Two weeks of docosahexaenoic acid (DHA) supplementation increases synthesis-secretion kinetics of n-3 polyunsaturated fatty acids compared to 8 weeks of DHA supplementation

Docosahexaenoic acid (DHA, 22:6n-3) must be consumed in the diet or synthesized from n-3 polyunsaturated fatty acid (PUFA) precursors. However, the effect of dietary DHA on the metabolic pathway is not fully understood. Presently, 21-day-old Long Evans rats were weaned onto one of four dietary protocols: 1) 8 weeks of 2% ALA (ALA), 2) 6 weeks ALA followed by 2 weeks of 2% ALA + 2% DHA (DHA), 3) 4 weeks ALA followed by 4 weeks DHA and 4) 8 weeks of DHA. After the feeding period, ²H₅-ALA and ¹³C₂₀-eicosapentaenoic acid (EPA, 20:5n-3) were co-infused and blood was collected over 3 h for determination of whole-body synthesis-secretion kinetics. The synthesis-secretion coefficient (ml/min, means ± SEM) for EPA (0.238±0.104 vs. 0.021±0.001) and DPAn-3 (0.194±0.060 vs. 0.020±0.008) synthesis from plasma unesterified ALA, and DPAn-3 from plasma unesterified EPA (2.04±0.89 vs. 0.163±0.025) were higher (P<.05) after 2 weeks compared to 8 weeks of DHA feeding. The daily synthesis-secretion rate (nmol/d) of DHA from EPA was highest after 4 weeks of DHA feeding (843±409) compared to no DHA (70±22). Liver gene expression of ELOVL2 and FADS2 were lower (P<.05) after 4 vs. 8 weeks of DHA. Higher synthesis-secretion kinetics after 2 and 4 weeks of DHA feeding suggests an increased throughput of the PUFA metabolic pathway. Furthermore, these findings may lead to novel dietary strategies to maximize DHA levels while minimizing dietary requirements.     

Some studies on dehydro-d-ascorbic acid 2-(phenylhydrazone)

Reaction of hydroxylamine with d-erythro-2,3-hexodiulosono-1, 4-lactone 2-(phenylhydrazone) (2) gave the 3-oxime 2-(phenylhydrazone) (3). On boiling with acetic anhydride, 3 gave 4-(d-erythro-2,3-diacetoxy-l-hydroxypropyl)-2-phenyl-1,2, 3-triazoIe-5-carboxylic acid 5,1′-lactone. Compound 3 was also converted into the related, unacetylated 2-(p-bromophenyl)triazole with bromine. Treatment of 2 with boiling acetic anhydride gave an optically inactive, olefinic compound, assigned the structure 4-(2-acetoxyethylidene)-4-hydroxy-2,3-dioxobutano-1,4-lactone 2-(phenylhydrazone). The 2-(phenylhydrazone) 2 gave the corresponding 2,3-bis(phenylhydrazone) on condensation with phenylhydrazine.     

Efficient synthesis of the cyclometalated complex fac-[Rh(ppy)3] (ppy = 2-phenylpyridinato)

A new convenient high-yield synthesis of the tris-cyclometalated complexes fac-[Rh(ppy)₃] (4; ppy = 2-phenylpyridinato) was developed. Complex 4 was prepared in a kind of one-pot synthesis starting from in situ prepared [Rh(acac)(coe)₂] (2) which was heated in refluxing 2-phenylpyridine for a short time. After purification by filtration over alumina, compound 4 was obtained in yields of 65%. Also [Rh(acac)(ppy)₂] (3) was prepared in a similar manner by oxidative addition of Hppy in refluxing toluene in high yields. In contrast to previous findings with the analogous iridium compounds, there was not any hint at the formation of the isomer mer-[Rh(ppy)₃] using similar reaction conditions as applied for iridium. Furthermore the compound [{Rh(μ-Cl)(ppy)₂}₂] (5) was prepared from [{Rh(μ-Cl)(coe)₂}₂] (1) and Hppy in refluxing toluene in nearly quantitative yield.     

Omega-3 polyunsaturated fatty acid has an anti-oxidant effect via the Nrf-2/HO-1 pathway in 3T3-L1 adipocytes

Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid (ω3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of ω3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treated with ω3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). ω3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with ω3-PUFA prevented H₂O₂-induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress.     

Nonenzymatic lipid mediators,neuroprostanes, exert the antiarrhythmic properties of docosahexaenoic acid

Neuroprostanes are lipid mediators produced by nonenzymatic free radical peroxidation of docosahexaenoic acid (DHA). DHA is associated with a lower atherosclerosis risk, suggesting a beneficial role in cardiovascular diseases. The aim of this study was to investigate the influence of DHA peroxidation on its potentially antiarrhythmic properties (AAP) in isolated ventricular cardiomyocytes and in vivo in post-myocardial infarcted mice. Calcium imaging and biochemical experiments indicate that cardiac arrhythmias induced by isoproterenol are associated with Ca²⁺ leak from the sarcoplasmic reticulum after oxidation and phosphorylation of the type 2 ryanodine receptor (RyR2) leading to dissociation of the FKBP12.6/RyR2 complex. Both oxidized DHA and 4(RS)-4-F_4t-NeuroP prevented cellular arrhythmias and posttranslational modifications of the RyR2 leading to a stabilized FKBP12.6/RyR2 complex. DHA per se did not have AAP. The AAP of 4(RS)-4-F_4t-NeuroP was also observed in vivo. In this study, we challenged the paradigm that spontaneously formed oxygenated metabolites of lipids are undesirable as they are unconditionally toxic. This study reveals that the lipid mediator 4(RS)-4-F_4t-neuroprostane derived from nonenzymatic peroxidation of docosahexaenoic acid can counteract such deleterious effects through cardiac antiarrhythmic properties. Our findings demonstrate 4(RS)-4-F_4t-NeuroP as a mediator of the cardioprotective AAP of DHA. This discovery opens new perspectives for products of nonenzymatic oxidized ω3 polyunsaturated fatty acids as potent mediators in diseases that involve ryanodine complex destabilization such as ischemic events.     

Beta-eliminative cleavage of 3-ketocerebroside and 3-ketosphingomyelin

3-Ketosphingolipids derived from cerebroside and sphingomyelin were converted to a less polar compound in as weak alkali as 0.0005 M Na₂CO₃ in chloroform-methanol 2 : 1 (v/v). The product was identified as 2-acylamido-3-keto-1,4-octadecadiene by several spectroscopic procedures and cleavage was concluded to occur by beta-elimination. The beta-elimination occurred at above pH 11.5 in aqueous solution. At higher pH values deacylation also occurred. The influence of solvent effects on beta-elimination was studied. This reaction can be used to obtain d oligosaccharides, phosphorycholine and other hydrophilic moieties from sphingolipids.     

Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa₃ in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230).2. The K_m for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 μM in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation.3. The apparent K_m for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts “competitively” towards oxygen.4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H⁺ gradients modify the oxidase activity in reconstituted vesicles.     

Rainbow trout (Oncorhynchus mykiss) Elovl5 and Elovl2 differ in selectivity for elongation of omega-3 docosapentaenoic acid

The synthesis of the omega-3 long-chain polyunsaturated fatty acids (LCPUFA)  eicosapentaenoic acid (EPA; 20:5n− 3) and docosahexaenoic acid (DHA; 22:6n − 3) from dietary α-linolenic acid (ALA; 18:3n − 3) requires three desaturation and three elongation steps in vertebrates. The elongation of EPA to docosapentaenoic acid (DPA; 22:5n − 3) can be catalysed by the elongase enzymes Elovl5 or Elovl2, but further elongation of DPA to 24:5n − 3, the penultimate precursor of DHA, is limited to Elovl2, at least in mammals. Elovl5 enzymes have been characterised from seventeen fish species but Elovl2 enzymes have only been characterised in two of these fish. The essentiality of Elovl2 for DHA synthesis is unknown in fish. This study is the first to identify an Elovl2 in rainbow trout (Oncorhynchus mykiss) and functionally characterise the Elovl5 and Elovl2 using a yeast expression system. Elovl5 was active with C_18–20 PUFA substrates and not C₂₂ PUFA. In contrast, Elovl2 was active with C_20–22 PUFA substrates and not C₁₈ PUFA. Thus, rainbow trout is dependent on Elovl2 for DPA to 24:5n − 3 synthesis and ultimately DHA synthesis. The expression of elovl5 was significantly higher than elovl2 in liver. Elucidating this dependence on Elovl2 to elongate DPA and the low elovl2 gene expression compared with elovl5 are critical findings in understanding the potential for rainbow trout to synthesize DHA.     

Regulation of rat brain polyunsaturated fatty acid (PUFA) metabolism during graded dietary n-3 PUFA deprivation

Knowing threshold changes in brain lipids and lipid enzymes during dietary n-3 polyunsaturated fatty acid deprivation may elucidate dietary regulation of brain lipid metabolism. To determine thresholds, rats were fed for 15 weeks DHA-free diets having graded reductions of α-linolenic acid (α-LNA). Compared with control diet (4.6% α-LNA), plasma DHA fell significantly at 1.7% dietary α-LNA while brain DHA remained unchanged down to 0.8% α-LNA, when plasma and brain docosapentaenoic acid (DPAn-6) were increased and DHA-selective iPLA₂ and COX-1 activities were downregulated. Brain AA was unchanged by deprivation, but AA selective-cPLA₂, sPLA₂ and COX-2 activities were increased at or below 0.8% dietary α-LNA, possibly in response to elevated brain DPAn-6. In summary, homeostatic mechanisms appear to maintain a control brain DHA concentration down to 0.8% dietary DHA despite reduced plasma DHA, when DPAn-6 replaces DHA. At extreme deprivation, decreased brain iPLA₂ and COX-1 activities may reduce brain DHA loss.     

Termination of asynchronous contractile activity in rat atrial myocytes by n-3 polyunsaturated fatty acids

A protective effect of the n-3 polyunsaturated fatty acids (PUFAs) in preventing ventricular fibrillation in experimental animals and cultured cardiomyocytes has been demonstrated in a number of studies. In this study, a possible role for the n-3 PUFAs in the treatment of atrial fibrillation (AF) was investigated at the cellular level using atrial myocytes isolated from young adult rats as the experimental model. Electrically-stimulated, synchronously-contracting myocytes were induced to contract asynchronously by the addition of 10 M isoproterenol. Asynchronous contractile activity was reduced following acute addition of the n-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) at 10 M, compared with no fatty acid addition (from 99.0 ±: 1.0% to 30.7 ± 5.2% (p < 0.05) for DHA and 23.8 ± 2.8% (p < 0.01) for EPA), while the saturated fatty acid, docosanoic acid (DA) and the methyl ester of DHA (DHA m.e.) did not exert a significant effect on asynchronous contractile activity. Asynchronous contractile activity was also reduced to 1.7 ± 1.7% in the presence of the membrane fluidising agent, benzyl alcohol (p < 0.001 vs no fatty acid addition). Cell membrane fluidity was determined by steady state fluorescence anisotropy using the fluorescent probe, TMAP-DPH. Addition of DHA, EPA or benzyl alcohol significantly increased sarcolemmal membrane fluidity (decreased anisotropy, r_ss) of atrial myocytes compared with no addition of fatty acid (control) (from r_ss = 0.203 ±0.004 to 0.159 ± 0.004 (p < 0.01) for DHA, 0.166 ± 0.001 (p < 0.01) for EPA and 0.186 ±0.003 (p < 0.05) for benzyl alcohol, while DA and DHA m.e. were without effect. It is concluded that the n-3 PUFAs exert anti-asynchronous effects in rat atrial myocytes by a mechanism which may involve changes in membrane fluidity.     

Dihydroxyacetone, the active browning ingredient in sunless tanning lotions, induces DNA damage, cell-cycle block and apoptosis in cultured HaCaT keratinocytes

Dihydroxyacetone (DHA), the active substance in sunless tanning lotions reacts with the amino groups of proteins to form a brown-colored complex. This non-enzymatic glycation, known as the Maillard reaction, can also occur with free amino groups in DNA, raising the possibility that DHA may be genotoxic. To address this issue we investigated the effects of DHA on cell survival and proliferation of a human keratinocyte cell line, HaCaT. Dose- and time-dependent morphological changes, chromatin condensation, cytoplasmic budding and cell detachment were seen in cells treated with DHA. Several dead cells were observed after long-time (24 h) incubation with 25 mM DHA or more. Furthermore, an extensive decline in proliferation was observed 1 day after DHA exposure for 24 h. When applied in different concentrations (5–50 mM) and for different time periods (1, 3 or 24 h) DHA caused a G₂/M block after the cyclin B₁ restriction point. Exit from this cell-cycle block was associated with massive apoptosis, as revealed by a clonogenic assay, TUNEL staining and electron microscopy. Furthermore, DHA caused DNA damage as revealed by the alkaline comet assay. Preincubation with antioxidants prevented the formation of DNA strand breaks. The DHA toxicity may be caused by direct redox reactions, with formation of ROS as the crucial intermediates. The genotoxic capacity of DHA raises a question about the long-term clinical consequences of treatment of the skin with this commonly used compound.     

Biological action of docosahexaenoic acid in a 3D tissue-engineered psoriatic skin model: Focus on the PPAR signaling pathway

N-3 polyunsaturated fatty acids (n-3 PUFAs), and in particular docosahexaenoic acid (DHA), have many beneficial metabolic effects, including reducing epidermal thickness in patients with psoriasis. The positive impacts of DHA in psoriasis could be mediated by its interactions with the PPAR signaling pathway, as well as by its secretion of anti-inflammatory bioactive metabolites, but the detailed metabolism is still not understood. In the present study, we evaluated the influence of DHA on the main features of psoriasis and its effects on the PPAR signaling pathway, in a psoriatic in vitro skin model. Healthy and psoriatic skin substitutes were produced according to the tissue-engineered self-assembly method, using culture media supplemented with 10 μM of DHA. The presence of DHA led to a reduction in the abnormal cell differentiation of psoriatic keratinocytes, seen in the increased expression of filaggrin and keratin 10. DHA was incorporated into the membrane phospholipids of the epidermis and transformed principally into eicosapentaenoic acid (EPA). Furthermore, the addition of DHA into the culture medium led to a decrease in the levels of lipid mediators derived from n-6 PUFAs, mainly prostaglandin E₂ (PGE₂) and 12-hydroxyeicosatetraenoic acid (12-HETE). Finally, DHA supplementation rebalanced the expression of PPAR receptors and caused a decrease in the secretion of TNF-α. Altogether, our results show that DHA possesses the ability to attenuate the psoriatic characteristics of psoriatic skin substitutes, mostly by restoring epidermal cell differentiation and proliferation, as well as by reducing inflammation.     

The reaction of carbonyl cyanide phenylhydrazones with thiols

Carbonyl cyanide phenylhydrazone and its ring-substituted analogs react with thiols (thioglycolic acid, 2-mercaptoethanol, dithiothreitol) and amino-thiols (cysteine, glutathione) to give corresponding N-(substituted phynyl)-N′-(alkythiodicyano)-methylhydrazine derivatives. These addition products decompose to the original components in alkaline solution. On the other hand, in the presence of an excess of thiols in aqueous buffered systems the addition reactions are practically quantitative with respect to phenylhydrazones, follow pseudo-first-order kinetics and can be investigated spectrophotometrically. These reactions are of the bimolecular Ad_N type where the non-dissociated form of carbonyl cyanide phenylhydrazones function as an electrophilic component, while the RS^? ion plays the role of nucleophilic component in the case of thiols (the attack of the azomethine group).The reactivity of carbonyl cyanide phenylhydrazones with respect to thiols increases in the order carbonyl cyanide phenylhydrazone < carbonyl cyanide m-chlorophenylhydrazone < carbonyl cyanide p-trifluoromethoxyphenylhydrazone which corresponds to the order of decreasing values of the pK_a constants. On the other hand, the reactivity of thiols increases with their basicity.The reactivity of carbonyl cyanide phynylhydrazone with thiols is comparable with the reactivity of phynyl isothiocyanate and N-ethylmaleimide. It was demostrated that carbonyl cyanide phenylhydrazone is an efficient inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12).The results obtained are discussed in relation to the biological activity of carbonyl cyanide phenylhydrazones.     

Neuroprotective Effects of n-3 Polyunsaturated Fatty Acid-Enriched Phosphatidylserine Against Oxidative Damage in PC12 Cells

Neurodegenerative diseases are defined by progressive loss of specific neuronal cell populations and are associated with protein aggregates. Oxidative stress has been implicated in their pathological processes. Previous studies revealed that docosahexaenoic acid (DHA) is beneficial in neurodegenerative diseases. Phospholipids (PLs) derived from marine products are rich in DHA and eicosapentaenoic acid (EPA). In the present study, we investigated the neuroprotective effects of DHA-enriched and unenriched phosphatidylcholine (PC) and phosphatidylserine (PS) on oxidative stress induced by hydrogen peroxide (H₂O₂) and tert-butylhydroperoxide in PC12 cells. Cell viability and leakage of lactate dehydrogenase results showed that the neuroprotective effect of PS was superior to that of PC. DHA- and EPA-enriched PC and PS were superior to that without DHA or EPA; in addition, the improvement with n-3 polyunsaturated fatty acid-enriched PS (n-3 PS) was dose dependent. Acridine orange/ethidium bromide staining showed that DHA- and EPA-enriched PS (DHA/EPA-PS) could significantly inhibit apoptosis. Mechanistic studies revealed that EPA-PS and DHA-PS were effective to increase superoxide dismutase (SOD) levels by 48.4 and 58.2 % and total antioxidant capacity (T-AOC) level by 51 and 94 %, respectively, in the H₂O₂ model. Similar results for SOD and T-AOC levels were shown in the t-BHP model. EPA/DHA-PS could downregulate the messenger RNA level of Caspase-3, Caspase-9, and Bax, upregulate Bcl-2, inhibit Bax, and increase Bcl-2 at protein level. In conclusion, EPA/DHA-PS could protect PC12 cells from oxidative stress and prevent mitochondrial-mediated apoptosis. Our findings indicate that the neuroprotective effects of DHA/EPA-PLs depend on the molecular form. Further studies are necessary to reveal detailed mechanisms and structure–effect relationships.     

Mechanism of alcohol‐enhanced lucigenin chemiluminescence in alkaline solution

The chemiluminescence (CL) of lucigenin (Luc²⁺) can be enhanced by different alcohols in alkaline solution. The effect of different fatty alcohols on the CL of lucigenin was related to the carbon chain length and the number of hydroxyl groups. Glycerol provides the greatest enhancement. UV/Vis absorption spectra and fluorescence spectra showed that N‐methylacridone (NMA) was produced in the CL reaction in the presence of different alcohols. The peak of the CL spectrum was located at 470 nm in all cases, indicating that the luminophore was always the excited‐state NMA. The quenching of lucigenin CL by superoxide dismutase (SOD) and the electron spin resonance (ESR) results with the spin trap of 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) demonstrated that superoxide anions (O₂^?–) were generated from dissolved oxygen in the CL reaction and that glycerol and dihydroxyacetone (DHA) can promote O₂^?? production by the reduction of dissolved oxygen in alkaline solution. It was assumed that the enhancement provided by different alcohols was related to the solvent effect and reducing capacity. Glycerol and DHA can also reduce Luc²⁺ into lucigenin cation radicals (Luc^?+), which react with O₂^?? to produce CL, and glycerol can slowly transform into DHA, which is oxidized quickly in alkaline solution. Copyright © 2015 John Wiley & Sons, Ltd.     

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2�� (VIA)-deficient mice

Ca²⁺-independent phospholipase A₂β (iPLA₂β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA₂β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M_1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA₂β^−/−, iPLA₂β^+/−, and iPLA₂β^+/+ mice, and [1-¹⁴C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J_in, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA₂β^−/− or iPLA₂β^+/− compared with iPLA₂β^+/+ mice showed widespread and significant baseline reductions in k* and J_in for DHA. Arecoline increased both parameters in brain regions of iPLA₂β^+/+ mice but quantitatively less so in iPLA₂β^−/− and iPLA₂β^+/− mice. Consistent with iPLA₂β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA₂β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M_1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.     

Docosahexaenoic acid withstands the Aβ25-35-induced neurotoxicity in SH-SY5Y cells

Background

Docosahexaenoic acid (DHA, C22:6, n-3) ameliorates the memory-related learning deficits of Alzheimer's disease (AD), which is characterized by fibrillar amyloid deposits in the affected brains. Here, we have investigated whether DHA-induced inhibition of Amyloid β-peptide_25-35 (Aβ_25-35) fibrillation limits or deteriorates the toxicity of the human neuroblastoma cells (SH-SY5Y).

Experimental methods

In vitro fibrillation of Aβ_25-35 was performed in the absence or presence of DHA. Afterwards, SH-SY5Y cells were incubated with Aβ_25-35 in absence or presence 20 μM DHA to evaluate its effect on the Aβ_25-35-induced neurotoxicity by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)]-redox and TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assay and immunohistochemistry. The level of Aβ_25-35-induced lipid peroxide (LPO) was determined in the absence or presence of oligomer-specific antibody. Fatty acid profile was estimated by gas chromatography.

Results

DHA significantly reduced the Aβ_25-35 in vitro fibrillation, as indicated by fluorospectroscopy and transmission electron microscopy. Aβ_25-35 decreased the MTT-redox activity and increased the apoptotic damage and levels of LPO when compared with those of the controls. However, when the SH-SY5Y cells were treated with Aβ_25-35 in the presence of DHA, MTT redox potential significantly increased and the levels LPO decreased, suggesting an inhibition of the Aβ_25-35-induced neurotoxity. DHA improved the Aβ_25-35 induced DNA damage and axodendritic loss, with a concomitant increase in the cellular level of DHA, suggesting DHA protects the cell from neurotoxic degeneration.

Conclusion

DHA not only inhibits the in vitro fibrillation but also resists the Aβ_25-35-induced toxicity in the neuronal cells. This might be the basis of the DHA-induced amelioration of Aβ-induced neurodegeneration and related cognitive deficits.     

Docosahexaenoic acid down-regulates endothelial Nox 4 through a sPLA2 signalling pathway

We investigated the anti-inflammatory and antioxidant activities of docosahexaenoic acid (DHA) by evaluating its modulation of the two enzymes most involved in vascular inflammation, i.e. endothelial secreted phospholipase A₂ (sPLA₂) and NADPH oxidase 4 (Nox) 4. Exposure of human aortic endothelial cells (HAECs) to DHA led to its preferential incorporation into outer leaflet phospholipids. Pre-treatment with DHA abolished HAECs stimulation induced by A23187 and Ang II, whereas the effects on IL-1β treatment were less pronounced. Group V sPLA₂ RNA was similarly modulated by DHA supplementation. In addition, DHA decreased Nox 4 expression and activity; this effect was associated with reduced production of reactive oxygen species. Further, the use of specific inhibitors allowed demonstrating that group V sPLA₂ is involved in the down-regulation of Nox 4 expression and activity by DHA. This interplay is mediated by ERK and PKC.