Caractérisation de métabolites oxygénés issus de l'acide alpha-linolénique (Effets anti-agrégants et anti-inflammatoires)

Les acides gras de la série n-3 et notamment l acide docosahexaénoïque (DHA) jouent un rôle important dans la prévention des maladies cardiovasculaires. Un de ses métabolites, la protectine DX (PDX), qui est un isomère de la protectine D1 (PD1), inhibe l agrégation des plaquettes sanguines. D autres composés similaires appelés "poxytrins", qui possèdent aussi un triène conjugué avec une géométrie E,Z,E, ont également été synthétisés à partir d'autres acides gras polyinsaturés (AGPI) via la lipoxygénase de soja. Ces composés présentent des propriétés anti-agrégantes en inhibant la cyclo-oxygénase plaquettaire et le récepteur du thromboxane A2. Dans cette thèse, nous décrivons de nouveaux composés dihydroxylés synthétisés par la 15-lipoxygénase de soja à partir de l acide alpha-linolénique (18:3n-3), un acide gras polyinsaturé indispensable consommé au niveau du gramme chez l Homme adulte. Il est converti en acides gras monohydroxylés et dihydroxylés. Ces composés ont été séparés par HPLC en phase inverse et caractérisés par GC-MS après dérivation adéquate. Un acide gras monohydroxylé, majoritaire, l acide 13(S)-octadécatriénoïque et quatre acides gras dihydroxylés ont été détectés. Ces derniers présentent tous un spectre UV caractéristique avec une absorption maximale à 270 nm et deux épaulements à 260 et 280 nm. Les spectres UV de deux d'entre eux sont superposables à celui de la PDX, ce qui suggère une géométrie E,Z,E des doubles liaisons de leur triène. La caractérisation complète de ces composés a été réalisée par RMN à haut champ et par GC-MS. Ce sont les acides 9(R),16(S)-dihydroxy-octadéca-10E,12E,14E-triénoïque, 9(S),16(S)-dihydroxy-octadéca-10E,12E,14E-triénoïque, 9(S),16(S)-dihydroxy-octadéca-10E,12Z,14E-triénoïque et 9(R),16(S)-dihydroxy-octadéca-10E,12Z,14E-triénoïque. Ils sont également synthétisés par la 15 lipoxygénase recombinante humaine de type 2. Ces composés dihydroxylés 9,16-diHOTEs ont été testés sur les plaquettes isolées à partir du sang humain. Nous avons observé que seules les molécules ayant la géométrie E,Z,E du triène conjugué inhibent l'agrégation plaquettaire induite par le collagène et inhibent la cyclooxygénase-1 (COX-1) de mouton. Les propriétés anti-inflammatoires de ces produits ont également été étudiés. Tous les isomères 9,16-diHOTEs, possédant un triène conjugué avec une géométrie E,Z,E, inhibent la COX-2 recombinante humaine et seul l acide 9(R),16(S)-dihydroxy-octadéca-10E,12Z,14E-triénoïques inhibe la 5-lipoxygénase des leucocytes, siège de la synthèse des leucotriènes issus de l acide arachidonique. En conclusion, les composés dihydroxlés possédant un triène conjugué E,Z,E, issus du 18:3n-3, ainsi que la PDX, inhibent l activité des COX-1 et 2, et seraient anti-agrégants et anti-inflammatoires. Ces résultats donnent des perspectives pharmacologiques aux recommandations nutritionnelles promouvant la consommation d acide alpha linolénique.N-3 fatty acids, especially docosahexaenoic acid (DHA), play an important role in the prevention of cardiovascular diseases. One metabolite of DHA, protectin DX (PDX), an isomer of protectin D1 (PD1) (Chen P et al., 2009),possesses inhibits blood platelet aggregation. Similar compounds called "poxytrins", which have a conjugated triene with a E,Z,E geometry have also been synthesized from other polyunsaturated fatty acids (PUFA) by soybean lipoxygenase. They have anti-aggregating properties by inhibiting platelet cyclooxygenase and thromboxane A2 receptor (Chen P et al., 2011). In this thesis, we describe new dihydroxy compounds synthesized by the soybean 15-lipoxygenase from alpha-linolenic acid (18:3n-3), an essential PUFA that is consumed in the gram range in human adults . It is converted into monohydroxylated and dihydroxylated derivatives. These compounds were separated by reverse phase high performance liquid chromatography (HPLC) and characterized by gas chromatography-mass spectrometry (GC-MS) after appropriate derivatization. A main monohydroxylated fatty acid, 13(S)-octadecatrienoic acid (13(S)-OH-18:3) and four dihydroxylated fatty acids were detected. The last ones have all a characteristic UV spectrum with a maximum absorbance at 270 nm with two shoulder peaks at 260 and 280 nm. The UV spectra from two of them are superimposable to that of PDX, suggesting a E,Z,E geometry for their conjugated triene. The complete characterization of these compounds was performed by high field nuclear magnetic resonance (NMR) and by GC-MS. These are the 9(R),16(S)-dihydroxy-octadeca-10E,12E,14E-trienoic, 9(S),16(S)-dihydroxy-octadeca-10E,12E,14E-trienoic, 9(S),16(S)-dihydroxy-octadeca-10E,12Z,14E-trienoic and 9(R),16(S)-dihydroxy-octadeca-10E,12Z,14E-trienoic acids. They can also be synthesized by the (type 2) 15 human recombinant lipoxygenase. These dihydroxylated compounds (9,16-diHOTEs)were tested on isolated human blood platelets. We observed that only molecules containing a conjugated triene with a E,Z,E geometry are able to inhibit platelet aggregation induced by collagen, and inhibit sheep cyclooxygenase-1 (COX-1). The anti-inflammatory properties of these products were also studied. All 9,16-diHOTEs isomers having a conjugated triene with a E,Z,E geometry, inhibit human recombinant cyclooxygenase-2 (COX-2) and only 9(R),16(S)-dihydroxy-octadeca-10E,12Z,14E-trienoic acid inhibits polymorphonuclear leukocytes (PMN) 5-lipoxygenase which is involved in the leukotriene synthesis from arachidonic acid. In conclusion, the E,Z,E dihydroxlated compounds from 18:3n-3, as well as PDX, inhibiting the COX-1 and 2 activities appear to be anti-aggregatory and anti-inflammatory agents. These results provide pharmacological perspectives to nutritional recommendations promoting the intake of alpha-linolenic acid.VILLEURBANNE-DOC'INSA-Bib. elec. (692669901) / SudocSudocFranceF

Docosahexaenoic acid-containing choline phospholipid modulates LPS-induced neuroinflammation in vivo and in microglia in vitro

BACKGROUND: Neuroinflammatory processes are considered a double-edged sword, having both protective and detrimental effects in the brain. Microglia, the brain's resident innate immune cells, are a key component of neuroinflammatory response. There is a growing interest in developing drugs to target microglia and control neuroinflammatory processes. In this regard, docosahexaenoic acid (DHA), the brain's n-3 polyunsaturated fatty acid, is a promising molecule to regulate pro-inflammatory microglia and cytokine production. Several works reported that the bioavailability of DHA to the brain is higher when DHA is acylated to phospholipid. In this work, we analyzed the anti-inflammatory activity of DHA-phospholipid, either acetylated at the sn-1 position (AceDoPC, a stable form thought to have superior access to the brain) or acylated with palmitic acid at the sn-1 position (PC-DHA) using a lipopolysaccharide (LPS)-induced neuroinflammation model both in vitro and in vivo. METHODS: In vivo, adult C57Bl6/J mice were injected intravenously (i.v.) with either AceDoPC or PC-DHA 24 h prior to LPS (i.p.). For in vitro studies, immortalized murine microglia cells BV-2 were co-incubated with DHA forms and LPS. AceDoPC and PC-DHA effect on brain or BV-2 PUFA content was assessed by gas chromatography. LPS-induced pro-inflammatory cytokines interleukin IL-1β, IL-6, and tumor necrosis factor (TNF) α production were measured by quantitative PCR (qPCR) or multiplex. IL-6 receptors and associated signaling pathway STAT3 were assessed by FACS analysis and western-blot in vitro. RESULTS: In vivo, a single injection of AceDoPC or PC-DHA decreased LPS-induced IL-6 production in the hippocampus of mice. This effect could be linked to their direct effect on microglia, as revealed in vitro. In addition, AceDoPC or PC-DHA reduced IL-6 receptor while only AceDoPC decreased IL-6-induced STAT3 phosphorylation. CONCLUSIONS: These results highlight the potency of administered DHA-acetylated to phospholipids-to rapidly regulate LPS-induced neuroinflammatory processes through their effect on microglia. In particular, both IL-6 production and signaling are targeted by AceDoPC in microglia.Metabolism in human of a structured phospholipid from marine origin and neural effec

Caractérisation d'un métabolite oxygéné dérivé de l'acide docosahexaénoïque (effet sur les plaquettes sanguines)

L acide docosahexaénoïque (DHA) est un acide gras d intérêt nutritionnel avec des potentialités bénéfiques dans la prévention des maladies cardiovasculaires. Des résultats récents montrent que le DHA est oxygéné en dérivés dotés de propriétés anti-inflammatoires puissantes appelés résolvines. Parmi ces dernières, la protectine D1 (PD1) a fait l objet de recherches approfondies. Cette thèse montre la caractérisation d un isomère, que nous avons nommé PDX, molécule capable d inhiber l agrégation des plaquettes sanguines humaines. La PDX a été préparée par oxygénation du DHA catalysée par la 15/ -6-lipoxygénase de soja. Une série d approches physico-chimiques à haute performance et différentes techniques RMN ont permis de caractériser la position des groupements hydroxyles, la stéréochimie des carbones oxygénés et la géométrie de chaque liaison du triène conjugué. L utilisation d 18O2 a également montré que la synthèse se fait via un mécanisme de double oxygénation. La PDX est donc l acide 10(S),17(S)-docosa-4Z,7Z,11E,13Z,15E,19Z- hexaénoïque qui diffère de la PD1 par la configuration du carbone 10 et par la géométrie du triène conjugué qui est 11E,13Z,15E. Connaissant les propriétés anti-agrégantes du DHA, nous avons aussi recherché si la PDX peut inhiber l agrégation plaquettaire. La PDX inhibe à des concentrations submicromolaires l agrégation induite par le collagène ou l acide arachidonique. Nous avons trouvé également que la PDX inhibe dans les plaquettes sanguines humaines la synthèse des prostaglandines sans altérer l activité 12-lipoxygénase. La PDX inhibe aussi l agrégation induite par l analogue stable de la prostaglandine H2 (U-46619) qui agit spécifiquement sur le récepteur du thromboxane A2. Par ailleurs, nous avons montré que tous les composés dihydroxylés contenant un triène conjugué et présentant une géométrie E/Z/E sont capables d inhiber l agrégation plaquettaire, alors que ceux qui ont une géométrie E/E/Z, ou E/E/E, sont inactifs. De plus, la position du triène conjugué sur la chaîne hydrocarbonée de l acide gras ainsi que le nombre de doubles liaisons extérieures au triène n affectent pas les propriétés inhibitrices de ces composés. En conclusion, la PDX s avère être un puissant agent anti-agrégant plaquettaire avec des potentialités anti-thrombotiques.VILLEURBANNE-DOC'INSA LYON (692662301) / SudocSudocFranceF

Biological relevance of double lipoxygenase products of polyunsaturated fatty acids, especially within blood vessels and brain

International audienceThe double lipoxygenation of polyunsaturated fatty acids (PUFA) is possible with PUFA having at least three methylene-interrupted double bonds. Several PUFA of the omega-3/n-3 and -6 families may be converted through this route, and the products show interesting inhibitory effects on blood platelet function and cyclooxygenase activities. This review focuses on two main omega-3 PUFA of nutritional interest, namely docosahexaenoic acid (DHA/22:6n-3) and alpha linolenic acid (ALA/18:3n-3). The chemical configuration of the double lipoxygenase end-product from DHA (protectin DX) is compared with that of protectin D1 which is produced through a mono-lipoxygenation step followed by an epoxidation and epoxide hydrolysis process. The different metabolic pathways are discussed as well as the different biological activities of both protectins

Anti-inflammatory and anti-virus potential of poxytrins, especially protectin DX

International audiencePoxytrins (Pufa Oxygenated Trienes) are dihydroxy derivatives from polyunsaturated fatty acids (PUFA) with adjacent hydroxyl groups to a conjugated triene having the specific E,Z,E geometry. They are made by the double action of one lipoxygenase or the combined actions of two lipoxygenases, followed by reduction of the resulting hydroperoxides with glutathione peroxidase. Because of their E,Z,E conjugated triene, poxytrins may inhibit inflammation associated with cyclooxygenase (COX) activities, and reactive oxygen species (ROS) formation. In addition of inhibiting COX activities, at least one poxytrin, namely protectin DX (PDX) from docosahexaenoic acid (DHA), has also been reported as able to inhibit influenza virus replication by targeting its RNA metabolism

Confusion between protectin D1 (PD1) and its isomer protectin DX (PDX). An overview on the dihydroxy-docosatrienes described to date.

International audienceThere is currently a growing interest in docosahexaenoic acid (DHA) oxygenated metabolites. Among them, protectin D1 (PD1), an endogenous dihydroxylated and non-cyclic docosatriene made through lipoxygenation and hydrolysis of an epoxide intermediate, shows appealing biological effects. However, with the present paper we wish to point out that results are sometimes assigned to PD1 while they are indeed related to its isomer protectin DX (PDX) made through double lipoxygenation only. These misleading conclusions urge us to review herein the structural/chemical and biological differences in the docosatrienes reported to date in the literature i.e. PD1, the related PD1n-3 DPA, AT-NPD1, maresin 1 (MaR1) and MaR1n-3 DPA, as well as their poxytrin analogs such as PDX, and some synthetic diastereoisomers. Hopefully, this will avoid further mistakes and confusion in the future

Poxytrins, a class of oxygenated products from polyunsaturated fatty acids, potently inhibit blood platelet aggregation.: PUFA conjugated trienes and platelet inhibition

International audienceDocosahexaenoic acid (DHA), an important component of marine lipids, exhibits anti-inflammatory activity related to some of its oxygenated metabolites, such as neuroprotectin/protectin D1 [NPD1/PD1; 10(R),17(S)-dihydroxy-docosa-4Z,7Z, 11E,13E,15Z,19Z-hexaenoic acid] produced through the 15-lipoxygenase pathway. However, other metabolites from DHA can be produced through this pathway, and other polyunsaturated fatty acids (PUFAs) of nutritional value may be oxygenated as well. Their biological activities remain unknown. Isomers of protectin D1 were synthesized using soybean lipoxygenase and tested for their ability to inhibit human blood platelet aggregation. A geometric isomer called PDX, previously described with the 11E,13Z,15E geometry, instead of 11E,13E,15Z in PD1, inhibited platelet aggregation at submicromolar concentrations when induced by either collagen, arachidonic acid, or thromboxane. The inhibition occurred at the level of both the cyclooxygenase activity and thromboxane receptor site. Interestingly, all the metabolites tested exhibiting the E,Z,E-conjugated triene were active, whereas E,E,Z trienes (as in PD1) or all-trans (E,E,E) trienes were inactive. We conclude that PDX and other oxygenated products from PUFAs of nutritional interest, having the E,Z,E-conjugated triene motif and collectively named poxytrins (PUFA oxygenated trienes), might have antithrombotic potential

In vitro and in vivo bimodal effects of docosahexaenoic acid supplements on redox status and platelet function

International audienceDocosahexaenoic acid (DHA) is a prominent nutrient of marine lipids. Together with eicosapentaenoic acid, it is recognized as a protective molecule against atherosclerosis and thrombosis through the regulation of blood cell functions, especially platelets. Its high unsaturation index may however make it prone to peroxidation, which is usually considered as deleterious. This short review takes into consideration this possibility related to DHA concentrations both in vitro and in vivo. It is suggested that protective effects of DHA on platelet activation depend on the reduction of oxidative stress, and appear bimodal with the abolishment of such a protection when DHA is used at relatively high concentrations. Introduction Docosahexaenoic acid (DHA) is one of the long-chain polyunsaturated fatty acids (PUFA) of the omega-3 family found in marine lipids. Together with eicosapentaenoic acid (EPA), DHA is well-known for its anti-atherothrombotic potential, in particular by decreasing the arachidonic acid (ArA) content of blood cells [1], inhibiting thromboxane production in platelets, as well as leukotriene B4 formation in leukocytes [2]. In addition to these inhibitory effects on ArA metabolism, DHA may decrease athero-thrombogenesis through the action of its lipoxygenase (LOX) end-products. Mono-hydroxylated derivatives produced through 12-, and 15-LOX, namely 11-, 14-, and 17-OH-22:6 (11-, 14-, and 17-HDoHE), respectively, inhibit the thromboxane-induced platelet aggregation (induced by the stable thromboxane A2 receptor agonist U-46619), even stronger than 12-OH-20:4 and-20:5 (12-HETE and-HEPE) [3]. More recently, the double lipoxygenase product of DHA, 10(S),17(S)-diHDoHE, named protectin DX, has been shown to inhibit platelet aggregation through the inhibition of cyclooxygenase-1, which initiates prostanoid synthesis including the potent pro-aggregatory thromboxane A2, as well as the response to the thromboxane A2 mimetic U-46619 [4]. In addition, DHA is known to be oxygenated into several di/tri-OH derivatives (protectin D1, maresins, and resolvins) that are potent inhibitors of the inflammation process [5]. Supplementation of humans with long-chain omega-3 PUFA is usually considered as a positive approach to decrease the risk of atherosclerosis and thrombosis [6], as recently reviewed [7], although some controversies arose in the recent years [8]. Among the controversial issues are the intake doses of those highly unsaturated PUFA, which may be prone to peroxidation [9]. We have been especially interested in the effects of different concentrations of DHA upon blood platelets and their blood environment both in vitro and in vivo, in particular regarding the redox status

Dose-effect and metabolism of docosahexaenoic acid: Pathophysiological relevance in blood platelets.

International audienceDocosahexaenoic acid (DHA) is known as a major nutrient from marine origin. Considering its beneficial effect in vascular risk prevention, the effect of DHA on blood components, especially platelets, will be reviewed here. Investigating the dose-effect of DHA in humans shows that daily intake lower than one gram/day brings several benefits, such as inhibition of platelet aggregation, resistance of monocytes against apoptosis, and reinforced antioxidant status in platelets and low-density lipoproteins. However, higher daily intake may be less efficient on those parameters, especially by losing the antioxidant effect. On the other hand, a focus on the inhibition of platelet aggregation by lipoxygenase end-products of DHA is made. The easy conversion of DHA by lipoxygenases and the formation of a double lipoxygenation product named protectin DX, reveal an original way for DHA to contribute in platelet inhibition through both the cyclooxygenase inhibition and the antagonism of thromboxane A(2) action

AceDoPC, a structured phospholipid to target the brain with docosahexaenoic acid

International audienceAceDoPCr (R) is a structured phospholipid or acetyl-LysoPC-DHA made to prevent docosahexaenoic acyl migrating from the sn-2 to sn-1 position of the phospholipid, however keeping the main physical-chemical properties of LysoPC-DHA. As previously shown for LysoPC-DHA, AceDoPCr (R) allows DHA crossing a re-constituted blood-brain barrier with higher efficiency than non-esterified DHA or PC-DHA. When injected to blood of rats, AceDoPCr (R) is processed within the brain to deliver DHA to phosphatidyl-choline and -ethanolamine. When injected to rats following the induction of an ischemic stroke, AceDoPCr (R) prevents the extension of brain lesions more efficiently than DHA. Overall, these properties make AceDoPCr (R) a promising phospholipid carrier of DHA to the brain