血清miR-21 及AFP 在大鼠肝癌发生过程中的动态表达比较分析

目的:分析血清miR-21在肝癌发生过程中的表达水平并将其与传统肝癌血清标志物甲胎蛋白(AFP)比较,探索其成为肝癌早期诊断血清标志物的可能性。方法:二乙基亚硝胺(DENA)腹腔注射诱导建立大鼠肝癌模型,建模过程中收集造癌各个阶段的血清。Realtime-PCR检测血清miR-21的表达情况,ELISA法检测血清AFP水平。结果:与正常组及纤维化期大鼠相比,miR-21在肝硬化期、肝癌早期、肝癌晚期的大鼠血清均有不同程度上调(P<0.05),AFP在肝癌早、晚期的大鼠血清明显上调(P<0.05);与肝硬化期大鼠比较,肝癌早、晚期大鼠体内的miR-21表达显著上调(P<0.05),AFP在肝癌早期、肝癌晚期均显著上调(P<0.05)。结论:血清miR-21参与了肝癌发生的过程,对于肝癌发生的各个阶段均有很大的指示作用,可能作为肝癌预防和早期诊断的一个潜在标志物。     

Metabolism of echitamine and plumbagin in rats

When administered to rats, echitamine was absorbed rapidly from the tissues and was detected in circulation within 30 min. The drug level reached a maximum value by 2 h and then decreased steadily. The drug had completely disappeared from the blood in 6 h. The presence of echitamine was observed within 2 h in urine and the maximum amount of drug was excreted between 2 and 4 h. About 90% of the drug was excreted in urine in 24 h and the drug could not be detected in urine after 48 h. Along with echitamine, its metabolites were also detected in the urine. Plumbagin was not detected in blood upto 24 h when administered into rats. The drug was detected in urine within 4 h after administration; a major portion of the drug was excreted in urine by 24 h and traces of the drug were observed in the urine even after 48 h.     

Comparative Excretion and Tissue Distribution of Selenium in Mice and Rats Following Treatment with Diphenyl Diselenide

The purpose of this study was to provide data about in vivo tissue distribution and excretion of diphenyl diselenide ((PhSe)₂) in rats and mice through determination of selenium levels in different biological samples. (PhSe)₂ (500?mg/kg, dissolved in canola oil) was administered to animals once a day per oral. After this, mice and rats were housed in metabolic cages (one animal per cage) and urine and feces were collected at specific times after treatment. Three to five animals per group (for each time-point) were anesthetized and blood samples were collected at 0 and 30?min, 24?h, at day 5, 15, and 30 after (PhSe)₂ administration. The plasma and red blood cells were separated. Brain, liver, lungs, kidneys, and adipose tissue were also collected. The determination of selenium levels was performed by inductively coupled plasma atomic emission spectrometry. The main results indicate that: (1) urine is an important route of excretion of selenium originated from (PhSe)₂ in mice and rats; (2) a large amount of (PhSe)₂ or some of its metabolites are stored in fat; (3) the content of selenium found in plasma was low; and (4) liver and kidneys are the tissues with high amounts of selenium.     

The antidiabetic and anticholinergic effects of chrysin on cyclophosphamide‐induced multiple organ toxicity in rats: Pharmacological evaluation of some metabolic enzyme activities

Chrysin (CH) or 5,7‐dihydroxyflavone is a flavonoid present in various plants, bee propolis, and honey. Cyclophosphamide (CYP) is a chemotherapeutic drug, which is extensively used in the treatment of multiple human malignancies. In our study, we aimed to investigate the effects of CYP and CH on some metabolic enzymes including carbonic anhydrase, aldose reductase, paraoxonase‐1, α‐glycosidase, acetylcholinesterase, and butyrylcholinesterase enzyme activities in the brain, heart, testis, liver, and kidney tissues of rats. Thirty‐five adult male Wistar rats were used. The animals were pretreated with CH (25 and 50 mg/kg b.w.) for seven days before administering a single dose of CYP (200 mg/kg b.w.) on the seventh day. In all the tissues, the treatment of CH significantly regulated these enzyme activities in CYP‐induced rats. These results showed that CH exhibited an ameliorative effect against CYP‐induced brain, heart, liver, testis, and kidney toxicity.     

Species differences in the metabolism of norephedrine in man, rabbit and rat

1. (+/-)-2-Amino-1-phenyl[1-(14)C]propan-1-ol ([(14)C]norephedrine) was administered orally to man, rat and rabbit and the metabolites excreted in the urine were identified and measured. Pronounced species differences in the metabolism of the drug were found. 2. Three male human subjects, receiving 25mg each of [(14)C]norephedrine hydrochloride, excreted over 90% of the (14)C in the first day. The main metabolite was the unchanged drug (86% of the dose) and minor metabolites were hippuric acid and 4-hydroxynorephedrine. 3. In rats given 12mg of the drug/kg almost 80% of the (14)C administered was excreted in the first day. The major metabolites in the urine were the unchanged drug (48% of the dose), 4-hydroxynorephedrine (28%) and trace amounts of side-chain degradation products. 4. Rabbits given 12mg of the drug/kg excreted 85-95% of the dose of (14)C in the urine in the first 24h after dosing. The major metabolites in the urine were conjugates of 1,2-dihydroxy-1-phenylpropane (31% of the dose) and of 1-hydroxy-1-phenylpropan-2-one (27%) and hippuric acid (20%). The unchanged drug was excreted in relatively small amounts (8%).     

Stereoselective disposition of hydroxychloroquine and its metabolites in rats

Racemic hydroxychloroquine-sulfate (HCQ-sulfate) was administered to rats orally. Groups of 9 male and 9 female rats received doses of 0, 8, 16, or 24 mg/kg/day for 6 weeks, followed by a reduction of the higher doses to 8 mg/kg/day for the duration of the study. Whole blood samples were collected at 0, 3, 6, 8, and 10 weeks, and eleven tissues were harvested after the tenth week. The concentrations and enantiomer ratios of the parent drug and three metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ), were determined. The highest concentration of HCQ was found in the intestinal smooth muscle, and the lowest in the brain and adipose tissue. The highest concentrations of the metabolites were found in the liver, adrenals, and lung tissue. The metabolism of HCQ in the rats was found to be stereoselective with R/S > 1 for the drug and < 1 for the metabolites. Gender-specific differences in the proportions of the drug and its metabolites and their enantiomers in blood and tissue were found. Varying dosages appeared to have only a temporary influence on blood concentrations and not to effect the enantiomer ratios in blood. Only a limited number of tissues exhibited significant differences between dose groups. There were no observed differences in enantiomer ratios among the blood collection times. © 1995 Wiley-Liss, Inc.     

Studies of the metabolism of the antihistaminic drug dimethindene by high-performance liquid chromatography and capillary electrophoresis including enantioselective aspects

A reversed-phase high-performance liquid chromatography method for the determination of dimethindene and its main metabolites N-demethyldimethindene, 6-hydroxydimethindene and 6-hydroxy-N-demethyldimethindene in human urine was developed. The assay was also applied to the quantification of dimethindene-N-oxide in rat urine. Conjugates of the hydroxylated metabolites were determined after enzymatic deconjugation. Moreover the direct determination of dimethindene and its metabolites without prior extraction from urine was performed by capillary electrophoresis. The direct simultaneous determination of the enantiomers of dimethindene and N-demethyldimethindene was achieved on a Chiralcel OD column. Urinary data after oral administration of dimethindene are presented. The assays were used to study dimethindene and it metabolites in urine upon oral administration of the drug to rats and human volunteers.     

小鼠MKP4基因组织、细胞表达谱研究

为了探讨MKP4基因mRNA在正常雄性C57小鼠的组织表达分布及其在3T3-L1脂肪细胞诱导进程中的表达谱。采用经典诱导分化方案诱导3T3-L1前脂肪细胞,分别收集前脂肪细胞、诱导分化day0、day2、day5、day8和day9的细胞,同时取正常C57雄性小鼠(鼠龄12周,数量n=3)多处组织如心、胰、肝、脂肪、脑、肾及睾丸等,并提取组织、细胞总RNA,RT-QPCR(荧光染料法)检测MKP4基因mRNA表达水平。结果显示MKP4基因在C57小鼠体内存在较广的表达谱,在肝脏、脂肪的表达量最高,其次为脑组织,其它组织存在低度表达;在3T3-L1前脂肪细胞、诱导分化day0、day2中表达极低,在day5中表达骤然升高,随后表达相对增高,于day9表达水平趋于平稳。MKP4基因组织表达谱结果表明其在维持正常生理功能可能发挥一定的作用,在胰岛素敏感相关组织肝脏、脂肪中表达量较高,提示可能参与胰岛素抵抗(IR)的发生与发展过程。此外,MKP4在3T3-L1前脂肪细胞诱导进程中表达上调,表明MKP4可能参与了脂质积累、肥胖等代谢疾病的发生与发展。     

Distribution and breakdown of labeled coenzyme Q10 in rat

Radioactive coenzyme Q(10) ([(3)H]CoQ) was synthesized in a way that the metabolites produced retained the radioactivity. Administration of the lipid to rats intraperitoneally resulted in an efficient uptake into the circulation, with high concentrations found in spleen, liver, and white blood cells; lower concentrations in adrenals, ovaries, thymus, and heart; and practically no uptake in kidney, muscle, and brain. In liver homogenate most [(3)H]CoQ appeared in the organelles, but it was also present in the cytosol and transport vesicles. Mitochondria, purified on a metrizamide gradient, had a very low concentration of [(3)H]CoQ, which was mainly present in the lysosomes. All organs that took up the labeled lipid also contained water-soluble metabolites. The majority of metabolites excreted through the kidney and appeared in the urine. Some metabolites were also present in the feces, which further contained nonmetabolized [(3)H]CoQ, excreted through the bile. The major metabolites were purified from the urine, and the mass spectrometric fragmentation showed that these compounds, containing the ring with a short side chain, are phosphorylated. Thus, the results demonstrate that CoQ is metabolized in all tissues, the metabolites are phosphorylated in the cells, transported in the blood to the kidney, and excreted into the urine.     

Oral administration of paclitaxel affects the distribution and metabolism of 2-aminofluorene in various tissues of Sprague-Dawley rats

To evaluate the question of whether or not paclitaxel affects the distribution and metabolism of chemical carcinogens such as 2-aminofluorene (AF) on Sprague-Dawley rats were examined. The AF, acetylated AF and AF metabolites were determined and examined by using high performance liquid chromatography. After having received AF only, AF with paclitaxel at the same time and paclitaxel pretreated for 24 h then treated with AF for 24 h, urine, stool and tissues such as liver, kidneys, stomach, colon, bladder and blood were collected and assayed for AF and its metabolites. Compared to the control group, paclitaxel caused an increase of the metabolites excreted in urine and stool. The major metabolite excreted in urine and stool was 9-OH-AAF. The liver is the major metabolism center and the major residual metabolite of AF in the liver was also 9-OH-AAF.     

Gene expression profiles in genetically different mice infected with Toxoplasma gondii: ALDH1A2, BEX2, EGR2, CCL3 and PLAU

Toxoplasma gondii can modulate host cell gene expression; however, determining gene expression levels in intermediate hosts after T. gondii infection is not known much. We selected 5 genes (ALDH1A2, BEX2, CCL3, EGR2 and PLAU) and compared the mRNA expression levels in the spleen, liver, lung and small intestine of genetically different mice infected with T. gondii. ALDH1A2 mRNA expressions of both mouse strains were markedly increased at day 1-4 postinfection (PI) and then decreased, and its expressions in the spleen and lung were significantly higher in C57BL/6 mice than those of BALB/c mice. BEX2 and CCR3 mRNA expressions of both mouse strains were significantly increased from day 7 PI and peaked at day 15-30 PI (P<0.05), especially high in the spleen liver or small intestine of C57BL/6 mice. EGR2 and PLAU mRNA expressions of both mouse strains were significantly increased after infection, especially high in the spleen and liver. However, their expression patterns were varied depending on the tissue and mouse strain. Taken together, T. gondii-susceptible C57BL/6 mice expressed higher levels of these 5 genes than did T. gondii-resistant BALB/c mice, particularly in the spleen and liver. And ALDH1A2 and PLAU expressions were increased acutely, whereas BEX2, CCL3 and EGR2 expressions were increased lately. Thus, these demonstrate that host genetic factors exert a strong impact on the expression of these 5 genes and their expression patterns were varied depending on the gene or tissue.     

Identification of potassium dehydroandrographolidi succinas and its major metabolites in rat urine by liquid chromatography-tandem mass spectrometry

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for identification of potassium dehydroandrographolidi succinas and its metabolites in rat urine. Five male rats were administrated a single dose (100 mg/kg) of potassium dehydroandrographolidi succinas by i.v. injection. The urine were sampled from 0 to 24 h and purified by using Oasis? HLB extraction cartridge, then the purified urine samples were separated on a reversed-phase C18 column with a linear gradient and detected by an on-line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular mass (Δm) and MS/MS spectra with those of the parent drug. Seven metabolites and the parent drug were found in rat urine. All these metabolites were reported for the first time.     

Metabolism of [14C]methamphetamine in man, the guinea pig and the rat

1. The metabolites of (+/-)-2-methylamino-1-phenyl[1-(14)C]propane ([(14)C]methamphetamine) in urine were examined in man, rat and guinea pig. 2. In two male human subjects receiving the drug orally (20mg per person) about 90% of the (14)C was excreted in the urine in 4 days. The urine of the first day was examined for metabolites, and the main metabolites were the unchanged drug (22% of the dose) and 4-hydroxymethamphetamine (15%). Minor metabolites were hippuric acid, norephedrine, 4-hydroxyamphetamine, 4-hydroxynorephedrine and an acid-labile precursor of benzyl methyl ketone. 3. In the rat some 82% of the dose of (14)C (45mg/kg) was excreted in the urine and 2-3% in the faeces in 3-4 days. In 2 days the main metabolites in the urine were 4-hydroxymethamphetamine (31% of dose), 4-hydroxynorephedrine (16%) and unchanged drug (11%). Minor metabolites were amphetamine, 4-hydroxyamphetamine and benzoic acid. 4. The guinea pig was injected intraperitoneally with the drug at two doses, 10 and 45mg/kg. In both cases nearly 90% of the (14)C was excreted, mainly in the urine after the lower dose, but in the urine (69%) and faeces (18%) after the higher dose. The main metabolites in the guinea pig were benzoic acid and its conjugates. Minor metabolites were unchanged drug, amphetamine, norephedrine, an acid-labile precursor of benzyl methyl ketone and an unknown weakly acidic metabolite. The output of norephedrine was dose-dependent, being about 19% on the higher dose and about 1% on the lower dose. 5. Marked species differences in the metabolism of methamphetamine were observed. The main reaction in the rat was aromatic hydroxylation, in the guinea pig demethylation and deamination, whereas in man much of the drug, possibly one-half, was excreted unchanged.     

PTEN在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨存检测肿瘤抑制基因PTEN(phosphatase andtensinhomologdeletedonchromosometen)在早孕小鼠子宫内膜中的表达规律,探讨PTEN在小鼠胚胎着床过程中的作用.采用实时荧光定量聚合酶联反应(real.time fluorescent quantitative PCR.FQ.PCR)和免疫组织化学方法分别检测未孕及孕1、3、4、5、7 d小鼠子宫内膜PTEN mRNA和蛋白的表达;子宫角注射PTEN反义寡核苷酸观察胚泡着床数.FQ-PCR结果显示,妊娠小鼠子宫内膜组织PTENmRNA的表达高于未妊娠小鼠,且随着妊娠天数的增加表达逐渐增强,到妊娠第5天达最高.免疫组织化学分析显示,PTEN蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射PTEN反义寡核苷酸后胚泡着床数明显减少.结果提示,PTEN在妊娠早期子宫内膜持续表达,可能参与了胚泡着床.     

Differences in inhibitory avoidance,cortisol and brain gene expression in TL and AB zebrafish

Recently, we established an inhibitory avoidance paradigm in Tupfel Long‐Fin (TL) zebrafish. Here, we compared task performance of TL fish and fish from the AB strain; another widely used strain and shown to differ genetically and behaviourally from TL fish. Whole‐body cortisol and telencephalic gene expression related to stress, anxiety and fear were measured before and 2 h post‐task. Inhibitory avoidance was assessed in a 3‐day paradigm: fish learn to avoid swimming from a white to a black compartment where a 3V‐shock is given: day 1 (first shock), day 2 (second shock) and day 3 (no shock, sampling). Tupfel Long‐Fin fish rapidly learned to avoid the black compartment and showed an increase in avoidance‐related spatial behaviour in the white compartment across days. In contrast, AB fish showed no inhibitory avoidance learning. AB fish had higher basal cortisol levels and expression levels of stress‐axis related genes than TL fish. Tupfel Long‐Fin fish showed post‐task learning‐related changes in cortisol and gene expression levels, but these responses were not seen in AB fish. We conclude that AB fish show higher cortisol levels and no inhibitory avoidance than TL fish. The differential learning responses of these Danio strains may unmask genetically defined risks for stress‐related disorders.     

Kinetics of 8-2 fluorotelomer alcohol and its metabolites, and liver glutathione status following daily oral dosing for 45 days in male and female rats

Fluorotelomer alcohols (FTOHs) are raw materials used in the manufacture of polymeric and surfactant products. Based on previous findings from single oral dosing in rats with radiolabeled 8-2 FTOH, glutathione (GSH) depletion and/or the presence of perfluorinated/polyfluorinated acids and aldehyde metabolites was hypothesized to account for the hepatocellular lesions observed in male rats from a 90-day subchronic oral dosing study. Further, the reported nephropathy in female rats from the subchronic experiment was hypothesized to have been initiated by a thiol metabolite produced by degradation of GSH conjugates. In the current investigation, the kinetics of 8-2 FTOH and its metabolites along with liver GSH status were evaluated in the rat following daily oral dosing with 8-2 FTOH for 45 days at 5 and 125 mg/kg/day. Liver GSH stores 1-2 h after dosing were unaffected, suggesting that GSH depletion is not likely a relevant mode of action in the liver. The tissue metabolite data indicate that the liver toxicity mode of action is likely associated with elevated levels of perfluoroalkyl acids found in males, since other polyfluorinated metabolites and 8-2 FTOH were present in livers from female rats at comparable or higher levels. Detection of the N-acetyl cysteine conjugate of the unsaturated parent telomer alcohol in urine from female rats and not male rats provides some evidence to support the mechanistic basis for the observed kidney effects. Further, the increasing levels of perfluorooctanoic acid (PFOA) in plasma from female rats over the 45-day dosing phase, while unexpected, may reflect an increased net absorption of 8-2 FTOH, slow elimination of intermediates in the metabolic pathway between 8-2 FTOH and PFOA, or altered kidney clearance. The results of this study have enhanced our understanding of 8-2 FTOH kinetics and metabolism and potential modes of action in the rat, which will guide the design of future studies for FTOHs and our need to define the mechanistic basis for the observed effects.