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Metabotropic glutamate receptor subtype 1 (mGluR1) is a crucial target in the development of new medications to treat central nervous system (CNS) disorders. Recently, we developed N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-4-[11C]methoxy-N-methyl-benzamide ([11C]ITMM) as a useful positron emission tomography (PET) probe for mGluR1 in clinical studies. Here, we aimed to improve visualization and threshold of specific binding for mGluR1 using [11C]ITMM with ultra-high specific activity (SA) of > 3,500 GBq/μmol in rat brains. A two-tissue compartment model indicated large differences between the two SAs in the constants k3 and k4, representing binding ability for mGluR1, while constants K1 and k2 showed no differences. The total distribution volume (VT) values of conventional and ultra-high SA were 9.1 and 11.2 in the thalamus, 7.7 and 9.7 in the striatum, and 6.4 and 8.5 mL/cm3 in the substantia nigra, respectively. The specific binding of [11C]ITMM with ultra-high SA was significantly higher than the conventional SA, especially in the basal ganglia. Parametric PET images scaled with VT of the ultra-high SA clearly identified regional differences in the rat brain. In conclusion, PET studies using [11C]ITMM with ultra-high SA could sufficiently improve visualization and specific binding for mGluR1, which could help further understanding for mGluR1 functions in CNS disorders. [ABSTRACT FROM AUTHOR]

The aim of this study is to investigate the effects of subchronic exposure to chlorpyrifos on reproductive toxicology of male rats. Forty healthy male rats were divided into four groups: three exposure groups and a control group. Chlorpyrifos was administered orally to male rats at 0, 2.7, 5.4, and 12.8mg/kg for 90 days to evaluate the toxic alterations in testicular histology, testicular marker enzyme activities and related genes expression levels, sperm dynamics, and testosterone levels. The body weight and the testis weight of animals did not show any significant changes. Chlorpyrifos brought about marked reduction in testicular sperm counts, sperm motility, and significant growth of sperm malformation rate in exposed males. Histopathological examination of testes showed mild to severe degenerative changes in seminiferous tubules at various dose levels. The levels of testosterone (T) showed a decreasing tendency, and there was a statistical difference between the 5.4, 12.8 mg/kg groups, and the control group. The levels of follicle stimulating hormone (FSH) were significantly increased in 5.4 and 12.8 mg/kg groups, but there were no obvious effects on the levels of luteinizing hormone (LH) and estradiol (E2). A significant increase in the activities of LDH and LDH-x was observed in chlorpyrifos exposed rats in 5.4 and12.8 mg/kg groups, but the expression levels of related genes had no significant differences between chlorpyrifos exposure groups and the control group. These results suggest that chlorpyrifos has adverse effects on reproductive system of male rats. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1083-1088, 2014. [ABSTRACT FROM AUTHOR]

We developed the novel positron emission tomography (PET) ligand 2-[5-(4-[11C]methoxyphenyl)-2-oxo-1,3-benzoxazol-3( 2H)-yl]- N-methyl- N-phenylacetamide ([11C]MBMP) for translocator protein (18 kDa, TSPO) imaging and evaluated its efficacy in ischemic rat brains. [11C]MBMP was synthesized by reacting desmethyl precursor ( 1) with [11C]CH3I in radiochemical purity of ≥ 98% and specific activity of 85 ± 30 GBq/μmol ( n = 18) at the end of synthesis. Biodistribution study on mice showed high accumulation of radioactivity in the TSPO-rich organs, e.g., the lungs, heart, kidneys, and adrenal glands. The metabolite analysis in mice brain homogenate showed 80.1 ± 2.7% intact [11C]MBMP at 60 min after injection. To determine the specific binding of [11C]MBMP with TSPO in the brain, in vitro autoradiography and PET studies were performed in an ischemic rat model. In vitro autoradiography indicated significantly increased binding on the ipsilateral side compared with that on the contralateral side of ischemic rat brains. This result was supported firmly by the contrast of radioactivity between the ipsilateral and contralateral sides in PET images. Displacement experiments with unlabelled MBMP or PK11195 minimized the difference in uptake between the two sides. In summary, [11C]MBMP is a potential PET imaging agent for TSPO and, consequently, for the up-regulation of microglia during neuroinflammation. [ABSTRACT FROM AUTHOR]

Purpose: Clinical studies suggest a correlation between changes in activity of the contralesional cerebral cortex and spontaneous recovery from stroke, but whether this is a causal relationship is uncertain. Methods: Young adult Sprague-Dawley male rats underwent unilateral or bilateral permanent distal middle cerebral artery occlusion (dMCAO). Infarct volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 hr after dMCAO, and functional outcome was assessed 1-28 days after dMCAO using the ladder rung walking and limb placing tests. Results: Infarct volume was unchanged, but functional neurological deficits were reduced 1 day after bilateral compared to unilateral dMCAO. Conclusions: Activity in the contralesional cerebral cortex may inhibit functional motor recovery after experimental stroke. [ABSTRACT FROM AUTHOR]

Physical and chemical constraints imposed by the periinfarct glial scar may contribute to the limited clinical improvement often observed after ischémie brain injury. To investigate the role of some of these mediators in outcome from cerebral ischemia, we treated rats with the growth-inhibitory chondroitin sulfate proteoglycan neurocan, the growth-stimulating heparan sulfate proteoglycan glypican, or the chondroitin sulfate proteoglycandegrading enzyme chondroitinase ABC. Neurocan, glypican, or chondroitinase ABC was infused directly into the infarct cavity for 7 d, beginning 7 d after middle cerebral artery occlusion. Glypican and chondroitinase ABC reduced glial fibrillary acidic protein immunoreactivity and increased microtubule-associated protein-2 immunoreactivity in the periinfarct region, and glypican- and chondroitinase ABC-treated rats showed behavioral improvement compared with neurocan- or saline-treated rats. Glypican and chondroitinase ABC also increased neurite extension in cortical neuron cultures. Glypican increased fibroblast growth factor-2 expression and chondroitinase ABC increased brain-derived neurotrophic factor expression in these cultures, whereas no such effects were seen following neurocan treatment. Thus, treatment with glypican or enzymatic disruption of neurocan with chondroitinase ABC improves gross anatomical, histological, and functional outcome in the chronic phase of experimental stroke in rats. Changes in growth factor expression and neuritogenesis may help to mediate these effects. [ABSTRACT FROM AUTHOR]

Aim:To investigate the changes of expression and function of P-glycoprotein (P-GP) in cerebral cortex, hippocampus, liver, intestinal mucosa and kidney of streptozocin-induced diabetic rats.Methods:Diabetic rats were prepared via a single dose of streptozocin (65 mg/kg, ip). Abcb1/P-GP mRNA and protein expression levels in tissues were evaluated using quantitative real time polymerase chain reaction (QRT-PCR) analysis and Western blot, respectively. P-GP function was investigated via measuring tissue-to-plasma concentration ratios and body fluid excretion percentages of rhodamine 123.Results:In 5- and 8-week diabetic rats, Abcb1a mRNA levels were significantly decreased in cerebral cortices and intestinal mucosa, but dramatically increased in hippocampus and kidney. In liver, the level was increased in 5-week diabetic rats, and decreased in 8-week diabetic rats. Abcb1b mRNA levels were increased in cerebral cortex, hippocampus and kidney, but reduced in liver and intestinal mucosa in the diabetic rats. Western blot results were in accordance with the alterations of Abcb1a mRNA levels in most tissues examined. P-GP activity was markedly decreased in most tissues of diabetic rats, except kidney tissues.Conclusion:Alterations in the expression and function of Abcb1/P-GP under diabetic conditions are tissue specific, Abcb1 specific and diabetic duration-dependent. [ABSTRACT FROM AUTHOR]

Abstract: Little is known about the relationship between neuronal cell transplantation and endogenous neurogenesis after experimental stroke. We found previously that transplantation of neuronal precursors derived from BG01 human embryonic stem cells reduced infarct volume and improved behavioral outcome after distal middle cerebral artery occlusion (MCAO) in rats. In this study, transplantation was performed 14days after distal MCAO and doublecortin (Dcx)-expressing cells in the subventricular zone (SVZ) and subgranular zone of dentate gyrus (SGZ) were counted 60days post-transplant. Transplantation increased neurogenesis (Dcx expression) in ipsilateral SVZ, but not in contralateral SVZ or either SGZ, in both young adult (3-month-old) and aged (24-month-old) rats. These findings suggest that cell-based therapy for stroke may be associated with changes in endogenous adaptive processes, including neurogenesis. [Copyright &y& Elsevier]

Neuroglobin (Ngb) is an intracellular, oxygen-binding neuronal protein with protective effects against ischemia and related pathological processes. To identify small molecules capable of inducing Ngb protein expression, which might have therapeutic benefit, we examined Ngb expression by Western blot in cultured HN33 (mouse hippocampal neuron ! N18TG2 neuroblastoma) cells. In addition to deferoxamine, which was shown previously to enhance Ngb levels, Ngb expression was increased by the short-chain fatty acids cinnamic acid and valproic acid (≥100 μmol/l), but not by other short-chain fatty acids, histone deacetylase inhibitors, or anticonvulsants. Drugs that stimulate the expression of neuroprotective proteins like Ngb may have therapeutic potential in the treatment of stroke and other neurological disorders. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background and purpose: The multidrug resistance of epilepsy may result from the overexpression of P-glycoprotein, but the mechanisms are unclear. We investigated whether the overexpression of P-glycoprotein in the brains of subjects with pharmacoresistant epilepsy resulted from both drug effects and seizure activity. Experimental approach: Kindled rats were developed by injecting a subconvulsive dose of pentylenetetrazole (33 mg·kg−1·day−1, i.p.) for 28 days. Groups were then treated with an oral dose of phenobarbital (45 mg·kg−1·day−1) for 40 days. In accord with behavioural observations, P-glycoprotein activity in brain was assessed using brain-to-plasma concentration ratios of rhodamine 123. P-glycoprotein levels in the brain regions were further evaluated using RT-PCR and Western blot analysis. The distribution of phenobarbital in the brain was assessed by measuring phenobarbital concentrations 1 h following its oral administration. Key results: The kindling significantly increased P-glycoprotein activity and expression. Good associations were found among P-glycoprotein activity, expression and phenobarbital concentration in the hippocampus. Short-term treatment with phenobarbital showed good anti-epileptic effect; the maximum effect occurred on day 14 when overexpression of P-glycoprotein was reversed. Continuous treatment with phenobarbital had a gradually reduced anti-epileptic effect and on day 40, phenobarbital exhibited no anti-epileptic effect; this was accompanied by both a re-enhancement of P-glycoprotein expression and decreased phenobarbital concentration in the hippocampus. P-glycoprotein function and expression were also increased in age-matched normal rats treated with phenobarbital. Conclusions and implications: The overexpression of P-glycoprotein in the brain of subjects with pharmacoresistant epilepsy is due to a combination of drug effects and epileptic seizures. [ABSTRACT FROM AUTHOR]

Transplantation of neural cells is a potential approach for stroke treatment, but disruption of tissue architecture may limit transplant efficacy. One strategy for enhancing the ability of transplants to restore brain structure and function is to administer cells together with biomaterial scaffolding. We electrocoagulated the distal middle cerebral artery in adult rats and, 3 weeks later, injected one of the following into the infarct cavity: artificial cerebrospinal fluid, Matrigel scaffolding, human embryonic stem cell-derived neuronal precursor cells, scaffolding plus cells, or cells cultured in and administered together with scaffolding. Five weeks after transplantation, the latter two groups showed ∼50% and ∼60% reductions, respectively, in infarct cavity volume. Rats given cells cultured in and administered together with scaffolding also showed (1) survival and neuronal differentiation of transplanted cells shown by immunostaining for neuronal marker proteins and cleaved caspase-3, and by patch-clamp recording, 8 weeks after transplantation and (2) improved outcome on tests of sensorimotor and cognitive functions, 4 to 9 weeks after transplantation. These results indicate that transplantation of human neural cells together with biomaterial scaffolding has the potential to improve the outcome from stroke, even when treatment is delayed for several weeks after the ischemic event. [ABSTRACT FROM AUTHOR]

Abstract: Basic fibroblast growth factor (FGF-2) has been reported to protect against ischemic injury in the brains of young adult rodents. However, little is known about whether FGF-2 retains this capability in the aged ischemic brain. Since stroke in human is much more common in older people than among younger adults, to address this question is clinically important. In this study, aged (24-month-old) rats were treated with intracerebroventricular infusion of FGF-2 or vehicle for 3 days, beginning 48 h before (pre-ischemia), 24 h after (early post-ischemia), or 96 h after (late post-ischemia) 60 min of middle cerebral artery occlusion, and were killed 10 days after ischemia. Aged rats given FGF-2 pre-ischemia showed better symmetry of movement and forepaw outstretching, and reduced infarct volumes, compared to rats treated with vehicle, but no significant improvement was found in aged rats given FGF-2 after focal ischemia. In contrast, young adult (3-month-old) rats treated with FGF-2 for 3 days beginning 24 h post-ischemia showed significant neurobehavioral improvement and better histological outcome. In addition, we also found that newborn neurons in the rostral subventricular zone (SVZ) were increased in aged rats treated with FGF-2 prior to ischemia. However, unlike in young adult ischemic rats, only a few of newly generated cells migrated into the damaged region in aged brain after focal ischemia. These findings point to differences in the response of aged versus young adult rats to FGF-2 in cerebral ischemia, and suggest that such differences need to be considered in the development of neuroprotective agents for stroke. [Copyright &y& Elsevier]

Diabetic retinopathy (DR) is a key reason for legal blindness worldwide. Currently, it is urgently necessary to determine the etiology and pathological molecular mechanism of DR to search for resultful therapies. Dickkopf-1 (DKK1) is inhibitive for canonical Wnt signaling via negative feedback, and has been reported as a biomarker for DR. However, the related mechanisms are still unclear. In this work, our data showed that DKK1 was decreased in the vitreous tissues at an early stage of diabetes triggered by streptozotocin (STZ) injection in rats. We subsequently found that DKK1 intravitreal injection significantly ameliorated the physiological function of retina in STZ-challenged rats, accompanied by improved retinal structure. Surprisingly, our results indicated that DKK1 injection remarkably suppressed PANoptosis in retinal tissues of STZ-challenged rats with DR, as proved by ameliorated pyroptosis, apoptosis and necroptosis, which were mainly through the blockage of cleaved Gasdermin-D (GSDMD), Caspase-3 and receptor-interacting protein kinase-3 (RIPK3). Additionally, Wnt signaling including the expression of Wnt, β-catenin and LDL receptor-related protein 5/6 (LRP5/6) was also highly prohibited in retina of DKK1-injected rats with DR. Furthermore, retinal neovascularization and acellular vessel in DR rats were also considerably abolished after DKK1 injection, accompanied by reduced expression levels of retinal vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9). More in vitro experiments showed that DKK1 treatment markedly repressed the proliferative and migratory ability of endothelial cells via inhibiting angiogenesis-related molecules. Together, all our results broaden the knowledge of the correlation between DKK1 and DR, and then provide a novel therapeutic strategy for the suppression of management of DR. • DKK1 ameliorates diabetic retinopathy in STZ-treated rats. • DKK1 restrains PANoptosis and Wnt signaling activation in the retinas of STZ-challenged rats. • DKK1 attenuates retinal neovascularization in STZ rats. • DKK1 inhibits cell proliferation and migration in HRMVECs in vitro. [ABSTRACT FROM AUTHOR]

Notoginsenosides, the main active gradients of Chinese traditional medicine Panax notoginseng, possesses a variety of biological activities including antioxidant property, anti-hyperglycemic, anti-obese, etc . However, pharmacokinetic evaluation for notoginsenosides is still a formidable task due to their low concentrations and complex components in vivo . The summation of this work generated a rapid and sensitive method for quantitative analysis of multi-notoginsenoside in rat plasma based on ultra fast liquid chromatographic-tandem mass spectrometric. After liquid–liquid extraction by n -butanol, notoginsenoside R1, Rg3, Rd, Rg2, Rb2, Rf, Rg1, Rb1 and Re were simultaneously monitored in negative ionization mode after separating on a Thermo ODS C 18 column (5 mm 50 mm × 2.1 mm) by a binary gradient elution, and all compounds were analyzed within 9 min. Multiple reaction monitoring (MRM) was performed as follows: R1 ( m / z 967.7 → 637.4), Rg3 ( m / z 819.6 → 621.4), Rd ( m/z 981.6 → 783.5), Rg2 ( m / z 819.6 → 475.4), Rb2 ( m / z 1113.4 → 783.4), Rf ( m / z 835.6 → 475.4), Rg1 ( m / z 835.6 → 637.6), Rb1 ( m / z 1143.7 → 945.6), Re ( m / z 981.6 → 637.4), internal standard (digoxin, m / z 815.5 → 779.4). Validation parameters (linearity, sensitivity, intra-and inter-assay precision and accuracy, recovery and matrix effect) were within acceptable ranges and biological extracts were stable during the entire storing and preparing process. This UFLC–MS/MS approach was further validated by being applied to the pharmacokinetic study for P. Notoginseng extract in rats, and the pharmacokinetic parameters were calculated by Winolin software. Thus, the presently developed methodology was simple, robust, accurate, precise, and would be useful for the pharmacokinetic studies for all kinds of notoginsenosides and other herbal saponins. [ABSTRACT FROM AUTHOR]

Abstract: 1-[2-(4-Methoxyphenyl)phenyl]piperazine (4) is a potent serotonin 5-HT7 receptor antagonist (K i =2.6nM) with a low binding affinity for the 5-HT1A receptor (K i =476nM). As a potential positron emission tomography (PET) radiotracer for the 5-HT7 receptor, [11C]4 was synthesized at high radiochemical yield and specific activity, by O-[11C]methylation of 2′-(piperazin-1-yl)-[1,1′-biphenyl]-4-ol (6) with [11C]methyl iodide. Autoradiography revealed that [11C]4 showed in vitro specific binding with 5-HT7 in the rat brain regions, such as the thalamus which is a region with high 5-HT7 expression. Metabolite analysis indicated that intact [11C]4 in the brain exceeded 90% of the radioactive components at 15min after the radiotracer injection, although two radiolabeled metabolites were found in the rat plasma. The PET study of rats showed moderated uptake of [11C]4 in the brain (1.2SUV), but no significant regional difference in radioactivity in the brain. Pretreatment with 5-HT7-selective antagonist SB269970 (3) did not decrease the uptake of [11C]4 in the rat brain. Further studies are warranted that focus on the development of PET ligand candidates with higher binding affinity for 5-HT7 and higher in vivo stability in brain than 4. [Copyright &y& Elsevier]

The aim of this study is to determine whether Sertoli cells are involved in spermatogenic failure induced by carbendazim, a benzimidazole fungicide that is widely used in agriculture. Total 40 rats were treated with carbendazim by oral gavage at dosages of 0, 20, 100 and 200mg/kg for 60 days. The testis weight, sperm counts, sperm motility and Sertoli cell morphology and secretions including vimentin, ABP, inhibin B, SCFs and SCFm in testis were examined. In 100 and 200mg/kg groups, the testis weight, the sperm counts and motility and SCFm levels were significantly decreased, the sloughing of germ cells and ABP levels were increased, and the vimentin filaments and Sertoli cell morphology were altered. Inhibin B and SCFs levels were unchanged. The results suggested that alterations of Sertoli cell morphology and function were involved in spermatogenic failure induced by carbendazim. [Copyright &y& Elsevier]

Background: It has been suggested that pregnancy-related hormones play a critical role in mediating selective immune tolerance during pregnancy. An understanding of why a woman''s body normally does not reject the fetus is highly relevant to the prevention of transplant rejection. Methods: The hearts of female inbred F344 rats (RT-1 lvl ) were transplanted into naive Lewis (RT-1 l ; nLewis) or pregnant (pLewis; Day 6, 12 and 18 of pregnancy) rats. The mean survival time (MST) of the cardiac allografts between the nLewis and pLewis rats was compared. We determined the rate of proliferation of the T cells isolated from nLewis and pLewis rats in response to concanavalin A (ConA), anti-CD3 and -CD28 antibody and alloantigen stimulation ex vivo. mRNA expression of several cytokines in these T cells was analyzed using quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). In addition, the effect of estriol on the cardiac allograft was tested. Results: The pLewis rats with transplant on Day 12 of pregnancy had the most significantly prolonged F344 cardiac graft survival (MST 13.3 days) as compared with nLewis recipients (MST 8 days). pLewis T-cell proliferation was stimulated by alloantigen and antibody but ConA was reduced, whereas Th1/Th2 cytokine mRNA profiles in the T cells were similar for nLewis and pLewis rats. Likewise, estriol also significantly prolonged survival of cardiac allografts. Conclusions: The results of this study demonstrate that pregnancy hormones not only appear to play a critical role in maternal acceptance of the fetus, but also have therapeutic potential for prolonging the survival of major histocompatibility complex (MHC)-compatible allografts during pregnancy. [Copyright &y& Elsevier]

The purpose of this study was to develop three new radiotracers, 1-(cyclopropylmethyl)-4-([ 11 C/ 18 F]substituted-phenyl)piperidin-1-yl-2-oxo-1,2-dihydropyridine-3-carbonitrile ([ 11 C] 1 , [ 11 C] 2 , and [ 18 F] 4 ), and to examine their specific bindings with metabotropic glutamate receptor subtype 2 (mGluR2) in rat brain sections by using in vitro autoradiography. These compounds were found to possess potent in vitro binding affinities ( K i : 8.0–34.1 nM) for mGluR2 in rat brain homogenate. [ 11 C] 1 , [ 11 C] 2 , and [ 18 F] 4 were synthesized by [ 11 C/ 18 F]alkylation of the corresponding phenol precursors with [ 11 C]methyl iodide or [ 18 F]fluoroethyl bromide with >98% radiochemical purity and 80–130 GBq/μmol specific activity at the end of synthesis. In vitro autoradiography indicated that these radiotracers showed heterogeneous specific bindings in mGluR2-rich brain regions, such as the cerebral cortex, striatum, hippocampus, and granular layer of the cerebellum. [ABSTRACT FROM AUTHOR]

Ziyuglycoside I is one of the major active ingredients in Sanguisorba officinalis , a popular medicinal plant in China. In the present study, the metabolites of ziyuglycoside I in rat liver microsome and intestinal flora were identified and structurally characterized, and the metabolic rules were summed based on the LC–Q-TOF/MS system. Then, the metabolites in rat excreta samples were rapidly screened and identified according to the in vitro metabolic rules. Finally, ziyuglycoside I was incubated with fresh liver/lung/kidney/stomach homogenates to further explore the source of the metabolites and reveal the possible metabolic organs involved. Four metabolites in liver microsome were identified as M0-Glu, M0-CH 2 OH, M0-Glu + CH 3 , M0-Glu-Ara + CH 3 . In intestinal flora incubation system, 6 degradation products including M0-Glu-Ara + O, M0-Ara, M0-Glu-COOH, M0-Glu, M0-Glu-Ara + O and M0-Ara + H 2 O were tentatively identified by interpretation of their accurate MS 1 and MS 2 data. Fifteen metabolites in rat urine and feces were identified, and most of the metabolites were attributed to the transformation in liver microsome and intestinal flora. Specifically, more than a dozen of new metabolites were identified in rat fresh tissues, and ziyuglycoside II was confirmed as the major metabolite in rats. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive and simple liquid chromatography–mass spectrometry (LC–MS) method has been developed and validated for the quantification of solamargine, a steroidal glycoalkaloid, in rat plasma. Vincristine was selected as the internal standard. Sample preparation involved simple liquid–liquid extraction by ethyl acetate with high efficiency. The chromatographical separation was performed on a Shimadzu C18 column (150mm×2.0mm, 5μm) with a gradient elution of acetonitrile and 0.02% (v/v) formic acid. The elutes were detected under positive electrospray ionization (ESI) and the target analytes quantified by selected ion monitoring (SIM) mode. The method was sensitive with the lowest limit of quantitation (LLOQ) at 0.5ng/mL in 50μL of rat plasma. Good linearity (r 2 =0.9996) was obtained covering the concentration of 0.5–2000.0ng/mL. The intra- and inter-day assay precision ranged from 2.87 to 3.60% and 0.52 to 6.81%, respectively. In addition, the stability, extraction recovery and matrix effect involved in the method were also validated. The practical utility of the aforementioned method was successfully confirmed in the pharmacokinetic evaluation of solamargine in Sprague-Dawley rats after intravenous administration. [Copyright &y& Elsevier]

Abstract: We report herein, a facile metabolite identification workflow on the antimicrobial strictosamide, which is derived from accurate mass measurement by a hybrid ion trap-TOF mass spectrometer. In step 1, the parent drug and metabolites in rat bile were separated on an HPLC column followed by ion trap-TOF mass spectrometer analysis after a single oral dose of 50mg/kg strictosamide. In step 2, mass defect filter technique, which enables high-resolution mass spectrometers to be utilized for detecting drug metabolites based on well-defined mass defect ranges, was used to find metabolites in the mass spectrum. In step 3, the differences of accurate masses and their mass fragmentation pattern among the parent drug and metabolites used to assign structures for the metabolites successfully. As a result, five metabolites of strictosamide were found in rat bile, and all the metabolites were reported for the first time. [Copyright &y& Elsevier]

Abstract: Quadrupole (Q) mass spectrometers are the most popular analytical tools due to their reliability, effectiveness, and low cost. However, they are not suitable for quantitative analysis of multi-component since the sensitivity will get worse rapidly with the increasing number of m/z detected. The present work, for the first time, attempted to analyze of 16 saponins simultaneously using an approach of segmental and selected ion monitoring (SSIM) based on LC–Q/MS, and systematically investigated the influence of different SSIM modes on signal level/noise level (S/N), lower limits of quantification (LLOQ), upper limits of quantification (ULOQs), etc. Our results showed that a proper SSIM mode could not only provide much higher sensitivity for all the targeting analytes, but also dramatically broadened their dynamic ranges. The developed methodology could effectively break the application bottleneck on the quantitative analysis of multi-component with LC–Q/MS, and would be applied widely in related fields for multi-component analysis, such as environmental monitoring, metabonomics, Chinese herbal medicine research. [Copyright &y& Elsevier]

Abstracts: Glucagon-like peptide (GLP)-1 is a potent glucose-dependent insulinotropic gut hormone released from intestinal L cells. Our previous studies showed that berberine increased GLP-1 secretion in streptozotocin-induced diabetic rats. The aim of this study was to investigate whether berberine affected GLP-1 release in normal rats and in NCI-H716 cells. Proglucagon and prohormone convertase 3 genes regulating GLP-1 biosynthesis were analyzed by RT-PCR. Effects of pharmacological inhibitors on berberine-mediated GLP-1 release were studied. In vivo, 5-week treatment of berberine enhanced GLP-1 secretion induced by glucose load and promoted proglucagon mRNA expression as well as L cell proliferation in intestine. In vitro, berberine concentration-dependently stimulated GLP-1 release in NCI-H716 cells. Berberine also promoted both prohormone convertase 3 and proglucagon mRNA expression. Chelerythrine (inhibitor of PKC) concentration-dependently suppressed berberine-mediated GLP-1 secretion. Compound C (inhibitor of AMPK) also inhibited berberine-mediated GLP-1 secretion. But only low concentrations of H89 (inhibitor of PKA) showed inhibitory effects on berberine-mediated GLP-1 release. The present results demonstrated that berberine showed its modulation on GLP-1 via promoting GLP-1 secretion and GLP-1 biosynthesis. Some signal pathways including PKC-dependent pathway were involved in this process. Elucidation of mechanisms controlling berberine-mediated GLP-1 secretion may facilitate the understanding of berberine''s antidiabetic effects. [Copyright &y& Elsevier]

Abstract: We describe herein, a very effective way in the rapid identification of metabolites for antofloxacin based on a hybrid ion trap (IT)/time-of-flight (TOF) mass spectrometer technique. The purified samples were separated by a reversed-phase C18 column under a gradient elution, antofloxacin and its metabolites were detected by the on-line IT/TOF detector in scan mode. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (ΔM), calculating compound-based component by Formula Predictor software, and defining sites of biotransformation based upon mass shifts of diagnostic fragment ions according to the accurate MS n spectral information. In this case, we used such strategies for the identification of the metabolism for antofloxacin, and six metabolites of antofloxacin were found in rats for the first time. [Copyright &y& Elsevier]

Abstract: The characteristics of gatifloxacin transport across blood brain barrier were investigated using primary cultured rat brain microvessel endothelial cells (rBMECs) as an in vitro model. Gatifloxacin uptake by rBMECs was time-, temperature- and energy-dependent. Gatifloxacin uptake by rBMECs was not influenced by P-glycoprotein (P-GP) inhibitor cyclosporine A or multidrug resistance associated-proteins (MRPs) inhibitor probenecid. However, verapamil inhibited the uptake in a concentration-dependent manner. Transendothelial transport study showed that transport of gatifloxacin across rBMEC monolayer was bidirectional, verapamil concentration-dependently inhibited transport from the apical to basolateral side, but did not significantly affect transport from basolateral to apical side. Gatifloxacin uptake was decreased in Ca2+-deprived medium but increased in Mg2+-deprived medium significantly. Furthermore, organic Ca2+ channel blockers nifedipine and diltiazem had no effect on gatifloxacin uptake, but inorganic Ca2+ channel blockers Ni2+ and Mg2+ inhibited the gatifloxacin uptake. The present study suggests that gatifloxacin transport across rBMECs involves a Na+/Ca2+ exchange mechanism and extracellular Ca2+ but not P-GP and MRPs. [Copyright &y& Elsevier]

Abstract: Aim of the study: Huang-Lian-Jie-Du-Decoction (HLJDD), a well-known Chinese herbal formula, has been used for diabetic treatment. The purpose of the study was to investigate whether HLJDD affected glucagon-like peptide (GLP)-1 (7–36) amide level in diabetic rats. Materials and methods: Streptozotocin (STZ)-induced diabetic rats were treated with HLJDD at low dose (2g/kg/day) or high dose (4g/kg/day). After 5-week treatment, GLP-1 (7–36) amide level and insulin level in portal vein and tissues stimulated by oral glucose load were measured by ELISA kits. The proglucagon gene expression in intestinal tracts and the proliferation of intestinal L cell and pancreatic beta cell were measured using RT-PCR and immunohistochemistry techniques, respectively. Results: It was found that 5-week HLJDD treatment attenuated alteration of glucose level and insulin level in plasma and tissues of diabetic rats induced by STZ, accompanied by improvement of diabetic syndrome. 5-week HLJDD treatment increased GLP-1 (7–36) amide level in portal vein plasma and distal ileum. Further studies showed that 5-week HLJDD treatment increased the mRNA level of proglucagon gene in distal ileum, promoted pancreatic beta cell and intestinal L cell proliferation in a dose-dependent manner. Conclusion: All the results indicated that HLJDD exerted its anti-diabetic effects partly via modulating GLP-1 (7–36) amide level. [Copyright &y& Elsevier]

Abstract: We herein describe the development of an LC–MS method for simultaneous determination of astilbin and 3′-O-methylastilbin in rat plasma. A simple liquid–liquid extraction procedure was followed by injection of the extracts on to a Shim-pack C18 column (150mm×2.0mm I.D., 5μm) with gradient elution and detection in negative ionization mode. Initially, the method was validated regarding linearity, accuracy and precision. The correlation coefficients of all the calibration curves showed good linearity (r >0.999) within test ranges, and the relative deviation was less than 10% for intra- and inter-day assays. Besides, this method was also validated for its stability, extraction efficiency, matrix effect and so on. Finally, this proposed method was successfully applied to rat pharmacokinetic study and yielded the most comprehensive data on systemic exposure of them to date. [Copyright &y& Elsevier]

Abstract: Our previous study showed that insulin restored impaired function and expression of P-glycoprotein in diabetic blood-brain barrier, and further study showed that insulin up-regulated P-glycoprotein expression and function in normal blood-brain barrier, so insulin might be one of the factors that regulated the function and expression of P-glycoprotein in blood-brain barrier of diabetes. In this study, the intracellular pathways that insulin regulated the P-glycoprotein were investigated using primarily cultured rat brain microvessel endothelial cells model. The rat brain microvessel endothelial cells were incubated in normal culture medium containing 50 mU/l insulin and different concentrations of inhibitors for 72 h. The P-glycoprotein function and expression in the rat brain microvessel endothelial cells were assessed using the uptake of P-glycoprotein substrate rhodamine 123 and western blot assay, respectively. It was found that treatment of 50 mU/l insulin significantly increased P-glycoprotein function and expression in rat brain microvessel endothelial cells. This induced effect was blocked by insulin receptor antibody, insulin receptor tyrosine kinase inhibitor I-OMe-AG538, PKC inhibitor chelerythrine and NF-κB inhibitor pyrrolidine dithiocarbamate ammonium (PDTC). But this induced effect was not inhibited by phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor LY294002. These results indicated that insulin regulated P-glycoprotein function and expression through signal transduction pathways involving activation of PKC/NF-κB but not PI3K/Akt pathway. [Copyright &y& Elsevier]

Abstract: A sensitive liquid chromatography–electrospray ionization mass spectrometric (LC–ESI-MS) method for the quantification of a newly active quinolone carboxylic acid caderofloxacin lactate in rat plasma was developed and validated after precipitation method with methanol. Chromatographic separation was achieved on a reversed-phase Shimadzu 2.0μm C18 column (150mm×2.00mm) with the mobile phase of methanol–0.02% formic acid and step gradient elution resulted in a total run time of about 10.0min. The analytes were detected by using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (5–2000ng/mL) (r =0.9998). The lowest limit of quantification (LLOQ) was 5ng/mL and the lowest limit of detection (LLOD) was 2ng/mL. Average recoveries ranged from 88.80 to 93.05% in plasma at the concentrations of 10, 100 and 1000ng/mL. Intra- and inter-day relative standard deviations were 4.01–7.30 and 4.15–7.51%, respectively. This method was successfully applied in the pharmacokinetic studies in rats. [Copyright &y& Elsevier]

Neuroglobin (Ngb), a recently discovered O2-binding heme protein related to hemoglobin and myoglobin, protects neurons from hypoxic-ischemic injury in vitro and in vivo. In immunostained mouse brain sections, we found widespread expression of Ngb protein in neurons, but not astrocytes, of several brain regions that are prominently involved in age-related neurodegenerative disorders. Western blots from young adult (3 month), middle-aged (12 month), and aged (24 month) rats showed an age-related decline in Ngb expression in cerebral neocortex, hippocampus, caudate-putamen, and cerebellum. Loss of this neuroprotective protein may have a role in increasing susceptibility to age-related neurological disorders. [Copyright &y& Elsevier]