Docosahexaenoic Acid (DHA) Reduces LPS-Induced Inflammatory Response Via ATF3 Transcription Factor and Stimulates Src/Syk Signaling-Dependent Phagocytosis in Microglia.

BACKGROUND/AIMS: Microglial cells play a crucial role in the development of neuroinflammation in response to harmful stimuli, such as infection, ischemia or injury. Their chronic activation, however, is associated with a progression of neurodegenerative diseases. Therefore, looking for potential factors limiting microglial activation, the effect of docosahexaenoic acid (DHA) on the inflammatory response and TREM2-dependent phagocytic activity in microglia was investigated. METHODS: In LPS-induced primary microglia preincubated with DHA, or without preincubation the expression of ATF3 and TREM2 genes and TREM2, Syk, Akt proteins were determined by RT-PCR and WB, respectively. Cell viability was assayed by MTT and cytokine and chemokine expression was determined by the Proteome Profiler assay. Moreover, the phagocytic activity of microglia was assayed using immunofluorescence. RESULTS: We found that DHA significantly increased the expression of ATF3 , and decreased the levels of CINC-1, CINC-2αß, CINC-3 chemokines, IL-1α and IL-1ß cytokines, and ICAM-1 adhesion protein. Additionally, preincubation of microglia with DHA resulted in increased Src/Syk kinases activation associated with increased phagocytic microglia activity. CONCLUSION: These findings indicate that DHA efficiently inhibits ATF3-dependent release of proinflammatory mediators and enhances phagocytic activity of microglia. The study provides a new mechanism of DHA action in reactive microglia, which may help limit neuronal damage caused by the pro-inflammatory milieu in the brain.

EPA stronger than DHA increases the mitochondrial membrane potential and cardiolipin levels but does not change the ATP level in astrocytes.

Astrocytes are highly energy-consuming glial cells critical for metabolic support to neurons. A growing body of evidence suggests that mitochondrial dysfunction in astrocytes is involved in age-related neurodegenerative disorders and that fish oil, rich in docosahexaenoic (DHA) and eicosapentaenoic (EPA) fatty acids, may alleviate cognition impairment in Parkinson's and Alzheimer's diseases. The present study examines the effect of DHA and EPA on mitochondrial membrane potential (MMP), apoptosis activation and ATP levels in astrocytes cultured in medium containing glucose or galactose, which limits oxidative phosphorylation (OXPHOS). MMP, expressed as the ratio of red to green JC-10 and MitoTracker fluorescence, increased in EPA-incubated cells in a dose dependent manner and was higher than in DHA-incubated astrocytes, also after uncoupling of OXPHOS by carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In cells cultured in glucose and galactose medium mitochondrial hyperpolarization had no impact on intracellular ATP level. Furthermore, both EPA and DHA elevated mitochondrial cardiolipin content, however only EPA did so in a dose-dependent manner and reduced apoptosis which was analyzed by flow cytometry.

An Overview of the Nrf2/ARE Pathway and Its Role in Neurodegenerative Diseases.

Nrf2 is a basic region leucine-zipper transcription factor that plays a pivotal role in the coordinated gene expression of antioxidant and detoxifying enzymes, promoting cell survival in adverse environmental or defective metabolic conditions. After synthesis, Nrf2 is arrested in the cytoplasm by the Kelch-like ECH-associated protein 1 suppressor (Keap1) leading Nrf2 to ubiquitin-dependent degradation. One Nrf2 activation mechanism relies on disconnection from the Keap1 homodimer through the oxidation of cysteine at specific sites of Keap1. Free Nrf2 enters the nucleus, dimerizes with small musculoaponeurotic fibrosarcoma proteins (sMafs), and binds to the antioxidant response element (ARE) sequence of the target genes. Since oxidative stress, next to neuroinflammation and mitochondrial dysfunction, is one of the hallmarks of neurodegenerative pathologies, a molecular intervention into Nrf2/ARE signaling and the enhancement of the transcriptional activity of particular genes are targets for prevention or delaying the onset of age-related and inherited neurogenerative diseases. In this study, we review evidence for the Nrf2/ARE-driven pathway dysfunctions leading to various neurological pathologies, such as Alzheimer's, Parkinson's, and Huntington's diseases, as well as amyotrophic lateral sclerosis, and the beneficial role of natural and synthetic molecules that are able to interact with Nrf2 to enhance its protective efficacy.

Omega-3 PUFAs Suppress IL-1ß-Induced Hyperactivity of Immunoproteasomes in Astrocytes.

The role of immunoproteasome (iP) in astroglia, the cellular component of innate immunity, has not been clarified. The results so far indicate that neuroinflammation, a prominent hallmark of Alzheimer's disease, strongly activates the iP subunits expression. Since omega-3 PUFAs possess anti-inflammatory and pro-resolving activity in the brain, we investigated the effect of DHA and EPA on the gene expression of constitutive (ß1 and ß5) and inducible (iß1/LMP2 and iß5/LMP7) proteasome subunits and proteasomal activity in IL-1ß-stimulated astrocytes. We found that both PUFAs downregulated the expression of IL-1ß-induced the iP subunits, but not the constitutive proteasome subunits. The chymotrypsin-like activity was inhibited in a dose-dependent manner by DHA, and much strongly in the lower concentration by EPA. Furthermore, we established that C/EBPα and C/EBPß transcription factors, being the cis-regulatory element of the transcription complex, frequently activated by inflammatory mediators, participate in a reduction in the iP subunits' expression. Moreover, the expression of connexin 43 the major gap junction protein in astrocytes, negatively regulated by IL-1ß was markedly increased in PUFA-treated cells. These findings indicate that omega-3 PUFAs attenuate inflammation-induced hyperactivity of iPs in astrocytes and have a beneficial effect on preservation of interastrocytic communication by gap junctions.

Docosahexaenoic fatty acid reduces the pro-inflammatory response induced by IL-1ß in astrocytes through inhibition of NF-κB and AP-1 transcription factor activation.

BACKGROUND: Astrocytes are responsible for a broad range of functions that maintain homeostasis in the brain. However, their response to the pro-inflammatory cytokines released by activated microglia in various neurological pathologies may exacerbate neurodegenerative processes. Accumulating evidence suggests that omega-3 docosahexaenoic fatty acid (DHA) has an anti-inflammatory effect in various cell cultures studies and in a variety of neurological disorders. In this study we examined the mechanism involved in the inhibition of the pro-inflammatory response by DHA in astrocytes treated with IL-1ß. METHODS AND RESULTS: Activation of the transcription factors NF-κB and AP-1 was measured in IL-1ß-treated primary astrocytes incubated with various concentrations of DHA. COX-2 and iNOS protein expression was determined by Western blot, and TNF-α and IL-6 secretion was measured using ELISA-based assays. DHA treatment inhibited translocation of p65NF-κB to the nucleus, significantly lowered p65NF-κB protein level and fluorescence of p65NF-κB in the nucleus, reduced dose-dependently IκB protein phosphorylation, and the binding of the AP-1 transcription factor members (c-Jun/c-Fos) to the specific TPA-response element (TRE) of DNA. In addition, the expression of pro-inflammatory COX-2 and iNOS proteins was downregulated and TNF-α and IL-6 secretion was also reduced. CONCLUSIONS: These results indicate that DHA is a powerful factor that reduces the pro-inflammatory response in astrocytes. Consequently, successful introduction of DHA into the astrocyte membranes can attenuate neuroinflammation, which is a key factor of age-related neurodegenerative disorders.

Docosahexaenoic Acid (DHA) Inhibits FADS2 Expression in Astrocytes but Increases Survival of Neurons Co-cultured with DHA-enriched Astrocytes.

Docosahexaenoic acid (DHA), the most abundant n-3 polyunsaturated fatty acid (n-3PUFA) in the brain, has attracted great importance for a variety of neuronal functions such as signal transduction through plasma membranes, neuronal plasticity, and neuroprotection. Astrocytes that provide structural, functional, and metabolic support for neurons, express ∆6- desaturase encoded by FADS2 gene that can be, next to the plasma DHA pool, additional source of DHA in the brain. Furthermore, the genetic variations of FADS gene cluster has been found in children with developmental disorders, and are associated with cognitive functions. Since, the regulation of DHA biosynthesis in astrocytes remains poorly studied the aim of this study was to determine the effect of palmitic acid (PA), α-linolenic acid (ALA) or docosahexaenoic acid (DHA), on the transcription of FADS2 gene in astrocytes and survival of neurons challenged with oxidative compounds after co-culture with astrocytes exposed to DHA. The lipid profile in cell membranes after incubation with fatty acids was determined by gas chromatography, and FADS2 expression was analyzed using real-time PCR. The viability of neurons cocultured with PUFA-enriched astrocytes was investigated by flow cytometry after staining cells with annexin V-FITC and PI. The results showed that DHA suppressed (P <0.01), PA stimulated (P <0.01), while ALA did not change the FADS2 gene expression after 24 h incubation of astrocytes with fatty acids. Although FADS2 mRNA was down-regulated by DHA, its level in astrocytic membranes significantly increased (P <0.01). Astrocytes with DHA-enriched membrane phospholipids markedly enhanced neuronal resistance to cytotoxic compounds and neuronal survival. These results suggest that beneficial effects of supplementation with n-3 PUFA in Alzheimer disease and in psychiatric disorders is caused, in part, by increased efficacy of DHA-enriched astrocytes to protect neurons under adverse conditions in the brain.

Cytotoxicity of titanium carbonitride coatings for prostodontic alloys with different amounts of carbon and nitrogen.

The aim of our work was to examine the cytotoxicity of NiCr alloys coated with Ti(C,N) with different amounts of C and N in the layer on human gingival fibroblasts. Cells were cultured for 24 hours in the alloy extracts or on the surface of tested materials. The viability of the cells exposed to 1-, 3-, 5- and 7-day extracts did not change in comparison to the viability of cells cultured in a control medium assayed by an MTT test. Moreover, the flow cytometry analysis of fibroblasts cultured in direct contact with tested alloys revealed that all coatings except TiC did not induce apoptosis or necrosis. Interestingly, 24 hour fibroblast culture on alloys with Ti(C,N) coatings showed that the number of fibroblasts adhered to these alloys, evaluated by scanning electron microscope, increased with an increase in the content of nitrogen in the layer. The present study demonstrates that Ti(C,N) coatings were not cytotoxic and did not induce apoptosis in Ti(C,N) extracts, nor in direct contact with gingival fibroblasts, and can be considered for biomedical applications in the future.

BanI polymorphism of cytosolic phospholipase A2 gene and somatic symptoms in medication-free acute depressed patients.

Somatic symptoms are commonly seen in patients with major depressive disorder (MDD) and might be associated with inflammatory activation. Cytosolic phospholipase A2 (cPLA2) and cyclo-oxygenase-2 (COX-2) are the key enzymes in the metabolism of polyunsaturated fatty acids (PUFAs), which in turn may play an important role in inflammation and somatic symptoms in depression. This study investigated the effects of BanI polymorphism of cPLA2 gene and COX-2 rs4648308 genotypes on somatic symptoms and inflammatory marker in patients with MDD. Eighty-two patients with MDD were assessed for their psychopathology including psychiatric and somatic symptoms, BanI polymorphism of cPLA2 and COX-2 rs4648308 genotypes and CRP levels. The results revealed that MDD patients with the cPLA2 BanI GG genotypes had higher somatic symptoms and higher levels of C-reactive protein (CRP), while no differences were found among the COX-2 rs4648308 genotypes. Inflammatory process, such as arachidonic acid cascade pathway, might help explain the effect of cPLA2 BanI polymorphism on the somatic symptoms, and may be a potential target for future investigation on treatment for MDD with somatic symptoms. However, the interpretation of the findings in this study is limited since we analyzed the data from a subset data from a larger study.

Eicosapentaenoic and docosahexaenoic acids have different effects on peripheral phospholipase A2 gene expressions in acute depressed patients.

INTRODUCTION: Omega-3 polyunsaturated fatty acids (PUFAs) have been proven critical in the development and management of major depressive disorder (MDD) by a number of epidemiological, clinical and preclinical studies, but the molecular mechanisms underlying this therapeutic action are yet to be understood. Although eicosapentaenoic acid (EPA) seems to be the active component of omega-3 PUFAs' antidepressant effects, the biological research about the difference of specific genetic regulations between EPA and docosahexaenoic acid (DHA), the two main components of omega-3 PUFAs, is still lacking in human subjects. METHODS: We conducted a 12-week randomized-controlled trial comparing the effects of EPA and DHA on gene expressions of phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX2), serotonin transporter (5HTT), and Tryptophan hydroxylase 2 (TPH-2) in 27 MDD patients. In addition, the erythrocyte PUFA compositions and the candidate gene expressions were also compared between these 27 MDD patients and 22 healthy controls. RESULTS: EPA was associated with a significant decrease in HAM-D scores (CI: -13 to -21, p<0.001) and significant increases in erythrocyte levels of EPA (CI: +1.0% to +2.9%, p=0.001) and DHA (CI: +2.9% to +5.6%, p=0.007). DHA treatment was associated with a significant decrease in HAM-D scores (CI: -6 to -14, p<0.001) and a significant increase in DHA levels (CI: +0.2% to +2.3%, p=0.047), but not of EPA levels. The cPLA2 gene expression levels were significantly increased in patients received EPA (1.9 folds, p=0.038), but not DHA (1.08 folds, p=0.92). There was a tendency for both EPA and DHA groups to decrease COX-2 gene expressions. The gene expressions of COX-2, cPLA2, TPH-2 and 5-HTT did not differ between MDD cases and healthy controls. CONCLUSIONS: EPA differentiates from DHA in clinical antidepressant efficacy and in upregulating cPLA2 gene regulations, which supports the clinical observation showing the superiority of EPA's antidepressant effects. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02615405.

Omega-3 polyunsaturated fatty acids improve the antioxidative defense in rat astrocytes via an Nrf2-dependent mechanism.

BACKGROUND: Neuronal tolerance to hypoxia and nutrient defficiency highly depends on GSH levels and antioxidant enzyme activity in astrocytes. Omega-3 polyunsaturated fatty acids (ω-3PUFA) enhance antioxidant defence in different cells. The aim of present study was to investigate if ω-3PUFA improve antioxidant status in astrocytes. METHODS: Rat primary astrocytes were incubated for 24h with DHA and EPA (30µM), then lysed, fractioned and fatty acids were determined by gas chromatography. GSH and protein thiols were assayed by enzymatic methods. Glutamate cysteine ligase (GCL), glutathione synthetase (GS), glutathione peroxidase 4 (GPx4) and Nrf2 protein expression was validated by Western blot. Intracellular ROS level using H2DCF-DA, and Nrf2 activation by ELISA were measured. RESULTS: Incubation of cells with DHA doubled DHA, not EPA content in the membranes, and incubation with EPA increased both fatty acids content compared to control. However, both ω-3PUFAs reduced ROS generation in dose-dependent manner in basal condition and in H2O2-treated cells, and significantly increased GSH, GCL and GPx4 levels. The thiols level was higher only in DHA-treated cells. DHA and EPA activated Nrf2 in a dose-dependent manner but p38MAPK-Nrf2 activation was found only in DHA-enriched astrocytes. CONCLUSION: Both ω-3PUFA improved the antioxidant defense in astrocytes via an Nrf2-dependent mechanism, however, upstream pathways of Nrf2 activation may depend on proportion of DHA to EPA incorporated into membrane phospholipids. These results suggest that enrichment of astrocytes with ω-3PUFA may better protect neurons during harmful conditions.

Polyunsaturated fatty acids levels and initial presentation of somatic symptoms induced by interferon-alpha therapy in patients with chronic hepatitis C viral infection.

OBJECTIVES: Somatic symptoms are common in depressive disorder and are similar to sickness behaviors due to inflammatory activation after cytokine administration. Omega-3 polyunsaturated fatty acids (PUFAs) are natural anti-inflammatory agents and may reduce inflammation-induced behavioral changes. The aim of this study was to investigate the role of PUFAs on the development of somatic symptoms and depression in patients of hepatitis C virus infection (HCV) receiving interferon-alpha therapy (IFN-α) in a prospective manner. METHODS: In this 24-week, prospective cohort study, 43 patients with chronic HCV ongoing IFN-α therapy were assessed with the mini-international neuropsychiatric interview for major depressive episodes and neurotoxicity rating scale (NRS) for somatic symptoms. RESULTS: One-third later developed IFN-α-induced depression (depression (DEP) group). As compared to subjects without depression, DEP group had higher NRS scores (P < 0.001), lower eicosapentaenoic acid (EPA) levels (P = 0.038) at week 2. Somatic symptoms, regardless of painful/non-painful characteristics, had positive association with arachidonic acid (P < 0.05), and negative association with EPA (P < 0.05). CONCLUSION: This study implies that early intervention with omega-3 PUFAs might be a promising strategy to prevent depression and somatic symptoms in patients receiving cytokine therapy.

Docosahexaenoic acid attenuates oxidative stress and protects human gingival fibroblasts against cytotoxicity induced by hydrogen peroxide and butyric acid.

OBJECTIVE: The oxidative burst of the host cells associated with bacterial pathogen infection contributes to the destruction of periodontal tissue. The present study investigates the effect of docosahexaenoic acid (DHA) on human gingival fibroblast (HGF) viability and ROS generation. METHODS: The cell viability by MTT assay, ROS level using H2DCF-DA probe, and protein thiol content were measured in HGFs after 24h preincubation with different concentrations of DHA followed by treatment with H2O2. The cell death rate was determined by Annexin V/propidium iodide staining, and mitochondrial membrane potential (ΔΨm) was examined by MitoTracker Red probe in H2O2- and butyric acid-treated HGFs. The fatty acid composition of plasma membranes after incubation with DHA was determined by gas chromatography mass spectrometry. RESULTS: DHA preincubation in a dose-dependent manner increased the viability of HGFs exposed to H2O2 and decreased ROS generation compared to the control cells. In HGFs preincubated with 30µM DHA, the ΔΨm significantly increased in both H2O2- and butyric acid-treated cells. Moreover, incubation with DHA preserved the protein thiol level as effectively as N-acetylcysteine. Application of 50µM DHA increased the quantity of viable cells, decreased the number of necrotic cells after H2O2 treatment, and protected HGFs from apoptosis induced by butyric acid. DHA in the plasma membranes of these HGFs represented about 6% of the total amount of fatty acids. CONCLUSIONS: These results demonstrate that enrichment of HGFs with DHA reduces ROS generation and enhances the mitochondrial membrane potential protecting the fibroblasts against cytotoxic factors.

[The role of docosahexaenoic acid in neuronal function]. / Rola kwasu dokozaheksaenowego w czynnosci komórek nerwowych.

Docosahexaenoic acid (DHA, C22: 6n-3) is the most abundant polyunsaturated fatty acid in neuronal phospholipids, particularly in the cortex. The main source of DHA for neural cells is food, and hepatic and astroglia DHA synthesis from essential a-linolenic acid (C18: 3n-3). Accretion of DHA in the brain is most intensive during fetal life and the first two years of life. An adequate level of DHA in cell membranes is important for many functions of neural cells and this is presumably the reason for DHA saving in the adult mammalian brain during dietary a-linolenic acid deficiency. DHA-containing phospholipids in membranes are flexible and membranes possessing a high content of them are quite thin, more permeable to ions and small molecules, have looser lipid packing, and finally are more "dynamic" than membranes composed of other fatty acid containing phospholipids. Furthermore, these membranes create an appropriate environment for integral proteins highly condensed in neurons, such as receptors, ion channels, enzymes, and peripheral proteins. The quantity of phosphatidylserine in the inner membrane lipid layer depends on the availability of DHA to neurons. Phosphatidylserine promotes neuronal survival by translocation/activation of kinase Akt and Raf-1/MEK. DHA present in membrane phospholipids facilitates v-SNARE/t-SNARE complex formation, which is necessary for fusion of synaptic vesicles and plasma membranes necessary for transmitter exocytosis, and neurite outgrowth-dependent plasticity. DHA plays an important neuroprotective role. DHA has been shown to inhibit PGE2 synthesis and COX-1 expression in astrocytes, and DHA derivatives, especially neuroprotectins D, can suppress inflammatory responses, preventing neuronal damage or apoptosis. The results of high DHA content in neuronal membranes and formation of DHA derivates, as well as the function of DHA-dependent phosphatidylserine, may explain the promising results supporting beneficial DHA supplementation in neurodegenerative diseases and improvement of brain function.

Effect of dietary fats on oxidative-antioxidative status of blood in rats.

This study was performed to examine the effect of different fat sources, lard, sunflower oil (SO), and fish oil (FO) in high-fat and low-fat diet on reactive oxygen species generation by blood phagocytes, glutathione redox status in erythrocytes, and total plasma antioxidant ability in rats. Whole blood chemiluminescence (CL) did not differ between three low-fat fed groups. However, baseline and phorbol myristate acetate (PMA)-stimulated CL in blood of high-lard fed rats were lower than in low-lard and high-SO fed animals. Phagocyte-stimulated oxidative burst was higher in rats fed high-SO diet than in those fed low-SO and high-FO diets. The highest level of oxidize glutathione (GSSH), the lowest reduce glutathione (GSH)/GSSG ratio in erythrocytes, and the highest plasma activity to reduce ferric ions were observed in rats fed both diets contaning linoleic acid-rich sunflower oil compared to animals fed the corresponding energy from other fats. 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of plasma was lower in high-lard and high-FO fed rats compared to the corresponding low-fat diets, and the lowest in low-FO fed rats among low-fat fed animals. We presume from our results that linoleic acid may have dual effect, prooxidative in blood cells but maintaining total antioxidant plasma ability.

Comparison of cadmium and enzyme-catalyzed nitrate reduction for determination of NO2-/NO3- in breath condensate.

BACKGROUND: Analysis of NO2-/NO3- in expired breath condensate (EBC) has been proposed as a marker of inflammation in various lung diseases. METHODS: NO2- and total NO3-/NO2- concentrations were determined in EBC collected from healthy and asthmatic subjects. The NO3- was first reduced to NO2-, and total NO2- was detected by colorimetric Griess reaction. Two methods of NO3- reduction were compared. To reduce NO3-, cadmium (600 microl EBC-macromethod) and enzyme-NADPH-nitrate reductase (60 microl EBC-micromethod) were used. RESULTS: Macromethod: Mean NO2- concentrations in EBC were 1.64 +/- 0.24 micromol/l in healthy subjects and 0.42 +/- 0.17 micromol/l in asthmatic patients. Mean total NO2-/NO3- levels were 3.64 +/- 0.43 micromol/l in healthy subjects and 3.27 +/- 0.34 micromol/l in asthmatic. Micromethod: NO2- level: 1.69 +/- 0.23 micromol/l in healthy subjects and 0.53 +/- 0.21 micromol/l in asthmatics. Total NO2-/NO3- levels: 3.56 +/- 0.37 micromol/l in healthy subjects and 3.57 +/- 1.17 micromol/l in asthmatics. Variability index was 27% and 6% for macro- and micromethod, respectively. Recovery of NO3- added to EBC was 100% for enzymatic and almost 88% for cadmium reduction. There was no correlation between total NO2-/NO3- levels determined by macro- and micromethod. CONCLUSIONS: We recommend enzymatic reduction as a better method for NO3- determination in EBC.

The role of nitric oxide (NO) in control of LHRH release that mediates gonadotropin release and sexual behavior.

Nitric oxide (NO) plays a crucial role in reproduction at every level in the organism. In the brain, it activates the release of luteinizing hormone-releasing hormone (LHRH). The axons of the LHRH neurons project to the mating centers in the brain stem and by efferent pathways, evoke the lordosis reflex in female rats. In males, there is activation of NOergic terminals that release NO in the corpora cavernosa penis to induce erection by generation of cyclic guanosine monophosphate (cGMP). NO also activates the release of LHRH which reaches the pituitary and activates the release of gonadotropins by activating neural NO synthase (NOS) in the pituitary gland. Follicle stimulating hormone (FSH)RH selectively releases FSH also by activating NOS. Leptin releases LHRH by activating NOS to release FSH and LH with the same potency as LHRH. These actions are mediated by specific receptors on the gonadotropes for LHRH, FSHRH and leptin. The responsiveness of the pituitary is controlled by gonadal steroids. In the gonad, NO plays an important role inducing ovulation and in causing luteolysis; whereas in the reproductive tract, it relaxes uterine muscle via cGMP and constricts it by prostaglandins.