Comparative effects between electronic cigarette and tobacco cigarette smoke on oxidative markers in cultured immune cells isolated from rats.

BACKGROUND: Tobacco cigarette smoke (TCS) was previously demonstrated to affect the innate and adaptive immune responses as a consequence of oxidant generation which play a pivotal role in neutrophilic airway inflammation. Aim of this paper was to investigate whether electronic cigarette smoke (ECS) generates reactive oxygen species (ROS) similarly to cigarette smoke. METHOD: By means of a house made apparatus, ECS and TCS were collected in fetal bovine serum (FBS) which was used to grow immune cells isolated from rats. As index of oxidative products nitrite, superoxide, and thiobarbituric acid-reactive substances (TBARS) were determined in the medium before and after cell growth. RESULTS: The results showed that: i) ECS caused a remarkable increase of nitrites and TBARS although in lesser extension than TCS; ii) the spleen and lymph node cells grown in ECS and TCS-exposed medium were able to reduce TBARS but not nitrites present in the medium; iii) PBMC in TCS-exposed medium were able to reduce nitrites and TBARS more efficiently than spleen and lymph node cells, but released more superoxide anion; iv) TCS and ECS not influence the PBMC and spleen T cell subtype populations (CD4+, CD8+). CONCLUSIONS: As ECS nicotine-free gave the same results of unexposed medium, we can support the hypothesis that the increase of ROS in ECS exposed medium was prevalently due to nicotine.

The adenosine A2A receptor agonist T1-11 ameliorates neurovisceral symptoms and extends the lifespan of a mouse model of Niemann-Pick type C disease.

Niemann-Pick C is a fatal neurovisceral disorder caused, in 95% of cases, by mutation of NPC1 gene. Therapeutic options are extremely limited and new "druggable" targets are highly warranted. We previously demonstrated that the stimulation of the adenosine A2A receptor (A2AR) normalized the pathological phenotype of cellular models of NPC1. Since the validation of A2ARs as a therapeutic target for NPC1 can be obtained only conducting studies in in vivo models of the disease, in the present paper, the effects of two agonists of A2ARs were evaluated in the mouse model Balb/c Npc1nih, hereafter indicated as NPC1-/-. The agonists CGS21680 (2.5 and 5mg/kg/day by intraperitoneal injection) and T1-11 (50mg/kg/day in drinking water) were administered at a presymptomatic stage of the disease of NPC1-/- mice (PN28 and PN30, respectively); the experimental groups were the following: vehicle-treated WT mice (N=16 for both CGS and T1-11 treatments); vehicle-treated NPC1-/- mice (N=14 for CGS and 12 for T1-11 treatment); CGS-treated NPC1-/- mice (N=7) and T1-11-treated NPC1-/- mice (N=11). The efficacy of the treatments was evaluated by comparing vehicle-treated and CGS or T1-11-treated NPC1-/- mice for their motor deficits (analyzed by both rotarod and footprint tests), hippocampal cognitive impairment (by Novel Object Recognition (NOR) test), cerebellar neurodegeneration (Purkinje neurons counting), and cholesterol and sphingomyelin accumulation in spleen and liver. Finally, the effect of both agonists on survival was evaluated by applying a humane late endpoint (weight loss >30% of peak weight, punched posture and reduced activity in the cage). The results demonstrated that, while CGS21680 only slightly attenuated cognitive deficits, T1-11 ameliorated motor coordination, significantly improved cognitive impairments, increased the survival of Purkinje neurons and reduced sphingomyelin accumulation in the liver. More importantly, it significantly prolonged the lifespan of NPC1-/- mice. In vitro experiments conducted in a neuronal model of NPC1 demonstrated that the ability of T1-11 to normalize cell phenotype was mediated by the selective activation of A2ARs and modulation of intracellular calcium levels. In conclusion, our results fully confirm the validity of A2ARs as a new target for NPC1 treatment. As soon as new ligands with improved pharmacokinetic characteristics (i.e. orally active, with brain bioavailability and metabolic stability) will be obtained, A2AR agonists could represent a breakthrough in the treatment of NPC.

Eicosapentaenoic acid modulates the synergistic action of CREB1 and ID/E2A family members in the rat pup brain and mouse embryonic stem cells.

The aim of this study was to investigate the molecular mechanism by which eicosapentaenoic acid (EPA) may exert neuroprotective effects through an "EPA-cyclic AMP response element-binding protein (CREB)" signaling pathway. The current study reveals that EPA modulates the exquisite interplay of interaction of CREB1 with the inhibitor of DNA binding (ID) and E2A family members, thereby delivering mechanistic insights into specific neural differentiation program. In this scenario, our work provides evidence for the capability of CREB1 to sequester ID:E2A family members in brain tissues and neural differentiating mouse embryonic stem cells (mESCs) through formation of a [CREB1]2:ID2:E47 tetrameric complex.In essence, the molecular function of CREB1 is to dynamically regulate the location-specific assembly or disassembly of basic-helix-loop-helix (bHLH):HLH protein complexes to mediate the activation of neural/glial target genes. Together, these findings support the one-to-many binding mechanism of CREB1 and indicate that EPA treatment potentiates the integration of CREB dependent signaling with HLH/bHLH transcriptional network, adding specificity to the CREB1-mediated gene regulation during neural/glial differentiation. Our current research on the EPA-CREB axis could reveal new molecular targets for treating neurogenerative disease.

Ethyl-eicosapentaenoic acid ameliorates the clinical course of experimental allergic encephalomyelitis induced in dark agouti rats.

Eicosapentaenoic acid (EPA), a fatty acid present in high amount in fish, modulates immune response and stimulates myelin gene expression. In the present paper, we investigated the effects of EPA in an established animal model for multiple sclerosis (MS): experimental autoimmune encephalomyelitis (EAE) induced in dark agouti rats. Diets supplemented either with 0.2% or 0.4% of EPA were administrated daily from the day of induction until the end of experiment. One group of rats received diet supplemented with 0.2% of EPA 10 days before induction. The control group (immunized rats) was fed with chow diet. The animals were analyzed at two different stages of the disease: during the acute phase (14 d.p.i.) and during the recovery phase (32 d.p.i.). We showed a delayed onset of clinical severity of disease in all groups of rats fed EPA-supplemented diets. This effect was associated to an increased expression of myelin proteins and an improved integrity of the myelin sheath as well as an up-regulation of FoxP3 expression in the central nervous system during the acute phase of EAE. No significant changes in T cell subsets were noted at the periphery. On the contrary, during the recovery phase of EAE, in animals assuming EPA-supplemented diet, an increase of CD4(+)CD25(+) and CD4(+)CD25(+)FoxP3(+) in peripheral lymphocytes was noted. Our results indicate that EPA-supplemented diets may provide benefits to MS patients.

Micronutrient-enriched rapeseed oils improve the brain oxidant/antioxidant system in rats fed a high-fat diet.

The main proposal of this study was to evaluate in vivo whether micronutrient-enriched rapeseed oils obtained using different crushing and refining procedures and characterized by different quantities and qualities of micronutrients (optimized oils) could have any beneficial effect on the antioxidant status of the brain. Sprague-Dawley rats were fed a high-fat diet for 4 weeks. The lipid source consisted of 20% optimized rapeseed oils with different quantities and qualities of micronutrients. The control group received traditional refined rapeseed oil. The experimental optimized oils decreased lipid peroxidation and increased endogenous antioxidant status in parallel with the enhancement of micronutrients. No alteration in acetylcholinesterase activity was induced by the high-fat diet in any experimental group. These results indicate that a regular intake of optimized rapeseed oils can prevent oxidative stress, providing evidence that optimized rapeseed oils could be a functional food with potentially important neuroprotective properties.

Micronutrient-enriched rapeseed oils reduce cardiovascular disease risk factors in rats fed a high-fat diet.

Many epidemiological studies have demonstrated that vegetable food consumption is associated with a reduced risk of cardiovascular diseases. The beneficial effects have been attributed to the content of bioactive molecules present in large quantities in plant food. The main proposal of this study was to evaluate in vivo whether micronutrient-enriched rapeseed oils (optimised oils) obtained using different crushing and refining procedures and characterised by different quantities and qualities of micronutrients, could have any beneficial effect on lipid profile and antioxidant status of plasma and liver. Sprague-Dawley rats were fed a high-fat diet for 4 weeks. The lipid source consisted of 20% optimised rapeseed oils with different quantities and qualities of micronutrients. The control group received traditional refined rapeseed oil. The experimental optimised oils all had a hypolipidaemic effect. In the group fed the highest levels of micronutrients, the reduction in plasma and hepatic triglycerides reached 25% and 17%, respectively, that of cholesterol 20% and 14%, respectively. In plasma, the ferric antioxidant capacity, superoxide dismutase, glutathione peroxidase and reduced glutathione significantly increased and lipid peroxidation decreased in parallel with the enhancement of micronutrients. The same trend was observed in the liver, except for glutathione peroxidase which was not affected by optimised oils. These results indicate that a regular intake of optimised rapeseed oils can help to improve lipid status and prevent oxidative stress, providing evidence that optimised oils could be a functional food with potentially important cardioprotective properties.

PMP70 knock-down generates oxidative stress and pro-inflammatory cytokine production in C6 glial cells.

By using RNA interference (RNAi) in rat C6 glial cells, we previously generated the cell line abcd3kd in which the peroxisomal half-transporter PMP70 was stably knocked-down. The observations that abcd3kd cells had peroxisomal beta-oxidation impairment and an increase of hexacosenoic acid in cholesterol ester fraction, indicated an overlapping function of PMP70 with adrenoleukodystrophy protein (ALDP), the peroxisomal half-transporters involved in X-linked adrenoleukodystrophy (X-ALD). The objective of the present study was to investigate whether PMP70 could affect some oxidative and inflammatory parameters, since many findings indicate oxidative damage in the brain of ALD patients and inflammation is a hallmark of the cerebral forms of X-ALD. We thus measured parameters indicative of oxidative stress, the expression or activity of antioxidant enzymes, and the production of some pro-inflammatory cytokines. Our results show that, due to inducible nitric oxide synthase up-regulation, abcd3kd cell line produces higher levels of nitrites than native C6 cells. The enhanced production of superoxide and thiobarbituric acid-reactive substances, the increased expression of mitochondrial superoxide dismutase, and the reduction of catalase and glutathione peroxidase activities confirm the presence of an oxidative process. We then measured the concentrations of TNFalpha, IFNgamma, and IL-12 and we observed that abcd3kd cells produce higher amounts of pro-inflammatory cytokines compared to native C6 cells. By using neutralizing antibodies against IL-12, not only inflammatory parameters significantly decrease, but nitrite and superoxide production is also affected. This demonstrates that oxidative status of abcd3kd cells is not a direct PMP70 knock-down consequence, but depends on IL-12 release. The scenery induced by the knock-down of PMP70 in C6 cells recall the oxidative and inflammatory status observed in human X-ALD and thus reinforce the idea that PMP70 could affect the clinical course of the disease.

RNAi-mediated silencing of ABCD3 gene expression in rat C6 glial cells: a model system to study PMP70 function.

The function of PMP70, one of the four ABC half-transporters of mammalian peroxisomes, encoded by ABCD3 gene, is still unclear. The finding that PMP70 over-expression partially corrected very long-chain fatty acid oxidation defects in fibroblasts of X-linked adrenoleukodystrophy patients, has unveiled its potential clinical relevance, prompting us to set up a model system to study PMP70 function. We used the RNA interference technique, a powerful approach to loss-of-function gene expression analysis, to knockdown the ABCD3 gene in the rat glial C6 cell line, since glia could represent the target tissue of X-linked adrenoleukodystrophy disease. Cells were transfected with a vector for RNA interference generating small interfering RNAs that specifically target the ABCD3 mRNA. By using a puromycin-selectable version of the plasmid, we generated a stable cell line (abcd3kd), in which we observed a stable decrease of PMP70 protein expression greater than 70%. We thus examined the effect of ABCD3 knockdown on lignoceric and palmitic acids beta-oxidation and we found that in abcd3kd cells the rate of peroxisomal and mitochondrial beta-oxidation activities were both reduced about one-third compared with control cells. The mitochondrial membrane potential, determined by cytofluorometric analysis, was also affected. Lipid and fatty acid analyses of abcd3kd cells showed an increase of hexacosenoic acid (C26:0) in the cholesteryl-ester fraction. These results add another clue about the overlapping function of PMP70 and ALDP, the peroxisomal protein involved in X-linked adrenoleukodystrophy, since C26:0 is the biochemical marker of the disease and in the brain lesions it is accumulated in the cholesteryl-ester fraction. Considered as a whole, our results indicate that the abcd3kd cell line is a valuable tool to further study the function of PMP70 and eventually its role in X-linked adrenoleukodystrophy.

Eicosapentaenoic acid stimulates the expression of myelin proteins in rat brain.

We have previously demonstrated that, in C6 glioma cells, eicosapentaenoic acid (EPA) stimulates the expression of proteolipid protein (PLP) via cAMP-mediated pathways. In this study, we investigated whether n-3 polyunsaturated fatty acids can affect myelinogenesis in vivo. A single dose of either EPA or docosahexaenoic acid (DHA) was injected intracerebroventricularly into 2-day-old rats, which were then killed after 3 days post-injection (p.i.). Total RNA was isolated from the medulla, cerebellum, and cortex, and the expression of myelin-specific mRNAs was analyzed by real-time PCR. The levels of PLP, myelin basic protein, and myelin oligodendrocyte protein mRNAs increased in nearly all brain regions of DHA- and EPA-treated animals, but the effect was more pronounced in EPA-treated rats. The enhancement in PLP transcript levels was followed by an increase in PLP translation in EPA-treated rats. A further indicator of accelerated myelination was the increase in 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) protein levels. In EPA-treated rats, the increased expression of myelin genes coincided with a decrease of cAMP-response element-binding protein (CREB)-DNA binding in the cerebellum and cortex (1 hr p.i.). After 16 hr, this effect was still present in the same cerebral regions even though the decrease in EPA-treated rats was less pronounced than in controls. The down-regulation of CREB activity was due to a decrease in the levels of CREB phosphorylation. In conclusion, our data suggest that EPA stimulates the expression of specific myelin proteins through decreased CREB phosphorylation. These results corroborate the clinical studies of the n-3 PUFA beneficial effects on several demyelinating diseases.

Docosahexaenoic acid supplementation induces dose and time dependent oxidative changes in C6 glioma cells.

In view of the promising use of n-3 polyunsaturated fatty acids (PUFAs) in the prevention and treatment of neurological diseases, it is necessary to ascertain the lack of detrimental oxidative effects. We evaluated short- and long-term effects of 25, 50 and 75 muM docosahexaenoic acid (DHA) supplementation on the oxidative status of C6 glial cells. DHA was incorporated into cells dose and time dependently without any cytotoxic effect. Reactive oxygen species (ROS) level was related to DHA dose and supplementation time. At the lowest dose no significant increase in ROS values was observed at hour 24. Low doses of DHA strengthened the cellular antioxidant defence system as highlighted by a raise in both GPX and catalase activity, and the decreased levels of lipid peroxidation. This effect was pronounced at 24 h of supplementation, almost disappeared at hour 48, while after 72 h an opposite effect was observed: lipid peroxidation increased concomitantly with DHA doses. Therefore, the final effect of DHA on cellular redox status is dependent on dose and time supplementation.

Eicosapentaenoic acid stimulates leptin receptor gene expression in the hypothalamus of newborn rats.

Body weight and obesity are controlled by the binding leptin (Ob) receptor, but in newborn rats, despite high Ob levels, hypothalamic leptin receptors (Ob-Rb) are only weakly expressed. In this study we have attempted to stimulate expression of the Ob-Rb gene by administering 2 polyunsaturated fatty acids (PUFAs) recommended for the maternal diet and known as gene regulators: docosahexaenoic acid and eicosapentaenoic acid (EPA). We studied the effects of a single dose injected into a cerebral ventricle of newborn rats on postnatal day 2. On days 1, 2, and 3 after administration, we dissected the hypothalamus and analyzed Ob-Rb and Ob messenger RNAs by polymerase chain reaction and protein expression by Western blot immunoassay. Our results demonstrate that EPA, but not docosahexaenoic acid, caused an early messenger RNA expression of the gene, 24 hours earlier than in the controls, and the protein was also detected earlier. Our data corroborate the observations regarding the role of PUFAs, EPA in particular, in the regulation of gene expression. In addition, they support the recommendation to enrich the maternal diet with fish and seafood rich in n-3 PUFA, because the concentrations of n-6 and n-3 PUFA in human milk reflect the composition of fat in the mother's diet.

Effect of arachidonic, eicosapentaenoic and docosahexaenoic acids on the oxidative status of C6 glioma cells.

n-3 polyunsaturated fatty acids (PUFAs) have been described to have beneficial effects on brain development and in the prevention and treatment of brain damage. C6 glioma cells were incubated with 100 microM of either C20:4n-6 (ARA), or C20:5n-3 (EPA), or C22:6n-3 (DHA) for different time periods to assess whether these acids altered the cellular oxidative state. The ARA and EPA were promptly metabolised to C22:4n-6 and C22:5n-3, respectively, whereas DHA treatment simply increased the amount of DHA in the cells. Cell viability was not affected by ARA, while a cytotoxic effect was observed 72 h after n-3 PUFAs supplementation. The levels of reactive oxygen species and thiobarbituric acid-reactive substances were significantly higher in DHA-treated cells than in EPA- and ARA-treated groups. This modification in the oxidative cellular status was also highlighted by a significant increase in catalase activity and a decrease in glutathione content in DHA-supplemented cells. Glucose-6-phosphate dehydrogenase activity, an enzyme involved in redox regulation, and O2*- release were significantly increased both in EPA and DHA groups. The effect of DHA was more severe than that of EPA. No significant changes were observed in the ARA group with respect to untreated cells. These data show that EPA and DHA induce alterations in the oxidative status that could affect the glial function.

Effects of L-mono methyl-arginine, N-acetyl-cysteine and diphenyleniodonium on free radical release in C6 glial cells enriched in hexacosenoic acid.

Previously, we have shown that C6 glial cells enriched in hexacosenoic acid (HA) incubated with oxidative stressors released higher amounts of nitric oxide (NO) products and superoxide (O2(-)), compared to native C6 cells. In the present study, we examined the effects of pretreatment with some of free radical release inhibitors. The aim was to determine the origin of the enhanced generation of NO and superoxide, and to test the possibility of preventing it. Pre-treatment with L-mono-methyl-arginine and N-acetyl-cysteine in oxidized low-density lipoprotein (ox-LDL) exposed HA cells, inhibited not only nitrite but also superoxide production suggesting that O2(-) anion could partially derive from inducible NO synthase. We also observed that ox-LDL treatment of HA cells reduced the intracellular glutathione levels and activated extracellular signal-related kinases. Since this signalling is related to neurotoxic effect, our data substantiate the role of the free radicals in X-linked adrenoleukodystrophy pathogenesis, as HA cells have been used as an in vitro model for this disease.

Free radical release in C6 glial cells enriched in hexacosanoic acid: implication for X-linked adrenoleukodystrophy pathogenesis.

Free radicals have been implicated in the etiopathology of some neurological and demyelinating diseases. To evaluate their involvement in the cerebral form of X-linked adrenoleukodystrophy (cerALD) disorder, characterised by very long chain fatty acid (VLCFA) accumulation, we utilised an in vitro model using rat C6 glial cells, enriched in hexacosenoic acid (C26:0, HA). Modified cells were incubated in presence of oxidative stressors, such as bacterial endotoxin lipopolisaccharides (LPS) and human oxidised low-density lipoprotein (ox-LDL), and the production of proinflammatory cytokines, nitrite, nitrate and superoxide was determined in the supernatants. The results show that modified cells produce higher amounts of nitric oxide (NO) products and superoxide compared to native C6 cells, supporting the role of free radicals as important pathophysiological modulator of the neuroinflammatory response in ALD. This hypothesis suggests that the cerebral damage in ALD could be due to intracellular signalling activated by interaction of exogenous factors with the particular membrane fatty acid composition.

Stimulation of myelin proteolipid protein gene expression by eicosapentaenoic acid in C6 glioma cells.

In this study, the role of exogenous fatty acids in the regulation of proteolipid protein (PLP) gene expression was investigated using the following model culture system: C6 glioma cells expressing the green-fluorescent protein (eGFP) driven by different segments of PLP promoter. Eicosapentanoic acid (EPA; 20:5 n-3), but not arachidonic acid (AA; 20:4 n-6), induced a significant increase in medium fluorescence intensity (MFI) determined by fluorescence-activated cell sorting (FACS). The induction of PLP promoter was time-dependent showing maximal activity between 24 and 48 h after EPA exposure. PLP promoter activation was dependent on fatty acid concentration, with maximum activation at 200 microM. Northern blot analysis confirmed the fluorescence data in C6 cells incubated with EPA. Furthermore, this treatment increased the adenylyl cyclase-cyclic AMP (cAMP) levels and the mitogen-activated protein kinase (MAPK) activation in C6 cells. PLP promoter activity was inhibited by pre-treatment with H89 (protein kinase A (PKA) inhibitor), but not with PD98059 (MAPK inhibitor), suggesting that EPA stimulates the expression of PLP via cAMP-mediated pathways.

Dietary prenatal lipids affect myelin gene expression in postnatal undernourished rats.

The effects on myelin gene expression of prenatal diets with a different lipid content and fatty acid composition were investigated in undernourished pups. Three groups of rats were fed ad libitum during gestation diets containing either 10% of margarine or 10% of microbial lipids or a standard diet containing 3.5% of lipids. After birth, all groups were switched to a reduced intake (60%) of the standard diet. A control group received ad libitum, the standard diet during pregnancy and throughout lactation. At birth no difference in body and brain weight was observed among the groups and the only significant difference in the brain fatty acid composition was the presence of odd-chain fatty acids in the group fed microbial lipids. Milk was removed from the stomach of pups at 1, 5 and 9 days of lactation for fatty acid analysis. During undernourishment, the monoenoic fatty acid and polyenoic fatty acid percentage was always higher and lower, respectively, in the groups fed 10% than in the group fed 3.5% of lipids during pregnancy. The expression of myelin genes at 11 days of undernutrition was determined in different brain regions by Northern analysis. In the standard group, the proteolipid protein and myelin oligodendrocyte glycoprotein transcripts were well detected only in the medulla whereas in groups fed 10% of lipids the transcripts were also visible in the cerebellum. These data suggest that the high lipid content of the prenatal diet independently from its fatty acid composition affects the myelin gene expression decreasing myelin susceptibility to postnatal undernutrition.