Marika Cordaro

Neuroprotection by Association of Palmitoylethanolamide with Luteolin in Experimental Alzheimer’s Disease Models: The Control of Neuroinflammation

Alzheimers disease (AD) is the most common neurodegenerative disorder. Its neuropathological hallmarks include deposition of beta amyloid (Aβ) fibrils in senile plaques. Numerous biochemical events, leading to Aβ neurotoxicity in AD, have been proposed and it seems that neuroinflammation plays a prominent role among these. Thus, since inflammatory processes and oxidative stress are considered to play an important role in neuroinflammatory disorders and in AD pathology, in the present work we decided to test a new composite, which is a formulation constituted of an anti-inflammatory compound such as palmitoylethanolamide (PEA) and the well recognized antioxidant flavonoid luteolin (Lut), subjected to an ultra-micronization process, here designated co-ultraPEALut. We investigated the effect of co-ultraPEALut in both an in vitro and ex vivo organotypic model of AD. For the in vitro model, we used human neuronal cells, obtained by differentiating SH-SY5Y neuroblastoma cells into sustainable neuronal morphology. These well differentiated cells express features specific to mature neurons, such as synaptic structures and functional axonal vesicle transport, making this new concept for in vitro differentiation valuable for many neuroscientific research areas, including AD. Differentiated SH-SY5Y cells were pre-treated with co-ultraPEALut (reference concentrations: 27, 2.7 and 0.27 µM PEA) for 2 h. AD features were induced by Aβ₁₋₄₂ stimulation (1 µM). Twenty-four hours later cell vitality was evaluated by the colorimetric MTT assay, whereas the neuroinflammation underling AD was observed by Western blot analysis for IκBα degradation and nuclear factor-κB traslocation, as well as glial fibrillary acidic protein expression. For the organotypic model of AD, hippocampal slice cultures were prepared from mice at postnatal day 6 and after 21 days of culturing the slices were pre-treated with co-ultraPEALut (reference concentrations: 27, 2.7 and 0.27 µM PEA) for 2 h and then incubated with Aβ₁₋₄₂ (1 µg/ml) for 24 h. Pre-treatment with co-ultraPEALut significantly reduced inducible nitric oxide synthase and glial fibrillary acidic protein expression, restored neuronal nitric oxide synthase and brainderived neurotrophic factor and reduced the apoptosis. Taken together our results clearly showed that co-ultraPEALut is able to blunt Aβ-induced astrocyte activation and to exert a marked protective effect on glial cells. These findings suggest that the association of co-ultraPEALut may provide an effective strategy for AD.

Roles of fatty acid ethanolamides (FAE) in traumatic and ischemic brain injury

Ethanolamides of long-chain fatty acids are a class of endogenous lipid mediators generally referred to as N-acylethanolamines (NAEs). NAEs include anti-inflammatory and analgesic palmitoylethanolamide, anorexic oleoylethanolamide, stearoylethanolamide, and the endocannabinoid anandamide. Traumatic brain injury (TBI), associated with a high morbidity and mortality and no specific therapeutic treatment, has become a pressing public health and medical problem. TBI is a complex process evoking systemic immune responses as well as direct local responses in the brain tissues. The direct (primary) damage disrupts the blood-brain barrier (BBB), injures the neurons and initiates a cascade of inflammatory reactions including chemokine production and activation of resident immune cells. The effect of TBI is not restricted to the brain; it can cause multi-organ damage and evoke systemic immune response with cytokine and chemokine production. This facilitates the recruitment of immune cells to the site of injury and progression of the inflammatory reaction. Depending on severity, TBI induces immediate neuropathologic effects that, for the mildest form, may be transient; however, with increasing severity, these injuries cause cumulative neural damage and degeneration. Moreover, TBI leads to increased catabolism of phospholipids, resulting in a series of phospholipid breakdown products, some of which have potent biological activity. Ischemia-reperfusion (I/R) injury resulting from stroke leads to metabolic distress, oxidative stress and neuroinflammation, making it likely that multiple therapeutic intervention strategies may be needed for successful treatment. Current therapeutic strategies for stroke need complimentary neuroprotective treatments to provide a better outcome. Prior studies on NAEs have demonstrated neurotrophic/neuroprotective activities across a broad spectrum of cellular and animal models of neurodegenerative and acute cerebrovascular disorders. The present review will summarize our knowledge of the biological role of these lipid signaling molecules in brain and highlights their therapeutic effect from multipotential actions on neuronal cell death and neuroinflammatory pathways.

Protective effect of polyphenols in an inflammatory process associated with experimental pulmonary fibrosis in mice

Polyphenols have been described to have a wide range of biological activities, and many reports, published during recent years, have highlighted the beneficial effects of phenolic compounds, illustrating their promising role as therapeutic tools in several acute and chronic disorders. The purpose of study was to evaluate, in an already-assessed model of lung injury caused by bleomycin (BLM) administration, the role of resveratrol and quercetin, as well as to explore the potential beneficial properties of a mango leaf extract, rich in mangiferin, and a grape leaf extract, rich in dihydroquercetin (DHQ), on the same model. Mice were subjected to intra-tracheal administration of BLM, and polyphenols were administered by oral route immediately after BLM instillation and daily for 7 d. Treatment with resveratrol, mangiferin, quercetin and DHQ inhibited oedema formation and body weight loss, as well as ameliorated polymorphonuclear infiltration into the lung tissue and reduced the number of inflammatory cells in bronchoalveolar lavage fluid. Moreover, polyphenols suppressed inducible nitric oxide synthase expression, and prevented oxidative and nitroxidative lung injury, as shown by the reduced nitrotyrosine and poly (ADP-ribose) polymerase levels. The degree of apoptosis, as evaluated by Bid and Bcl-2 balance, was also suppressed after polyphenol treatment. Finally, these natural products down-regulated cyclo-oxygenase-2, extracellular signal-regulated kinase phosphorylated expression and reduced NF-κBp65 translocation. Our findings confirmed the anti-inflammatory effects of resveratrol and quercetin in BLM-induced lung damage, and highlight, for the first time, the protective properties of exogenous administration of mangiferin and DHQ on experimental pulmonary fibrosis.

Anti-inflammatory and Antioxidant Effects of Flavonoid-Rich Fraction of Bergamot Juice (BJe) in a Mouse Model of Intestinal Ischemia/Reperfusion Injury

The flavonoid-rich fraction of bergamot juice (BJe) has demonstrated anti-inflammatory and antioxidant activities. The aim of work was to test the beneficial effects of BJe on the modulation of the ileum inflammation caused by intestinal ischemia/reperfusion (I/R) injury in mice. To understand the cellular mechanisms by which BJe may decrease the development of intestinal I/R injury, we have evaluated the activation of signaling transduction pathways that can be induced by reactive oxygen species production. Superior mesenteric artery and celiac trunk were occluded for 30 min and reperfused for 1 h. The animals were sacrificed after 1 h of reperfusion, for both histological and molecular examinations of the ileum tissue. The experimental results demonstrated that BJe was able to reduce histological damage, cytokines production, adhesion molecules expression, neutrophil infiltration and oxidative stress by a mechanism involved both NF-κB and MAP kinases pathways. This study indicates that BJe could represent a new treatment against inflammatory events of intestinal I/R injury.

The Association of Palmitoylethanolamide with Luteolin Decreases Neuroinflammation and Stimulates Autophagy in Parkinson's Disease Model

Parkinsons disease (PD) is a disorder resulted by degeneration of dopaminergic neurons. To counteract the neuroinflammation and oxidative stress of PD, we decided to test a new composite constituted by palmitoylethanolamide (PEA) and luteolin (Lut), in a mass ratio of 10:1, respectively (co-ultraPEALut). In this study the neuroprotective property of the new compound was investigated. For the in vivo model of PD, mice received four injections of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). Starting 24 h after the first administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we treated animals with co-ultraPEALut daily until 7 days. On day 8, brains were processed for Western blotting and immunohistochemical analysis. Treatment with co-ultraPEALut reduced the specific markers of PD (tyrosine hydroxylase immunopositive), and the increased levels of activated astrocytes and pro-inflammatory cytokines as well as inducible nitric oxide synthase. Further, the possible association of autophagy with the beneficial effects of coultraPEALut. Western blot analysis and immunofluorescence staining showed that co-ultraPEALut administration increased autophagy process. These data were confirmed by an in vitro model, using SH-SY5Y neuroblastoma cells. Western blot analysis showed that co-ultraPEALut pre-treatment maintained high Beclin-1 and p62 expression, while continued to inhibit the p70S6K expression. Altogether, these results put forward that treatment with co-ultraPEALut is able to modulate both the neuroinflammatory process and the autophagic pathway involved in PD, actions which may underlie its neuroprotective effect.

Hydrogen sulfide-releasing cyclooxygenase inhibitor ATB-346 enhances motor function and reduces cortical lesion volume following traumatic brain injury in mice

BackgroundTraumatic brain injury (TBI) induces secondary injury mechanisms, including dynamic interplay between ischemic, inflammatory and cytotoxic processes. We recently reported that administration of ATB-346 (2-(6-methoxynapthalen- 2-yl)-propionic acid 4-thiocarbamoyl-phenyl ester), a hydrogen sulfide-releasing cyclooxygenase inhibitor, showed marked beneficial effects in an animal model of spinal cord injury, significantly enhancing recovery of motor function and reducing the secondary inflammation and tissue injury.MethodsHere we evaluated the neuroprotective potential of ATB-346, a hydrogen sulfide-releasing derivative of naproxen, using the controlled cortical impact (CCI) injury model in mice, one of the most common models of TBI. Moreover, the aim of the present study was to carefully investigate molecular pathways and subtypes of glial cells involved in the protective effect of ATB-346 on inflammatory reaction associated with an experimental model of TBI. In these studies, TBI was induced in mice by CCI and mice were orally administered ATB-346, naproxen (both at 30 μmol/kg) or vehicle (dimethylsulfoxide:1% carboxymethylcellulose [5:95] suspension) one and six hours after brain trauma and once daily for 10 days.ResultsResults revealed that ATB-346 attenuated TBI-induced brain edema, suppressed TBI-induced neural cell death and improved neurological function. ATB-346 also significantly reduced the severity of inflammation and restored neurotrophic factors that characterized the secondary events of TBI.ConclusionsThese data demonstrate that ATB-346 can be efficacious in a TBI animal model by reducing the secondary inflammation and tissue injury. Therefore, ATB-346 could represent an interesting approach for the management of secondary damage following CNS diseases, counteracting behavioral changes and inflammatory process.

KU0063794, a Dual mTORC1 and mTORC2 Inhibitor, Reduces Neural Tissue Damage and Locomotor Impairment After Spinal Cord Injury in Mice

Autophagy is an intracellular catabolic mechanism for the degradation of cytoplasmic constituents in the autophagosomal–lysosomal pathway. This mechanism plays an important role in homeostasis and it is defective in certain diseases. Preceding studies have revealed that autophagy is developing as an important moderator of pathological responses associated to spinal cord injury (SCI) and plays a crucial role in secondary injury initiating a progressive degeneration of the spinal cord. Thus, based on this evidence in this study, we used two different selective inhibitors of mTOR activity to explore the functional role of autophagy in an in vivo model of SCI as well as to determine whether the autophagic process is involved in spinal cord tissue damage. We treated animals with a novel synthetic inhibitor temsirolimus and with a dual mTORC1 and mTORC2 inhibitor KU0063794 matched all with the well-known inhibitor of mTOR the rapamycin. Our results demonstrated that mTOR inhibitors could regulate the neuroinflammation associated to SCI and the results that we obtained evidently demonstrated that rapamycin and temsirolimus significantly diminished the expression of iNOS, COX2, GFAP, and re-established nNOS levels, but the administration of KU0063794 is able to blunt the neuroinflammation better than rapamycin and temsirolimus. In addition, neuronal loss and cell mortality in the spinal cord after injury were considerably reduced in the KU0063794-treated mice. Accordingly, taken together our results denote that the administration of KU0063794 produced a neuroprotective function at the lesion site following SCI, representing a novel therapeutic approach after SCI.

A new co-micronized composite containing palmitoylethanolamide and polydatin shows superior oral efficacy compared to their association in a rat paw model of carrageenan-induced inflammation

Palmitoylethanolamide (PEA), a special food for medical purposes, has anti-inflammatory and neuroprotective effects. Nevertheless, PEA lacks direct ability to prevent free radical formation. Polydatin (PLD), a natural precursor of resveratrol, has antioxidant activity. The combination of PEA and PLD could have beneficial effects on oxidative stress induced by inflammatory processes. In the present study, we compared the effects of micronized PEA (PEA-m) and PLD association (PEA-m+PLD) with a new co-micronized composite containing PEA and PLD (m(PEA/PLD)) in the rat paw model of carrageenan (CAR)-induced acute inflammation. Intraplantar injection of CAR led to a time-dependent development of peripheral inflammation, in terms of paw edema, cytokine release in paw exudates, nitrotyrosine formation, inducible nitric oxide synthase and cyclooxygenase-2 expression. m(PEA/PLD) reduced all measured parameters. Thermal hyperalgesia and mechanical allodynia were also markedly reduced. At the spinal cord level, manganese superoxide dismutase (MnSOD) was found to be nitrated and subsequently deactivated. Further, m(PEA/PLD) treatment increased spinal MnSOD expression, prevented IkB-α degradation and nuclear factor-κB translocation, suggesting a possible role on central sensitization. m(PEA/PLD) showed more robust anti-inflammatory and anti-hyperalgesic effects compared to the simple association of PEA-m and PLD. This composite formulation approach opens a new therapeutic strategy for the development of novel non-narcotic anti-hyperalgesic agents.

Traumatic Brain Injury Leads to Development of Parkinson's Disease Related Pathology in Mice

Traumatic brain injury (TBI) is a major health and socio-economic problem that affects all societies. This condition results from the application of external physical strength to the brain that leads to transitory or permanent structural and functional impairments. Moreover, TBI is a risk factor for neurodegeneration and can e.g., increase the risk for Parkinsons disease (PD), a late-onset neurodegenerative disorder with loss of dopaminergic neurons in substantia nigra. In this study, we wanted to explore the possible development of PD-related pathology within the context of an experimental model of TBI. Traumatic brain injury was induced in mice by controlled cortical impact. At different time points behavioral tests (open field, elevated plus maze tests, and Barnes maze) were performed: The animals were sacrificed 30 days after the impact and the brains were processed for Western blot and immunohistochemical analyses. Following TBI there was a significant decrease in expression of tyrosine hydroxylase and dopamine transporter in the substantia nigra as well as significant behavioral alterations. In addition, a strong increase in neuroinflammation was evident, as shown by increased levels of cyclooxygenase-2 and inducible nitric oxide synthase as well as IκB-α degradation and nuclear-κB translocation. Moreover, neurotrophic factors such as brain-derived neurotrophic factor, neurotrophin-3, nerve growth factor, and glial cell line-derived neurotrophic factor were decreased 30 days post-TBI. Interestingly, we observed a significant accumulation of α-synuclein in microglia compared to astrocytes. This study suggests that PD-related molecular events can be triggered upon TBI. The biological mechanisms linking brain trauma and neurodegenerative diseases need to be further investigated.

2-pentadecyl-2-oxazoline: Identification in coffee, synthesis and activity in a rat model of carrageenan-induced hindpaw inflammation.

N-acylethanolamines (NAEs) comprise a family of bioactive lipid molecules present in animal and plant tissues, with N-palmitoylethanolamine (PEA) having received much attention owing to its anti-inflammatory, analgesic and neuroprotective activities. 2-Pentadecyl-2-oxazoline (PEA-OXA), the oxazoline of PEA, reportedly modulates activity of N-acylethanolamine-hydrolyzing acid amidase (NAAA), which catabolizes PEA. Because PEA is produced on demand and exerts pleiotropic effects on non-neuronal cells implicated in neuroinflammation, modulating the specific amidases for NAEs (NAAA in particular) could be a way to preserve PEA role in maintaining cellular homeostasis through its rapid on-demand synthesis and equally rapid degradation. This study provides the first description of PEA-OXA in both green and roasted coffee beans and Moka infusions, and its synthesis. In an established model of carrageenan (CAR)-induced rat paw inflammation, PEA-OXA was orally active in limiting histological damage and thermal hyperalgesia 6h after CAR intraplantar injection in the right hindpaw and the accumulation of infiltrating inflammatory cells. PEA-OXA appeared to be more potent compared to ultramicronized PEA given orally at the same dose (10mg/kg). PEA-OXA markedly reduced also the increase in hindpaw myeloperoxidase activity, an index of polymorphonuclear cell accumulation in inflammatory tissues. NAAA modulators like PEA-OXA may serve to maximize availability of NAEs (e.g. PEA) while providing for recycling of the NAE components for further resynthesis.