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The normal wound healing process is characterized by a complex, highly integrated cascade of events, requiring the interactions of many cell types, including inflammatory cells, fibroblasts, keratinocytes and endothelial cells, as well as the involvement of growth factors and enzymes. However, several diseases such as diabetes, thermal injury and ischemia could lead to an impaired wound healing process characterized by wound hypoxia, high levels of oxygen radicals, reduced angiogenesis, decreased collagen synthesis and organization. Polydeoxyribonucleotide (PDRN) has been used to improve wound healing through local and systemic administration thanks to its ability to promote cell migration and growth, angiogenesis, and to reduce inflammation on impaired wound healing models in vitro, in vivo and clinical studies. In light of all these observations, the aim of this review is to provide a full overview of PDRN applications on skin regeneration. We reviewed papers published in the last 25 years on PubMed, inserting “polydeoxyribonucleotide and wound healing” as the main search term. All data obtained proved the ability of PDRN in promoting physiological tissue repair through adenosine A2A receptor activation and salvage pathway suggesting that PDRN has proven encouraging results in terms of healing time, wound regeneration and absence of side effects.

Proteasome inhibition is a promising strategy for the treatment of multiple myeloma; unfortunately, this disease is often associated with an increasing chemoresistance. One novel approach may be to target the immunoproteasome, a proteasomal isoform mainly present in cells of hematopoietic origin. We investigated the activity of a panel of amides against immunoproteasome core particles as potential agents for the treatment of multiple myeloma (MM). Amide 6 showed an ideal profile since it was able to inhibit both the chymotrypsin-like activities of the immunoproteasome with Ki values of 4.90 µM and 4.39 µM for β1i and β5i, respectively, coupled with an EC50 =17.8 µM against MM.1R cells. Compound 6 inhibited also ubiquitinated protein degradation and was able to act on different phases of MM cell cycle reducing the levels of cyclin A/CDK1, cyclin B/CDK1 and cyclin D/CDK4/6 complexes, which turns in cell cycle arrest.

Inflammatory bowel diseases (IBDs) are chronic conditions that can benefit from the combined treatment of adenosine receptor agonists and hyaluronic acid (HA), which, binding the CD44, has pro-survival effects. Therefore, this study investigated the effects of a mixture of polynucleotides and HA in an experimental model of dinitrobenzenesulfonic acid (DNBS)-induced colitis. A group of 40 rats received a single intra-colonic instillation of DNBS, and after 6 h, animals were randomized to receive daily: (i) saline solution, (ii) polynucleotides (Poly, 8 mg/kg), (iii) polynucleotides (8 mg/kg) plus hyaluronic acid (HA, 15 mg/kg), and (iv) hyaluronic acid (HA, 15 mg/kg). Rats in the control group (n = 10) received saline solution only. Seven days after induction, animals receiving Poly plus HA showed reduced clinical signs, weight loss and colon shortening, ameliorated macroscopic and histological damage, and apoptosis. Moreover, the combined treatment reduced the positivity in the colonic infiltrate of CD3 positive T cells, CD20 positive B cells and CD44. Furthermore, Poly plus HA reduced colonic myeloperoxidase activity and malondialdehyde, indicating a dampening of the inflammatory infiltrate and oxidation products. Our research demonstrated that a combined treatment of polynucleotides with hyaluronic acid had a protective effect in a model of ulcerative colitis, suggesting that this association deserves further attention for the treatment of IBDs.

Traumatic brain injury (TBI) represents an important problem of global health. The damage related to TBI is first due to the direct injury and then to a secondary phase in which neuroinflammation plays a key role. NLRP3 inflammasome is a component of the innate immune response and different diseases, such as neurodegenerative diseases, are characterized by NLRP3 activation. This review aims to describe NLRP3 inflammasome and the consequences related to its activation following TBI. NLRP3, caspase-1, IL-1β, and IL-18 are significantly upregulated after TBI, therefore, the use of nonspecific, but mostly specific NLRP3 inhibitors is useful to ameliorate the damage post-TBI characterized by neuroinflammation. Moreover, NLRP3 and the molecules associated with its activation may be considered as biomarkers and predictive factors for other neurodegenerative diseases consequent to TBI. Complications such as continuous stimuli or viral infections, such as the SARS-CoV-2 infection, may worsen the prognosis of TBI, altering the immune response and increasing the neuroinflammatory processes related to NLRP3, whose activation occurs both in TBI and in SARS-CoV-2 infection. This review points out the role of NLRP3 in TBI and highlights the hypothesis that NLRP3 may be considered as a potential therapeutic target for the management of neuroinflammation in TBI.

Human gingival fibroblasts (GF) and human oral mucosa epithelial cells (EC) with an inflammatory phenotype represent a valuable experimental paradigm to explore the curative activity of agents to be used in oral mucositis. The role of cannabinoid receptor 2 (CB2) has not yet been investigated in oral mucositis. The aim of this study was to evaluate the therapeutic potential of &beta, Caryophyllene (BCP), a CB2 agonist, in an in vitro model of oral mucositis. GF and EC were stimulated with LPS (2 &micro, g/mL) alone or in combination with BCP, a group of LPS challenged GF and EC were treated with BCP and AM630, a CB2 antagonist. LPS increased the inflammatory cytokines TNF-&alpha, IL-1&beta, IL-6 and IL-17A whereas it decreased the anti-inflammatory cytokine IL-13. The upstream signals were identified in an augmented expression of NF-&kappa, B and STAT-3 and in reduced mRNA levels of PPAR&gamma, and PGC-1&alpha, BCP blunted the LPS-induced inflammatory phenotype and this effect was reverted by the CB2 antagonist AM630. These results suggest that CB2 receptors are an interesting target to develop innovative strategies for oral mucositis and point out that BCP exerts a marked curative effect in a preclinical model of oral mucositis which deserves to be confirmed in a clinical setting.

Nuclear factor-&kappa, B (NF-&kappa, B) plays a central role in psoriasis and canonical Wnt/&beta, catenin pathway blunts the immune-mediated inflammatory cascade in psoriasis. Adenosine A2A receptor activation blocks NF-&kappa, B and boosts the Wnt/&beta, catenin signaling. PDRN (Polydeoxyribonucleotide) is a biologic agonist of the A2A receptor and its effects were studied in an experimental model of psoriasis. Psoriasis-like lesions were induced by a daily application of imiquimod (IMQ) on the shaved back skin of mice for 7 days. Animals were randomly assigned to the following groups: Sham psoriasis challenged with Vaseline, IMQ animals challenged with imiquimod, and IMQ animals treated with PDRN (8 mg/kg/ip). An additional arm of IMQ animals was treated with PDRN plus istradefylline (KW6002, 25 mg/kg/ip) as an A2A antagonist. PDRN restored a normal skin architecture, whereas istradefylline abrogated PDRN positive effects, thus pointing out the mechanistic role of the A2A receptor. PDRN decreased pro-inflammatory cytokines, prompted Wnt signaling, reduced IL-2 and increased IL-10. PDRN also reverted the LPS repressed Wnt-1/&beta, catenin in human keratinocytes and these effects were abolished by ZM241385, an A2A receptor antagonist. Finally, PDRN reduced CD3+ cells in superficial psoriatic dermis. PDRN anti-psoriasis potential may be linked to a &ldquo, dual mode&rdquo, of action: NF-&kappa, B inhibition and Wnt/&beta, catenin stimulation.

Varicocele is one of the main causes of infertility in men. Oxidative stress and consequently apoptosis activation contribute to varicocele pathogenesis, worsening its prognosis. Natural products, such as lycopene, showed antioxidant and anti-inflammatory effects in several experimental models, also in testes. In this study we investigated lycopene effects in an experimental model of varicocele. Male rats (n = 14) underwent sham operations and were administered with vehicle (n = 7) or with lycopene (n = 7, 1 mg/kg i.p., daily). Another group of animals (n = 14) underwent surgical varicocele. After 28 days, the sham and 7 varicocele animals were euthanized, and both operated and contralateral testes were weighted and processed. The remaining rats were treated with lycopene (1 mg/kg i.p., daily) for 30 days. Varicocele rats showed reduced testosterone levels, testes weight, Bcl-2 mRNA expression, changes in testes structure and increased malondialdehyde levels and BAX gene expression. TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) assay showed an increased number of apoptotic cells. Treatment with lycopene significantly increased testosterone levels, testes weight, and Bcl-2 mRNA expression, improved tubular structure and decreased malondialdehyde levels, BAX mRNA expression and TUNEL-positive cells. The present results show that lycopene exerts beneficial effects in testes, and suggest that supplementation with the tomato-derived carotenoid might be considered a novel nutraceutical strategy for the treatment of varicocele and male infertility.

Systemic sclerosis (SSc) is a connective tissue disorder characterized by fibroblast activation and fibrosis of the skin and internal organs. Alterations in cell–integrin interaction are sufficient to initiate profibrotic processes. SSc fibroblasts express both αvβ3 and αvβ5 integrins and their activation induces myofibroblasts differentiation. The aim of the present study was to evaluate the effect of the anb3 and anb5 inhibitor, cilengitide, on the development of vascular and fibrotic changes in the chronic oxidant stress murine model of systemic sclerosis. SSc was induced in BALB/c mice by daily s.c. injections of HOCl for 6 weeks. Mice were randomized in three arms: HOCl alone (n=8), HOCl + Cilengitide (n=8), or Vehicle alone (n=8). Treatment with cilengitide 20 (mg/kg/i.p./day) was started 4 weeks after the first administration of HOCl and maintained throughout the remaining experimental period (2 weeks). Lung, skin, and heart fibrosis were evaluated by histology while kidney morphology by PAS staining. Collagen type I, focal adhesion kinase (FAK), and a-SMA were evaluated by immunostaining and p-FAK and TGF-β1 by Western blot and gene expression. Both cutaneous and pulmonary fibrosis induced by HOCl were attenuated by cilengitide treatment. Cilengitide administration reduced a-SMA, TGF-β1, and p-FAK expression and the increased deposition of fibrillar collagen in the heart and prevented glomeruli collapse in the kidneys. The inhibition of aνβ3 and aνβ5 integrin signaling prevented systemic fibrosis and renal vascular abnormalities in the reactive oxygen species model of SSc. Integrins aνβ3 and aνβ5 could prove useful as a therapeutic target in SSc.

Background and purpose Diabetic patients with non-healing ulcers have a reduced expression of VEGF. Genetically diabetic mice have an altered expression pattern of VEGF and its receptor, VEGF receptor 2 (VEGFR-2). In diabetic wounds, the microRNAs, miR15b and miR200b, which respectively inhibit VEGF and VEGF-R2 mRNAs, are up-regulated, further affecting the impaired angiogenesis. We investigated whether anti-miRs directed toward miR15b and miR200b could improve wound repair in genetically diabetic mice. Experimental approach Skin wounds were produced on the backs of female diabetic mice. The anti-miRs (antimiR15b, antimiR200b or antimiR15b/200b) at 10 mg·kg-1 , or vehicle were applied to the wound edge. Mice were killed on days 7, 14 and at time of complete wound closure. Levels of mRNA and protein of angiogenic mediators and their receptors were measured with RT-qPCR and Western blotting. Wounds were examined by histological and immunochemical methods. Key results mRNA expression of VEGF, VEGFR-2, angiopoietin-1 and its receptor TEK were evaluated after 7 and 14 days. Protein levels of VEGF and transglutaminase II were measured at day 7, while VEGFR-2 and Angiopoietin-1 were measured at day 14. Histological features and the time to achieve a complete wound closure were also examined. Treatment with the anti-miRs improved the analysed parameters and the co-treatment resulted the most effective. Conclusion and implications The results suggest that the inhibition of miR15b and miR200b may have a potential application in diabetes-related wound disorders.

Inflammatory Bowel Disease (IBD) comprises a group of disorders, in particular Crohn’s disease (CD) and ulcerative colitis (UC), characterized by chronic inflammation affecting the gastrointestinal tract. The treatment of these conditions is primarily based on anti-inflammatory drugs, although the use of biological drugs with lower side effects quickly increased in the last decade. However, the presence of certain polymorphisms in the population may determine a different outcome in response to therapy, reflecting the heterogeneity of the efficacy in patients. Considering that several studies showed important correlations between genetic polymorphisms and response to biological treatments in IBD patients, this systematic review aims to summarize the pharmacogenetics of biologicals approved for IBD, thus highlighting a possible association between some polymorphisms and drug response. With this purpose, we reviewed PubMed papers published over the past 21 years (2000–2021), using as the search term “drug name and IBD or CD or UC and polymorphisms” to underline the role of pharmacogenetic tests in approaching the disease with a targeted therapy.

Vitiligo is a chronic autoimmune dermatosis of which the pathogenesis remains scarcely known. A wide variety of clinical studies have been proposed to investigate the immune mediators which have shown the most recurrency. However, such trials have produced controversial results. The aim of this review is to summarize the main factors involved in the pathogenesis of vitiligo, the latest findings regarding the cytokines involved and to evaluate the treatments based on the use of biological drugs in order to stop disease progression and achieve repigmentation. According to the results, the most recurrent studies dealt with inhibitors of IFN-gamma and TNF-alpha. It is possible that, given the great deal of cytokines involved in the lesion formation process of vitiligo, other biologics could be developed in the future to be used as adjuvants and/or to entirely replace the treatments that have proven to be unsatisfactory so far.

Oral mucositis is a side effect hard to treat following high dose chemotherapy or radiotherapy. Adenosine A2A receptor stimulation blocks NF-κB and boosts the Wnt/β-catenin signaling, thus blunting inflammation and triggering growth factor codifying genes. Polydeoxyribonucleotide (PDRN) is a registered drug that activates the A2A receptor. Therefore, the aim of this study was to evaluate PDRN effects in an “in vitro” model of oral mucositis induced by prompting an inflammatory phenotype in human gingival fibroblasts (GF) and human oral mucosal epithelial cells (EC). GF and EC were stimulated with LPS (2 μg/ml) alone or in combination with i) PDRN (100 μg/ml); ii) PDRN plus ZM241385 (1 μM) as an A2AR antagonist; iii) CGS21680 (1 μM) as an A2AR agonist. LPS boosted NF-κB, TNF-α and IL-6 expression, decreased IL-10 levels and downregulated both Wnt/β-catenin, VEGF and EGF expression. PDRN reverted the LPS-induced phenotype as well as CGS21680. Co-incubation with ZM241385 abolished PDRN effects, thus confirming A2A receptor involvement in PDRN mechanism of action. These results suggest that PDRN efficacy may be due to a “dual mode” of action: NF-κB inhibition and Wnt/β-catenin signaling activation. However, these interesting findings need to be confirmed by animal and clinical studies.

Alzheimer’s disease (AD) is associated with amyloid plaques (Aβ) and hyperphosphorylated tau protein tangles in the brain. We investigated the possible neuroprotective role of flavocoxid, a dual inhibitor of cyclooxygenases-1/2 (COX-1/2) and 5-Lipoxygenase (5-LOX), in triple-transgenic (3xTg-AD) mice. Mice were 3 months at the beginning of the study. Animals received once daily for 3-month saline solution or flavocoxid (20 mg/kg/ip). Morris water maze was used to assess learning and memory. Histology was performed to evidence Aβ plaques and neuronal loss, while inflammatory proteins were determined by western blot analysis. Saline-treated 3xTg-AD mice showed an impairment in spatial learning and memory (assessed at 6 months of age), and increased expression of inflammatory and apoptotic molecules. Treatment of 3xTg-AD mice with flavocoxid reduced: (1) learning and memory loss; (2) the increased eicosanoid production and the phosphorylation level of amyloid precursor protein (APP-pThr668), Aβ 1–42, p-tau (pThr181), pERK, and the activation of the NLRP3 inflammasome; (3) Aβ plaques; and (4) neuronal loss, compared to saline-treated animals. Pharmacological blockade of both COX-1/2 and 5-LOX was able to counteract the progression of AD by targeting pathophysiological mechanisms up- and downstream of Aβ and tau.

&beta, caryophyllene (BCP) is a cannabinoid receptor 2 (CB2) agonist that tempers inflammation. An interaction between the CB2 receptor and peroxisome proliferator-activated receptor gamma (PPAR-&gamma, ) has been suggested and PPAR-&gamma, activation exerts anti-arthritic effects. The aim of this study was to characterize the therapeutic activity of BCP and to investigate PPAR-&gamma, involvement in a collagen antibody induced arthritis (CAIA) experimental model. CAIA was induced through intraperitoneal injection of a monoclonal antibody cocktail and lipopolysaccharide (LPS, 50 &mu, g/100 &mu, L/ip). CAIA animals were then randomized to orally receive either BCP (10 mg/kg/100 &mu, L) or its vehicle (100 &mu, L of corn oil). BCP significantly hampered the severity of the disease, reduced relevant pro-inflammatory cytokines, and increased the anti-inflammatory cytokine IL-13. BCP also decreased joint expression of matrix metalloproteinases 3 and 9. Arthritic joints showed increased COX2 and NF-ĸB mRNA expression and reduced expression of the PPAR&gamma, coactivator-1 alpha, PGC-1&alpha, and PPAR-&gamma, These conditions were reverted following BCP treatment. Finally, BCP reduced NF-ĸB activation and increased PGC-1&alpha, expression in human articular chondrocytes stimulated with LPS. These effects were reverted by AM630, a CB2 receptor antagonist. These results suggest that BCP ameliorates arthritis through a cross-talk between CB2 and PPAR-&gamma

Background: Inflammation is a key hallmark of psoriasis and pro-inflammatory cytokines are produced by the transcription factor Nuclear factor-κB (NF-κB). Canonical Wnt/β-catenin pathway blunts the immune-mediated inflammatory cascade in psoriasis. Adenosine A2A receptor activation either blocks NF-κB and boosts the Wnt/β-catenin signalling. PDRN (Polydeoxyribonucleotide) is a biologic agonist of the A2A receptor and its effects were studied in an experimental model of psoriasis. Methods: Psoriasis-like lesions were induced by a topical daily application of imiquimod cream (IMQ; 62.5 mg/day for 7 days) on the shaved back skin of mice. Animals were randomly assigned to the following groups: Sham psoriasis challenged with vaseline cream; IMQ animals challenged with imiquimod and IMQ animals challenged with IMQ and treated with PDRN (8 mg/kg/ip). In histological experiments an additional arm of IMQ animals was treated with PDRN plus istradefylline (KW6002; 25 mg/kg/ip) as an A2A antagonist. Findings: PDRN administration reduced squamous lesions, erythema, epidermal thickness and acanthosis, trying to restore a normal skin architecture. Istradefylline administration abrogated PDRN positive effects, thus pointing out the mechanistic role of the A2A receptor. PDRN decreased pNF-κB and TNF-α IL-6 and IL-12 expression compared to untreated psoriatic animals. Furthermore, A2Areceptor activation prompted Wnt signalling, reduced IL-2 expression and increased IL-10 mRNA expression in psoriatic skin. Finally, PDRN reduced CD3+ cells in superficial psoriatic dermis. Interpretations: PDRN anti-psoriasis potential may be linked to a "dual mode" of action: NF-κB pathway inhibition and Wnt/β-catenin signalling stimulation. Funding: The work has been performed with Departmental funding assigned to FS. Competing Interest Declaration: The authors declare that the research was conducted in the absence of any commercial or financial conflict of interest. Ethics: The procedures were evaluated and approved by the Ethics Committee of the University of Messina (OPBA, #820/2016).

Obesity is a worldwide growing problem for the health care systems and its treatment is strongly recommended. Orlistat, naltrexone/bupropion, and liraglutide are approved for weight loss in Italy in patients with a Body Mass Index (BMI) ≥ 30 kg/m2 or ≥ 27 kg/m2 with concomitant diseases. However, the prescription of these drugs is significantly low worldwide. General practitioners (GPs) play a key role in the early diagnosis and appropriate management of obesity. The aim of the study was to investigate the management of obesity and the prescriptive attitude of anti-obesity drugs in a general practice setting.All patients registered in lists of 8 GPs with a recorded diagnosis of obesity or BMI values ≥ 30 kg/m2 in the period 2017–2018, were recruited. A descriptive analysis of demographic and clinical characteristic was carried out. The Spearman’s correlation rank test was applied to identify correlations between BMI and all the variables of interest.Among 1301 obese patients, only 66.1 % had been diagnosed and 29.4 % had no registered BMI value. Patients with recorded BMI, were overweight (7.8 %) or in the obesity class I (38.8 %), class II (14.1 %), and class III (7.1 %), respectively.The obese patients (class 1–3) were older [66 (55–76) vs 49 (32–59); p

Spinal cord injury (SCI) is a complex clinical and progressive condition characterized by neuronal loss, axonal destruction and demyelination. In the last few years, adenosine receptors have been studied as a target for many diseases, including neurodegenerative conditions. The aim of this study was to investigate the effects of an adenosine receptor agonist, PDRN, in an experimental model of SCI. Moreover, since adenosine receptors stimulation may also activate the Wnt pathway, we wanted to study PDRN effects on Wnt signaling following SCI. Spinal trauma was induced by extradural compression of spinal cord at T5-T8 level in C57BL6/J mice. Animals were randomly assigned to the following groups: Sham (n = 10), SCI (n = 14), SCI+PDRN (8 mg/kg/i.p.; n = 14), SCI+PDRN+DMPX (8 and 10 mg/kg/i.p., respectively; n = 14). DMPX was used as an adenosine receptor antagonist to evaluate whether adenosine receptor block might prevent PDRN effects. PDRN systemically administered 1 h following SCI, protected from tissue damage, demyelination, and reduced motor deficits evaluated after 10 days. PDRN also reduced the release of the pro-inflammatory cytokines TNF-α and IL-1β, reduced BAX expression and preserved Bcl-2. Furthermore, PDRN stimulated Wnt/β-catenin pathway and decreased apoptotic process 24 h following SCI, whereas DMPX administration prevented PDRN effects on Wnt/β-catenin signaling. These results confirm PDRN anti-inflammatory activity and demonstrate that a crosstalk between Wnt/β-catenin signaling is possible by adenosine receptors activation. Moreover, these data let us hypothesize that PDRN might promote neural repair through axonal regeneration and/or neurogenesis.

Objective and design: The late stages of carotid atherosclerosis are responsible for increased local stiffness, suggesting the need for a therapeutic target that affect either plaque composition and arterial stiffness. There is a lack of data on the role of the local arterial stiffness, assessed by radio-frequency based system and its relationship with the molecular profile of plaques. Subjects: In this study we enrolled 18 consecutive patients undergoing carotid endoarterectomy, with homogeneus or heterogeneous plaques, as established by Doppler-ultrasound and local pulse-wave velocity was assessed before surgery. Methods: In carotid plaque specimens, we evaluated inflammasome (NLRP3), Receptor Activator of Nuclear Factor κB (RANK) and its natural ligand (RANK-L), Osteoprotegerin (OPG), and other inflammatory and apoptotic molecules by Western Blotting and qPCR analysis. In addition, lipid peroxidation of arterial specimens was assessed by TBARS assay. Results: In heterogeneus plaques we observed increased OPG expression (p=0.04), positively correlated with lipid peroxidation values (r=0.511, p=0.03); increased levels of RANK (p=0.02), and other inflammatory and apoptotic molecules. RANK-L protein was augmented in homogeneus plaques (p=0.01) and correlated to s-index (r=0.514, p=0.03) and PWV (r=0.525, p=0.03) values. Conclusions: Our data provide evidence that increased local PWV and s-index might identify plaque evolution towards calcification.

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.

Dystrophin lack in DMD causes neuronal nitric oxide synthase (nNOS) membrane delocalization which in turn promotes functional muscle ischemia, and exacerbates muscle injury. Apoptosis and the exhaustion of muscle regenerative capacity are implicated in Duchenne muscular dystrophy (DMD) pathogenesis and therefore are relevant therapeutic targets. Genistein has been reported to have pro-proliferative effects, promoting G1/S cell phase transition through the induction of cyclin D1, and anti-apoptotic properties. We previously showed that genistein could reduce muscle necrosis and enhance regeneration with an augmented number of myogenin-positive satellite cells and myonuclei, ameliorating muscle function in mdx mice. In this study we evaluated the underlying mechanisms of genistein effect on muscle specimens of mdx and wild type mice treated for five weeks with genistein (2 mg/kg/i.p. daily) or vehicle. Western blot analysis show that genistein increased cyclin D1 and nNOS expression; and showed an antiapoptotic effect, modulating the expression of BAX and Bcl-2. Our results suggest that this isoflavone might enhance the regenerative spurt in mdx mice muscle restoring nNOS, promoting G1/S phase transition in muscle cell, and inhibiting apoptosis. Further studies with longer time treatment or using different experimental approaches are needed to better investigate the underlying mechanisms of such results.

Laparoscopy (LS) has been shown to decrease the inflammatory sequelae of endotoxemia.β-arrestin 2 plays an important function in signal transduction pathway of TLR4. High mobility group box-1 (HMGB-1) is involved in the delayed systemic inflammatory response. We investigated the effects of CO2insufflation on liver, lung, and kidney expression of bothβ-arrestin 2 and HMGB-1 during sepsis. Cecal ligation and puncture (CLP) was performed in male rats and 6 h later the animals were randomly assigned to receive a CO2pneumoperitoneum or laparotomy. Animals were euthanized; liver, lung, and kidney were removed for the evaluation ofβ-arrestin 2 and HMGB-1 expression. Immunohistochemical detection of myeloperoxidase (MPO) was investigated in lung and liver and bacterial load was determined in the peritoneal fluid. CO2pneumoperitoneum reduced peritoneal bacterial load, increased the expression ofβ-arrestin 2, and blunted the expression of the potent proinflammatory HMGB-1 in liver, lung, and kidney compared with laparotomy. Liver and lung MPO was markedly reduced in rats subjected to LS compared with laparotomy. We believe that CO2exerts an early protective effect by reducing bacterial load and likely toll-like receptor activation which in turn leads to a preservedβ-arrestin 2 expression and a reduced HMGB-1 expression.

Nitric oxide (NO) is involved in several biological processes, but its role in human melanogenesis is still not well understood. Exposure to UVA and UVB induces nitric oxide production in keratinocytes and melanocytes through the activation of constitutive nitric oxide synthase, increasing tyrosinase activity and melanin synthesis, whereas inducible nitric oxide synthase over expression might be involved in hypopigmentary disorders. The aim of this study was to evaluate whether inducible nitric oxide synthase and neuronal nitric oxide synthase expression were modified in vitiligo skin compared to healthy controls. Skin biopsies were obtained from inflammatory/lesional and white/lesional skin in 12 patients with active, non-segmental vitiligo; site-matched biopsies of normal skin from eight patients were used as controls. Nitric oxide synthase isoforms expression was evaluated by confocal laser scanning microscopy and Western Blot analysis. Inducible nitric oxide synthase expression was significantly increased in inflammatory/lesional skin compared to healthy skin; melanocytes showed a moderate neuronal nitric oxide synthase expression in white/lesional skin, demonstrating that metabolic function still goes on. The obtained data demonstrated that vitiligo lesions were characterized by modifications of nitric oxide synthase isoforms, thus confirming the hypothesis that nitric oxide imbalance is involved in vitiligo and supporting the idea that nitric oxide synthase inhibitors might be used as a possible therapeutic approach for the management of vitiligo.

Glucocorticoid-induced osteoporosis (GIO) is a secondary cause of bone loss. Bisphosphonates approved for GIO, might induce jaw osteonecrosis; thus additional therapeutics are required. Adenosine receptor agonists are positive regulators of bone remodeling, thus the efficacy of adenosine receptor stimulation for treating GIO was tested. In a preventive study GIO was induced in Sprague-Dawley rats by methylprednisolone (MP) for 60 days. Animals were randomly assigned to receive polydeoxyribonucleotide (PDRN), an adenosine A2 receptor agonist, or PDRN and DMPX (3,7-dimethyl-1-propargylxanthine, an A2 antagonist), or vehicle (0.9% NaCl). Another set of animals was used for a treatment study, following the 60 days of MP-induction rats were randomized to receive (for additional 60 days) PDRN, or PDRN and DMPX (an adenosine A2 receptor antagonist), or zoledronate (as control for gold standard treatment), or vehicle. Control animals were administered with vehicle for either 60 or 120 days. Femurs were analyzed after treatments for histology, imaging, and breaking strength analysis. MP treatment induced severe bone loss, the concomitant use of PDRN prevented the developing of osteoporosis. In rats treated for 120 days, PDRN restored bone architecture and bone strength; increased b-ALP, osteocalcin, osteoprotegerin and stimulated the Wnt canonical and non-canonical pathway. Zoledronate reduced bone resorption and ameliorated the histological features, without significant effects on bone formation. Our results suggest that adenosine receptor stimulation might be useful for preventing and treating GIO.

Treatment for traumatic brain injury (TBI) remains elusive despite compelling evidence from animal models for a variety of therapeutic targets. The activation of the NLRP3 (Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome has been proposed as key point in the brain damage associated with TBI. NLRP3 was tested as potential target for reducing neuronal loss and promoting functional recovery in a mouse model of TBI. Male NLRP3-/- (n = 20) and wild type (n = 27) mice were used. A closed TBI model was performed and inflammatory and apoptotic markers were evaluated. A group of WT mice also received BAY 11-7082, a NLRP3 inhibitor, to further evaluate the role of this pathway. At 24 h following TBI NLRP3-/- animals demonstrated a preserved cognitive function as compared to WT mice, additionally brain damage was less severe and the inflammatory mediators were reduced in brain lysates. The administration of BAY 11-7082 in WT animals subjected to TBI produced overlapping results. At day 7 histology revealed a more conserved brain structure with reduced damage in TBI NLRP3-/- animals compared to WT. Our data indicate that the NLRP3 pathway might be exploited as molecular target for the short-term sequelae of TBI.

Diabetes is characterized by poor wound healing which currently lacks an efficacious treatment. The innate repair receptor (IRR) is a master regulator of tissue protection and repair which is expressed as a response injury or metabolic stress, including in diabetes. Activation of the IRR might provide benefit for diabetic wound healing. A specific IRR agonist cibinetide was administered in an incisional wound healing model performed mice with genetic diabetes (db+/db+) and compared to the normal wild-type. Animals were treated daily with cibinetide (30μg/kg/s.c.) or vehicle and euthanized 3, 7, and 14days after the injury to quantitate vascular endothelial growth factor (VEGF), malondialdehyde (MAL), phospho-Akt (pAkt), phospho e-NOS (p-eNOS), and nitrite/nitrate content within the wound. Additional evaluations included quantification of skin histological change, angiogenesis, scar strength, and time to complete wound closure. Throughout the wound healing process diabetic animals treated with vehicle exhibited increased wound MAL with reduced VEGF, pAkt, peNOS and nitrite/nitrate, all associated with poor re-epitheliziation, angiogenesis, and wound breaking strength. Cibenitide administration significantly improved these abnormalities. The results suggest that cibinetide-mediated IRR activation may represent an interesting strategy to treat diabetes-associated wound healing.

In women, aging and declining estrogen levels are associated with several cutaneous changes, many of which can be reversed or improved by estrogen supplementation. Two estrogen receptors—α and β—have been cloned and found in various tissue types. Epidermal thinning, declining dermal collagen content, diminished skin moisture, decreased laxity, and impaired wound healing have been reported in postmenopausal women. Experimental and clinical studies in postmenopausal conditions indicate that estrogen deprivation is associated with dryness, atrophy, fine wrinkling, and poor wound healing. The isoflavone genistein binds to estrogen receptor β and has been reported to improve skin changes. This review article will focus on the effects of genistein on skin health.

Genistein has a preventive role against bone mass loss during menopause. However, experimental data in animal models of osteoporosis suggest an anti-osteoporotic potential for this isoflavone. We performed a post-hoc analysis of a previously published trial investigating the effects of genistein in postmenopausal women with low bone mineral density. The parent study was a randomized, double-blind, placebo-controlled trial involving postmenopausal women with a femoral neck (FN) density

During menopause, an increased prevalence of metabolic syndrome (MetS) and central obesity seems to increase hot flashes (HFs). Visfatin is an inflammatory adipokine secreted by visceral fat. We investigated visfatin levels and its relationship with hot flash number and BMI, in postmenopausal women with MetS. We also evaluated the effect of genistein, an isoflavone effective in reducing HFs, on visfatin levels and HFs after 1 year of treatment. This was a randomized, double-blind, placebo-controlled trial. Postmenopausal women with MetS were randomly assigned to receive placebo (n = 60) or 54 mg genistein (n = 60), daily for 1 year. As main outcome measures, hot flashes number and circulating visfatin levels were evaluated. Visfatin significantly correlated with BMI and HFs number in women with MetS at basal. After 6 and 12 months, our results indicate a strong correlation and a significant effect of genistein in reducing both HFs and visfatin in women with MetS. The present study suggests that visfatin plays a role in the vasomotor symptoms, at least in postmenopausal women with metabolic syndrome. Genistein may reduce HFs decreasing the circulating levels of this inflammatory adipokine.

Experimental evidence suggests that cadmium (Cd) boosts oxidative stress that may result in toxicity on the endocrine system also in humans. The aim of this study was to investigate the glycemic control and oxidative stress markers in male adolescents with increased urinary levels of cadmium. We investigated 111 males, aged 12–14 years, living in a polluted area of Sicily and a control age-matched population (n=60) living 28–45 km far from the polluted site. Malondialdehyde (MDA), total antioxidant activity (TAC), metallothionein-1A (MT-1A) gene expression, insulin resistance by the homeostatic model assessment (HOMA-IR), and urinary cadmium were investigated. Cd levels were significantly higher in adolescents living in the polluted area than in control age-matched subjects. Adolescents with elevated Cd levels had a significant increase in MDA, MT-1A, and HOMA-IR and reduced TAC compared to the control group. A robust correlation was found between urinary cadmium and MT-1A, HOMA-IR, and MDA whereas an inverse correlation was identified between urinary cadmium and TAC. This study indicates that cadmium burden alters glycemic control in adolescents and suggests that oxidative stress plays a key role in cadmium-induced insulin resistance, increasing the risk of developing metabolic disorders.

Background and Purpose Type 2 diabetes impairs the healing process because of an exaggerated and persistent inflammatory response, and an altered expression pattern of angiogenic molecules. We investigated the effects of inflammasome blockade in diabetes-related wound-healings defects, in genetically diabetic mice. Experimental Approach An incisional skin wound model was produced on the back of female diabetic C57BL/KsJ-m +/+ Leptdb mice (db+/db+) and their normal littermates (db+/m+). Animals were treated daily with two inflammasome blocking agents, BAY 11-7082 (20 mg·kg−1 i.p.), or Brilliant Blue G (BBG, 45.5 mg·kg−1 i.p.), or vehicle. Mice were killed on 3, 6 and 12 days after skin injury to measure expression of the NOD-like receptor NLRP3, caspase-1, VEGF, the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and the chemokine CXCL12. Wound levels of IL-1β and IL-18 were also measured, along with histological assessments of wound tissue and the time to complete wound closure. Key Results During healing, the diabetic mice exhibited increased activation of NLRP3, caspase-1, ASC, IL-1β and IL-18. They also showed a reduced expression of VEGF and CXCL12.Treatment with BAY 11-7082 or BBG, to block activation of the inflammasome, decreased the levels of pro-inflammatory molecules. Histological evaluation indicated that inflammasome blockade improved the impaired healing pattern, at day 12 in diabetic mice, along with a decreased time to complete skin healing. Conclusions and Implications These data strongly suggest that activation of the NLRP3 inflammasome is one of the key contributors to the delayed healing of wounds in diabetic mice.

Benign prostatic hyperplasia (BPH) is a major health concern that is likely to have an increasing impact in line with the gradual aging of the population. BPH is characterized by smooth muscle and epithelial proliferation primarily within the prostatic transition zone that can cause a variety of problems for patients, the most frequent are the lower urinary tract symptoms. BPH is thought to involve in disruption of dihydrotestosterone (DHT)-supported homeostasis between cell proliferation and cell death, and, as a result, proliferative processes predominate and apoptotic processes are inhibited. Phytotherapeutic supplements, mainly based on Saw Palmetto-derived Serenoa Repens (SeR), are numerous and used frequently. Serenoa Repens reduces inflammation and decreases in vivo the androgenic support to prostatic cell growth. Furthermore, SeR stimulates the apoptotic machinery; however, data supporting efficacy is limited, making treatment recommendations difficult. Besides SeR, selenium (Se), an essential trace element mainly functioning through selenoproteins and able to promote an optimal antioxidant/oxidant balance, and lycopene (Ly), a dietary carotenoid synthesized by plants, fruits, and microorganisms with a strong antioxidant activity, has been shown to exert beneficial effects in prostate disease. SeR is frequently associated with Ly and Se, in order to increase its therapeutic activity in benign prostatic hyperplasia (BPH). It has been shown that the Ly-Se-SeR association has a greater and enhanced antiinflammatory activity that might be of particular interest in the treatment of BPH. The Ly-Se-SeR association is also more effective than SeR alone in reducing prostate weight and hyperplasia, in augmenting the pro-apoptotic Bax and caspase-9 and blunting the anti-apoptotic Bcl-2 mRNA. In addition, Ly-Se-SeR more efficiently suppresses the EGF and Vascular Endothelial Growth Factor (VEGF) expressions in hyperplastic prostates. Therefore, SeR particularly when combined with Se and Ly may have a greater potential for the management of benign prostate hyperplasia.

BAY 11-7082 antagonizes I-κB kinase-β preventing nuclear translocation of nuclear factor-κB (NF-κB); it also inhibits NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation. NF-κB is involved in psoriasis, whereas the role of NLRP3 is controversial. We investigated BAY 11-7082 effects in an experimental model of psoriasis-like dermatitis. Psoriasis-like lesions were induced by a topical application of imiquimod (IMQ) cream (62.5 mg/day) on the shaved back skin of C57BL/6 and NLRP3 knockout (KO) mice for 7 consecutive days. Sham psoriasis animals were challenged with Vaseline cream. Sham and IMQ animals were randomized to receive BAY 11-7082 (20 mg/kg/i.p.) or its vehicle (100 μl/i.p of 0.9% NaCl). Skin of IMQ animals developed erythema, scales, thickening and epidermal acanthosis. IMQ skin samples showed increased expression of pNF-κB and NLRP3 activation. BAY 11-7082 blunted epidermal thickness, acanthosis and inflammatory infiltrate. BAY 11-7082 reduced pNF-κB, NLRP3, tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β expression, blunted the phosphorylation of signal transducer and activators of transcription 3 (STAT3) and decreased IL-23 levels. In addition, BAY 11-7082 reawakened the apoptotic machinery. NLRP3 KO animals showed a reduced total histological score but persistent mild acanthosis, dermal thickness and expression of pNF-κB and pSTAT3, following IMQ application. Our data suggest that BAY 11-7082 might represent an interesting approach for the management of psoriasis-like dermatitis depending on the dual inhibition of NF-κB and NLRP3.

We reported a case of an 11-year-old girl admitted to our hospital for goiter, tachycardia, sweating, and visible and palpable thyroid. Thyroid function tests revealed a low thyrotropin level (

Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) play an important role in inflammatory bowel diseases (IBDs). We investigated the effects of flavocoxid, a dual COX/LOX inhibitor, in experimental colitis induced with either dinitrobenzenesulfonic acid (DNBS) or dextrane sulphate sodium (DSS) In the first model, colitis was induced in rats by a single intra-colonic instillation (25mg in 0.8ml 50% ethanol) of DNBS; after 24h animals were randomized to receive orally twice a day, flavocoxid (10mg/kg), zileuton (50mg/kg), or celecoxib (5mg/kg). Sham animals received 0.8ml of saline by a single intra-colonic instillation. Rats were killed 4 days after induction and samples were collected for analysis. In the second model, colitis was induced in rats by the administration of 8% DSS dissolved in drinking water; after 24h animals were randomized to the same above reported treatments. Sham animals received standard drinking water. Rats were killed 5 days after induction and samples were collected for analysis. Flavocoxid, zileuton and celecoxib improved weight loss, reduced colonic myeloperoxydase activity, macroscopic and microscopic damage, and TNF-α serum levels. Flavocoxid and celecoxib also reduced malondialdheyde, 6-keto PGF1α and PGE-2 levels while flavocoxid and zileuton decreased LTB-4 levels. In addition, flavocoxid treatment improved histological features and apoptosis as compared to zileuton and celecoxib; moreover only flavocoxid reduced TXB2, thus avoiding an imbalance in eicosanoids production. Our results show that flavocoxid has protective effect in IBDs and may represents a future safe treatment for inflammatory bowel diseases.

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.

Activation of the adenosine receptor pathway has been demonstrated to be effective in improving tissue remodeling and blunting the inflammatory response. Active colitis is characterized by an intense inflammatory reaction resulting in extensive tissue damage. Symptomatic improvement requires both control of the inflammatory process and repair and remodeling of damaged tissues. We investigated the ability of an A2A receptor agonist, polydeoxyribonucleotide (PDRN), to restore tissue structural integrity in two experimental colitis models using male Sprague-Dawley rats. In the first model, colitis was induced with a single intra-colonic instillation of dinitrobenzenesulfonic acid (DNBS), 25 mg diluted in 0.8 ml 50% ethanol. After 6 h, animals were randomized to receive either PDRN (8 mg/kg/i.p.), or PDRN + the A2A antagonist [3,7-dimethyl-1-propargylxanthine (DMPX); 10 mg/kg/i.p.], or vehicle (0.8 ml saline solution) daily. In the second model, dextran sulfate sodium (DSS) was dissolved in drinking water at a concentration of 8%. Control animals received standard drinking water. After 24 h animals were randomized to receive PDRN or PDRN+DMPX as described above. Rats were sacrificed 7 days after receiving DNBS or 5 days after DSS. In both experimental models of colitis, PDRN ameliorated the clinical symptoms and weight loss associated with disease as well as promoted the histological repair of damaged tissues. Moreover, PDRN reduced expression of inflammatory cytokines, myeloperoxidase activity, and malondialdehyde. All these effects were abolished by the concomitant administration of the A2A antagonist DMPX. Our study suggests that PDRN may represent a promising treatment for improving tissue repair during inflammatory bowel diseases.

Aims Systemic sclerosis (SSc) is characterized by vasculopathy and organ fibrosis. Although microvascular alterations are very well characterized, structural and functional abnormalities of large vessels are not well defined. Therefore, we evaluated the effect of simvastatin administration on aortic and small renal arteries thickening, and on myofibroblasts differentiation in a murine model of SSc. Methods and results SSc was induced in BALB/c mice by daily subcutaneous injections of hypochlorous acid (HOCl, 100 μl) for 6 weeks. Mice (n = 23) were randomized to receive: HOCl (n = 10); HOCl plus simvastatin (40 mg/kg; n = 8); or vehicle (n = 5). Simvastatin administration started 30 min after HOCl injection, and up to week 6. Aortic and small renal arteries intima-media thickness was evaluated by histological analysis. Immunostaining for α-smooth muscle actin (SMA), vascular endothelial growth factor receptor 2 (VEGFR2), and CD31 in aortic tissues was performed to evaluate myofibroblast differentiation and endothelial markers.In HOCl-treated mice, intima-media thickening with reduced lumen diameter was observed in the aorta and in small renal arteries and simvastatin administration prevented this increase. Aortic and renal myofibroblasts count, as expressed by α-SMA + density, was lower in the group of mice treated with simvastatin compared to HOCl-treated mice. Simvastatin prevented the reduction in VEGFR2 and CD31 expression induced by HOCl. Conclusions The administration of simvastatin regulates collagen deposition in the aortic tissues and in the small renal arteries by modulating myofibroblasts differentiation and vascular markers. Further studies are needed to better address the effect of statins in the macrovascular component of SSc.

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.

AIM Adenosine receptors modulate inflammation in periodontal tissues. No data are available regarding the effects of adenosine A(2A) receptor stimulation in experimental periodontitis (EPD). The aim of this study was to investigate the effects of polynucleotides (also known as polydeoxyribonucleotide, PDRN), a ligand of A(2A) receptor, in EPD in rats. MATERIALS AND METHODS EPD was induced ligating the cervix of the lower left first molar. Sham-EPD had no ligature. After 7 days, EPD animals were randomized to a daily treatment with vehicle gel or 0.75% PDRN gel or PDRN gel with a specific A(2A) antagonist (DMPX). Treatments lasted 7 days. Animals were then euthanized and the periodontium and surrounding gingival tissue were excised for histological evaluation and bio-molecular analysis of inflammatory (p-JNK, p-ERK, TNF-α, IL-6, HMGB-1) and apoptotic proteins (BAX and Bcl-2). RESULTS Vehicle-treated EPD rats showed severe inflammatory infiltrate in both gingival and periodontal ligament, as well as an enhanced expression of p-JNK, p-ERK, TNF-α, IL-6, HMGB-1 and BAX and a reduction in Bcl-2. PDRN gel restored the histological features, blunted inflammatory and apoptotic proteins expression and preserved Bcl-2 expression. DMPX abrogated PDRN positive effects. CONCLUSION Our data suggest that adenosine receptor stimulation by PDRN might represent a new therapeutic strategy for periodontitis.

Background and purpose 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. Experimental approach Rats were treated daily with testosterone propionate (3 mg·kg(-1) s.c.) or its vehicle for 14 days to induce BPH. Animals receiving testosterone were randomized to receive vehicle (1 mL·kg(-1) , i.p.) or flavocoxid (20 mg·kg(-1) , i.p.) for 14 days. Histological changes, eicosanoid content and mRNA and protein levels for apoptosis-related proteins and growth factors were assayed in prostate tissue. The effects of flavocoxid were also tested on human prostate carcinoma PC3 cells. Key results Flavocoxid reduced prostate weight and hyperplasia, blunted inducible expression of COX-2 and 5-LOX as well as the increased production of PGE(2) and leukotriene B(4) (LTB(4) ), enhanced pro-apoptotic Bax and caspase-9 and decreased the anti-apoptotic Bcl-2 mRNA. Flavocoxid also reduced EGF and VEGF expression. In PC3 cells, flavocoxid stimulated apoptosis and inhibited growth factor expression. Flavocoxid-mediated induction of apoptosis was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, in PC3 cells, suggesting an essential role of caspases in flavocoxid-mediated apoptosis during prostatic growth. Conclusion and implications Our results show that a 'dual inhibitor' of the COX and 5-LOX enzymes, such as flavocoxid, might represent a rational approach to reduce BPH through modulation of eicosanoid production and a caspase-induced apoptotic mechanism.

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.Randomized experiment.Research laboratory at a university hospital.Male Sprague-Dawley rats (n = 215).Experimental rat model of diffuse traumatic brain injury, the impact-acceleration model.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-α, 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 [Nle, D-Phe]-α-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-α, 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 [Nle, D-Phe]-α-melanocyte-stimulating hormone.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.

Ischemia is a major factor contributing to failure of skin flap surgery, which is routinely used for coverage of wounds to prevent infection and to restore form and function. An emerging concept is that adenosine A(2A) receptors can improve tissue oxygenation by stimulating angiogenesis, likely through vascular endothelial growth factor (VEGF). This study assessed the ability of polydeoxyribonucleotide (PDRN) to restore blood flow and improve wound healing, acting through the A(2A) receptor, in a rat model of ischemic skin flaps.The H-shaped double-flap model was used in male Sprague-Dawley rats. After surgical procedures, the animals were randomized to receive intraperitoneal PDRN (8 mg/kg) or vehicle (NaCl 0.9%). Rats were euthanized 3, 5, and 10 days after skin injury, after the evaluation of skin perfusion by laser Doppler. The wounds underwent histologic analysis and were measured for VEGF messenger RNA and protein expression, hypoxia inducible factor-1-α (HIF-1α), and inducible nitric oxide synthase (iNOS) protein expression, and nitrite content.Blood flow markedly increased in blood flow in ischemic flaps treated with PDRN, with a complete recovery starting from day 5 (ischemic flap + vehicle, 1.80 ± 0.25; ischemic flap + PDRN, 2.46 ± 0.25; P.001). Administration of PDRN enhanced the expression of VEGF (ischemic flap + vehicle, 5.3 ± 0.6; ischemic flap + PDRN, 6.2 ± 0.5; P.01) at day 5, and iNOS (ischemic flap + vehicle, 3.9 ± 0.6; ischemic flap + PDRN, 5.3 ± 1; P.01), but reduced HIF-1α expression (ischemic flap + vehicle, 7 ± 1.1; ischemic flap + PDRN, 4.8 ± 0.5; P.05) at day 3. Histologically, the PDRN-treated group showed complete re-epithelialization and well-formed granulation tissue rich in fibroblasts.These results suggest that PDRN restores blood flow and tissue architecture, probably by modulating HIF-1α and VEGF expression, and may be an effective therapeutic approach in improving healing of ischemic skin flaps.

BACKGROUND AND PURPOSE Ovariectomy accelerates age-related skin changes as adequate oestrogen levels are required to control structural integrity and functional capacity of skin. Genistein, a soy-derived isoflavone, has been tested in anti-ageing cosmetic preparations with interesting results on skin elasticity, photoaging and skin cancer prevention. We investigated the effects of genistein aglycone and compared them with systemic raloxifene hydrochloride and 17-α-ethinyloestradiol on skin changes in aged, ovariectomized (OVX) rats. EXPERIMENTAL APPROACH Six months after ovariectomy, rats were randomly allocated to different groups and treated, daily, with genistein aglycone (1 and 10 mg·kg−1 s.c.), raloxifene hydrochloride (0.05 and 0.5 mg·kg−1 s.c.) or 17-α-ethinyloestradiol (0.003 and 0.03 mg·kg−1 s.c.) for 12 weeks. Controls were untreated OVX and sham OVX rats. At the end of the treatment period, a skin biopsy was carried out and skin samples were assessed for molecular, histological and functional changes. KEY RESULTS Skin samples of untreated OVX rats showed a decrease in TGF-β1, VEGF, MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 compared with sham OVX rats. All the treatments significantly restored this depressed molecular profile revealed in OVX rats. Genistein aglycone, 1 mg·kg−1, also significantly increased the thickness of collagen and breaking strength of skin in the OVX rats. CONCLUSIONS AND IMPLICATIONS Relatively long-term, systemic treatment with genistein aglycone shows comparable efficacy to oestrogen in reversing some molecular, histological and functional changes of the skin associated with ovariectomy in aged rats. This suggests that genistein aglycone might be an effective alternative therapy for the management of age-related skin changes in postmenopausal women.

Objective To study the effect of PDRN on angiogenesis in a model of varicocele in rats. Design After the creation of experimental varicocele, rats were randomized to one of the four treatments: vehicle, PDRN, DMPX, and PDRN plus DMPX. Twenty-one days after randomization, all animals were euthanized and the left testis was harvested. Setting Academic hospital. Animal(s) Male Sprague-Dawley rats were used. Intervention(s) A clamp was passed behind the left renal vein distally to the spermatic vein insertion. A silk ligature was placed around the left renal vein at this site and was tied over the top of a probe. The latter was then withdrawn and the vein was allowed to expand. In shams, a suture was placed but it was not tied. Main Outcome Measure(s) To assess testicular microvascular density using CD34 immunostaining. Result(s) Microvascular density in the varicocele plus PDRN group was significantly higher than in other groups. Conclusion(s) PDRN could represent a novel therapeutic strategy for varicocele treatment in subfertile patients, improving the innate pathophysiologic mechanism of neoangiogenesis, through compensatory oxygen and metabolite supply to tubular and extratubular testicular compartments.

Multiple factors may contribute to the pathogenesis of postmenopausal osteoporosis including environmental, life-style and genetic factors. Common variants in ESR2 gene encoding for ER-beta, highly expressed in bone tissue, have recently been proposed as candidates for affecting bone phenotype at the population level, particularly in postmenopausal women. In this study, we examined the genetic background at ESR2 AluI (rs4986938, 1730G>A) locus in 89 osteopenic, postmenopausal women (age range 49–56 years) together with BMD at lumbar spine and femoral neck sites as well as variations in plasma levels of bone metabolism and turnover markers. Genotyping for ESR2 G1730A polymorphism showed that the frequency of A mutated allele accounted for 0.4 in our cohort of postmenopausal women; moreover, the GA1730 heterozygous individuals were the most represented (50.6%) compared with GG (37.8%) and AA homozygous ones (14.6%). A regression analysis showed that lumbar spine BMD values were significantly associated with both ESR2 AA1730 genotype (p = 0.044) and time since the onset of menopause (p = 0.031), while no significant association was detected between biochemical markers and genetic background. Interestingly, 85% of patients with AA1730 genotype presented the smallest lumbar spine BMD values. These findings first indicate a worsening effect of ESR2 AluI polymorphism on lumbar spine BMD reduction in postmenopause, suggesting that the detection of this ESR2 variant should be recommended in postmenopausal women, particularly in populations with a high prevalence of ESR2 AA1730 homozygous genotype.

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 Freund's 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.

Serenoa repens is frequently combined with other natural compounds, such as the carotenoid lycopene and the essential trace element Se, to increase its therapeutic activity in benign prostatic hyperplasia. We noted that the lycopene-Se-Serenoa repens combination has greater, enhanced anti-inflammatory activity, which might be of particular interest for benign prostatic hyperplasia treatment. Testosterone administration in rats is a suitable tool for investigating hormone dependent benign prostatic hyperplasia. We performed a comparison experiment between Serenoa repens and the lycopene-Se-Serenoa repens combination on prostate growth induced in rats by testosterone administration.Rats were treated daily with testosterone propionate (3 mg/kg subcutaneously) or its vehicle for 14 days. Testosterone administered animals were randomized to receive vehicle, Serenoa repens (25 mg/kg subcutaneously) or the combination of lycopene (3 mg/kg subcutaneously), Se (3 mg/kg subcutaneously) and Serenoa repens for 14 days. The rats were sacrificed and the prostate was removed for analysis.Lycopene-Se-Serenoa repens was more effective than Serenoa repens alone for decreasing prostate weight and hyperplasia, augmenting pro-apoptotic Bax and caspase-9, and blunting anti-apoptotic Bcl-2 mRNA. Lycopene-Se-Serenoa repens also markedly decreased epidermal growth factor and vascular endothelial growth factor expression.The data indicate that the lycopene-Se-Serenoa repens combination is superior to Serenoa repens alone for decreasing hormone dependent prostatic growth.

Summary The effects of polydeoxyribonucleotide (PDRN), an agonist of the A2A adenosine receptors which when activated positively influences sperm activity, were tested in an experimental testicular ischaemia/reperfusion injury model. Anaesthetized male Sprague-Dawley rats were subjected to testicular torsion-induced ischaemia, followed by reperfusion (TI/R). Immediately after detorsion, randomized animals, including SHAM, received intraperitoneal injections of: (i) vehicle (1 mL/kg 0.9% NaCl solution); (ii) PDRN (8 mg/kg); (iii) DMPX (3,7-dimethyl-1-propargilxanthine, 0.1 mg/kg); or (iv) PDRN (8 mg/kg) + DMPX (0.1 mg/kg). Animals were euthanized at 1, 7 and 30 days following reperfusion. Vascular endothelial growth factor (VEGF) expression is normally associated with adenosine A2A receptor stimulation. After treatment, VEGF mRNA/protein expression quantified by qPCR and Western blot, vascular endothelial growth factor receptor-1 (VEGFR1) and endothelial nitric oxide synthase (eNOS) mRNA measured by qPCR, VEGF and VEGFR1 assessed using immunohistochemical methods, histological staining and spermatogenic activity were all analysed. Testis ischaemia-reperfusion (TI/R) injury caused increases in VEGF mRNA and protein, VEGFR1 and eNOS mRNA, histological damage and reduced spermatogenic activity. Immunostaining showed a lower expression of VEGF in germinal epithelial cells and a strong expression of VEGFR1 in Leydig cells after TI/R. PDRN administration increased significantly VEGF message/protein, VEGFR1 and eNOS message, decreased histological damage and ameliorated spermatogenic activity. PDRN might be useful in the management of testicular torsion.

BACKGROUND AND PURPOSE Melanocortins reverse circulatory shock and improve survival by counteracting the systemic inflammatory response, and through the activation of the vagus nerve-mediated cholinergic anti-inflammatory pathway. To gain insight into the potential therapeutic value of melanocortins against multiple organ damage following systemic inflammatory response, here we investigated the effects of the melanocortin analogue [Nle4, D-Phe7]α-MSH (NDP-α-MSH) in a widely used murine model of multiple organ dysfunction syndrome (MODS). EXPERIMENTAL APPROACH MODS was induced in mice by a single intraperitoneal injection of lipopolysaccharide followed, 6 days later (= day 0), by zymosan. After MODS or sham MODS induction, animals were randomized to receive intraperitoneally NDP-α-MSH (340 µg·kg−1 day) or saline for up to 16 days. Additional groups of MODS mice were concomitantly treated with the melanocortin MC4 receptor antagonist HS024, or the nicotinic acetylcholine receptor antagonist chlorisondamine, and NDP-α-MSH. KEY RESULTS At day 7, in the liver and lung NDP-α-MSH, significantly reduced mRNA expression of tumour necrosis factor-α (TNF-α), increased mRNA expression of interleukin-10 and improved the histological picture, as well as reduced TNF-α plasma levels; furthermore, NDP-α-MSH dose-dependently increased survival rate, as assessed throughout the 16 day observation period. HS024 and chlorisondamine prevented all the beneficial effects of NDP-α-MSH in MODS mice. CONCLUSIONS AND IMPLICATIONS These data indicate that NDP-α-MSH protects against experimental MODS by counteracting the systemic inflammatory response, probably through brain MC4 receptor-triggered activation of the cholinergic anti-inflammatory pathway. These findings reveal previously undescribed effects of melanocortins and could have clinical relevance in the MODS setting.