Rossana Morabito

Molecular evidence for the involvement of PPAR-δ and PPAR-γ in anti-inflammatory and neuroprotective activities of palmitoylethanolamide after spinal cord trauma

BackgroundPalmitoylethanolamide (PEA) is an endogenous fatty acid amide displaying anti-inflammatory and analgesic actions. Moreover, several data have suggested that PEA reduced inflammation and tissue injury associated with spinal cord trauma and showed a regulatory role for peroxisome proliferator-activated receptor (PPAR)-α signaling in the neuroprotective effect of PEA. However, several other mechanisms could explain the anti-inflammatory and anti-hyperalgesic effects of PEA, including the activation of PPAR-δ and PPAR-γ. The aim of the present study was to carefully investigate the exact contribution of PPAR-δ and PPAR-γ in addition to PPAR-α, in the protective effect of PEA on secondary inflammatory damage associated with an experimental model of spinal cord injury (SCI).MethodsSCI was induced in mice through a spinal cord compression by the application of vascular clips (force of 24 g) to the dura via a four-level T5 to T8 laminectomy, and PEA (10 mg/kg, intraperitoneally, 1 and 6 hours after SCI) was injected into wildtype mice and into mice lacking PPAR-α (PPAR-αKO). To deepen the ability of specific PPAR-δ and PPAR-γ antagonists to reverse the effect of PEA, mice were administered GSK0660 or GW9662, 30 minutes before PEA injection.ResultsGenetic ablation of PPAR-α in mice exacerbated spinal cord damage, while PEA-induced neuroprotection seemed be abolished in PPARαKO mice. Twenty-four hours after spinal cord damage, immunohistological and biochemical studies were performed on spinal cord tissue. Our results indicate that PPAR-δ and PPAR-γ also mediated the protection induced by PEA. In particular, PEA was less effective in PPAR-αKO, GSK0660-treated or GW9662-pretreated mice, as evaluated by the degree of spinal cord inflammation and tissue injury, neutrophil infiltration, proinflammmatory cytokine, inducible nitric oxide synthase expression and motor function. PEA is also able to restore PPAR-δ and PPAR-γ expression in spinal cord tissue.ConclusionThis study indicates that PPAR-δ and PPAR-γ can also contribute to the anti-inflammatory activity of PEA in SCI.

Effect of various factors on Pelagia noctiluca (Cnidaria, Scyphozoa) crude venom-induced haemolysis

The haemolytic power of isolated nematocysts from the scyphozoan Pelagia noctiluca was studied with attention to the effect of osmotic protectants as carbohydrates at different MW, cations as Mg2+, Ca2+, Ba2+,Cu2+, K+; proteases as collagenase, trypsin, alpha-chymotrypsin, papain; and antioxidants. Crude venom was at first obtained by sonication of holotrichous-isorhiza nematocysts previously isolated from oral arms of P. noctiluca and then haemolytically tested upon human erythrocytes. Osmotic protectants were effective in inhibiting the haemolytic power depending on their molecular weight so that total inhibition of crude venom-induced haemolysis was observed after PEG treatment (polyethyleneglycol 6000Da). Amongst divalent cations only Ba2+ and Cu2+ significantly inhibited the haemolytic power of crude venom. Proteases seem not to alter the haemolytic activity while antioxidant compounds only slightly reduced the haemolytic power. Such findings may suggest a pore-forming mechanism for P. noctiluca crude venom rather than an oxidative damage to the cell membrane.

Oxidative stress induced by crude venom from the jellyfish Pelagia noctiluca in neuronal-like differentiated SH-SY5Y cells

Marine toxins are a suitable research model and their mechanism of action is intriguing and still under debate. Either a pore formation mechanism or oxidative stress phenomena may explain the damage induced by toxins. The effect of crude venom from isolated nematocysts of the jellyfish Pelagia noctiluca on neuronal-like cells derived from human neuroblastoma SH-SY5Y has been here studied. To prove the possible oxidative stress events, cell viability, assessed by MTT quantitative colorimetric assay, intracellular reactive oxygen species (ROS) quantified by the non-fluorescent probe H2DCF-DA and changes in mitochondrial transmembrane potential (ΔΨm) measured by the incorporation of a cationic fluorescent dye rhodamine-123 were verified on venom-treated cells (0.05-0.5μg/ml doses). A dose- and time-dependent reduction of all parameters was observed after venom treatment. NAC (N-acetyl-cysteine), antioxidant applied before crude venom application, significantly counteracted the decrease in cell viability and ROS production, while ΔΨm was only partially restored. The disruption of mitochondrial membrane by P. noctiluca crude venom may thus induce oxidative stress by inhibiting mitochondrial respiration and uncoupling oxidative phosphorylation, sensitizing mitochondria in SH-SY5H cells and facilitating membrane permeability. In sum, our findings suggest that P. noctiluca crude venom directly induces ΔΨm collapse with further generation of ROS and add novel information to the understanding of such toxins, still not completely clarified.

Combination therapy with melatonin and dexamethasone in a mouse model of traumatic brain injury

Traumatic brain injury (TBI) is a major cause of preventable death and morbidity in young adults. This complex condition is characterized by a significant blood-brain barrier leakage that stems from cerebral ischemia, inflammation, and redox imbalances in the traumatic penumbra of the injured brain. Recovery of function after TBI is partly through neuronal plasticity. In order to test whether combination therapy with melatonin and dexamethasone (DEX) might improve functional recovery, a controlled cortical impact (CCI) was performed in adult mice, acting as a model of TBI. Once trauma has occurred, combating these exacerbations is the keystone of an effective TBI therapy. The therapy with melatonin (10  mg/kg) and DEX (0.025  mg/kg) is able to reduce edema and brain infractions as evidenced by decreased 2,3,5-triphenyltetrazolium chloride staining across the brain sections. Melatonin- and DEX-mediated improvements in tissue histology shown by the reduction in lesion size and an improvement in apoptosis level further support the efficacy of combination therapy. The combination therapy also blocked the infiltration of astrocytes and reduced CCI-mediated oxidative stress. In addition, we have also clearly demonstrated that the combination therapy significantly ameliorated neurological scores. Taken together, our results clearly indicate that combination therapy with melatonin and DEX presents beneficial synergistic effects, and we consider it an avenue for further development of novel combination therapeutic agents in the treatment of TBI that are more effective than a single effector molecule.

Protective effect of melatonin against the inflammatory response elicited by crude venom from isolated nematocysts of Pelagia noctiluca (Cnidaria, Scyphozoa).

Abstract:  Melatonin (N‐acetyl‐5‐methoxytryptamine) is an efficient free radical scavenger and antioxidant, both in vitro and in vivo. The role of melatonin as an immunomodulator is, in some cases, contradictory. In this study we have investigated the therapeutic efficacy of melatonin in rats subjected to Pelagia noctiluca crude venom (of the familia Pelaguiidae; and genus Pelagia) induced acute paw inflammation. In particular, injection of the venom into the paw of rats elicited an acute inflammatory response characterized by accumulation of fluid containing a large number of polymorphonuclear neutrophils in the paw and subsequent lipid peroxidation. Furthermore, the venom promoted an expression of iNOS, nitrotyrosine and the activation of the nuclear enzyme poly (ADP‐ribose) polymerase as determined by immunohistochemical analysis of paw tissues. Administration of melatonin 30 min, 1 and 6 hr after the challenge with the venom, caused a significant reduction in all the parameters of inflammation measured. Thus, based on these findings we propose that melatonin may be useful a treatment of local acute inflammation induced by P. noctiluca crude venom.

Nematocyst discharge in Pelagia noctiluca (Cnidaria, Scyphozoa) oral arms can be affected by lidocaine, ethanol, ammonia and acetic acid

Nematocyst discharge and concomitant delivery of toxins is triggered to perform both defence and predation strategies in Cnidarians, and may lead to serious local and systemic reactions in humans. Pelagia noctiluca (Cnidaria, Scyphozoa) is a jellyfish particularly abundant in the Strait of Messina (Italy). After accidental contact with this jellyfish, not discharged nematocysts or even fragments of tentacles or oral arms may tightly adhere to the human skin and, following discharge, severely increase pain and the other adverse consequences of the sting. The aim of the present study is to verify if the local anesthetic lidocaine and other compounds, like alcohols, acetic acid and ammonia, known to provide pain relief after jellyfish stings, may also affect in situ discharge of nematocysts. Discharge was induced by a combined physico-chemical stimulation of oral arms by chemosensitizers (such as N-acetylated sugars, aminoacids, proteins and nucleotides), in the presence or absence of 1% lidocaine, 70% ethanol, 5% acetic acid or 20% ammonia, followed by mechanical stimulation by a non-vibrating test probe. The above mentioned compounds failed to induce discharge per se, and dramatically impaired the chemosensitizer-induced discharge response. We therefore suggest that prompt local treatment of the stung epidermis with lidocaine, acetic acid, ethanol and ammonia may provide substantial pain relief and help in reducing possible harmful local and systemic adverse reaction following accidental contact with P. noctiluca specimens.

Factors altering the haemolytic power of crude venom from Aiptasia mutabilis (Anthozoa) nematocysts

The effect of different agents upon the haemolytic power of Aiptasia mutabilis crude venom was studied inhuman erythrocytes to determine its toxicity and stability. Nematocysts were isolated from acontia of the Anthozoan A. mutabilis and submitted to sonication for extracting crude venom. Aliquots of venom were tested in 0.05% erythrocyte suspensions in the presence of various factors such as proteases (papain,collagenase, trypsin, alpha-chymotrypsin); cations (Ca2+, Mg2+, Ba2+, K+ and Cu2+), osmotic protectants as polyethylenglycole (PEG) of different MW and antioxidant compounds (GSH, cysteine and ascorbic acid).Results demonstrate the dose-response of the haemolytic effect of A. mutabilis. Haemolysis by the crude venom was prevented by Ca2+, Ba2+ and Cu2+ treatment, and to a minor extent by Mg2+ and K+. Papain and PEG with a molecular mass exceeding 1000 Da also prevented haemolysis. These findings are consistent with a pore-forming mechanism of crude venom in erythrocytes rather than an oxidative damage at the employed doses.

Pelagia noctiluca (Scyphozoa) Crude Venom Injection Elicits Oxidative Stress and Inflammatory Response in Rats

Cnidarian toxins represent a rich source of biologically active compounds. Since they may act via oxidative stress events, the aim of the present study was to verify whether crude venom, extracted from the jellyfish Pelagia noctiluca, elicits inflammation and oxidative stress processes, known to be mediated by Reactive Oxygen Species (ROS) production, in rats. In a first set of experiments, the animals were injected with crude venom (at three different doses 6, 30 and 60 µg/kg, suspended in saline solution, i.v.) to test the mortality and possible blood pressure changes. In a second set of experiments, to confirm that Pelagia noctiluca crude venom enhances ROS formation and may contribute to the pathophysiology of inflammation, crude venom-injected animals (30 µg/kg) were also treated with tempol, a powerful antioxidant (100 mg/kg i.p., 30 and 60 min after crude venom). Administration of tempol after crude venom challenge, caused a significant reduction of each parameter related to inflammation. The potential effect of Pelagia noctiluca crude venom in the systemic inflammation process has been here demonstrated, adding novel information about its biological activity.

Crude Venom from Nematocysts of the Jellyfish Pelagia noctiluca as a Tool to Study Cell Physiology

Marine animals represent a source of novel bioactive compounds considered as a good research model, whose mechanism of action is intriguing and still under debate. Among stinging animals, Cnidarians differentiated highly specialized cells, termed nematocytes, containing a capsule fluid with toxins and an inverted tubule, synergistically responsible for mechanisms of defence and predation. Such compounds include proteins and secondary metabolites with toxic action. With the aim of better elucidating the effects of Cnidarian venom upon cell targets, this short review reports on the current knowledge about the toxicological activity of venom extracted from nematocysts of the jellyfish Pelagia noctiluca, whose notable blooming is well known in the Strait of Messina (Italy). The effects on cultured cells, from both mammals and invertebrates, and erythrocytes are here being considered. What is known about the biological activity of Pelagia noctiluca crude venom accounts for a powerful biological activity at different levels, suggesting that cell damage may be due to a pore formation mechanism on cell membrane target leading to osmotic lysis, and /or to oxidative stress events. In this light, the study of venom activity may contribute to: i) validate suitable biological assays for venom testing; ii) elucidate cell function features; iii) understand the pathophysiology of envenoming.

Heavy Metals Affect Nematocysts Discharge Response and Biological Activity of Crude Venom in the Jellyfish Pelagia noctiluca (Cnidaria, Scyphozoa)

Background: Pollution of marine ecosystems and, specifically, heavy metals contamination may compromise the physiology of marine animals with events occurring on a cellular and molecular level. The present study focuses on the effect of short-term exposure to heavy metals like Zinc, Cadmium, Cobalt and Lanthanum (2-10 mM) on the homeostasis of Pelagia noctiluca (Cnidaria, Scyphozoa), a jellyfish abundant in the Mediterranean sea. This species possesses stinging organoids, termed nematocysts, whose discharge and concomitant delivery of venom underlie the survival of all Cnidaria. Methods: Nematocysts discharge response, elicited by combined chemico-physical stimulation, was verified on excised oral arms exposed to heavy metals for 20 min. In addition, the hemolytic activity of toxins, contained in the crude venom extracted from nematocysts isolated from oral arms, was tested on human erythrocytes, in the presence of heavy metals or their mixture. Results: Treatment with heavy metals significantly inhibited both nematocysts discharge response and hemolytic activity of crude venom, in a dose-dependent manner, not involving oxidative events, that was irreversible in the case of Lanthanum. Conclusion: Our findings show that the homeostasis of Pelagia noctiluca, in terms of nematocysts discharge capability and effectiveness of venom toxins, is dramatically and rapidly compromised by heavy metals and confirm that this jellyfish is eligible as a model for ecotoxicological investigations.