A pilot study on transient ischemic stroke induced with endothelin-1 in the rhesus monkeys.

Endothelin-1 (ET-1), a vasoconstrictor, has recently been used to induce focal ischemia in rodents and marmoset monkeys. The rhesus monkey, however, has numerous advantages to the rodent and marmoset that make it a superior and irreplaceable animal model for studying stroke in the brain. In the present study, after mapping the preferred hand representation in two healthy male monkeys with intracortical micro-stimulation, ET-1 was microinjected into the contralateral motor cortex (M1) to its preferred hand. The monkeys had been trained in three manual dexterity tasks before the microinjection and were tested for these tasks following the ET-1 injection. Brain Magnetic Resonance Imaging scans were performed 1, 7, 14 and 28 days post ischemia. It was found that ET-1 impaired the manual dexterity of the monkeys in the vertical slot and rotating Brinkman board tasks 3-8 days after the injection. Brain imaging found that severe edema was present 7 days after the focal ischemia. This data suggest that ET-1 can induce transient ischemic stroke in rhesus monkey and that ET-1 induced focal ischemia in non-human primates is a potential model to study the mechanism of stroke and brain repair after stroke.

Species- and gender-dependent differences in the glucuronidation of a flavonoid glucoside and its aglycone determined using expressed UGT enzymes and microsomes.

Flavonoids occur naturally as glucosides and aglycones. Their common phenolic hydroxyl groups may trigger extensive UDP-glucuronosyltransferase (UGT)- catalysed metabolism. Unlike aglycones, glucosides contain glucose moieties. However, the influence of these glucose moieties on glucuronidation of glucosides and aglycones remains unclear. In this study, the flavonoid glucoside tilianin and its aglycone acacetin were used as model compounds. The glucuronidation characteristics and enzyme kinetics of tilianin and acacetin were compared using human UGT isoforms, liver microsomes and intestinal microsomes obtained from different animal species. Tilianin and acacetin were metabolized into different glucuronides, with UGT1A8 produced as the main isoform. Assessment of enzyme kinetics in UGT1A8, human liver microsomes and human intestinal microsomes revealed that compared with tilianin, acacetin displayed lower Km (0.6-, 0.7- and 0.6-fold, respectively), higher Vmax (20-, 60- and 230-fold, respectively) and higher clearance (30-, 80- and 300-fold, respectively). Furthermore, glucuronidation of acacetin and tilianin showed significant species- and gender-dependent differences. In conclusion, glucuronidation of flavonoid aglycones is faster than that of glucosides in the intestine and the liver. Understanding the metabolism and species- and gender-dependent differences between glucosides and aglycones is crucial for the development of drugs from flavonoids.

A combined strategy of mass fragmentation, post-column cobalt complexation and shift in ultraviolet absorption spectra to determine the uridine 5'-diphospho-glucuronosyltransferase metabolism profiling of flavones after oral administration of a flavone mixture in rats.

The use of dietary flavones is becoming increasingly popular for their prevention of cancers, cardiovascular diseases, and other diseases. Despite many pharmacokinetic studies on flavone mixtures, the position(s) of glucuronidation sites on the flavone skeleton in vivo remain(s) uncertain because of the lack of a convenient method to differentiate the isomers in biological samples. Accordingly, this study aimed to develop a new strategy to identify the position of the mono-O-glucuronide of flavones in vivo and to simultaneously determine the parent agent and its major metabolites responsible for complex pharmacokinetic characteristics. The novel strategy involves accurate mass measurements of flavone glucuronides, their [Co(II) (flavone glucuronide-H) (4,7-diphenyl-1,10-phenanthroline)2](+) complexes generated via the post-column addition of CoBr2 and 4,7-diphenyl-1,10-phenanthroline, and their mass spectrometric fragmentation by UPLC-DAD-Q-TOF and the comparison of retention times with biosynthesized standards of different isomers that were identified by analyzing the shift in UV spectra compared with the spectra of their respective aglycones. We successfully generated a metabolite profiling of flavones in rat plasma after oral administration of a flavone mixture from Dracocephalum moldavica L., which was used here as the model to demonstrate the strategy. Twelve flavone glucuronides, which were glucuronidated derivatives of acacetin, apigenin, luteolin, diosmetin, chrysoeriol and cirsimaritin, were detected and identified. Glucuronidation of the flavone skeleton at the 3'-/7-position was more prevalent, however, luteolin 4'-glucuronide levels exceeded luteolin 7-glucuronide levels. Based on the UDP-glucuronosyltransferase (UGT) metabolism profiling of flavones in rat plasma, six main compounds (tilianin, acacetin 7-glucuronide, apigenin 7-glucuronide, luteolin 3'-glucuronide, acacetin, and apigenin) were selected as pharmacokinetic markers. Pharmacokinetic results indicated that their maximal concentrations in blood were obtained within 0.4h, except for the concentration of luteolin 3'-glucronide (approximately 9h). Rat exposure was practically non-linear under the studied dosages (200 to 400mg/kg).

UDP-Glucuronosyltransferases 1A6 and 1A9 are the Major Isozymes Responsible for the 7-O-Glucuronidation of Esculetin and 4-Methylesculetin in Human Liver Microsomes.

Esculetin (6,7-dihydroxycoumarin, ET) and 4-methylesculetin (6,7-dihydroxy-4-methylcoumarin, 4-ME) are typical coumarin derivatives that are attracting considerable attention because of their wide spectrum of biologic activities, but their metabolism remains unknown. This study aimed to elucidate the in vitro UDP-glucuronosyltransferase (UGT) metabolism characteristics of ET and 4-ME. 7-O-monoglucuronide esculetin (ET-G) and 7-O-monoglucuronide 4-methylesculetin (4-ME-G) were identified by liquid chromatography-mass spectrometry (LC-MS) and (1)H-nuclear magnetic resonance ((1)HNMR) when ET or 4-ME was incubated with human liver (HLM) in the presence of UDP-glucuronic acid. Screening assays with 12 human expressed UGTs demonstrated that the formations of ET-G and 4-ME-G were almost exclusively catalyzed by UGT1A6 and UGT1A9. Phenylbutazone and carvacrol (UGT1A6 and UGT1A9 chemical inhibitors, respectively) at different concentrations (50, 100, and 200 µM) significantly inhibited the formation of glucuronidates of ET and 4-ME in HLM, UGT1A6, and UGT1A9 when the concentrations of ET and 4-ME ranged from 10 to 300 µM (P < 0.05). Clearance rates of ET in HLM, HIM, UGT1A6, and UGT1A9 were 0.54, 0.16, 0.69, and 0.14 ml/min/mg, respectively. Corresponding clearance rates values of 4-ME were 0.59, 0.03, 0.14, and 0.04 ml/min/mg, respectively. In conclusion, 7-O-monoglucuronidation by UGT1A6 and UGT1A9 was the predominant UGT metabolic pathway for both ET and 4-ME in vitro. The liver is probably the major contributor to the glucuronidation metabolism of ET and 4-ME. ET showed more rapid metabolism than 4-ME in glucuronidation.

Multidrug resistance-associated protein 2 is involved in the efflux of Aconitum alkaloids determined by MRP2-MDCKII cells.

AIMS: Aconitum alkaloids mainly contain highly toxic aconitine (AC), mesaconitine (MA), and hypaconitine (HA) and less toxic benzoylaconine (BAC), benzoylmesaconine (BMA), benzoylhypaconine (BHA), aconine, mesaconine, and hypaconine. The efflux transporters including P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) can efflux toxicants to prevent poisoning. Our previous publication has proved that P-gp and BCRP contributed to the efflux of AC, MA and HA, which is demonstrated in the human colonic adenocarcinoma cell lines (Caco-2 cells), Mardin-Darby canine kidney cell lines transfected with MDR1 or BCRP (MDR1-MDCKII and BCRP-MDCKII cells). However, the role of MRP2 remains uncertain. MAIN METHODS: The MRP2-MDCKII cells were used to determine the efflux ratios (Er) and intracellular amounts of Aconitum alkaloids. In addition, the importance of MRP2 was further investigated with or without the MRP2 inhibitor, LTC4. KEY FINDINGS: The Er values of AC, MA, HA, BAC, BMA and BHA in MRP2-MDCKII cells (6.4 ± 0.3, 5.9 ± 0.5, 2.2 ± 0.2, 1.6 ± 0.3, 1.7 ± 0.2 and 1.9 ± 0.2 respectively) were significantly higher than those in MDCKII cells, which were close to 1. In the presence of LTC4, the Er values of AC, MA, HA, BAC, BMA and BHA were reduced to approximately 1 and their intracellular amounts were also significantly increased in MRP2-MDCKII cells. SIGNIFICANCE: MRP2 was involved in the efflux of AC, MA, HA, BAC, BMA and BHA, which would be useful for the safe application of these components or their herbs.

Triple Recycling Processes Impact Systemic and Local Bioavailability of Orally Administered Flavonoids.

Triple recycling (i.e., enterohepatic, enteric and local recycling) plays a central role in governing the disposition of phenolics such as flavonoids, resulting in low systemic bioavailability but higher gut bioavailability and longer than expected apparent half-life. The present study aims to investigate the coexistence of these recycling schemes using model bioactive flavonoid tilianin and a four-site perfused rat intestinal model in the presence or absence of a lactase phlorizin hydrolase (LPH) inhibitor gluconolactone and/or a glucuronidase inhibitor saccharolactone. The result showed that tilianin could be metabolized into tilianin glucuronide, acacetin, and acacetin glucuronide, which are excreted into the bile and luminal perfusate (highest in the duodenum and lowest in the colon). Gluconolactone (20 mM) significantly reduced the absorption of tilianin and the enteric and biliary excretion of acacetin glucuronide. Saccharolactone (0.1 mM) alone or in combination of gluconolactone also remarkably reduced the biliary and intestinal excretion of acacetin glucuronide. Acacetin glucuronides from bile or perfusate were rapidly hydrolyzed by bacterial ß-glucuronidases to acacetin, enabling enterohepatic and enteric recycling. Moreover, saccharolactone-sensitive tilianin disposition and glucuronide deconjugation, which was more active in the small intestine than the colon, points to the small intestinal origin of the deconjugation enzyme and supports the presence of local recycling scheme. In conclusion, our studies have demonstrated triple recycling of a bioactive phenolic (i.e., a model flavonoid), and this recycling may have an impact on the site and duration of polyphenols pharmacokinetics in vivo.

Pharmacokinetic comparisons of benzoylmesaconine in rats using ultra-performance liquid chromatography-tandem mass spectrometry after administration of pure benzoylmesaconine and Wutou decoction.

Wutou decoction is widely used in China because of its therapeutic effect on rheumatoid arthritis. Benzoylmesaconine (BMA), the most abundant component of Wutou decoction, was used as the marker compound for the pharmacokinetic study of Wutou decoction. The aim of the present study was to compare the pharmacokinetics of BMA in rats after oral administration of pure BMA and Wutou decoction. Pure BMA (5 mg/kg) and Wutou decoction (0.54 g/kg, equivalent to 5 mg/kg BMA) were orally administered to rats with blood samples collected over 10 h. Quantification of BMA in rat plasma was achieved using sensitive and validated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Specifically, the half-life (T1/2) and mean residence time values of pure BMA were 228.3 ± 117.0 min and 155.0 ± 33.2 min, respectively, whereas those of BMA in Wutou decoction were decreased to 61.8 ± 35.1 min and 55.8 ± 16.4 min, respectively. The area under the curve (AUC) of BMA after administration of Wutou decoction was significantly decreased (five-fold) compared with that of pure BMA. The results indicate that the elimination of BMA in rats after the administration of Wutou decoction was significantly faster compared with that of pure BMA.

Nanog expression is negatively regulated by protein kinase C activities in human cancer cell lines.

Nanog is a transcription factor that is essential for the maintenance of pluripotency of the embryonic stem cells. Nanog has been shown to be expressed in various kinds of human tumors, suggesting a role in tumorigenesis. In this study, we found that Nanog expression was upregulated by inhibition of protein kinase C (PKC) activity in six human cancer cell lines examined. In a Nanog non-expressing human nasopharyngeal carcinoma cell line, NPC-076, Nanog mRNA level and protein level were both induced and dose-dependently promoted by exposure to PKC inhibitors. Knockdown experiments showed that PKCα and PKCδ were two subtypes exerted most of the effect. The reporter assay showed that Nanog promoter activity was promoted by exposure of the cells to PKC inhibitors and the effect was dependent on the presence of the Octamer-Sox composite element. The involvement of Octamer-Sox composite element was further supported by the observation that silencing of Oct4 and Sox2 in NPC-076 cells attenuated the effects of PKC inhibitors. In Nanog-expressing human embryonal carcinoma cell lines, NT2/D1 and NCCIT, Nanog expression was suppressed by exposure to PKC activator Phorbol-12-myristate-13-acetate (PMA). Further study showed that overexpression of PKCα elicited a repressive effect on Nanog expression in NT2/D1 cells. Consistently, mutation of the Octamer-Sox composite element abolished the suppressive effect by PKC activator. Nanog expression was of cellular significance in that ectopic expression in NPC-076 stimulated cell proliferation and knockdown of the endogenous Nanog expression in NT2/D1-suppressed cell proliferation.

Glycogen synthase kinase-3beta regulates DeltaNp63 gene transcription through the beta-catenin signaling pathway.

Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role for DeltaNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase-3beta (GSK-3beta) by lithium chloride (LiCl) elicited a stimulatory effect on DeltaNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced DeltaNp63 promoter activation as well as DeltaNp63 protein expression in the cells. The effect of GSK-3beta on DeltaNp63 expression was further confirmed by the use of two highly specific GSK-3beta inhibitors, SB216763 and SB415286. Further study showed the presence of a putative beta-catenin responsive element (beta-catenin-RE) in the DeltaNp63 promoter region, and the stimulation of DeltaNp63 promoter activity by GSK-3beta inhibitor is markedly abolished by mutation or deletion of the putative beta-catenin-RE. Data are also presented to show that beta-catenin acts together with Lef-1 to influence DeltaNp63 promoter activity and protein expression.

Transcriptional activity of the DeltaNp63 promoter is regulated by STAT3.

The N terminus-truncated splicing variant of TAp63 is known as DeltaNp63. DeltaNp63 lacks transactivation function and is thought to antagonize the transcriptional regulation of the p53 and TAp63 target genes. Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role in carcinogenesis. In the present study we present data showing that the DeltaNp63 gene promoter activity is positively regulated by DeltaNp63alpha, and such positive autoregulation is mediated via activation of STAT3 activity. We show that expression of DeltaNp63alpha in Hep3B cells induces Stat3 phosphorylation on Tyr-705 and Ser-727. A putative STAT3-responsive element (STAT3-RE) is identified in the DeltaNp63 promoter region. Electrophoretic mobility shift and avidin biotin-Conjugated DNA assays show direct binding of STAT3 to STAT3-RE of the DeltaNp63 promoter, and such binding is stimulated by DeltaNp63alpha. Binding of the endogenous STAT3 to the DeltaNp63 promoter in Hep3B cells was demonstrated by a chromatin immunoprecipitation assay. The stimulation of the DeltaNp63 transcriptional activity by DeltaNp63alpha is abolished by Janus kinase 2 (JAK2)/STAT3 inhibitor AG490, dominant-negative STAT3, STAT3 small interfering RNA, and deletion of the STAT3-RE sequence from DeltaNp63 promoter. Taken together these observations clearly indicated that autoregulation of DeltaNp63 gene transcription is mediated through activation of STAT3 and its subsequent binding to the STAT3RE. Because the activation of STAT3 by interleukin-6 also leads to DeltaNp63 up-regulation and the blockade of DeltaNp63 or STAT3 expression by siRNA leads to repression of the cell growth, the identified regulatory pathway is presumably of cell physiological significance.