Effect of 15-Deoxy-△(12,14)-prostaglandin J2 on Expression of Macrophage Migration Inhibitory Factor in Mouse Monocyte/macrophage Cell Line J774A.1 / 中国医学科学院学报

Objective To investigate the effect of 15-Deoxy-△(12,14)-prostaglandin J2 (15 d-PGJ2) on the expression of macrophage migration inhibitory factor (MIF) and its underlying mechanism in J774A.1. Methods The murine monocyte/macrophage cell line J774A.1 were divided into six groups:lipopolysaccharide (LPS) group,incubated with 1 μg/ml LPS for 1 h;normal control group,incubated with PBS for 1 h;negative control group,incubated with 5 μmol/L 15 d-PGJ2 for 1 h;15 d-PGJ2 group,incubated with 5 μmol/L 15 d-PGJ2 for 1 h followed by 1 μg/ml LPS for 1 h;GW9662 group,incubated with 5 μmol/L 15 d-PGJ2 for 1 h following GW9662 10 μmol/L for 1 h,and then incubated with 1 μg/ml LPS for 1 h;and Vehicle group,control of GW9662,GW9662 was replaced by its solvent DMSO. The expression of MIF was detected via immunofluorescence and agarose gel electrophoresis. RT-qPCR and Western blotting were used to test whether 15 d-PGJ2 could regulate mRNA and protein expression of MIF in J774A.1 upon LPS challenge. The effect of peroxisome proliferator-activated receptor-γ (PPAR-γ) antagonist GW9662 on the regulation of MIF by 15 d-PGJ2 was observed. The effects of 15 d-PGJ2 on the nuclear translocation of PPAR-γ upon LPS challenge were detected via high content screening analysis. Results MIF DNA and protein expressions were detected in J774A.1. MIF mRNA expression was up-regulated (1.75±0.09,P=0.037) when challenged with LPS and 15 d-PGJ2 inhibited its upregulation (0.84±0.08,P=0.026) in J774A.1. The protein level was consistent with the mRNA level. PPAR-γ antagonist GW9662 reversed the effect of 15 d-PGJ2 (mRNA,1.48±0.06,P=0.016;protein,1.28). Furthermore,nuclear translocation of PPAR-γ was regulated by 15 d-PGJ2 in J774A.1 upon LPS challenge(1.39±0.02 vs. 1.01±0.03,P=0.003). Conclusion 15 d-PGJ2 may down-regulate the MIF expression in J774A.1 in a PPAR-γ-dependent manner.

Discovery of novel and potent leukotriene B4 receptor antagonists. Part 1.

The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4)) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found to be highly potent inhibitors of LTB(4) binding to BLT1 and BLT2 receptors, with varying levels of selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB(4) and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity experiment provided the rationale for differentiation and selection of one compound (33) for clinical development.

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

Quantification of diacylglycerol molecular species in biological samples by electrospray ionization mass spectrometry after one-step derivatization.

Diacylglycerols (DAGs) are important lipid intermediates in cellular trafficking and signaling. Their concentrations are altered in diabetes, cancer, and other disease states. Quantification of DAGs in biological samples may provide critical information to uncover molecular mechanisms leading to various cellular functional disorders. Recent advances in lipidomics using mass spectrometry have greatly accelerated global lipid analysis and quantification. Quantification of DAGs by electrospray mass spectrometry (ESI/MS), however, is challenged by the absence of a permanent charge on the molecule, its low proton affinity and acidity, and its low abundance under normal biological conditions. We describe here the introduction of a quaternary ammonium cation to DAG molecules, using N-chlorobetainyl chloride, to afford a derivatized DAG that gives 2 orders of magnitude higher signal intensities than their underivatized sodium adducts. A linear calibration curve in which peak intensity ratios are plotted versus molar ratios can be achieved by using ESI/MS with dilauroyl glycerol as the internal standard. Employing this new approach to this analyte, we found a 9-fold increase of total DAGs in the livers of obese db/db mice as compared to their heterozygous lean controls. This proven strategy can be used to detect and quantify DAG molecular species from biological samples using ESI/MS after one-step derivatization.

Ionic-liquid matrices for improved analysis of phospholipids by MALDI-TOF mass spectrometry.

The use of ionic liquid matrices (ILMs) for phospholipids (PLs) affords higher signal intensity, smaller spot size, improved spot homogeneity, better signal reproducibility, and comparable or better detection limits compared to that of the crystalline matrix 2,5-dihydroxybenzoic acid (2,5-DHB). The ionization products are comparable to those with 2,5-DHB although the use of ILMs gives a stronger tendency to produce alkali-metal-ion adducts and a lower extent of prompt fragmentation.

Ionic-liquid matrices for quantitative analysis by MALDI-TOF mass spectrometry.

Ionic liquid matrices (ILMs) were tested as MALDI matrices for quantification of oligodeoxynucleotides (ODNs), peptides, and small proteins. Good calibrations with high linearity and reproducibility were achieved over a broad concentration range for all the tested ILMs in spite of their different physical states. However, the standard deviation is higher for ILMs that are solid with visible crystals. The experimental results indicate various ILMs have different sensitivity owing to changes in their cation components. More importantly, we found that the slopes of the calibration curves correlate with the inverse of the peptide molecular weights, presenting an opportunity to predict a priori, the relative sensitivities (slopes of calibration plots) for various analytes that have similar hydrophobicites.