Activation of Wnt signaling reduces high-glucose mediated damages on skin fibroblast cells

Objective(s): High-glucose (HG) stress, a mimic of diabetes mellitus (DM) in culture cells, alters expression of a large number of genes including Wnt and NF-κB signaling-related genes; however, the role of Wnt signaling during HG-mediated fibroblast damage and the relationship between Wnt and NF-κB signaling have not been understood. In this study, we aimed to investigate the ffects of Wnt signaling on HG-mediated damages. Materials and Methods: Wnt3a was treated to HG-stressed human primary foreskin fibroblasts and the levels of Wnt signaling markers and cell proliferation were monitored. In addition, Wnt3a and NF-κB signaling inhibitor were assisted to analyze the relationship between two pathways. Results: The results indicated that HG treatment repressed β-catenin level, and Wnt3a treatment increased the levels of β-catenin and FZD8 as well as cell proliferation. RNA-seq based transcriptome analysis identified 207 up-regulated and 200 down-regulated genes upon Wnt3a supply. These altered genes are distributed into 20 different pathways. In addition, gene ontology (GO) analysis indicates that 20 GO terms are enriched. Wnt signaling genes were further verified by qRT-PCR and the results were similar with RNA-seq assay. Since NF-κB signaling negatively regulates Wnt marker gene expression, Bay117082, a typical NF-κB signaling inhibitor and Wnt3a were supplemented for testing β-catenin and phosphorylated IκBα (p-IκBα), respectively. Conclusion: HG positively inhibits Wnt signaling, and signaling activation via supplementation of Wnt3a rescued the defect caused by HG. NF-κB signaling negatively regulates accumulation of β-catenin, but Wnt signaling has no effects on IκBα activation

Development and Validation of LC-MS/MS Method for Determination of Ondansetron in rat Plasma and its Application

A selective and sensitive liquid chromatography-mass spectrometry (LC–MS) method for determination of ondansetron in rat plasma was developed and validated. After addition of midazolam as internal standard (IS), protein precipitation by acetonitrile was used as sample preparation, and chromatography involved Agilent SB-C18 column (2.1 x 150 mm, 5 μm) using 0.1 % formic acid in water and acetonitrile as a mobile phase with gradient elution. Detection involved positive ion mode electrospray ionization (ESI), and multiple reaction monitoring (MRM) mode was used for quantification of target fragment ions m/z 294.0→169.7 for ondansetron and m/z 326.0→291.0 for midazolam (internal standard, IS). The assay was linear over the range of 5–1000 ng/mL for ondansetron, with a lower limit of quantitation (LLOQ) of 5 ng/mL for ondansetron. Intra- and inter-day precisions were less than 14 % and the accuracies were in the range of 94.7-113.5 % for ondansetron. This developed method was successfully applied for the determination of ondansetron in rat plasma for pharmacokinetic study.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Determination of ramosetron in rat plasma by LC-ESI-MS and its application

A sensitive and simple liquid chromatography/electrospray mass spectrometry (LC-ESI-MS) method for determination of ramosetron in rat plasma using one-step protein precipitation was developed and validated. After addition of midazolam as internal standard (IS), protein precipitation by acetonitrile was used as sample preparation. Chromatographically separation was achieved on an SB-C18 (2.1 mm × 150 mm, 5 μm) column with acetonitrile-0.1 % formic acid as the mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selected ion monitoring (SIM) mode was used to quantification using target fragment ions m/z 280 for ramosetron and m/z 326 for the IS. Calibration plots were linear over the range of 10-1000 ng/mL for ramosetron in rat plasma. Lower limit of quantification (LLOQ) for Ramosetron was 10 ng/mL. Mean recovery of ramosetron from plasma was in the range of 88.5-92.8 %. CV of intra-day and inter-day precision were both less than 15 %. This method is simple and sensitive enough to be used in pharmacokinetic study for determination of ramosetron in rat plasma.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Application of citrate as a tricarboxylic acid (TCA) cycle intermediate, prevents diabetic-induced heart damages in mice

Objective(s):Higher cellular reactive oxygen species (ROS) levels is important in reducing cellular energy charge (EC) by increasing the levels of key metabolic protein, and nitrosative modifications, and have been shown to damage the cardiac tissue of diabetic mice. However, the relation between energy production and heart function is unclear. Materials and Methods:Streptozotocin (STZ, 150 mg/kg body weight) was injected intraperitoneally once to mice that had been fasted overnight for induction of diabetes. After diabetic induction, mice received citrate (5 µg/kg) through intraperitoneal injection every other day for 5 weeks. The caspase-3, plasminogen activator inhibitor 1 (PAI1), protein kinase B (PKB), commonly known as AKT and phosphorylated-AKT (p-AKT) proteins were examined to elucidate inflammation and apoptosis in the heart. For histological analysis, heart samples were fixed with 10% formalin and stained with hematoxylin-eosin (HE) and Sirius red to assess pathological changes and fibrosis. The expression levels[AGA1]  of marker proteins, tyrosine nitration, activity of ATP synthase and succinyl-CoA:3-ketoacid coenzyme A transferase-1 (SCOT), and EC were measured. Results:Intraperitoneal injection of citrate significantly reduced caspase-3 and PAI-1 protein levels and increased p-AKT level on the 5th week; EC in the heart was found to be increased as well. Further, the expression level, activity, and tyrosine nitration of ATP synthase and SCOT were not affected after induction of diabetes. Conclusion: Results indicate that application of citrate, a tricarboxylic acid (TCA) cycle intermediate, might alleviate cardiac dysfunction by reducing cardiac inflammation, apoptosis, and increasing cardiac EC

Cyclodextrin-modified polycarboxylate superplasticizers as dispersant agents for multiwalled carbon nanotubes

A new type poly(AA-co-beta-CD-A-co-TPEG) (PACD) copolymer was prepared by the copolymerization of a novel monovinyl beta-cyclodextrin monomer (beta-CD-A), acrylic acid, and isoprenyl oxy poly(ethylene glycol) (TPEG-2400). This copolymer could be used as dispersant for multiwalled carbon nanotubes (MWCNTs), which exhibit an excellent dispersion ability. This study mainly investigated the dispersing behavior of the PACD as well as the best ratio between these dispersants and MWCNTs. Transmission electron microscopy was employed to observe the morphology of the dispersed of MWCNTs. ultraviolet-visible-near infrared spectra were utilized to determine the dispersion of MWCNTs and the optimum concentration of PACD. It was found that the polycarboxylate superplasticizer decreased MWCNTs aggregative tendency in water but not as well as PACD. Moreover, the optimum mass ratio of the PACD to the MWCNTs is 5:1. In addition, the effect of PACD on the dispersion ability of MWCNTs was evaluated in cement samples. Our results indicated that MWCNTs could still disperse well with the help of PACD even with the presence of cement. (c) 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47311

Insight into the Effects of Adipose Tissue Inflammation Factors on miR-378 Expression and the Underlying Mechanism

Background/Aims: Obesity and the related metabolic syndrome have emerged as major public health issues in modern society. miRNAs have been shown to play key roles in regulating obesity-related metabolic syndrome, and some miRNAs regulated by adiponectin were identified as novel targets for controlling adipose tissue inflammation. miR-378 is a candidate target that was shown to be involved in adipose differentiation, mitochondrial metabolism and systemic energy homeostasis. However, little is known about the regulatory mechanisms of miR-378 expression. To better understand the physiological role of miR-378 in obesity and metabolic syndrome, it is crucial that we understand the regulation of miR-378 gene expression in human adipocytes. Methods: In this study, we investigated the effects of adipokines and inflammatory cytokines on miR-378 expression using Real-time PCR and the potential regulatory mechanisms using luciferase reporter assays and electrophoretic mobility shift assay (EMSA). Results: We found that adipokines and cytokines upregulated miR-378 expression primarily through SREBP and C/EBP binding sites in the miR-378 promoter region. Conclusion: Our findings showed that adipokines induced miR-378 expression and revealed the most likely mechanism of adipokine-induced miR-378 dysregulation in human adipocytes. miRNAs have been shown to function in regulating obesity-related metabolic syndrome, and miR-378 may be a novel target for controlling adipose tissue inflammation. This study offers a theoretical basis for understanding systemic adipose tissue inflammation and may provide new strategies for clinical treatment