代谢组学方法研究水飞蓟宾对四氯化碳致小鼠肝损伤的保护作用

运用代谢组学方法研究四氯化碳(CCl4)致小鼠肝损及水飞蓟宾肝保护作用的机理. 通过气相色谱-质谱(GC-MS)技术分析CCl4及水飞蓟宾作用下的小鼠肝组织匀浆及血浆代谢物谱. 使用正交偏最小二乘判别分析法研究正常组与CCl4模型组之间的代谢物谱差异, 并通过变量重要性投影(VIP)选取肝组织与血浆中各12种标志性代谢物. 使用主成分分析法研究水飞蓟宾预防性及治疗性给药对CCl4肝损的干预效果. 通过比较选取的标志性代谢物的含量的差异, 探讨了水飞蓟宾对CCl4致小鼠肝损的保护机理. 结果表明, CCl4作用后, 小鼠机体能量代谢、氨基酸代谢及脂类代谢都受到不同程度的影响. 水飞蓟宾能有效地缓解CCl4所造成的体内线粒体功能及氨基酸代谢紊乱. 采用代谢组学方法能较全面地反应生物体的生理及代谢状态, 并可应用于物质毒性和药效评价研究.     

甲亢患者血清和尿液的核磁共振代谢组学研究

应用基于核磁共振(NMR)的代谢组学方法, 研究甲状腺功能亢进(简称甲亢)患者和健康人群的血清和尿液, 分析甲亢疾病的特征代谢物. 实验收集33个甲亢患者和17个健康志愿者的血清样品以及53个甲亢患者和58个健康志愿者的尿液样品, 采用多元统计分析方法研究甲亢组和对照组血清和尿液中的内源性代谢差异. 结果表明, 甲亢组血清中的胆碱、葡萄糖和三甲胺等物质的含量升高, 而VLDL, LDL和胆固醇等脂质以及乳酸、糖蛋白和丙氨酸等代谢物的含量下降; 甲亢组尿液中的葡萄糖、柠檬酸、牛磺酸以及肌氨酸等代谢物的含量升高, 而马尿酸、TMAO、甲酸和琥珀酸等代谢物的含量下降. 结果表明, 甲亢病不仅影响了糖类、脂类和蛋白质三大物质的代谢, 还对能量代谢、肝肠循环和肠道微生物等多个生理系统产生显著影响, 并且可能造成肝脏及肾脏等器官的损伤.     

小剂量丙泊酚联合氯胺酮镇静对骨科小儿认知功能影响及安全性研究

目的分析应用小剂量丙泊酚(PPF)联合氯胺酮(KTM)镇静麻醉对于骨科患儿认知功能的影响情况并评价镇静治疗用药的安全性。方法将7~12岁的骨科患儿64例随机分成观察组(36例,0.25 mg/kg的PPF+0.25 mg/kg的KTM)和参照组(28例,0.5 mg/kg的PPF+0.5 mg/kg的KTM),在换药镇静前后各12 h采用自制认知功能量表对两组患儿进行评价,并记录、比较镇静后12 h内两组患儿的意识恢复时间、不良反应发生情况。结果认知功能评分方面,两组患儿在换药镇静前无显著差异(P0.05),而在换药镇静后观察组明显高于对照组(P0.05);在安全性上,与对照组相比,观察组患儿的意识恢复时间更短、不良反应发生率更低,且存在显著差异(P0.05)。结论采用小剂量的PPF联合KTM在换药时对骨科患儿进行镇静治疗,可有效减少PPF对患儿神经系统、认知能力的影响及相关不良反应,并促使患儿在镇静治疗后快速恢复意识,值得在骨科临床中应用、实践。     

比格犬血浆中盐酸羟考酮和罗通定GC-MS测定法及其在药代动力学研究中的应用

盐酸羟考酮是阿片类镇痛剂,罗通定具有良好的镇静止痛作用,近年来的研究发现罗通定能抑制羟考酮躯体依赖的形成,提示罗通定与羟考酮等阿片类药物在临床合用可以达到镇痛的协同作用,同时减少成瘾等不良反应.本文建立了同时检测比格犬血浆样品中这两种药物的GC-MS方法,并应用于两药联合用药的药代动力学研究.     

N-乙酰半胱氨酸保护下给药钆-二乙三胺五乙酸后慢性肾衰大鼠的尿液代谢组学研究

N-乙酰半胱氨酸(NAC)有减轻造影剂引发肾损伤的作用,但其作用机制尚未明确.本研究采用基于1H NMR的代谢组学方法,结合正交偏最小二乘法判别分析(OPLS-DA),在NAC保护下对慢性肾衰大鼠给药造影剂钆-二乙三胺五乙酸(Gd-DTPA), 通过分析大鼠尿液中内源性代谢物的变化,研究了NAC对慢性肾衰大鼠的保护机制.结果表明,慢性肾衰大鼠能量代谢、尿素循环等代谢通路发生紊乱.给药Gd-DTPA后,大鼠尿液中胆碱、N-氧三甲胺、邻羟基苯乙酸苯酯、对羟基苯乙酸苯酯、马尿酸、甘氨酸、烟酸、牛磺酸减少,尿囊素增加;而在NAC保护下相关代谢产物向模型组的恢复,说明NAC对Gd-DTPA引发的大鼠肠道细菌代谢、肝线粒体代谢、犬尿氨酸代谢紊乱及氧化损伤具有一定修复作用.NAC对尿素循环代谢的改善可能减轻大鼠体内的肾损伤,而其对细胞中谷胱甘肽的补充可能减轻Gd-DTPA造成的氧化损伤.     

硫酸氧钒毒性的核磁共振代谢组学方法研究

采用基于核磁共振(NMR)的代谢组学方法,结合生化指标分析及组织病理学检测,研究了具有类胰岛素活性的硫酸氧钒(VOSO4)对Wistar大鼠的毒性作用.通过不同剂量的VOSO4对Wistar大鼠连续灌胃给药16d,收集大鼠的血清和尿液,并采集样品的1H NMR谱进行多变量数据统计分析来辨识其特征代谢物,然后采用TICL(a web Tool for automatic Interpretation of Compound List)方法建立特征代谢物的代谢网络模型,分析受影响的主要代谢途径及其相互关系.研究结果表明:高剂量组(45mg/kg)和低剂量组(15mg/kg)的特征代谢物含量与对照组存在明显的差异;与对照组相比,高剂量和低剂量组血清中乳酸、肌氨酸酐以及牛磺酸等代谢物的含量增加,尿液中氧化三甲胺(TMAO)、肌酐、牛磺酸和甘氨酸等代谢物的含量增加,并呈现显著的剂量依赖关系;给药组中乙酸和琥珀酸的含量都降低.这些结果说明VOSO4可能影响大鼠体内的糖代谢、脂类代谢及肠道菌群代谢等多个代谢系统,高剂量的VOSO4会导致肝脏毒性和肾脏损伤.     

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。     

大鼠尿液中壬基酚的代谢轮廓

运用代谢组学方法研究了壬基酚对大鼠尿液代谢的影响, 通过高效液相色谱-飞行时间质谱技术建立了大鼠尿样的代谢指纹图谱, 用主成分分析法分析给药组与对照组代谢物指纹图谱的差异, 通过t检验选取潜在的生物标志物及效应标志物, 并结合代谢物数据库检索对其进行鉴定. 结果表明, 壬基酚给药后, 尿液中含量变化显著的成分苯基葡萄糖苷酸、L-高半胱氨酸、3-硝基丙酸、肌酸酐、左旋肉碱及5-羟色胺等构成了大鼠尿液代谢的轮廓, 它们在生物体内的变化可能对生殖系统、免疫系统、神经系统及脂肪的代谢造成一定的影响, 从代谢的角度解释了环境雌激素壬基酚对生物体的危害, 为毒理学研究提供了依据.     

代谢组学方法研究奶牛注射氯霉素后牛奶中的生物标志物

建立了高效液相色谱-质谱(HPLC-MS)技术研究牛奶代谢组学的方法.样品用乙腈提取,经浓缩富集,采用HPLC-MS技术研究了注射氯霉素后牛奶中内源性代谢物随时间的变化情况.结果表明,注射氯霉素后,牛奶的代谢指纹图谱中8个谱峰的变化随给药及休药时间呈现一定升降规律;采用偏最小二乘判别分析法(PLS-DA)可将给药组与空白组样品完全分开;鉴定了对组间差异贡献较大的化合物为单羟基十八碳二烯酸(HODE),HODE可以作为注射氯霉素后奶牛体内代谢应激产生的内源性生物标志物.     

N-苄基十六碳酰胺促小鼠Leydig细胞增殖和分泌睾酮的1H NMR代谢组学研究

采用基于核磁共振氢谱(¹H NMR)的细胞代谢组学技术探讨了玛咖有效成分N-苄基十六碳酰胺(NBH)促进小鼠Leydig细胞增殖和分泌睾酮的作用机制. 测定了小鼠Leydig细胞在给药前后的细胞增殖率和细胞培养液中的睾酮含量, 采用主成分分析和正交偏最小二乘判别分析, 研究了小鼠Leydig细胞在给药前后细胞破碎液中的代谢物差异, 并进行了代谢通路分析. 实验结果表明, NBH能提高小鼠Leydig细胞增殖率和睾酮分泌量, 给药后小鼠Leydig细胞破碎液中的代谢轮廓明显改变, 共鉴定亮氨酸、 赖氨酸、 鲨肌醇、 缬氨酸和丙氨酸等24种差异代谢物. 经Metaboanalyst分析发现, 差异代谢物主要涉及丙氨酸/天冬氨酸/谷氨酸代谢、 甘氨酸/丝氨酸/苏氨酸代谢、 谷胱甘肽代谢及丙酮酸代谢等10条新陈代谢和遗传信息处理代谢通路, 初步阐明了NBH通过调节上述代谢通路的相关节点发挥促进小鼠Leydig细胞增殖和分泌睾酮作用.     

Interaction of surface-active drugs in rabbits

The interaction of chlorpromazine and promethazine in vivo has been investigated. The drugs were administered to the rabbit orally as a single dose (100 mg of each drug) as well as simultaneously with an interval of 15 min. The presence of multiple peaks at the separate administration of promethazine and chlorpromazine on the one hand, and increase of number of peaks, symbathic character of kinetic curves of mentioned drugs and its prolonged appearance in the systemic circulation of the blood by simultaneous administration on the other hand, may be explained by the intensive presystem metabolism and surface-activity ability of these drugs, and by the periodic 'lassitude' of liver for their capture and elimination (either presystem or systemic). The micelle formation from these drugs in the gastro-intestinal tract and formation of the mixed micelles on simultaneous administration were also taken into consideration. Chlorpromazine is more strongly captured by the liver at its first pass through it than promethazine, from comparison of pharmacokinetics of these drugs administered separately. Therefore, chlorpromazine on simultaneous administration occupies the sites of the liver which were covered by promethazine at single dose, thereby substituting promethazine and promoting its transferral into the systemic blood circulation. This results in a large increase in promethazine content in blood, additional peaks appear and the presence of promethazine in the blood is prolonged. The influence of chlorpromazine on the kinetics of promethazine is especially obvious when chlorpromazine enters the organism first and more easily occupies those sites in the liver which participate in the capture and elimination of both drugs. Concerning influence of promethazine on the kinetics of chlorpromazine, promethazine reinforces in some way the ability of liver to capture chlorpromazine, thereby intensifying the presystem metabolism of chlorpromazine and inhibiting its own metabolism. The analogous effect was observed in the study of the influence of promethazine on the kinetics of carbamazepine.     

Interaction of carbamazepine and promethazine in rabbits

The interaction of carbamazepine and promethazine in rabbits has been investigated. The influence of this interaction on the processes of biotransformation in the liver was revealed. The drugs were administered as single oral doses (100 mg of each drug) as well as simultaneously with an interval of 15 min. The sequence of administration of the drugs was varied. The influence of promethazine on the pharmacokinetics of carbamazepine is expressed by: (a) strong suppression of carbamazepine's level in plasma and appearance of multiple peaks of carbamazepine; (b) suppression of biotransformation of carbamazepine into carbamazepine-10,11-epoxide at the initial stages and its increase in the intermediate stages. These data are explained by the active capture of carbamazepine by liver at its primary transferal through the liver and sufficient presystem elimination of carbamazepine in the presence of promethazine. The character of kinetic curves of promethazine varies substantially under the influence of carbamazepine. However, this change is not as strong as in case of carbamazepine. The concentration of promethazine in plasma varies slightly and multiple peaks are not observed. The rate of terminal elimination of promethazine varies and abrupt prolonged segments of elimination appear at the initial and terminal stages of the process in return. These data perhaps indicate the induction of biotransformation of promethazine in the presence of carbamazepine-an inductor of microsomal liver enzymes. The changes of kinetics of promethazine and carbamazepine by simultaneous administration as compared with their administration separately, as well as a comparative consideration of pharmacokinetics of promethazine and carbamazepine by simultaneous administration show the existence of competition in the elimination between these drugs and the periodic saturation of liver for their biotransformation.     

Effect of caffeine on UVB-induced carcinogenesis, apoptosis, and the elimination of UVB-induced patches of p53 mutant epidermal cells in SKH-1 mice

Oral administration of green tea or caffeine to SKH-1 mice during UVB irradiation for several months inhibited the formation of skin cancer. Similar effects were observed when green tea or caffeine was given to tumor-free UVB-initiated mice with a high risk of developing skin tumors in the absence of further UVB irradiation (high risk mice). Mechanistic studies indicated that topical application of caffeine stimulated UVB-induced apoptosis as well as apoptosis in UVB-induced focal hyperplasia and tumors in tumor-bearing mice. Oral or topical administration of caffeine enhanced the removal of patches of epidermal cells with a mutant form of p53 protein that appeared early during the course of UVB-induced carcinogenesis, and oral administration of caffeine altered the profile of p53 mutations in the patches. In additional studies, topical application of caffeine was shown to have a sunscreen effect, and topical application of caffeine sodium benzoate was more active than caffeine as a sunscreen and for stimulating UVB-induced apoptosis. Caffeine sodium benzoate was also highly active in inhibiting carcinogenesis in UVB-pretreated high risk mice. Our studies indicate that caffeine and caffeine sodium benzoate may be useful as novel inhibitors of sunlight-induced skin cancer.     

固相萃取/超高效液相色谱-串联质谱法测定畜肉中16种镇静剂类兽药残留

建立了畜肉中16种镇静剂类兽药残留的固相萃取净化/超高效液相色谱-串联质谱(LC-MS/MS)的同时测定方法。样品均质后经氢氧化钠溶液水解,加入盐酸溶液提取。提取液经固相萃取柱MCX净化,洗脱液氮气吹干后用流动相溶解,经0.22μm滤膜过滤,采用Waters ACQUITY UPLC BEH C_(18)(2.1 mm×100mm,1.7μm)色谱柱分离,在电喷雾电离源(ESI)和多反应监测(MRM)正离子模式下测定,外标法定量。结果表明:采用基质匹配外标法测定,16种镇静剂类化合物在一定浓度范围内呈良好的线性关系(r~2≥0.996 8),在样品中的检出限和定量下限分别为0.01~0.05μg/kg和0.1~0.5μg/kg,在3个浓度加标水平下的平均回收率为71.6%~112%,相对标准偏差(RSD)为2.9%~15.8%。市售多种鲜肉制品的测定结果表明,该方法选择性好、操作简单快速、结果准确,适用于畜肉中16种镇静剂类兽药残留的同时快速测定。     

咖啡因分子印迹固相萃取柱的制备及应用

以咖啡因作为模板分子,α-甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂,制备了咖啡因分子印迹聚合物(MIP)。与非印迹聚合物(NIP)相比,MIP对咖啡因具有更高的吸附容量和选择性,MIP和NIP对咖啡因的最大静态吸附量分别为28.1和16.5mg/g,相对选择因子为1.25。以咖啡因分子印迹聚合物为固相萃取填料,结合高效液相色谱(HPLC),建立了茶水中咖啡因浓度及人饮茶后血清中咖啡因浓度的检测方法。考察了洗脱剂种类和用量对咖啡因回收率的影响。当萃取柱依次以2mL水活化,水溶液上样,2mL水淋洗,6mL甲醇-乙酸(9∶1,V/V)洗脱,咖啡因在MIP固相萃取柱上的回收率达到97.5%,而在NIP柱上的回收率仅为54.9%。     

A metabolomic study of Asian and American Ginseng based on RRLC-QTOF/MS methods

In order to understand the significant differences in activities and effects between Asian ginseng (ASG) and American ginseng (AMG), it is important to study the correlation between chemical structures and biological activities in the two types of herbs. However, more attention has been paid to the analysis of ginsenosides in previous reports distinguishing between ASG and AMG. There are some other bioactive compounds besides ginsenosides, however, few studies have focused on a systematic comparison of these types of compounds. Two metabolomic methods were developed in this study by qualitative data acquisition using normal phase liquid chromatography mass spectrometry (NPLC-MS) and reverse phase liquid chromatography mass spectrometry (RPLC-MS) respectively, in combination with principal component analysis (PCA). Results show that both NPLC-MS and RPLC-MS-based metabolomic methods are feasible in composition profiling, biomarker screening as well as in discrimination of ASG and AMG. 17 ginsenosides were identified as analytical markers in RPLC-MS-based metabolomic method. In comparison, using NPLC-MS-based method, 5 ginsonosides, two amino acids as well as 1 oligosaccharide were identified as analytical markers. Therefore, RPLC-MS-based metabolomic method exhibits better profiling in ginsenosides, while NPLC-MS-based metabolomic method offers the advantage that multiple active ingredients can be determined simultaneously. The two methods are both helpful in identification of biomarker as well as in discrimination of American ginsengs from Asian ginsengs.     

Studies on the detoxification effects and acute toxicity of a mixture of cis-sec-butyl-1-propoenyl disulphide and trans-sec-butyl-1-propoenyl disulphide isolated from crude essential oil of Ferula sinkiangensis K.M. Shen, a Chinese traditional herbal medicine

There have, so far, been no papers about the detoxification effects on morphine-dependent animals of a mixture of cis-sec-butyl-1-propoenyl disulphide and trans-sec-butyl-1-propoenyl disulphide (SBD), which is isolated from crude essential oil of Ferula sinkiangensis K.M. Shen, a Chinese traditional herbal medicine. Naloxone-precipitated abstinent morphine-dependent models and naturally abstinent morphine-dependent models were used to evaluate the detoxification effects of intraperitoneal injection of SBD. In addition, the writhing test for mice was used to assess the anti-nociceptive effects of SBD, and the spontaneous activity test was used to verify the sedative effects of SBD. Acute toxicity tests of the SBD in mice were performed and the LD?? values were calculated. The results indicated that SBD can inhibit the abstinent syndromes, and that SBD had good anti-nociceptive and sedative effects.     

Comparison of the sedative and hypnotic effects of flavonoids, saponins, and polysaccharides extracted from Semen Ziziphus jujube

Semen Ziziphus jujube (SZJ), the seeds of Ziziphus jujuba Mill. var. spinosa, is a kind of traditional Chinese medicine used for its action on insomnia. In order to analyze the effective component, we investigated and compared the sedative and hypnotic effects of three kinds of compounds, flavonoids, saponins, and polysaccharides. Flavonoids, saponins, and polysaccharides were extracted from SZJ and orally administered to mice separately at 17 g kg(-1) per day for certain days before animal tests. Spontaneous motility and coordinated movement tests were used to observe the effects of the three kinds of compounds on the mouse behavior, and sodium barbital-induced sleeping time of mouse were tested to analyze the effects of the three kinds of compounds on the sleep of mouse. Results show that flavonoids and saponins caused a significant reduction of walking time and coordinated movement ability of mouse, significantly prolonged its sleeping time at 40 mg kg(-1), ip, subthreshold dose and increased the sleeping number of animals at 50 mg kg(-1), ip, superthreshold dose induced by coeliac injection of sodium barbital. Polysaccharides did not show any significance in all animal tests. Comparative analysis showed that saponins had a more effective sedative and hypnotic function than that of flavonoids, polysaccharides did not show a sedative and hypnotic effect.     

Compatibility effects of herb pair Phellodendri chinensis cortex and Anemarrhenae rhizoma on benign prostatic hyperplasia using targeted metabolomics

Phellodendri chinensis cortex (P. C. cortex) and Anemarrhenae rhizoma (A. rhizoma) herb pair is a core component of traditional Chinese medicines used to treat inflammation and benign prostatic hyperplasia (BPH). The present study was designed to profile the arachidonic acid (AA) metabolomic characteristics in rat plasma and prostate after being treated with P. C. cortex and A. rhizoma as well as their combination. Plasma and prostate samples from sham group, BPH model group, herb pair group and two single herb groups were collected on days 7, 14, 21 and 28. Then, a systemic metabolomic analysis based on UFLC‐MS/MS was employed to quantify AA and its cyclooxygenase and lipoxygenase pathway metabolites (15‐HETE, 12‐HETE, 5‐HETE, AA, PGI₂, PGF_2α, 8‐HETE, PGD₂, PGE₂ and LTB₄). The results demonstrated that BPH led a significant increase of 10 biomarkers in plasma and tissue (p < 0.05). The clusters of herb pair group and single herb groups showed a tendency to return to the initial space, and the AA and its metabolites from those groups were differently downregulated to a healthier level, with the combination of single herbs most obvious. The present study demonstrated that P. C. cortex–A. rhizoma herb pair might produce synergistic or complementary compatibility effects on suppressing inflammatory processes occurring in BPH.     

Application of microdialysis for study of caffeine distribution into brain and cerebrospinal fluid in rats.

The usefulness of microdialysis was examined for the chronological determination of caffeine concentration in the brain and cerebrospinal fluids (CSF) following intravenous administration of caffeine in rats. The recovery percent of caffeine by microdialysis, the concentration ratio of caffeine in the dialysate against that in the brain tissue or CSF was determined. The recovery percent was proved to be constant at 5 different steady-state plasma concentrations of caffeine (0.1-280 nmol/ml) and in different collecting periods of dialysate ranging from 30 s to 10 min. The mean recovery percent in the brain and CSF were 10.9 and 13.1%, respectively. Thus, microdialysis was proved useful for determination of drug concentration in the tissue and biological fluids with time resolution of more than 30 s. The microdialysis method was then applied for the chronological determination of caffeine concentration in the brain and CSF following intravenous bolus administration. The estimated caffeine concentration in the brain and CSF was the same as those obtained by direct determination in isolated brain and CSF, respectively. Transfer of caffeine from plasma to brain and CSF were further pharmacokinetically analyzed using a modified 2-compartment model. In this kinetic model, the transfer of caffeine between the CSF and brain was neglected, since the mutual transfer of caffeine was not detected in in vivo experiments. Calculated curves were well fitted on observed caffeine concentrations in the plasma, brain and CSF.