mtt-formazan and Inflammation

Chronic systemic inflammation and repetitive damage of vascular endothelia by incompatible dialysis system are probable causes of cardiovascular disease in patients on dialysis. The present study aimed to assess in vitro biocompatibility and anti-inflammatory effect of hemodialysis fluid supplemented with rosmarinic acid (RA) using human umbilical vein endothelial cells (HUVEC). HUVECs (5×106 cells/mL) were pre-exposed to 1 μg/mL of lipopolysaccharides (LPS) and incubated with RA-supplemented hemodialysis fluid (HDF). Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by MTT assay. Expressions of proinflammatory mediators were assessed using quantitative real-time PCR and production of NO was quantified. Phosphorylation of AKT and nuclear localization of nuclear factor kappa B (NF-κB) were examined using western blotting. Exposure of HUVECs to RA-supplemented HDF had no influence on morphology and viability. Inhibition of proinflammatory mediator production in HUVECs by RA supplementation to HDF was significant in a dose-dependent manner. Exposure to RA-supplemented HDF resulted in a decrease in nitric oxide synthase expression and reduction of NO production in LPS-stimulated HUVECs. RA supplementation of HDF suppressed Akt activation in LPS-stimulated HUVECs. In addition, the level of cellular IκB was increased in parallel to a reduced nuclear translocation of NF-κB in LPS-induced endothelial cells. Our results suggest that RA-supplemented HDF is biocompatible and significantly suppressed inflammation induced in endothelial cells. In this respect, the use of HDF supplemented with RA could alleviate inflammation and improve long-term treatment of patients with renal failure on dialysis. Further clinical studies are required to confirm the effects.

Topics: Analysis of Variance; Anti-Inflammatory Agents, Non-Steroidal; Biocompatible Materials; Cell Survival; Cells, Cultured; Cinnamates; Cytokines; Depsides; Formazans; Hemodialysis Solutions; Human Umbilical Vein Endothelial Cells; Humans; Immunoblotting; Inflammation; Lipopolysaccharides; NF-kappa B; Nitric Oxide; Phosphorylation; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Rosmarinic Acid; Tetrazolium Salts

NPM is a major nucleolar multifunctional protein involved in ribosome biogenesis, centrosome duplication, cell-cycle progression, apoptosis, cell differentiation, and sensing cellular stress. Alarmins are endogenous molecules released from activated cells and/or dying cells, which activate the immune system and cause severe damage to cells and tissue organs. In the present work, stimulation of cells with the alarmin-inducible molecule endotoxin, for 16 h, resulted in NPM release into the culture supernatants of RAW264.7 cells, a murine macrophage cell line. Extracellular NPM was detected in the ascites of the CLP model. NPM was translocated into the cytoplasm from the nucleus in LPS -stimulated RAW264.7 cells; furthermore, NPM was detected in the cytosols of infiltrated macrophages in the CLP model. rNPM induced release of proinflammatory cytokines, TNF-alpha, IL-6, and MCP-1, from RAW264.7 cells and increased the expression level of ICAM-1 in HUVECs. NPM induced the phosphorylation of MAPKs in RAW264.7 cells. Our data indicate that NPM may have potent biological activities that contribute to systemic inflammation. Further investigations of the role of NPM may lead to new therapies for patients with septic shock or other inflammatory diseases.

Topics: Animals; Ascites; Cell Nucleus; Cell Survival; Cells, Cultured; Culture Media, Serum-Free; Cytokines; Dose-Response Relationship, Drug; Formazans; Immunohistochemistry; Immunologic Factors; Inflammation; Inflammation Mediators; Lipopolysaccharides; Macrophages; Male; Mice; Molecular Chaperones; Nuclear Proteins; Nucleoplasmins; Phosphoproteins; Rats; Rats, Sprague-Dawley; Sepsis; Tetrazolium Salts; Time Factors

Indomethacin is used as an anti-inflammatory drug and a nonselective cyclooxygenase inhibitor. When indomethacin in methanol was photo-irradiated with an Hg lamp, methyl ester, ethyl ester, and gamma-lactone derivatives of indomethacin were produced. In the present study, we found that the methyl ester derivative of indomethacin (M-IN) could more potently inhibit prostaglandin E(2) (PGE(2)) and nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX 2) protein expression from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells than indomethacin, similar to the effect of a non-steroidal anti-inflammatory drugs (NSAID). On the other hand, the results showed that M-IN with an IC(50) value maintained at 36.9 microg/ml for 12 h exhibited stronger cytotoxicity than ethyl ester, gamma-lactone derivatives of indomethacin, and indomethacin in promyelocytic leukemia HL-60 cells. Moreover, a series of biochemical analyses determined that M-IN caused apoptotic bodies, DNA fragmentation, and enhanced PARP and pro-caspase 3 degradation in HL-60 cells. These above results indicate that the photosynthesized product, M-IN, had stronger anti-inflammatory effects in LPS-stimulated RAW 264.7 cells and cytotoxicity effects in HL-60 cells than the parent drug, indomethacin.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Cyclooxygenase 2; Dinoprostone; DNA Fragmentation; Dose-Response Relationship, Drug; Formazans; G1 Phase; Gene Expression; HL-60 Cells; Humans; Indomethacin; Inflammation; Inhibitory Concentration 50; Leukemia, Promyelocytic, Acute; Lipopolysaccharides; Methods; Mice; Nitric Oxide Synthase Type II; Poly(ADP-ribose) Polymerases; Tetrazolium Salts