Tiziana Genovese

Protective Effects of Anthocyanins from Blackberry in a Rat Model of Acute Lung Inflammation

Anthocyanins are a group of naturally occuring phenolic compounds related to the coloring of plants, flowers and fruits. These pigments are important as quality indicators, as chemotaxonomic markers and for their antioxidant activities. Here, we have investigated the therapeutic efficacy of anthocyanins contained in blackberry extract (cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents) in an experimental model of lung inflammation induced by carrageenan in rats. Injection of carrageenan into the pleural cavity elicited an acute inflammatory response characterized by fluid accumulation which contained a large number of neutrophils as well as an infiltration of polymorphonuclear leukocytes in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx) and prostaglandin E2 (PGE2). All parameters of inflammation were attenuated in a dose-dependent manner by anthocyanins (10, 30 mg kg-1 30 min before carrageenan). Furthermore, carrageenan induced an upregulation of the adhesion molecule ICAM-1, nitrotyrosine and poly (ADP-ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues. The degree of staining was lowered by anthocyanins treatment. Thus, the anthocyanins contained in the blackberry extract exert multiple protective effects in carrageenan-induced pleurisy.

Immunomodulatory Effects of Etanercept in an Experimental Model of Spinal Cord Injury

Etanercept is a tumor necrosis factor antagonist with anti-inflammatory effects. The aim of our study was to evaluate for the first time the therapeutic efficacy of in vivo inhibition of tumor necrosis factor-α (TNF-α) in experimental model of spinal cord trauma, which was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5–T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and cytokine production that it is followed by recruitment of other inflammatory cells, such as production of a range of inflammation mediators, tissue damage, apoptosis, and disease. Treatment of the mice with etanercept significantly reduced the degree of 1) spinal cord inflammation and tissue injury (histological score); 2) neutrophil infiltration (myeloperoxidase evaluation); 3) inducible nitric-oxide synthase, nitrotyrosine, cyclooxygenase-2, and cytokines expression (TNF-α and interleukin-1β); and 4) apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and Bax and Bcl-2 expression). In a separate set of experiment, we have also clearly demonstrated that TNF-α inhibitor significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with etanercept reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Effect of rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2 on bleomycin-induced lung injury

Thiazolidinedione rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), are two peroxisome proliferator-activated receptor (PPAR)-γ ligands. The aim of this study was to investigate the effect of rosiglitazone and 15d-PGJ2 on the lung injury caused by bleomycin administration. Mice subjected to intratracheal administration of bleomycin developed significant lung injury. An increase in immunoreactivity to nitrotyrosine, poly(ADP ribose) polymerase (PARP) and inducible nitric oxide synthase as well as a significant loss of body weight and mortality was observed in the lung of bleomycin-treated mice. Administration of the two PPAR-γ agonists rosiglitazone (10 mg·kg−1 i.p.) and 15d-PGJ2 (30 µg·kg−1 i.p.) significantly reduced the: 1) loss of body weight, 2) mortality rate, 3) infiltration of the lung with polymorphonuclear neutrophils (myeloperoxidase activity), 4) oedema formation, and 5) histological evidence of lung injury. Administration of rosiglitazone and 15d-PGJ2 also markedly reduced the nitrotyrosine, PARP and inducible nitric oxide synthase formation. In addition, treatment with the PPAR-γ antagonist bisphenol A diglycidyl ether (1 mg·kg−1 i.p. 30 min before the rosiglitazone or 15d-PGJ2) significantly antagonised the effect of the two PPAR-γ agonists. These results demonstrate that the two peroxisome proliferator-activated receptor-γ agonists, rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2, significantly reduce lung injury induced by bleomycin in mice.

Rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2, ligands of the peroxisome proliferator-activated receptor-γ (PPAR-γ), reduce ischaemia/reperfusion injury of the gut

The peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is a member of the nuclear receptor superfamily of ligand‐dependent transcription factors related to retinoid, steroid and thyroid hormone receptors. The thiazolidinedione rosiglitazone and the endogenous cyclopentenone prostaglandin (PG)D2 metabolite, 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2), are two PPAR‐γ ligands, which modulate the transcription of target genes. The aim of this study was to investigate the effect of rosiglitazone and 15d‐PGJ2 on the tissue injury caused by ischaemia/reperfusion (I/R) of the gut. I/R injury of the intestine was caused by clamping both the superior mesenteric artery and the coeliac trunk for 45 min, followed by release of the clamp allowing reperfusion for 2 or 4 h. This procedure results in splanchnic artery occlusion (SAO) shock. Rats subjected to SAO developed a significant fall in mean arterial blood pressure, and only 10% of the animals survived for the entire 4 h reperfusion period. Surviving animals were killed for histological examination and biochemical studies. Rats subjected to SAO displayed a significant increase in tissue myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, significant increases in plasma tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β levels and marked injury to the distal ileum. Increased immunoreactivity to nitrotyrosine was observed in the ileum of rats subjected to SAO. Staining of sections of the ileum obtained from SAO rats with anti‐intercellular adhesion molecule (ICAM‐1) antibody resulted in diffuse staining. Administration at 30 min prior to the onset of gut ischaemia of the two PPAR‐γ agonists (rosiglitazone (0.3 mg kg−1 i.v.) and 15d‐PGJ2 (0.3 mg kg−1 i.v.)) significantly reduced the (i) fall in mean arterial blood pressure, (ii) mortality rate, (iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils (MPO activity), (iv) lipid peroxidation (MDA levels), (v) production of proinflammatory cytokines (TNF‐α and IL‐1β) and (vi) histological evidence of gut injury. Administration of rosiglitazone and 15d‐PGJ2 also markedly reduced the nitrotyrosine formation and the upregulation of ICAM‐1 during reperfusion. In order to elucidate whether the protective effects of rosiglitazone and 15d‐PGJ2 are related to the activation of the PPAR‐γ receptor, we also investigated the effect of a PPAR‐γ antagonist, bisphenol A diglycidyl ether (BADGE), on the protective effects of rosiglitazone and 15d‐PGJ2. BADGE (1 mg kg−1 administered i.v. 30 min prior to the treatment of rosiglitazone or 15d‐PGJ2) significantly antagonised the effect of the two PPAR‐γ agonists and thus abolished the protective effect against gut I/R. These results demonstrate that the two PPAR‐γ agonists, rosiglitazone and 15d‐PGJ2, significantly reduce I/R injury of the intestine.

The role of the peroxisome proliferator-activated receptor-α (PPAR-α) in the regulation of acute inflammation

The peroxisome proliferator‐activated receptor‐α (PPAR‐α) is a member of the nuclear receptor superfamily of ligand‐dependent transcription factors related to retinoid, steroid, and thyroid hormone receptors. The aim of the present study was to evaluate the role of the PPAR‐α receptor on the development of acute inflammation. To address this question, we used two animal models of acute inflammation (carrageenan‐induced paw edema and carrageenan‐induced pleurisy). We report here that when compared with PPAR‐α wild‐type mice, PPAR‐α knockout mice (PPAR‐αKO) mice experienced a higher rate of the extent and severity when subjected to carrageenan injection in the paw edema model or to carrageenan administration in the pleurisy model. In particular, the absence of a functional PPAR‐α gene in PPAR‐αKO mice resulted in a significant augmentation of various inflammatory parameters (e.g., enhancement of paw edema, pleural exudate formation, mononuclear cell infiltration, and histological injury) in vivo. Furthermore, the absence of a functional PPAR‐α gene enhanced the staining (immunohistochemistry) for FAS ligand in the paw and in the lung and the expression of tumor necrosis factor α and interleukin‐1β in the lungs of carrageenan‐treated mice. In conclusion, the increased inflammatory response observed in PPAR‐αΚΟ mice strongly suggests that a PPAR‐α pathway modulates the degree of acute inflammation in the mice.

Role of endogenous and exogenous ligands for the peroxisome proliferators activated receptors alpha (PPAR- α ) in the development of inflammatory bowel disease in mice

The peroxisome proliferator-activated receptor-α (PPAR-α) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors related to retinoid, steroid and thyroid hormone receptors. The aim of the present study was to examine the effects of endogenous and exogenous PPAR-α ligand on the development of experimental colitis. To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). When compared to DNBS-treated PPAR-α wild-type (WT) mice, DNBS-treated PPAR-α knockout mice (PPAR-αKO) mice experienced a higher rate of the extent and severity of the histological signs of colon injury. After administration of DNBS PPAR-αWT mice experienced hemorrhagic diarrhea, weight loss and large areas of necrosis in the mucosa of the colon were also observed. Neutrophil infiltration was associated with upregulation of ICAM-1. Immunohistochemistry for nitrotyrosine showed an intense staining in the inflamed colon. Absence of a functional PPAR-α gene in PPAR-αKO mice resulted in a significant augmentation of all the above-described parameters. On the contrary, the treatment of PPAR-αWT with Wy-14643 (1 mg/kg daily i.p) significantly reduced: (i) the degree of hemorrhagic diarrhea and weight loss, (ii) the degree of colon injury, (iii) the rise in MPO activity (mucosa), (iv) the increase in staining (immunohistochemistry) for nitrotyrosine, as well as (v) the upregulation of ICAM-1 caused by DNBS in the colon. In order to elucidate whether the protective effects of Wy-14643 is related to activation of the PPAR-α receptor, we also investigated the effect the of Wy-14643 treatment on PPAR-α-deficient mice. The absence of the PPAR-α receptor significantly abolished the protective effect of the PPAR-α agonist against DNBS-induced colitis. Thus, endogenous and exogenous PPAR-α ligands reduce the degree of colitis caused by DNBS. We propose that PPAR-α ligand may be useful in the treatment of inflammatory bowel disease.

Role of glucocorticoid-induced TNF receptor family gene (GITR) in collagen-induced arthritis

In rheumatoid arthritis (RA), a widespread autoimmune/inflammatory joint disease, early activation of effector CD4+ T lymphocytes, and cytokine production is followed by recruitment of other inflammatory cells, production of a range of inflammation mediators, tissue damage, and disease. GITR (glucocorticoid‐induced TNFR family‐related gene), a costimulatory molecule for T lymphocytes, increases CD4+CD25− effector T cell activation while inhibiting suppressor activity of CD4+CD25+ T regulatory (Treg) cells. We analyzed the role of GITR in type II collagen (CII) ‐induced arthritis (CIA) using GITR− /− and GITR+ / + mice. Results indicate significantly less CIA induction in GITR−/− mice than in GITR+/+ mice, with marked differences in erythema, edema, neutrophil infiltration, joint injury, and bone erosion. Production of IFNγ, IL‐6, TNFα, MIP‐1α, and MIP‐2, inducible NOS (iNOS), COX‐2, and nitrotyrosine poly‐ADP‐ribose (PAR) were also less in CII‐treated GITR−/− mice. Although CD4+CD25+ Treg cells from GITR+/+ and GITR−/− CII‐challenged mice exerted similar suppressor activity in vitro, GITR triggering abrogated GITR+/+ Treg suppressor activity and costimulated CD4+CD25− GITR+/+ effector cells. Furthermore, Treg cells from GITR−/− protected more than Treg cells from GITR+/+ mice against CIA when cotransferred with Treg‐depleted splenocytes from arthritic GITR+/+ animals into severe combined immunodeficient (SCID) mice. In conclusion, GITR plays a critical role in the immunological response against CII and in the development of CIA. Cuzzocrea, S., Ayroldi, E., Di Paola, R., Agostini, M., Mazzon, E., Bruscoli, S., Genovese, T., Ronchetti, S., Caputi, A. P., Riccardi, C. Role of glucocorticoid‐induced TNF receptor family gene (GITR) in collagen‐induced arthritis. FASEB J. 19, 1253–1265 (2005)

Effect of Aminoguanidine in Ligature-induced Periodontitis in Rats

The role of nitric oxide and reactive oxygen species is well-demonstrated in inflammation. In this study, we evaluated the effect of aminoguanidine, a nitric oxide synthase inhibitor, in a rat model of periodontitis. We induced periodontitis in rats by placing a piece of 2/0 braided silk around the lower left 1st molar. At day 8, the gingivomucosal tissue encircling the mandibular 1st molar was removed for biochemical and histological analysis. Ligation significantly increased inducible nitric oxide synthase activity and expression, and damaged tissue revealed increased neutrophil infiltration, lipid peroxidation, and positive staining for nitrotyrosine formation and poly (ADP-ribose) polymerase activation. Ligation significantly increased Evans blue extravasation in gingivomucosal tissue and alveolar bone destruction. Aminoguanidine (100 mg/kg i.p., daily for 8 days) treatment significantly reduced all these inflammatory parameters, indicating that it protects against the tissue damage associated with periodontitis by reducing nitric oxide production and oxidative stress.

Effects of Palmitoylethanolamide on Signaling Pathways Implicated in the Development of Spinal Cord Injury

Activation of peroxisome proliferator-activated receptor (PPAR)-α, a member of the nuclear receptor superfamily, modulates inflammation and tissue injury events associated with spinal cord trauma in mice. Palmitoylethanolamide (PEA), the naturally occurring amide of palmitic acid and ethanolamine, reduces pain and inflammation through a mechanism dependent on PPAR-α activation. The aim of the present study was to evaluate the effect of the PEA on secondary damage induced by experimental spinal cord injury (SCI) in mice. SCI was induced by application of vascular clips to the dura mater via a four-level T5-T8 laminectomy. This resulted in severe trauma characterized by edema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, and apoptosis. Repeated PEA administration (10 mg/kg i.p.; 30 min before and 1 and 6 h after SCI) significantly reduced: 1) the degree of spinal cord inflammation and tissue injury, 2) neutrophil infiltration, 3) nitrotyrosine formation, 4) proinflammatory cytokine expression, 5) nuclear transcription factor activation-κB activation, 6) inducible nitric-oxide synthase expression, and 6) apoptosis. Moreover, PEA treatment significantly ameliorated the recovery of motor limb function. Together, the results indicate that PEA reduces inflammation and tissue injury associated with SCI and suggest a regulatory role for endogenous PPAR-α signaling in the inflammatory response associated with spinal cord trauma.

Attenuation in the evolution of experimental spinal cord trauma by treatment with melatonin

Abstract:   Melatonin is the principal secretory product of the pineal gland and its role as an immumo‐modulator is well established. Recent evidence shows that melatonin is a scavenger of oxyradicals and peroxynitrite and exerts protective effects in septic shock, hemorrhagic shock and inflammation. In the present study, we evaluated the effect of melatonin treatment, in a model of spinal cord injury (SCI). SCI was induced by the application of vascular clips (force of 50 g) to the dura via a four‐level T5‐T8 laminectomy. SCI in rats resulted in severe trauma characterized by edema, neutrophil infiltration and apoptosis (measured by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end‐labeling staining). Infiltration of spinal cord tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced lipid peroxidation (increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine and Poly(ADP‐ribose) (PAR) in the spinal cord tissue. In contrast, the degree of (a) spinal cord inflammation and tissue injury (histological score), (b) nitrotyrosine and PAR formation, (c) neutrophils infiltration and (d) apoptosis was markedly reduced in spinal cord tissue obtained from rats treated with melatonin (50 mg/kg i.p., 30 min before SCI, 30 min, 6 hr, 12 hr and 24 hr after SCI). In a separate set of experiment we have clearly demonstrated that melatonin treatment significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results demonstrate that treatment with melatonin reduces the development of inflammation and tissue injury events associated with spinal cord trauma.