- MeSH
- kardiologie trendy MeSH
- kardiovaskulární nemoci * prevence a kontrola MeSH
- lidé MeSH
- výzkum MeSH
- Check Tag
- lidé MeSH
Chloroquine, an antimalarial drug, can also be used in the regulation of the immune system, e.g. it is used in the treatment of autoimmune diseases. In this study we investigated the effects of chloroquine and its hydroxy-derivative on nitric oxide (NO) production in two different cell types: (i) immortalized mouse macrophage cell line RAW 264.7 and (ii) mouse bone marrow-derived macrophages (BMDM). The cells were treated with different concentrations (1-100 μM) of chloroquine or hydroxychloroquine and stimulated with lipopolysaccharide for 24 h to induce NO production. Measurement of nitrites by the Griess reaction was used to evaluate the production of NO. Expression of inducible NO synthase was evaluated with Western blot and ATPcytotoxicity test was used to measure the viability of the cells. Our results showed that both chloroquine and its hydroxy-derivative inhibited NO production in both cell types. However, based on the results of LD50 these inhibitory effects of both derivatives were due to their cytotoxicity. The LD50 values for chloroquine were 24.77 μM (RAW 264.7) and 24.86 μM (BMDM), the LD50 for hydroxychloroquine were 13.28 μM (RAW 264.7) and 13.98 μM (BMDM). In conclusion, hydroxychloroquine was more cytotoxic than its parent molecule. Comparing the two cell types tested, our data suggest that there are no differences in cytotoxicity of chloroquine or hydroxychloroquine for primary cells (BMDM) or immortalized cell line (RAW 264.7).
- MeSH
- buňky kostní dřeně účinky léků metabolismus MeSH
- chlorochin chemie farmakologie MeSH
- dusitany metabolismus MeSH
- hydroxychlorochin chemie farmakologie MeSH
- makrofágy účinky léků metabolismus MeSH
- myši MeSH
- oxid dusnatý biosyntéza MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In recent years, microsensor technologies have made a rapid expansion into different fields of physical sciences, engineering, and biomedicine. For analyses of various biomolecules, novel sensors and detection platforms in the electrochemical field have been reported recently. The most important applications based on microelectromechanical systems dramatically reduce the need of manipulation steps with samples, while improving data quality and quantitative capabilities. This is also the case of a special class of electrochemical sensors that allow direct, real-time and non-invasive measurements of nitric oxide, whose determination is crucial for the purposes of basic research, as well as of preclinical and clinical studies. Therefore, this minireview will focus on the description of recent discoveries in the electrochemical determination of nitric oxide, released in different in vitro systems.
- MeSH
- buňky metabolismus MeSH
- elektrochemické techniky přístrojové vybavení metody MeSH
- elektrody MeSH
- lidé MeSH
- oxid dusnatý metabolismus MeSH
- uhlík chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Lipid peroxidation induced by oxidants leads to the formation of highly reactive metabolites. These can affect various immune functions, including reactive oxygen species (ROS) and nitric oxide (NO) production. The aim of the present study was to investigate the effects of lipid peroxidation products (LPPs) - acrolein, 4-hydroxynonenal, and malondialdehyde - on ROS and NO production in RAW 264.7 macrophages and to compare these effects with the cytotoxic properties of LPPs. Macrophages were stimulated with lipopolysaccharide (0.1 μg/ml) and treated with selected LPPs (concentration range: 0.1-100 μM). ATP test, luminol-enhanced chemiluminescence, Griess reaction, Western blotting analysis, amperometric and total peroxyl radical-trapping antioxidant parameter assay were used for determining the LPPs cytotoxicity, ROS and NO production, inducible nitric oxide synthase expression, NO scavenging, and antioxidant properties of LPPs, respectively. Our study shows that the cytotoxic action of acrolein and 4-hydroxynonenal works in a dose- and time-dependent manner. Further, our results imply that acrolein, 4-hydroxynonenal, and malondialdehyde can inhibit, to a different degree, ROS and NO production in stimulated macrophages, partially independently of their toxic effect. Also, changes in enzymatic pathways (especially NADPH-oxidase and nitric oxide synthase inhibition) and NO scavenging properties are included in the downregulation of reactive species formation.
- MeSH
- akrolein toxicita MeSH
- aktivace makrofágů MeSH
- aldehydy toxicita MeSH
- časové faktory MeSH
- down regulace účinky léků MeSH
- dusitany metabolismus MeSH
- lipopolysacharidy imunologie MeSH
- makrofágy imunologie metabolismus MeSH
- malondialdehyd toxicita MeSH
- myši MeSH
- NADPH-oxidasy antagonisté a inhibitory MeSH
- osmolární koncentrace MeSH
- oxid dusnatý antagonisté a inhibitory metabolismus MeSH
- peroxidace lipidů MeSH
- reaktivní formy kyslíku antagonisté a inhibitory metabolismus MeSH
- scavengery volných radikálů farmakologie MeSH
- synthasa oxidu dusnatého, typ II metabolismus MeSH
- transformované buněčné linie MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
BACKGROUND: Polyunsaturated fatty acids (PUFAs) can affect various functions of the immune system including inflammatory responses. An oxidative burst of phagocytes accompanied by reactive oxygen species (ROS) and reactive nitrogen species (RNS) formation is one of the phagocyte functions that could be modulated by PUFAs. AIM OF THE STUDY: To investigate the effects of omega-3 (alpha-linolenic, docosahexaenoic, eicosapentaenoic) and omega-6 (arachidonic, linoleic) PUFAs on lipopolysaccharide (LPS)-stimulated ROS and RNS production by the murine macrophage cell line RAW 264.7. METHODS: Murine peritoneal macrophages RAW 264.7 were stimulated with LPS (0.1 microg/ml) and treated with 0.1-100 microM omega-3 or omega-6 PUFAs for either 8 (ROS production) or 20 h (RNS production). The cytotoxicity of PUFAs was evaluated by an ATP (adenosine triphosphate) test after both 8 and 20 h of treatment with PUFAs. Changes in ROS production by LPS-treated macrophages subsequently activated with phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP) were determined by luminol-enhanced chemiluminescence, whilst the production of RNS was determined as the concentration of nitrites in cell supernatants (Griess reaction). Changes in inducible nitric oxide synthase (iNOS) expression were evaluated by Western blot analysis. The antioxidant properties of PUFAs were tested by TRAP (total peroxyl radical-trapping antioxidant parameter) assay. RESULTS: All PUFAs in 100 microM concentration except eicosapentaenoic acid decreased ROS production. The effect was most significant when docosahexaenoic acid was used. Arachidonic acid decreased PMA-activated ROS production even in 1 and 10 microM concentrations. On the other hand, 10 and 100 microM eicosapentaenoic acid potentiated ROS production. As concerns RNS production, all the fatty acids that were tested in a concentration of 100 microM decreased iNOS expression and nitrite accumulation. Fatty acids had no significant effect on the viability and proliferation of RAW 264.7 cells. The TRAP assay confirmed that none of the tested PUFAs exerted any significant antioxidant properties. CONCLUSION: High concentrations of PUFAs of both omega-3 and omega-6 groups can inhibit ROS and RNS formation by stimulated macrophages. The expression of iNOS can also be inhibited. This effect, together with the absence of antioxidant activity and cytotoxic properties, indicates that PUFAs can participate in the regulation of enzymes responsible for reactive species production.
- MeSH
- časové faktory MeSH
- dusík metabolismus MeSH
- dusitany metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- kultivované buňky MeSH
- luminiscenční měření metody MeSH
- makrofágy účinky léků metabolismus MeSH
- myši MeSH
- nenasycené mastné kyseliny farmakologie MeSH
- oxidační stres účinky léků MeSH
- reaktivní formy kyslíku metabolismus MeSH
- synthasa oxidu dusnatého účinky léků metabolismus MeSH
- viabilita buněk účinky léků MeSH
- western blotting metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Elevated plasma uric acid indicates an increased risk of cardiovascular diseases associated with endothelial dysfunction. However, the role of uric acid in the pathogenesis of endothelial dysfunction is still a matter of debate. It is not clear whether uric acid is a real causative risk factor, an inert marker, or even a protective molecule with respect to its antioxidant properties. We have studied the effect of uric acid on intact endothelial cells as well as cells with homocysteine-induced endothelial dysfunction. DESIGN: Bovine aortic endothelial cells were treated with uric acid (100 - 600 muM) and homocysteine (100 muM) or with uric acid only. After 24 hours, the cells were stimulated with 1 mug/ml of calcium ionophore A23187, and nitric oxide (NO) production was measured electrochemically with the use of a NO-sensitive microelectrode. The expression of endothelial nitric oxide synthase (eNOS) and eNOS phosphorylation at Ser1179 was estimated with the use of Western blotting. Interaction between NO and uric acid was measured with a NO electrode. Superoxide generation was measured with the use of the fluorescence dye MitoSox Red. RESULTS: Homocysteine strongly diminished A23187-induced NO release. 100 muM uric acid slightly restored NO production; higher concentrations were ineffective. Interestingly, a dose-dependent decrease of NO release was observed in the cells treated only with uric acid. Uric acid did not scavenge NO and did not change eNOS protein expression or phosphorylation at Ser1179, but dose-dependently increased superoxide production in A23187-stimulated cells. CONCLUSION: In conclusion, uric acid decreased NO bioavailability and enhanced superoxide generation in A23187-stimulated bovine aortic endothelial cells.
- MeSH
- aorta MeSH
- calcimycin farmakologie MeSH
- časové faktory MeSH
- endoteliální buňky účinky léků fyziologie MeSH
- fosforylace MeSH
- homocystein metabolismus farmakologie MeSH
- ionofory farmakologie MeSH
- kinetika MeSH
- kultivované buňky MeSH
- kyselina močová metabolismus farmakologie MeSH
- mikroelektrody MeSH
- oxid dusnatý metabolismus MeSH
- skot MeSH
- superoxidy metabolismus MeSH
- synthasa oxidu dusnatého, typ III metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Hyaluronan, a linear glycosaminoglycan, is an abundant component of extracellular matrix. In its native form, the high-molar-mass hyaluronan polymers have an array of structural and regulatory, mainly anti-inflammatory and anti-angiogenic, functions. In contradiction, the biological effects of fragmented low molecular weight hyaluronan are suggested to be pro-angiogenic and pro-inflammatory. METHODS: The effects of highly purified pharmacological grade hyaluronan of defined molecular weights 11, 52, 87, 250 and 970 kilodaltons were tested on mouse macrophage cell lines RAW 264.7 and MHS. The surface expression of CD44 and Toll-like receptor 2, surface receptors for hyaluronan, was determined by flow cytometry. Activation of macrophages was determined based on nitric oxide and tumour necrosis factor alpha production, inducible nitric oxide synthase expression, and the activation of the nuclear factor kappa B transcriptional factor. RESULTS: Both macrophage cell lines expressed CD44 and Toll-like receptor 2, which were significantly increased by the pre-treatment of macrophages with bacterial lipopolysaccharide. Hyaluronan of any molecular weight did not activate production of nitric oxide or tumour necrosis factor alpha in any mouse macrophage cell lines. Correspondingly, hyaluronan of any tested molecular weight did not stimulate nuclear factor kappa B activation. Similarly, hyaluronan of any molecular weight neither exerted stimulatory nor inhibitory effects on macrophages pre-treated by lipopolysaccharide. CONCLUSION: Interestingly, the data does not support the current view of low molecular weight hyaluronan as a pro-inflammatory mediator for macrophages. Further studies are necessary to clarify the effects of different molecular weight hyaluronan on phagocytes.
- MeSH
- aktivace makrofágů účinky léků fyziologie MeSH
- antigeny CD44 metabolismus MeSH
- buněčné linie MeSH
- kyselina hyaluronová chemie metabolismus farmakologie MeSH
- lipopolysacharidy toxicita MeSH
- makrofágy chemie účinky léků fyziologie MeSH
- molekulová hmotnost MeSH
- myši MeSH
- NF-kappa B metabolismus MeSH
- oxid dusnatý metabolismus MeSH
- průtoková cytometrie MeSH
- synthasa oxidu dusnatého, typ II metabolismus MeSH
- TNF-alfa metabolismus MeSH
- toll-like receptor 2 metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this study, we realized the continual and long-term electrochemical detection of NO production by stimulated macrophages using modified porphyrinic microsensor. The NO release from RAW 264.7 cells stimulated by lipopolysaccharide started 5 h after the lipopolysaccharide administration. After reaching its maximum at the sixth hour, the stable level of NO production was observed between the seventh and 12th hour of the experiment. This phase was followed by a gradual decline in NO production. A close correlation between the NO signal detected with microelectrode and nitrite accumulation, which had been determined in supernatants removed from stimulated cells, was observed. This finding was utilized for the calibration of the electrochemical experiment. The presence of iNOS enzyme, which constitutes a main requirement for NO production by stimulated macrophages, was confirmed by Western blot analysis of iNOS protein expression at key time points of the corresponding electrochemical experiment. The capability of our microsensor to instantaneously monitor the changes in the NO production by stimulated RAW 264.7 cells was demonstrated by the immediate decrease in the signal due to NO as a response to the addition of iNOS inhibitor into the cell culture medium.
- MeSH
- buněčné linie MeSH
- časové faktory MeSH
- dusitany analýza chemie metabolismus MeSH
- elektrochemické techniky metody přístrojové vybavení MeSH
- lipopolysacharidy farmakologie MeSH
- makrofágy chemie metabolismus sekrece účinky léků MeSH
- myši MeSH
- oxid dusnatý analýza biosyntéza chemie sekrece MeSH
- synthasa oxidu dusnatého, typ II metabolismus MeSH
- thiaziny MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
H1-antihistamines are known to be important modulators of inflammatory response. However, the information about the influence of these drugs on reactive nitrogen species generation is still controversial. The main aim of the present study was to investigate the effects of selected H1-antihistamines on nitric oxide production by lipopolysaccharide-stimulated murine macrophages RAW 264.7, measured as changes in inducible nitric oxide synthase (iNOS) protein expression in cell lysates by Western blotting and nitrite formation in cell supernatants using the Griess reaction. In pharmacological non-toxic concentrations, H1-antihistamines significantly inhibited nitrite accumulation that was not caused by the scavenging ability of drugs against nitric oxide, measured amperometrically. The degree of inhibition of nitrite accumulation positively correlated with the degree of tested lipophilicity, measured by reversed-phase thin layer chromatography. Furthermore, H1-antihistamines differentially modulated the iNOS protein expression. In conclusion, as was shown in this study, the modulation of nitric oxide production could be caused by the downregulation of iNOS protein expression and/or the iNOS protein activity.
- MeSH
- buněčné extrakty MeSH
- buněčné linie MeSH
- down regulace MeSH
- dusitany metabolismus MeSH
- ethylendiaminy MeSH
- lipidy chemie MeSH
- lipopolysacharidy metabolismus MeSH
- makrofágy enzymologie patologie účinky léků MeSH
- myši MeSH
- nesedativní antagonisté histaminu H1 farmakologie chemie MeSH
- oxid dusnatý metabolismus MeSH
- sulfanilamidy MeSH
- synthasa oxidu dusnatého, typ II metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
