Corrigendum: Adenosine receptor stimulation improves glucocorticoid-induced osteoporosis in a rat model.

[This corrects the article DOI: 10.3389/fphar.2017.00558.].

Corrigendum: Adenosine receptor stimulation by polydeoxyribonucleotide improves tissue repair and symptomology in experimental colitis.

[This corrects the article DOI: 10.3389/fphar.2016.00273.].

Effects of the antagomiRs 15b and 200b on the altered healing pattern of diabetic mice.

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.

Adenosine Receptor Stimulation Improves Glucocorticoid-Induced Osteoporosis in a Rat Model.

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.

Ammonium tetrathiomolybdate following ischemia/reperfusion injury: Chemistry, pharmacology, and impact of a new class of sulfide donor in preclinical injury models.

BACKGROUND: Early revascularization of ischemic organs is key to improving outcomes, yet consequent reperfusion injury may be harmful. Reperfusion injury is largely attributed to excess mitochondrial production of reactive oxygen species (ROS). Sulfide inhibits mitochondria and reduces ROS production. Ammonium tetrathiomolybdate (ATTM), a copper chelator, releases sulfide in a controlled and novel manner, and may offer potential therapeutic utility. METHODS AND FINDINGS: In vitro, ATTM releases sulfide in a time-, pH-, temperature-, and thiol-dependent manner. Controlled sulfide release from ATTM reduces metabolism (measured as oxygen consumption) both in vivo in awake rats and ex vivo in skeletal muscle tissue, with a superior safety profile compared to standard sulfide generators. Given intravenously at reperfusion/resuscitation to rats, ATTM significantly reduced infarct size following either myocardial or cerebral ischemia, and conferred survival benefit following severe hemorrhage. Mechanistic studies (in vitro anoxia/reoxygenation) demonstrated a mitochondrial site of action (decreased MitoSOX fluorescence), where the majority of damaging ROS is produced. CONCLUSIONS: The inorganic thiometallate ATTM represents a new class of sulfide-releasing drugs. Our findings provide impetus for further investigation of this compound as a novel adjunct therapy for reperfusion injury.

Adenosine Receptor Stimulation by Polydeoxyribonucleotide Improves Tissue Repair and Symptomology in Experimental Colitis.

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.

Use of a balanced dual cyclooxygenase-1/2 and 5-lypoxygenase inhibitor in experimental colitis.

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.

Flavocoxid Protects Against Cadmium-Induced Disruption of the Blood­Testis Barrier and Improves Testicular Damage and Germ Cell Impairment in Mice [corrected].

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) -ß3, 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-ß3, 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.

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.

A dual inhibitor of cyclooxygenase and 5-lipoxygenase protects against kainic acid-induced brain injury.

Systemic administration of kainic acid causes inflammation and apoptosis in the brain, resulting in neuronal loss. Dual cyclooxygenase/5-lipoxygenase (COX/5-LOX) inhibitors could represent a possible neuroprotective approach in preventing glutamate excitotoxicity. Consequently, we investigated the effects of a dual inhibitor of COX/5-LOX following intraperitoneal administration of kainic acid (KA, 10 mg/kg) in rats. Animals were randomized to receive either the dual inhibitor of COX/5-LOX (flavocoxid, 20 mg/kg i.p.) or its vehicle (1 ml/kg i.p.) 30 min after KA administration. Sham brain injury rats were used as controls. We evaluated protein expression of phosphorylated extracellular signal-regulated kinase (p-ERK1/2) and tumor necrosis factor alpha (TNF-α) as well as levels of malondialdehyde (MDA), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the hippocampus. Animals were also observed for monitoring behavioral changes according to Racine Scale. Finally, histological analysis and brain edema evaluation were carried out. Treatment with the dual inhibitor of COX/5-LOX decreased protein expression of p-ERK1/2 and TNF-α in hippocampus, markedly reduced MDA, LTB4 and PGE2 hippocampal levels, and also ameliorated brain edema. Histological analysis showed a reduction in cell damage in rats treated with the dual inhibitor of COX/5-LOX, particularly in hippocampal subregion CA3c. Moreover, flavocoxid significantly improved behavioral signs following kainic acid administration. Our results suggest that dual inhibition of COX/5-LOX by flavocoxid has neuroprotective effects during kainic acid-induced excitotoxicity.

Blockade of the JNK signalling as a rational therapeutic approach to modulate the early and late steps of the inflammatory cascade in polymicrobial sepsis.

Cecal ligation and puncture (CLP) is an experimental polymicrobial sepsis induced systemic inflammation that leads to acute organ failure. Aim of our study was to evaluate the effects of SP600125, a specific c-Jun NH2-terminal kinase (JNK) inhibitor, to modulate the early and late steps of the inflammatory cascade in a murine model of CLP-induced sepsis. CB57BL/6J mice were subjected to CLP or sham operation. Animals were randomized to receive either SP600125 (15 mg/kg) or its vehicle intraperitoneally 1 hour after surgery and repeat treatment every 24 hours. To evaluate survival, a group of animals was monitored every 24 hours for 120 hours. Two other animals were sacrificed 4 or 18 hours after surgical procedures; lung and liver samples were collected for biomolecular and histopathologic analysis. The expression of p-JNK, p-ERK, TNF-α, HMGB-1, NF-κB, Ras, Rho, Caspase 3, Bcl-2, and Bax was evaluated in lung and liver samples; SP600125 improved survival, reduced CLP induced activation of JNK, NF-κB, TNF-α, and HMGB-1, inhibited proapoptotic pathway, preserved Bcl-2 expression, and reduced histologic damage in both lung and liver of septic mice. SP600125 protects against CLP induced sepsis by blocking JNK signalling; therefore, it can be considered a therapeutic approach in human sepsis.

Cadmium delays puberty onset and testis growth in adolescents.

OBJECTIVE: Cadmium (Cd) has been shown to impair pubertal development in experimental animals. However, no data are available for male adolescents with increased urinary cadmium levels. DESIGN: The aim of this cross-sectional study was to evaluate pubertal onset and pituitary-gonadal axis hormones in male adolescents with increased urinary levels of Cd. SUBJECTS: We studied 111 males, aged 12-14 years living in the Milazzo-Valle del Mela area. A control age-matched population (n = 60) living 28-45 km far from the industrial site was also enrolled. MEASUREMENTS: Pubertal stages were assessed by clinical examination according to Tanner's score. Mean testicular volume was also investigated by ultrasound examination. Urinary Cd concentration and blood levels of FSH, LH, testosterone and inhibin B were also investigated. RESULTS: Cd levels were significantly higher in adolescents living in the Milazzo-Valle del Mela area, compared to both age-matched subjects living far from the industrial plants and the reference values. Our population showed also a delayed onset of puberty, a smaller testicular volume and lower testosterone levels. An inverse correlation was found between urinary Cd and testicular volume (r = -0·25; P = 0·0008), testosterone levels (Spearman's r = -0·0·37; two-tailed P < 0·0001) and LH levels (Spearman's r = 0·048; P < 0·05). Testosterone levels were positively correlated with testicular volume (Spearman's r = 0·48; P < 0·0001). CONCLUSIONS: This study, for the first time, suggests that increased Cd burden is associated with delayed onset of puberty in male adolescents and impaired testicular growth.

Flavocoxid, a nutraceutical approach to blunt inflammatory conditions.

Flavonoids, from Scutellaria baicalensis (Chinese skullcap) and Acacia catechu (black catechu), have been shown to exert a variety of therapeutic effects, including anti-inflammatory, antiviral, antibacterial, and anticancer activities. Flavocoxid is a mixed extract containing baicalin and catechin and it acts as a dual balanced inhibitor of cyclooxygenase-1 (COX-1) and COX-2 peroxidase enzyme activities with a significant inhibition of 5-lipoxygenase (5-LOX) enzyme activity in vitro. Flavocoxid downregulates gene or protein expression of several inflammatory markers and exerts also strong antioxidant activity in several experimental models. Controlled clinical trials and a postmarketing study have clearly shown that flavocoxid is as effective as naproxen in managing the signs and symptoms of osteoarthritis of the knee and it has better upper gastrointestinal, renal, and respiratory safety profile than naproxen. Flavocoxid may therefore provide a potential therapeutic approach to the treatment of chronic inflammatory conditions.

Oxidative stress and DNA repair and detoxification gene expression in adolescents exposed to heavy metals living in the Milazzo-Valle del Mela area (Sicily, Italy).

BACKGROUND: The area of Milazzo-Valle del Mela (Sicily, Italy) is considered at high risk of environmental crisis by regional authorities. OBJECTIVE: To measure oxidative-stress, DNA repair and detoxification genes in school children living near the industrial area and in age-matched controls. METHODS: The parent study was a biomonitoring investigation evaluating heavy metal urine levels in 226 children aged 12-14 years, living in the high risk area, and in 29 age-matched controls living 45 km far from the industrial site. In the present study 67 exposed adolescents and 29 controls were included. Samples were analyzed for urinary 8-hydroxydeoxyguanosine (8OHdG) levels, and gene expression of OGG1 (DNA repair gene), NQO1, ST13, and MT1A (detoxifying genes). RESULTS: Urinary cadmium was higher (p = 0.0004) in exposed [geometric mean, 0.46 µg/L; 25th-75th percentile: 0.3-0.56] than in control adolescents [geometric mean, 0.26 µg/L; 25th-75th percentile: 0.2-0.3]. Chromium was also significantly elevated in exposed [geometric mean, 1.52 µg/L; 25th-75th percentile: 1.19-1.93] compared with controls [geometric mean, 1.25 µg/L; 25th-75th percentile: 1.05-1.48; p = 0.02]. Urinary 8-OHdG concentration was greater in exposed than in controls (71.49 vs 61.87 µg/L, p = 0.02), and it was correlated with cadmium levels (r = 0.46, p < 0.0001), and with the combined exposure index (r = 0.43, p < 0.0001). Moreover, cadmium levels showed a robust correlation with OGG1 and MT1A gene expression levels (r = 0.44, p < 0.0001; r = 0.39, p < 0.0001, respectively). Finally, OGG1 and MT1A were over-expressed in adolescents from Milazzo-Valle del Mela area compared with controls (p = 0.0004; p < 0.0001, respectively). CONCLUSIONS: Continuous exposure at relatively low concentrations of heavy metals is associated with increased oxidative DNA damage and impaired expression of DNA repair and detoxification genes in adolescents.

Epigenetic modifications due to heavy metals exposure in children living in polluted areas.

The aim of the present article is to provide a summary of the epigenetic modifications that might occur in children exposed to heavy metals pollutants. It is known that children are more susceptible to environmental pollutants, because their detoxification enzymes are less competent, and this may lead to alterations in chromatin structure or of DNA causing, in turn, epigenetic modifications. Little is currently known about the long-term effects of these changes when occur early in childhood, none-theless there are ethics and practical concerns that make the assessment of DNA modifications difficult to perform in large-scale.