Plaquettes sanguines et mégacaryocytes humains (impact de différentes concentrations d acide docosahexaénoïque sur leur activation et leur état redox)

Docosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid known for its protective effects against cardiovascular disease. However high doses of DHA may nevertheless counteract its beneficial effects owing to the presence of six double bounds in the fatty acyl chain. Our first objective was to determine the effects of increasing DHA consumption by healthy men on both platelet function and oxidative stress. The second objective was to analyse the effects of DHA on gene expression of enzymes controlling redox status and prostanoid formation, using human megakaryocytes MEG-01 as a model of platelet precursors. Our results show that ingestion of low doses of DHA (400 to 1600 mg) decreases the platelet aggregation response. A lower dose shows an anti-oxidant action. This result is reinforced by the transcriptional effects obtained on MEG-01 cells. Our results could explain the beneficial influence of n-3 polyunsaturated fatty acids at low doses on cardiovascular diseasesLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Stress oxydant au niveau des plaquettes sanguines humaines dans le contexte du diabète (étude du glutathion et de la glutathion peroxydase 4)

Les plaquettes sanguines jouent un rôle prépondérant dans les complications cardiovasculaires chez le diabétique. En raison de l hyperglycémie, la glycosylation non enzymatique ou glycation des protéines est augmentée. Des travaux ont déjà mis en évidence la glycation de la glutathion peroxydase cytosolique (cGPx ou GPx1) plaquettaire chez les diabétiques de type 1 ce qui entraîne une diminution de l activité enzymatique. De plus, la concentration plaquettaire du glutathion (GSH), est diminuée. Dans ce contexte, notre étude a porté sur deux antioxydants plaquettaires : le GSH et la phospholipid hydroperoxide glutathione peroxidase (PHGPx ou GPx4). Le rôle protecteur et détoxifiant du GSH résulte principalement de sa fonction de cosubstrat des GPx ; la GPx4 catalyse la réduction des hydroperoxydes lipidiques contenus dans les membranes. La finalité est d apporter ou de compléter des connaissances fondamentales sur l équilibre pro/antioxydant des plaquettes de diabétiques. Nous avons mis en évidence que le GSH peut être glyqué in vitro ; l adduit formé n est plus cosubstrat de la GPx1, ce qui pourrait ainsi contribuer à la diminution des défenses antioxydantes que nous avons observée dans les plaquettes des diabétiques. Nous avons caractérisé la GPx4 plaquettaire et étudié sa localisation subcellulaire par une double approche (dosage enzymatique et immunodétection). La synthèse d un substrat spécifique de l isoforme 4 des GPx (les hydroperoxydes de phosphatidylcholine) et le dosage enzymatique plaquettaire ont été mis au point. Ceci a permis de mettre en évidence une activité GPx4 dans les plaquettes de sujets sains, distincte et fonctionnellement complémentaire de l activité GPx1 déjà connue. Nous observons 83,5% de l activité GPx4 totale plaquettaire dans le cytosol, et respectivement 8,9% et 7,6% dans les fractions membranaire et mitochondriale. Parmi plusieurs cosubstrats testés (dérivés du GSH et dithiol), le GSH reste le cosubstrat de la GPx4 le plus utilisé. Enfin, nous avons étudié le métabolisme lipidique et le statut pro/antioxydant des plaquettes sanguines dans le diabète. Malgré un bon contrôle glycémique et en absence de manifestations cliniques de complications cardiovasculaires, un stress oxydant est observé dans les plaquettes sanguines des diabétiques (augmentation du taux de MDA, diminution du taux d a-tocophérol et des activités GPx). Une augmentation du taux de TXB2 basal ainsi qu une hyperagrégabilité ont été observées dans les plaquettes des diabétiques. La possibilité que l hyperactivité plaquettaire observée dans le diabète soit médiée, entre autres, par une augmentation de la libération d AA via une activation de la phospholipase A2 cytosolique est également discutéePlatelets contribute to the pathogenesis and progression of vascular complications in diabetes. One of the consequences of hyperglycaemia is oxidative stress which accelerates glycation of proteins. The glycation of platelet cytosolic glutathione peroxidase (cGPx or GPx1) has been reported in type 1 diabetics and results in a decreased activity. Moreover, platelet glutathione (GSH) concentration has been found to be lower. In this context, two platelet antioxidant species, GSH and phospholipid hydroperoxide glutathione peroxidase (PHGPx ou GPx4) were studied. GSH is a major endogenous antioxidant and is a cosubstrate of several enzymes including GPx. GPx4 catalyzes reduction of diverse hydroperoxides including phospholipid hydroperoxides and thus protects membranes against oxidative damage. The aim of our study is to better understand the pro/antioxidant balance in diabetic platelets. The present study shows that in vitro GSH can be glycated and interestingly, the glycated GSH fails to be a cosubstrate for GPx1. Thus, glycation of GSH might contribute to the platelet decreased antioxidant defenses that we have observed in diabetics. In addition, we have characterized platelet GPx4 and studied its subcellular localization by several methods (enzymatic assay and immunodetection). Setting up the synthesis of a specific substrate of GPx4 (hydroperoxy derivatives of phosphatidylcholine), the platelet GPx4 activity assay and western blot analyses allowed us to provide the evidence of GPx4 in human platelets. In addition, our data show a cytosolic, membrane and mitochondrial localization. Although GPx4 activity has been detected using either with cysteine, mercaptoethanol or dithiols as cosubstrate, our results support that GSH is the most effective cosubstrate. Finally, we have examined lipid metabolism and pro/antioxidant status of diabetic platelets. Despite a good glycemic control and in the absence of any cardiovascular complications of diabetic patients, an increased platelet oxidative stress was detected (increase of platelet MDA levels, decrease of vitamin E level and GPx activities). Moreover, our results show an increased platelet aggregation and basal arachidonic acid metabolism. It could be hypothesized that an increased of arachidonic acid release, via an activation of cytosolic phospholipase A2, mightly partly be responsible for platelet hyperactivity is diabetes.VILLEURBANNE-DOC'INSA LYON (692662301) / SudocSudocFranceF

Fatty acids platelets and oxidative markers following intravenous n-3 fatty acids administration in cystic fibrosis: An open pilot observational study

AbstractBackgroundAn imbalance in the ratio of arachidonic acid and docosahexaenoic acid (DHA) was found in cystic fibrosis (CF) affected tissues and was suggested to promote inflammation. Several studies have shown that the long chain n-3 fatty acids reduced inflammatory activity while others have highlighted prooxidant activity of DHA at high concentrations. The aim of our study was to evaluate the effects of an intravenous fish-oil emulsion enriched with n-3 FA in patients with CF on plasma and platelet FA composition and peroxidation markers.Methods13 patients with CF received one IV emulsion per week of 2 mL/kg fish-oil n-3 emulsion for 12 weeks.ResultsThere was a significant increase in 20:5 n-3 and 22:6 n-3 platelet FA composition, no variation in 20:4 n-6, a decrease in n-9. There was no variation in plasma FA composition. Specific urinary markers of lipid peroxidation derived from n-3 and n-6 showed a very high level before infusion compared with usual values in healthy subjects which was not affected by treatment. A significant weight loss and a decrease in reduced glutathione were observed in adult patients.ConclusionsThe intravenous administration of n-3 FA in CF patients induced a significant modification in platelet FA composition but no modification of oxidative markers. However, the weight loss and the decreased level in reduced glutathione observed in adult patients may suggest a potential deleterious activity for some patients. Further studies are necessary to determine the optimal dose and route for long chain FA administration required to reach a potential beneficial effect

Lipid Remodeling of Murine Epididymosomes and Spermatozoa During Epididymal Maturation1

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