Mariagrazia Rinaldi

Effects of flavocoxid, a dual inhibitor of COX and 5-lipoxygenase enzymes, on benign prostatic hyperplasia

Inflammation plays a key role in the development of benign prostatic hyperplasia (BPH). Eicosanoids derived from the COX and 5‐lipoxygenase (5‐LOX) pathways are elevated in the enlarging prostate. Flavocoxid is a novel flavonoid–based ‘dual inhibitor’ of the COX and 5‐LOX enzymes. This study evaluated the effects of flavocoxid in experimental BPH.

Polydeoxyribonucleotide reduces cytokine production and the severity of collagen‐induced arthritis by stimulation of adenosine A2A receptor

OBJECTIVE Broad antiinflammatory effects following adenosine A(₂A) receptor stimulation have been demonstrated in acute inflammatory diseases, including arthritis. Polydeoxyribonucleotide (PDRN) activates the adenosine A(₂A) receptor. This study was undertaken to investigate the effects of PDRN in collagen-induced arthritis (CIA) in mice. METHODS Arthritis was induced in DBA/1 mice by an intradermal injection of 100 μl of bovine type II collagen in Freunds complete adjuvant. Mice were immunized a second time 21 days later. Control animals received 100 μl of a saline solution. Animals with CIA were randomized to receive one of the following: vehicle (1 ml/kg); PDRN (8 mg/kg intraperitoneally daily); 3,7-dimethyl-propargylxanthine (DMPX), a specific adenosine A(₂A) receptor antagonist (0.1 mg/kg intraperitoneally daily); or PDRN plus DMPX. The treatment was initiated immediately after the second immunization and continued to day 45. Clinical evaluation of arthritis was performed throughout the study. On day 45, the animals were killed and the severity of arthritis was evaluated histologically. Cartilage expression and circulating levels of high mobility group box chromosomal protein 1 (HMGB-1), tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and IL-10 were investigated. Inflammatory cytokine production was also evaluated in stimulated human chondrocytes treated with PDRN. RESULTS PDRN treatment significantly ameliorated clinical signs of arthritis, improved histologic damage, reduced the cartilage expression and circulating levels of HMGB-1, TNFα, and IL-6, and enhanced IL-10 expression. The concomitant administration of DMPX and PDRN ablated the PDRN-induced protective effect in experimental arthritis. PDRN also reduced cytokine production from stimulated human chondrocytes. CONCLUSION Our findings indicate that PDRN may represent a new alternative for the treatment of arthritis.

Flavocoxid, a dual inhibitor of COX-2 and 5-LOX of natural origin, attenuates the inflammatory response and protects mice from sepsis

IntroductionCecal ligation and puncture (CLP) is an inflammatory condition that leads to multisystemic organ failure. Flavocoxid, a dual inhibitor of cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX), has been shown in vitro to possess antiinflammatory activity in lipopolysaccharide (LPS)-stimulated rat macrophages by reducing nuclear factor (NF)-κB activity and COX-2, 5-LOX and inducible nitric oxide synthase (iNOS) expression. The aim of this study was to evaluate the effects of flavocoxid in a murine model of CLP-induced polymicrobial sepsis.MethodsC57BL/6J mice were subjected to CLP or sham operation. In a first set of experiments, an intraperitoneal injection of flavocoxid (20 mg/kg) or vehicle was administered 1 hour after surgery and repeated every 12 hours. Survival rate was monitored every 24 hours throughout 120 hours. Furthermore, additional groups of sham and CLP mice were killed 18 hours after surgical procedures for blood-sample collection and the lung and liver were collected for biomolecular, biochemical and histopathologic studies.ResultsCOX-2, 5-LOX, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-10, extracellular-regulated-kinase 1/2 (ERK), JunN-terminal kinase (JNK), NF-κB, and β-arrestin 2 protein expression were evaluated in lung and liver with Western blot analysis. In addition, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), cytokines, and lipoxin A4 serum content were measured with an enzyme-linked immunosorbent assay (ELISA). Flavocoxid administration improved survival, reduced the expression of NF-κB, COX-2, 5-LOX, TNF-α and IL-6 and increased IL-10 production. Moreover, flavocoxid inhibited the mitogen-activated protein kinases (MAPKs) pathway, preserved β-arrestin 2 expression, reduced blood LTB4, PGE2, TNF-α and IL-6, and increased IL-10 and lipoxin A4 serum levels. The treatment with flavocoxid also protected against the histologic damage induced by CLP and reduced the myeloperoxidase (MPO) activity in the lung and liver.ConclusionsFlavocoxid protects mice from sepsis, suggesting that this dual inhibitor may represent a promising approach in such a life-threatening condition.

ROS-Mediated NLRP3 Inflammasome Activation in Brain, Heart, Kidney, and Testis Ischemia/Reperfusion Injury

Ischemia and reperfusion (I/R) causes a reduction in arterial blood supply to tissues, followed by the restoration of perfusion and consequent reoxygenation. The reestablishment of blood flow triggers further damage to the ischemic tissue through reactive oxygen species (ROS) accumulation, interference with cellular ion homeostasis, and inflammatory responses to cell death. In normal conditions, ROS mediate important beneficial responses. When their production is prolonged or elevated, harmful events are observed with peculiar cellular changes. In particular, during I/R, ROS stimulate tissue inflammation and induce NLRP3 inflammasome activation. The mechanisms underlying the activation of NLRP3 are several and not completely elucidated. It was recently shown that NLRP3 might sense directly the presence of ROS produced by normal or malfunctioning mitochondria or indirectly by other activators of NLRP3. Aim of the present review is to describe the current knowledge on the role of NLRP3 in some organs (brain, heart, kidney, and testis) after I/R injury, with particular regard to the role played by ROS in its activation. Furthermore, as no specific therapy for the prevention or treatment of the high mortality and morbidity associated with I/R is available, the state of the art of the development of novel therapeutic approaches is illustrated.

Protective effects of melanocortins on short-term changes in a rat model of traumatic brain injury*.

Objective:Treatment for traumatic brain injury remains elusive despite compelling evidence from animal models for a variety of therapeutic targets. Melanocortins have established neuroprotective effects against experimental ischemic stroke. We investigated whether melanocortin treatment of traumatic brain injury induces neuroprotection and promotes functional recovery. Design:Randomized experiment. Setting:Research laboratory at a university hospital. Subjects:Male Sprague-Dawley rats (n = 215). Interventions:Experimental rat model of diffuse traumatic brain injury, the impact-acceleration model. Measurement and Main Results:Brain tissue nitrites, phosphorylation level of extracellular signal-regulated kinases, and c-jun N-terminal kinases; and expression of active caspase-3, tumor necrosis factor-&agr;, BAX, and Bcl-2 as well as serum levels of interleukin-6, high mobility group box-1, interleukin-10, and brain histologic damage were evaluated 24 or 48 hrs after the insult. Sensorimotor orientation and limb use were evaluated at day 7 and learning and memory at days 23–30 after injury. Posttraumatic treatment every 12 hrs with the melanocortin analog [Nle4, D-Phe7]-&agr;-melanocyte-stimulating hormone (starting 3 or 6 hrs after injury) inhibited traumatic brain injury-induced upregulation of nitric oxide synthesis, phosphorylation level of extracellular signal-regulated kinases, phosphorylation level of c-jun N-terminal kinases, and active caspase-3; reduced expressions/levels of tumor necrosis factor-&agr;, BAX, interleukin-6, and high mobility group box-1; and increased those of Bcl-2 and interleukin-10. These molecular changes were associated with a reduction in brain tissue damage, as highlighted by histopathological findings and improved functional recovery. Pretreatment with the melanocortin MC4 receptor antagonist HS024 abated the positive effects of [Nle4, D-Phe7]-&agr;-melanocyte-stimulating hormone. Conclusions:Our data indicate that melanocortins protect against traumatic brain injury, in a broad time window and through activation of MC4 receptors, by counteracting the main traumatic brain injury-related mechanisms of damage. These findings could have major clinical implications.

Melanocortin 4 Receptor Activation Protects Against Testicular Ischemia-Reperfusion Injury by Triggering the Cholinergic Antiinflammatory Pathway

Melanocortins (MC) trigger a vagus nerve-mediated cholinergic-antiinflammatory pathway projecting to the testis. We tested whether pharmacological activation of brain MC receptors might protect the testis from the damage induced by ischemia-reperfusion. Adult male rats were subjected to 1-h testicular ischemia, followed by 24-h reperfusion [testicular ischemia-reperfusion (TI/R)]. Before TI/R, groups of animals were subjected to bilateral cervical vagotomy, or pretreated with the nicotinic acetylcholine receptor antagonist chlorisondamine or the selective MC(4) receptor antagonist HS024. Immediately after reperfusion, rats were ip treated with saline or the MC analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) (340 μg/kg). We evaluated testicular IL-6 and TNF-α by Western blot analysis and organ damage by light microscopy. Some experimental groups were prepared for neural efferent activity recording along the vagus nerve starting 30 min after treatment with NDP-α-MSH or saline, and for a 30-min period. Additional groups of TI/R rats were treated for 30 d with saline, NDP-α-MSH, chlorisondamine plus NDP-α-MSH, or HS024 plus NDP-α-MSH to evaluate spermatogenesis, organ damage, and the apoptosis machinery. After a 24-h reperfusion, in TI/R saline-treated rats, there was an increase in IL-6 and TNF-α expression and a marked damage in both testes. NDP-α-MSH inhibited IL-6 and TNF-α expression, decreased histological damage, and increased neural efferent activity. Furthermore, NDP-α-MSH administration for 30 d greatly improved spermatogenesis, reduced organ damage, and inhibited apoptosis. All positive NDP-α-MSH effects were abrogated by vagotomy, chlorisondamine, or HS024. Our data suggest that selective MC(4) receptor agonists might be therapeutic candidates for the management of testicular torsion.

ROS and Brain Gliomas: An Overview of Potential and Innovative Therapeutic Strategies

Reactive oxygen species (ROS) represent reactive products belonging to the partial reduction of oxygen. It has been reported that ROS are involved in different signaling pathways to control cellular stability. Under normal conditions, the correct function of redox systems leads to the prevention of cell oxidative damage. When ROS exceed the antioxidant defense system, cellular stress occurs. The cellular redox impairment is strictly related to tumorigenesis. Tumor cells, through the generation of hydrogen peroxide, tend to the alteration of cell cycle phases and, finally to cancer progression. In adults, the most common form of primary malignant brain tumors is represented by gliomas. The gliomagenesis is characterized by numerous molecular processes all characterized by an altered production of growth factor receptors. The difficulty to treat brain cancer depends on several biological mechanisms such as failure of drug delivery through the blood-brain barrier, tumor response to chemotherapy, and intrinsic resistance of tumor cells. Understanding the mechanisms of ROS action could allow the formulation of new therapeutic protocols to treat brain gliomas.

Flavocoxid Protects Against Cadmium-Induced Disruption of the Blood–Testis Barrier and Improves Testicular Damage and Germ Cell Impairment in Mice

Cadmium (Cd) causes male infertility. There is the need to identify safe treatments counteracting this toxicity. Flavocoxid is a flavonoid that induces a balanced inhibition of cyclooxygenase (COX)-1 and COX-2 peroxidase moieties and of 5-lipoxygenase (LOX) and has efficacy in the male genitourinary system. We investigated flavocoxid effects on Cd-induced testicular toxicity in mice. Swiss mice were divided into 4 groups: 2 control groups received 0.9% NaCl (vehicle; 1 ml/kg/day) or flavocoxid (20 mg/kg/day ip); 2 groups were challenged with cadmium chloride (CdCl2; 2 mg/kg/day ip) and administered with vehicle or flavocoxid. The treatment lasted for 1 or 2 weeks. The testes were processed for biochemical and morphological studies. CdCl2 increased phosphorylated extracellular signal-regulated kinase (p-ERK) 1/2, tumor necrosis factor (TNF)-α, COX-2, 5-LOX, malondialdehyde (MDA), B-cell-lymphoma (Bcl)-2-associated X protein (Bax), follicle-stimulating hormone (FSH), luteinizing hormone (LH), transforming growth factor (TGF) -β3, decreased Bcl-2, testosterone, inhibin-B, occludin, N-Cadherin, induced structural damages in the testis and disrupted the blood-testis barrier. Many TUNEL-positive germ cells and changes in claudin-11, occludin, and N-cadherin localization were present. Flavocoxid administration reduced, in a time-dependent way, p-ERK 1/2, TNF-α, COX-2, 5-LOX, MDA, Bax, FSH, LH, TGF-β3, augmented Bcl-2, testosterone, inhibin B, occludin, N-Cadherin, and improved the structural organization of the testis and the blood-testis barrier. Few TUNEL-positive germ cells were present and a morphological retrieval of the intercellular junctions was observed. In conclusion, flavocoxid has a protective anti-inflammatory, antioxidant, and antiapoptotic function against Cd-induced toxicity in mice testis. We suggest that flavocoxid may play a relevant positive role against environmental levels of Cd, otherwise deleterious to gametogenesis and tubular integrity.

Cadmium, organ toxicity and therapeutic approaches. A review on brain, kidney and testis damage

BACKGROUND Cadmium (Cd) is a heavy metal particularly hazardous for human health, as it is highly diffused and, therefore, a ubiquitous environmental toxicant. In fact, in the general population, the main sources of exposure are food, cigarette smoking, inhalation of ambient air, drinking water, contaminated soil or dust. Furthermore, an occupational exposure usually involves human during mining, fume inhalation or manufacturing nickel-cadmium battery, electroplating and paint pigments that utilize Cd. METHODS We undertook a structured search in literature about Cd. This metal is noxious on the cells of many organs, among which the kidney, the testis and the brain will be considered in this review. RESULTS The toxic effects induced by Cd include many specific mechanisms, such as the oxidative stress, cellular death and inflammation. As no specific therapy for the prevention or treatment of the morbidity and mortality associated with Cd exposure is available, the state of the art of the therapeutic approaches is illustrated. CONCLUSION Nowadays, a therapy able to counteract Cd toxicity is still lacking and the development of new therapeutic agents is requested.

Role of Inhibitors of Apoptosis Proteins in Testicular Function and Male Fertility: Effects of Polydeoxyribonucleotide Administration in Experimental Varicocele.

Neuronal apoptosis inhibitory protein (NAIP) and survivin might play an important role in testicular function. We investigated the effect of PDRN, an agonist of adenosine A2A receptor, on testicular NAIP and survivin expression in an experimental model of varicocele. After the creation of experimental varicocele (28 days), adolescent male Sprague-Dawley rats were randomized to one of the following treatments lasting 21 days: vehicle, PDRN (8 mg/kg i.p., daily), PDRN + 3,7-dimethyl-propargylxanthine (DMPX, a specific adenosine A2A-receptor antagonist, 0.1 mg/kg i.p., daily), varicocelectomy, and varicocelectomy + PDRN (8 mg/kg i.p., daily). Sham-operated animals were used as controls. Animals were then euthanized and testis expression of NAIP and survivin was evaluated through qRT-PCR, western blot, and immunohistochemical analysis. Spermatogenetic activity was also assessed. NAIP and survivin expressions were significantly reduced following varicocele induction when compared to sham animals whereas PDRN-treated rats showed an increase in NAIP and survivin levels. Immunohistochemistry revealed an enhanced expression of NAIP and survivin with a characteristic pattern of cellular localization following PDRN treatment. Moreover, administration of PDRN significantly restored spermatogenic function in varicocele rats. PDRN may represent a rational therapeutic option for accelerating recovery from depressed testicular function through a strategic modulation of apoptosis in experimental varicocele.