天麻醉后心室取血,用Varian UnityInova 600 M超导傅立叶变化核磁共振(nuclear magnetic resonance,NMR)波谱仪检测。自由感应衰减信号经过32 k傅立叶变换转为一维NMR谱图。调用VNMR软件中的程序将1H谱按默认值,分别从4.5~0.5 ppm(弛豫时间编辑)以及6.0~0 ppm(扩散编辑),每段为0.04 ppm,进行分段积分,将积分数据归一化后,以文本文件或Excel文件贮存,用于模式识别分析。将上述得到的数据文件利用Si mca-P 10.0软件包进行主成分分析。结果:大鼠血浆1H NMR谱的主成分分析,各组动物代谢谱各不相同,与已知应激状态下动物体内代谢的调节过程及结果相一致。正常对照组与模型组相比较发现,醋酸、乳酸、酪氨酸、低密度脂蛋白和3.44 ppm的未知化合物的谱峰峰形改变较为明显。这些发生改变的代谢物可以作为肝郁脾虚证的生物标志物做进一步的研究。结论:各组大鼠血浆1H NMR代谢谱之间存在差异,而且可能从代谢组学分析中找出特异的标志性代谢产物,阐释中医证候的生物学本质。代谢组学分析是一种有良好发展前景的中医证候学研究方法。     

A serum metabonomic study on the difference between alcohol- and HBV-induced liver cirrhosis by ultraperformance liquid chromatography coupled to mass spectrometry plus quadrupole time-of-flight mass spectrometry

Background  Liver cirrhosis is the fatal consequence of chronic hepatitis, making early diagnosis of liver cirrhosis critical. Liver biopsy is still the standard diagnostic method for liver cirrhosis, although its use in a broad population with alcoholism or hepatitis B virus (HBV) infection remains difficult. In this study, we used a metabonomic approach to detect potential biomarkers for early diagnosis of liver cirrhosis.

Methods  Serum specimens were collected prospectively from normal control subjects (n=22) and patients with alcoholic cirrhosis (n=18) or HBV-induced cirrhosis (n=19). The serum metabonome was analyzed using ultraperformance liquid chromatography (LC)/time-of-flight mass spectrometry (MS) integrated with chemometrics. The acquired LC-MS data were normalized and processed using principal component analysis (PCA) and partial least squares discrimination analysis (PLS-DA).

Results  Significant differences in the metabonomics among the three groups were observed. Lysophosphatidyl cholines (LPCs) (LPC C16:0, LPC C18:0, LPC C18:2, LPC C18:3, LPC C20:3, LPC C20:5) were decreased in the serum of patients with hepatic cirrhosis, whereas bile acids (glycocholic acid, glycochenodeoxycholic acid), hypoxanthine, and stearamide were increased in the serum of patients with hepatic cirrhosis. These metabolites are considered “common” biomarkers for hepatic cirrhosis. Oleamide and myristamide were increased in the serum of patients with alcoholic cirrhosis but decreased in those with HBV-induced cirrhosis. These could be specific biomarkers for differential diagnosis between alcohol- and HBV-induced hepatic cirrhosis.

Conclusions  There are significant metabonomic differences between alcohol- and HBV-induced liver cirrhosis. Metabonomics is a top-down systems biology tool for conducting research on clinical problems.

     

基于LC-QTOF-MS代谢组学研究甲基苯丙胺临床戒毒分期

甲基苯丙胺的滥用已成为全球公共卫生安全的一大隐患。为了更加科学可靠的判断甲基苯丙胺成瘾和戒毒阶段,本研究采用液相色谱-四极杆-飞行时间串联质谱（LC-QTOF-MS）分析技术,对甲基苯丙胺成瘾者、戒毒康复者和健康人的血浆、血清和尿中的内源性代谢物进行分析。运用单变量和多变量统计分析处理数据,筛选符合条件的潜在生物标志物,将鉴定后的生物标志物进行代谢物富集分析,寻找代谢物与代谢途径之间的联系。多变量统计结果表明,甲基苯丙胺急性期组、康复期组和健康组3组聚类分离明显;在血清、血浆和尿液中分别鉴定出3,18和6个差异代谢物,提示甲基苯丙胺急性期组脂质代谢异常,康复期组脂肪酸代谢、硫酸盐/亚硫酸盐代谢和性激素代谢异常。本研究筛选出的潜在生物标志物,为甲基苯丙胺临床戒毒分期提供科学依据。     

Erythrocyte sphingolipid species as biomarkers of Alzheimer's disease

Diagnosing Alzheimer's disease (AD) in the early stage is challenging. Informative biomarkers can be of great value for population-based screening. Metabolomics studies have been used to find potential biomarkers, but commonly used tissue sources can be difficult to obtain. The objective of this study was to determine the potential utility of erythrocyte metabolite profiles in screening for AD. Unlike some commonly-used sources such as cerebrospinal fluid and brain tissue, erythrocytes are plentiful and easily accessed. Moreover, erythrocytes are metabolically active, a feature that distinguishes this sample source from other bodily fluids like plasma and urine. In this preliminary pilot study, the erythrocyte metabolomes of 10 histopathologically confirmed AD patients and 10 patients without AD (control (CTRL)) were compared. Whole blood was collected post-mortem and erythrocytes were analyzed using ultra-performance liquid chromatography tandem mass spectrometry. Over 750 metabolites were identified in AD and CTRL erythrocytes. Seven were increased in AD while 24 were decreased (P<0.05). The majority of the metabolites increased in AD were associated with amino acid metabolism and all of the decreased metabolites were associated with lipid metabolism. Prominent among the potential biomarkers were 10 sphingolipid or sphingolipid-related species that were consistently decreased in AD patients. Sphingolipids have been previously implicated in AD and other neurological conditions. Furthermore, previous studies have shown that erythrocyte sphingolipid concentrations vary widely in normal, healthy adults. Together, these observations suggest that certain erythrocyte lipid phenotypes could be markers of risk for development of AD.     

Metabonomic analysis of plasma biochemical changes in pyrexia rats after treatment with Gegenqinlian decoction,aspirin and itraconazole by UHPLC-FT-ICR-MS

A metabonomic approach involving an ultrahigh-performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) was used to investigate the changes in the endogenous metabolites in the plasma of rats with yeast-induced pyrexia treated with Gegenqinlian decoction (GQLD), aspirin and itraconazole. The differences in the small molecule profiles of treatment using traditional Chinese medicine, etiological treatment and symptomatic treatment were elucidated. Thirty-six plasma metabolites were identified or putatively identified, and the effects of the three medicines on the thirty-six metabolites were studied. Their metabolic pathways indicated that GQLD, aspirin and itraconazole ameliorated the rats with yeast-induced pyrexia predominantly by regulating the metabolisms of phospholipid, sphingolipid, fatty acid oxidation, fatty acid amides, amino acid and glycerolipid in vivo. The pharmacodynamics and metabonomic results showed that the three medicines exhibited the therapeutic effects on pyrexia by regulating the perturbations of multiple metabolisms. The study provided a scientific basis for an in-depth understanding of the therapeutic effects of GQLD, aspirin and itraconazole on rats with yeast-induced pyrexia.     

心力衰竭患者血浆内皮素的变化及倍他乐克的影响

为探讨充血性心力衰竭（ＣＨＦ）患者血浆内皮素－１（ＥＴ－１）的变化及倍他乐克（Ｂｅｔａｌｏｃ）对之影响，采用放射免疫法及高效液相色谱法测定４３例ＣＨＦ患者血浆ＥＴ－１和去甲肾上腺素（ＮＥ）水平。４３例中２４例用倍他乐克＋常规治疗，１９例用常规治疗，疗程１月。结果表明：ＣＨＦ患者治疗前血浆ＥＴ－１和ＮＥ均显著升高，且与ＣＨＦ程度呈正相关；治疗后降低。常规治疗对ＥＴ－１和ＮＥ均无影响     

心力衰竭患者血浆皮素的变化及倍他乐克的影响

为探讨充血性心力衰竭（ＣＨＦ）患者血浆内皮素－１（ＥＴ－１）的变化及倍他乐克对之影响，采用放射免疫法及高效液相色谱法测定４３例ＣＨＦ患者血浆ＥＴ－１和去甲肾上腺素（ＮＥ）水平。４３例中２４例用倍他乐克＋常规治疗，１９例用常规治疗，疗程１个月。结果表明：ＣＨＦ患者治疗前血浆ＥＴ－１和ＮＥ均显著升高，且与ＣＨＦ程度呈正相关，治疗后降低。常规治疗对ＥＴ－１和ＮＥ均无影响。     

基于UHPLC/Q-TOF MS联用技术的顺铂肾毒性代谢组学研究

目的 构建顺铂诱导的急性肾损伤小鼠模型,利用代谢组学方法,筛选顺铂诱导急性肾损伤的潜在生物学标志物,进一步探索其可能形成机制。方法 通过对小鼠腹腔注射顺铂溶液,建立急性肾损伤的实验动物模型;利用超高效液相色谱-四极杆飞行时间串联质谱(UHPLC/Q-TOF MS)技术对健康对照组及急性肾损伤模型组的血清进行代谢轮廓分析,通过多元统计分析方法对两组间的数据进行分析,筛选顺铂诱导小鼠急性肾损伤的潜在生物学标志物。结果 主成分分析结果表明,模型组与对照组有明显的分离趋势,显示小鼠血清的内源性代谢物发生了较为显著地变化,进一步分析筛选,得到40种顺铂诱导急性肾损伤的差异代谢物,涉及氨基酸代谢、脂肪代谢、糖类代谢、三羧酸循环等多种代谢途径。结论 应用代谢组学的方法可以初步筛选顺铂诱导急性肾损伤的差异代谢物,并对其涉及的生物通路进行归属,初步探索顺铂肾毒性的形成机制。     

UPLC-Q-TOF-MS/MS代谢组学分析针刺对抑郁症患者血浆代谢物变化的影响

目的 运用高效液相色谱-飞行时间质谱联用(UPLC-Q-TOF-MS/MS)技术研究针刺治疗抑郁症的潜在生物标志物,探究针刺抗抑郁的潜在代谢调节机制。方法 选取2017年10月至2018年6月南京脑科医院的抑郁症患者25例,随机分为针刺药物组13例和药物组12例(脱落4例),药物组予SSRIs口服治疗,针刺药物组在药物组治疗基础上加予耳内皮刺治疗。收集治疗前后2组患者血浆样品,运用UPLC-Q-TOF/MS检测治疗前后2组患者的血浆代谢产物,比较2组治疗前后代谢物的差异。结果 针刺药物组筛选出16个潜在生物标记物,推测针刺药物组通过调节苯丙氨酸代谢、组氨酸代谢、酮体的合成与降解、赖氨酸降解4条代谢途径改善抑郁症患者的异常状态。药物组筛选出9个潜在生物标记物,推测药物组通过调节亚油酸代谢、咖啡因代谢、牛磺酸和亚牛磺酸的代谢、精氨酸和脯氨酸代谢4条代谢途径发挥治疗作用。结论 对抑郁症患者代谢物的调控可能是针刺发挥抗抑郁作用的内在机制。     

Multi-factor combined biomarker screening strategy to rapidly diagnose Alzheimer's disease and evaluate drug effect based on a rat model

Alzheimer's disease (AD) represents the main form of dementia; however, valid diagnosis and treatment measures are lacking. The discovery of valuable biomarkers through omics technologies can help solve this problem. For this reason, metabolomic analysis using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS) was carried out on plasma, hippocampus, and cortex samples of an AD rat model. Based on the metabolomic data, we report a multi-factor combined biomarker screening strategy to rapidly and accurately identify potential biomarkers. Compared with the usual procedure, our strategy can identify fewer biomarkers with higher diagnostic specificity and sensitivity. In addition to diagnosis, the potential biomarkers identified using our strategy were also beneficial for drug evaluation. Multi-factor combined biomarker screening strategy was used to identify differential metabolites from a rat model of amyloid beta peptide 1–40 (Aβ₁₋₄₀) plus ibotenic acid-induced AD (compared with the controls) for the first time; lysophosphatidylcholine (LysoPC) and intermediates of sphingolipid metabolism were screened as potential biomarkers. Subsequently, the effects of donepezil and pine nut were successfully reflected by regulating the levels of the abovementioned biomarkers and metabolic profile distribution in partial least squares-discriminant analysis (PLS-DA). This novel biomarker screening strategy can be used to analyze other metabolomic data to simultaneously enable disease diagnosis and drug evaluation.     

基于液质联用技术的白头翁汤治疗溃疡性结肠炎小鼠血清代谢组学分析

目的 研究白头翁汤给予溃疡性结肠炎小鼠前后内源性代谢物的变化,寻找与治疗溃疡性结肠炎有关的代谢生物标志物,探讨白头翁汤对溃疡性结肠炎模型的调节作用及其可能机制。方法 采用DSS复制小鼠溃疡性结肠炎模型,借助超高效液相色谱串联四级杆飞行时间质谱(UPLC-Q-TOF/MS)技术,对各组小鼠血清样本进行测定,采用主成分分析法(PCA)和正交偏最小二乘法判别分析(OPLS-DA)来筛选差异性代谢物。结果 经分析,对照组、模型组和白头翁汤治疗组血清样本能够得到很好的区分,共鉴定出6种潜在生物标志物,白头翁汤给药后溃结小鼠的内源性代谢物水平发生不同程度的回调。结论 白头翁汤可以使DSS诱导的溃结小鼠的异常代谢有所恢复,其治疗作用可能与机体内6个代谢物及3条相关代谢通路的调节有关。      

基于核磁共振1H谱的乳腺纤维腺瘤与乳腺浸润性导管癌血清代谢组学比较研究

目的 分析乳腺纤维腺瘤患者及乳腺浸润性导管癌患者在血清代谢组学上的差异,探讨术前通过代谢特征来辨别良恶性肿瘤的可能性。方法 收集经临床确诊的乳腺纤维腺瘤和乳腺浸润性导管癌患者血清各8例,通过核磁共振¹H谱（¹HNMR）技术检测其血清代谢组特征,分别采用主成分分析（PCA）和正交偏最小二乘判别法 （OPLS-DA）分析乳腺纤维腺瘤与乳腺浸润性导管癌之间的代谢差异。结果 PCA和OPLS-DA分析显示乳腺纤维腺瘤患者和乳腺浸润性导管癌患者的血清代谢特征存在明显差异。与乳腺纤维腺瘤患者相比,浸润性导管癌患者血清中脂质、乳酸盐、磷脂酰胆碱/甘油磷脂酰胆碱的含量升高,而谷氨酰胺、牛磺酸、肌酸和葡萄糖含量降低。结论 乳腺纤维腺瘤患者与乳腺浸润性导管癌患者在血清代谢水平上具有明显差异,这些具有明显变化的代谢物可作为潜在血清代谢标记物,为临床区分良恶性肿瘤提供参考。     

浅低温心脏跳动中瓣膜置换手术对空腹血糖受损患者胰岛素抵抗的影响

目的研究浅低温心脏跳动中瓣膜置换手术对空腹血糖受损患者胰岛素抵抗的影响。方法选择择期行体外循环(CPB)人工瓣膜置换手术的空腹血糖受损的风湿性心脏病患者30例,根据患者手术方式分为停跳组与不停跳组,停跳组在中度低温心脏停跳下完成心脏瓣膜置换手术,不停跳组在浅低温心脏跳动中完成手术。分别于手术前、麻醉诱导后、CPB开始后30 min、停CPB时、CPB后2 h和术后第1、3、5天共8时间点采集血样,检测空腹血糖、胰岛素和肿瘤坏死因子-α(TNF-α)浓度变化,并计算胰岛素抵抗指数。结果两组患者手术前和麻醉诱导后各指标差异无统计学意义,停跳组空腹血糖、胰岛素抵抗指数和TNF-α水平在CPB开始后30min至术后第3天较CPB前升高(P<0.05),不停跳组CPB开始后30 min至术后第1天较CPB前升高(P<0.05),两组间比较在CPB开始后30 min至术后第3天时差异有统计学意义(P<0.05);两组患者胰岛素水平在CPB开始后30 min至术后第1天较CPB前显著升高(P<0.05),不停跳组浓度较停跳组低(P<0.05)。结论在空腹血糖受损的患者使用心脏不停跳技术可以减轻机体炎症反应,有助于缓解胰岛素抵抗,维护葡萄糖代谢的稳态。     

赣南地区非综合征性唇腭裂患者血浆代谢组学研究

目的通过检测赣南地区非综合性唇腭裂（NSCL/P）患者与正常人血浆内源性代谢产物的变化,探讨基于核磁共振（NMR）的代谢组学方法在临床实践中的可行性。方法分别收集17例NSCL/P患者和16例正常人对照组的血浆样品,测定NSCL/P患者血浆的一维1H-NMR图谱,采用主成分分析法（PCA）分析NMR数据,以鉴别NSCL/P组和正常对照组血浆中内源血浆代谢产物的变化,获得NSCL/P患者的特征代谢物。结果 NSCL/P组和正常对照组间的血浆代谢产物存在明显差别。与正常对照组相比,NSCL/P患者血浆中的缬氨酸、苯丙氨酸和酪氨酸的含量升高;NSCL/P患者血浆中的丙氨酸、胆碱、磷酰胆碱等代谢物的含量降低。结论采用基于NMR的代谢组学技术能较好地反映赣南地区NSCL/P患者血浆的代谢变化,通过1H-NMR代谢图谱获得NSCL/P的特征代谢物,可以初步探讨NSCL/P的代谢机制,表明了该技术在临床应用中具有很大的潜能。     

芪苈强心胶囊治疗慢性心力衰竭临床疗效及其对血浆NT-proBNP水平的影响

目的观察辨证应用芪苈强心胶囊治疗慢性心力衰竭的疗效、安全性及对血浆NT-proBNP水平的影响。方法120例患者随机分3组,A组40例为辨病辨证结合治疗组,B组40例为辨病治疗组,C组40例为常规西药治疗组。A组在辨病基础上辨证加用芪苈强心胶囊,B组辨病加用芪苈强心胶囊,C组单用西药;疗程8周。观察血浆NT-proBNP水平、心功能分级、左室射血分数(LVEF)、中医证候疗效指标及安全性指标。结果3组治疗后心功能分级、LVEF、中医证候积分明显改善,血浆NT-proBNP水平明显下降;A组心功能分级、LVEF、中医证候积分改善和NT-proBNP水平下降均优于B、C组(P<0.05,P<0.01)。治疗后各组未见不良反应。结论芪苈强心胶囊对血浆NT-proBNP水平的抑制可能是其发挥治疗作用的机制之一。在辨病基础上辨证运用芪苈强心胶囊可提高临床疗效。     

血管紧张素转换酶抑制剂对慢性心力衰竭患者脑钠肽和去甲肾上腺素的作用

目的探讨不同剂量血管紧张素转换酶抑制剂(ACEI)对慢性心力衰竭患者脑钠肽和去甲肾上腺素(NE)的作用,以及较大剂量ACEI治疗的可行性与安全性。方法将66例慢性心力衰竭患者随机分为培哚普利较小剂量组(33例,2～4mg/d)和较大剂量组(33例,8～10mg/d)治疗12周,治疗前后测定各项指标[NE、脑钠肽浓度,左室舒张末内径(LVED),左室射血分数(LVEF)等]。并且选择30名年龄相仿的健康对照者,分别测定基础NE、脑钠肽浓度。结果慢性心力衰竭患者血浆中脑钠肽浓度随着纽约心脏病协会心功能分级增加而升高,与LVEF呈显著负相关(r=-0.327,P=0.012);脑钠肽与LVED和NE呈显著正相关(r=0.42,P=0.015;r=0.402,P=0.002)。治疗12周后7例患者因出现不能耐受的咳嗽反应而退出(其中较小剂量组3例,较大剂量组4例),其余59例完成研究。较小剂量组治疗后脑钠肽浓度为8μg/L±4μg/L,NE浓度为387ng/L±211ng/L,较大剂量组治疗后脑钠肽浓度为6μg/L±4μg/L,NE浓度为250ng/L±63ng/L,较大剂量组治疗后的脑钠肽、NE浓度明显低于较小剂量组治疗后(均P<0.05)。结论心力衰竭时血浆脑钠肽浓度与心力衰竭严重程度密切相关,且与NE浓度呈正相关。在慢性心力衰竭患者中采用小剂量逐渐递增剂量方式给予培哚普利可明显降低脑钠肽、NE,且耐受性良好。     

正常人血和尿中麦芽糖的廓清

目的建立麦芽糖分析方法,研究正常志愿者输入不同浓度麦芽糖溶液的廓清变化。方法用高效液相色谱仪分析血浆和尿样中的麦芽糖;12名男性健康志愿者,分别接受以0.2、0.3、0.5g.kg-1.h-1的给药速率静脉滴注麦芽糖注射液4h,在滴注前和滴注后不同时间点采集血样、不同时间段收集尿液测定麦芽糖和葡萄糖。结果血样分析麦芽糖的准确性和回收率的变异系数(RSD)分别在3.68%～4.58%和0.44%～4.83%之间;尿样分析准确性和回收率的RSD分别在2.91%～7.62%和0.95%～8.27%之间。健康志愿者以0.2、0.3、0.5g.kg-1.h-1的给药速率静脉滴注麦芽糖注射液4h后,血中麦芽糖的浓度随给药剂量的增加而成比例增加,具有良好的剂量依赖性(r>0.99)。3个高、中、低不同浓度的麦芽糖在输入12h后血中麦芽糖的浓度均回到起始点,麦芽糖在血中消除较快。在输入麦芽糖后2h,尿中麦芽糖和葡萄糖排出增加,2～4h达高峰,6h后减少,24h完全回到零点。结论在0.2～0.5g.kg-1.h-1的给药速率下,输注麦芽糖基本不会导致体内血糖浓度的明显升高,在上述浓度下具有很好的廓清能力。建议除需要立即补充大量能量时,可考虑采用较快的输注速率,常规补充能量时宜采用不超过0.3g.kg-1.h-1的输注速率。     

氯沙坦联合阿托伐他汀对慢性心衰患者左心功能和血浆IL-6、TNF-a浓度的影响

目的:观察氯沙坦联合阿托伐他汀对慢性左心衰患者心功能和血清IL-6、TNF-a浓度的影响。方法:102例心衰患者随机分为2组,对照组50例,治疗组52例。对照组给予氯沙坦及常规治疗,治疗组在此基础上加用阿托伐他汀,疗程均为8个月。比较两组心衰指标左室收缩末期内径(LVSD)、左室内径缩短率(FS)、射血分数(EF)和血浆IL-6、TNF-a浓度治疗前后及两组治疗后的差异。结果:两组各指标治疗前后及两组治疗后差别均有统计学意义。结论:氯沙坦联合阿托伐他汀对改善心功能和心衰患者的炎症状态具有显著效果,疗效优于单用氯沙坦。