Gold Nanoparticles Inhibit Steroid-Insensitive Asthma in Mice Preserving Histone Deacetylase 2 and NRF2 Pathways.

BACKGROUND: Gold nanoparticles (AuNPs) can inhibit pivotal pathological changes in experimental asthma, but their effect on steroid-insensitive asthma is unclear. The current study assessed the effectiveness of nebulized AuNPs in a murine model of glucocorticoid (GC)-resistant asthma. METHODS: A/J mice were sensitized and subjected to intranasal instillations of ovalbumin (OVA) once a week for nine weeks. Two weeks after starting allergen stimulations, mice were subjected to Budesonide or AuNP nebulization 1 h before stimuli. Analyses were carried out 24 h after the last provocation. RESULTS: We found that mice challenged with OVA had airway hyperreactivity, eosinophil, and neutrophil infiltrates in the lung, concomitantly with peribronchiolar fibrosis, mucus production, and pro-inflammatory cytokine generation compared to sham-challenged mice. These changes were inhibited in mice treated with AuNPs, but not Budesonide. In the GC-resistant asthmatic mice, oxidative stress was established, marked by a reduction in nuclear factor erythroid 2-related factor 2 (NRF2) levels and catalase activity, accompanied by elevated values of thiobarbituric acid reactive substances (TBARS), phosphoinositide 3-kinases δ (PI3Kδ) expression, as well as a reduction in the nuclear expression of histone deacetylase 2 (HDAC2) in the lung tissue, all of which sensitive to AuNPs but not Budesonide treatment. CONCLUSION: These findings suggest that AuNPs can improve GC-insensitive asthma by preserving HDAC2 and NRF2.

Increased levels of cortisol are associated with the severity of experimental visceral leishmaniasis in a Leishmania (L.) infantum-hamster model.

BACKGROUND: Several infectious diseases are associated with hypothalamic-pituitary-adrenal (HPA) axis disorders by elevating circulating glucocorticoids (GCs), which are known to have an immunosuppressive potential. We conducted this study in golden hamsters, a suitable model for human visceral leishmaniasis (VL), to investigate the relationship of Leishmania (L.) infantum infection on cortisol production and VL severity. METHODS: L. infantum-infected (n = 42) and uninfected hamsters (n = 30) were followed-up at 30, 120, and 180 days post-infection (dpi). Plasma cortisol was analyzed by radioimmunoassay and cytokines, inducible nitric oxide synthase (iNOS), and arginase by RT-qPCR. RESULTS: All hamsters showed splenomegaly at 180 dpi. Increased parasite burden was associated with higher arginase expression and lower iNOS induction. Cortisol levels were elevated in infected animals in all-time points evaluated. Except for monocytes, all other leucocytes showed a strong negative correlation with cortisol, while transaminases were positively correlated. Immunological markers as interleukin (IL)-6, IL-1ß, IL-10, and transforming growth-factor-ß (TGF-ß) were positively correlated to cortisol production, while interferon-γ (IFN-γ) presented a negative correlation. A network analysis showed cortisol as an important knot linking clinical status and immunological parameters. CONCLUSIONS: These results suggest that L. infantum increases the systemic levels of cortisol, which showed to be associated with hematological, biochemical, and immunological parameters associated to VL severity.

Oxidative Imbalance as a Crucial Factor in Inflammatory Lung Diseases: Could Antioxidant Treatment Constitute a New Therapeutic Strategy?

Inflammatory lung disease results in a high global burden of death and disability. There are no effective treatments for the most severe forms of many inflammatory lung diseases, such as chronic obstructive pulmonary disease, emphysema, corticosteroid-resistant asthma, and coronavirus disease 2019; hence, new treatment options are required. Here, we review the role of oxidative imbalance in the development of difficult-to-treat inflammatory lung diseases. The inflammation-induced overproduction of reactive oxygen species (ROS) means that endogenous antioxidants may not be sufficient to prevent oxidative damage, resulting in an oxidative imbalance in the lung. In turn, intracellular signaling events trigger the production of proinflammatory mediators that perpetuate and aggravate the inflammatory response and may lead to tissue damage. The production of high levels of ROS in inflammatory lung diseases can induce the phosphorylation of mitogen-activated protein kinases, the inactivation of phosphoinositide 3-kinase (PI3K) signaling and histone deacetylase 2, a decrease in glucocorticoid binding to its receptor, and thus resistance to glucocorticoid treatment. Hence, antioxidant treatment might be a therapeutic option for inflammatory lung diseases. Preclinical studies have shown that antioxidants (alone or combined with anti-inflammatory drugs) are effective in the treatment of inflammatory lung diseases, although the clinical evidence of efficacy is weaker. Despite the high level of evidence for the efficacy of antioxidants in the treatment of inflammatory lung diseases, the discovery and clinical investigation of safer, more efficacious compounds are now a priority.

Activation of PPARγ reduces N-acetyl-cysteine -induced hypercorticoidism by down-regulating MC2R expression into adrenal glands.

We previously demonstrated that oral supplementation with antioxidants induced hyperactivity of hypothalamus-pituitary-adrenal (HPA) axis, attested by hypercorticoidism, through an up-regulation of adrenocorticotrophic hormone (ACTH) receptors (MC2R) in adrenal. This study analyzed the role of peroxisome proliferator-activated receptor (PPAR)-γ on HPA axis hyperactivity induced by N-acetyl-cysteine (NAC). Male Swiss-Webster mice were orally treated with NAC for 1, 3, 5, 10, 15, or 18 consecutive days. The PPAR-γ agonist rosiglitazone and/or antagonist GW9662 were daily-injected i.p. for 5 consecutive days, starting concomitantly with NAC treatment. Rosiglitazone treatment inhibited NAC-induced adrenal hypertrophy and hypercorticoidism. Rosiglitazone also significantly reversed the NAC-induced increase in the MC2R expression in adrenal, but not steroidogenic acute regulatory protein (StAR). NAC treatment reduces the expression of PPARγ in the adrenals, but rosiglitazone did not restore the expression of this cytoprotective gene. In addition, GW9662 blocked the ability of rosiglitazone to decrease plasma corticosterone levels in NAC-treated mice. In conclusion, our findings showed that antioxidant supplementation induced a state of hypercorticoidism through down-regulation of PPARγ expression in the adrenals, in a mechanism probably related to a down-regulation of ACTH receptor expression.

Glucagon reduces airway hyperreactivity, inflammation, and remodeling induced by ovalbumin.

Glucagon has been shown to be beneficial as a treatment for bronchospasm in asthmatics. Here, we investigate if glucagon would prevent airway hyperreactivity (AHR), lung inflammation, and remodeling in a murine model of asthma. Glucagon (10 and 100 µg/Kg, i.n.) significantly prevented AHR and eosinophilia in BAL and peribronchiolar region induced by ovalbumin (OVA) challenge, while only the dose of 100 µg/Kg of glucagon inhibited subepithelial fibrosis and T lymphocytes accumulation in BAL and lung. The inhibitory action of glucagon occurred in parallel with reduction of OVA-induced generation of IL-4, IL-5, IL-13, TNF-α, eotaxin-1/CCL11, and eotaxin-2/CCL24 but not MDC/CCL22 and TARC/CCL17. The inhibitory effect of glucagon (100 µg/Kg, i.n.) on OVA-induced AHR and collagen deposition was reversed by pre-treatment with indomethacin (10 mg/Kg, i.p.). Glucagon increased intracellular cAMP levels and inhibits anti-CD3 plus anti-CD28-induced proliferation and production of IL-2, IL-4, IL-10, and TNF- α from TCD4+ cells in vitro. These findings suggest that glucagon reduces crucial features of asthma, including AHR, lung inflammation, and remodeling, in a mechanism probably associated with inhibition of eosinophils accumulation and TCD4+ cell proliferation and function. Glucagon should be further investigated as an option for asthma therapy.

Capsaicin inhibits lipopolysaccharide-induced adrenal steroidogenesis by raising intracellular calcium levels.

INTRODUCTION: Glucocorticoid release by adrenals has been described as significant to survive sepsis. The activation of transient receptor potential vanilloid type 1 (TRPV1) inhibited ACTH-induced glucocorticoid release by adrenal glands in vitro. OBJECTIVE: The aim of this study was to investigate if capsaicin, an activator of TRPV1, would prevent LPS-induced glucocorticoid production by adrenals. METHODS: Male Swiss-Webster mice were treated with capsaicin intraperitoneally (0.2 or 2 mg/kg) 30 min before LPS injection. All analyses were performed 2 h after the LPS stimulation, including plasma corticosterone and peritoneal IL-1ß and TNF-α levels. Furthermore, murine adrenocortical Y1 cells were used to assess the effects of capsaicin on LPS-induced corticosterone production in vitro. RESULTS: Capsaicin (2 mg/kg, i.p.) significantly reduced plasma corticosterone levels and adrenal hypertrophy induced by LPS without alter the levels of pro-steroidogenic cytokines IL-1ß and TNF-α in peritoneal cavity of mice, while the dose of 0.2 mg/kg of capsaicin did not interfere with adrenal steroidogenesis, attested by RIA and ELISA, respectively. Y1 cells express TRPV1, measured by immunofluorescence and western blot, and capsaicin decreased LPS-induced corticosterone production by these cells in vitro. Capsaicin also induces calcium mobilization in Y1 cells in vitro. CONCLUSIONS: These findings suggest that capsaicin inhibits corticosterone production induced by LPS by acting directly on adrenal cells producing glucocorticoids, in a mechanism probably associated with induction of a cytoplasmic calcium increase in these cells.

Glucocorticoids decrease the numbers and activation of mast cells by inducing the transactivation receptors of AGEs.

Glucocorticoids (GCs) are potent anti-allergic compounds that function, at least in part, by inhibiting signaling pathways in mast cells. We hypothesized that the GC-induced mastocytopenia and suppression of mast cell activation are mediated by the advanced glycation end products (AGEs)/receptors of AGEs (RAGEs) signaling axis. We evaluated the role of AGEs in GC-mediated mastocytopenia and impaired mast cell degranulation in male Wistar rats and Swiss-Webster mice subcutaneously injected with dexamethasone or prednisolone (0.1 mg/kg) once a day for 21 consecutive days. The animals were treated with either the AGE inhibitor aminoguanidine (250 mg/kg), the RAGE antagonist FPS-ZM1 (1 mg/kg) or the galectin-3 antagonist GSC-100 (1 mg/kg) daily for 18 days, starting 3 days following GC treatment. Aminoguanidine inhibited GC-induced mast cell apoptosis and restored mast cell numbers in the pleural cavity of GC-treated rats. Aminoguanidine also reversed the GC-induced reduction in histamine release triggered by allergens or compound 48/80 in vitro. GC treatment induced RAGE and galectin expression in mast cells, and blocking these agents by FPS-ZM1 or GSC-100 significantly reversed mast cell numbers in the peritoneal cavity and mesenteric tissue of GC-treated mice. In addition, the combination of GC and AGE-induced mast cell apoptosis in vitro was inhibited by both FPS-ZM1 and GSC-100. We concluded that the GC-induced mastocytopenia and suppression of mast cell stimulation are associated with the gene transactivation of RAGE and galectin-3.

The Neuropeptides Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Control HIV-1 Infection in Macrophages Through Activation of Protein Kinases A and C.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are highly similar neuropeptides present in several tissues, endowed with immunoregulatory functions and other systemic effects. We previously reported that both neuropeptides reduce viral production in HIV-1-infected primary macrophages, with the participation of ß-chemokines and IL-10, and now we describe molecular mechanisms engaged in this activity. Macrophages exposed to VIP or PACAP before HIV-1 infection showed resistance to viral replication, comparable to that observed when the cells were treated after infection. Also, multiple treatments with a suboptimal dose of VIP or PACAP after macrophage infection resulted in a decline of virus production similar to the inhibition promoted by a single exposure to the optimal inhibitory concentration. Cellular signaling pathways involving cAMP production and activation of protein kinases A and C were critical components of the VIP and PACAP anti-HIV-1 effects. Analysis of the transcription factors and the transcriptional/cell cycle regulators showed that VIP and PACAP induced cAMP response element-binding protein activation, inhibited NF-kB, and reduced Cyclin D1 levels in HIV-1-infected cells. Remarkably, VIP and PACAP promoted G-to-A mutations in the HIV-1 provirus, matching those derived from the activity of the APOBEC family of viral restriction factors, and reduced viral infectivity. In conclusion, our findings strengthen the antiretroviral potential of VIP and PACAP and point to new therapeutic approaches to control the progression of HIV-1 infection.

Repeated Allergen Exposure in A/J Mice Causes Steroid-Insensitive Asthma via a Defect in Glucocorticoid Receptor Bioavailability.

The importance of developing new animal models to assess the pathogenesis of glucocorticoid (GC)-insensitive asthma has been stressed. Because of the asthma-prone background of A/J mice, we hypothesized that asthma changes in these animals would be or become resistant to GCs under repeated exposures to an allergen. A/J mice were challenged with OVA for 2 or 4 consecutive d, starting on day 19 postsensitization. Oral dexamethasone or inhaled budesonide were given 1 h before challenge, and analyses were done 24 h after the last challenge. Airway hyperreactivity, leukocyte infiltration, tissue remodeling, and cytokine levels as well as phosphorylated GC receptor (p-GCR), p-GATA-3, p-p38, MAPK phosphatase-1 (MKP-1), and GC-induced leucine zipper (GILZ) levels were assessed. A/J mice subjected to two daily consecutive challenges reacted with airway hyperreactivity, subepithelial fibrosis, and marked accumulation of eosinophils in both bronchoalveolar lavage fluid and peribronchial space, all of which were clearly sensitive to dexamethasone and budesonide. Conversely, under four provocations, most of these changes were steroid resistant. A significant reduction in p-GCR/GCR ratio following 4- but not 2-d treatment was observed, as compared with untreated positive control. Accordingly, steroid efficacy to transactivate MKP-1 and GILZ and to downregulate p-p38, p-GATA-3 as well as proinflammatory cytokine levels was also seen after two but not four provocations. In conclusion, we report that repeated allergen exposure causes GC-insensitive asthma in A/J mice in a mechanism associated with decrease in GCR availability and subsequent loss of steroid capacity to modulate pivotal regulatory proteins, such as GATA-3, p-p38, MKP-1, and GILZ.

Diabetes Downregulates Allergen-Induced Airway Inflammation in Mice.

Previous studies described that allergic diseases, including asthma, occur less often than expected in patients with type 1 diabetes. Here, we investigated the influence of diabetes on allergic airway inflammation in a model of experimental asthma in mice. Diabetes was induced by intravenous injection of alloxan into 12 h-fasted A/J mice, followed by subcutaneous sensitization with ovalbumin (OVA) and aluminum hydroxide (Al(OH)3), on days 5 and 19 after diabetes induction. Animals were intranasally challenged with OVA (25 µg), from day 24 to day 26. Alloxan-induced diabetes significantly attenuated airway inflammation as attested by the lower number of total leukocytes in the bronchoalveolar lavage fluid, mainly neutrophils and eosinophils. Suppression of eosinophil infiltration in the peribronchiolar space and generation of eosinophilotactic mediators, such as CCL-11/eotaxin, CCL-3/MIP-1α, and IL-5, were noted in the lungs of diabetic sensitized mice. In parallel, reduction of airway hyperreactivity (AHR) to methacholine, mucus production, and serum IgE levels was also noted under diabetic conditions. Our findings show that alloxan diabetes caused attenuation of lung allergic inflammatory response in A/J mice, by a mechanism possibly associated with downregulation of IgE antibody production.

LASSBio-897 Reduces Lung Injury Induced by Silica Particles in Mice: Potential Interaction with the A2A Receptor.

Silicosis is a lethal fibro-granulomatous pulmonary disease highly prevalent in developing countries, for which no proper therapy is available. Among a small series of N-acylhydrazones, the safrole-derived compound LASSBio-897 (3-thienylidene-3, 4-methylenedioxybenzoylhydrazide) raised interest due to its ability to bind to the adenosine A2A receptor. Here, we evaluated the anti-inflammatory and anti-fibrotic potential of LASSBio-897, exploring translation to a mouse model of silicosis and the A2A receptor as a site of action. Pulmonary mechanics, inflammatory, and fibrotic changes were assessed 28 days after intranasal instillation of silica particles in Swiss-Webster mice. Glosensor cAMP HEK293G cells, CHO cells stably expressing human adenosine receptors and ligand binding assay were used to evaluate the pharmacological properties of LASSBio-897 in vitro. Molecular docking studies of LASSBio-897 were performed using the genetic algorithm software GOLD 5.2. We found that the interventional treatment with the A2A receptor agonist CGS 21680 reversed silica particle-induced airway hyper-reactivity as revealed by increased responses of airway resistance and lung elastance following aerosolized methacholine. LASSBio-897 (2 and 5 mg/kg, oral) similarly reversed pivotal lung pathological features of silicosis in this model, reducing levels of airway resistance and lung elastance, granuloma formation and collagen deposition. In competition assays, LASSBio-897 decreased the binding of the selective A2A receptor agonist [3H]-CGS21680 (IC50 = 9.3 µM). LASSBio-897 (50 µM) induced modest cAMP production in HEK293G cells, but it clearly synergized the cAMP production by adenosine in a mechanism sensitive to the A2A antagonist SCH 58261. This synergism was also seen in CHO cells expressing the A2A, but not those expressing A2B, A1 or A3 receptors. Based on the evidence that LASSBio-897 binds to A2A receptor, molecular docking studies were performed using the A2A receptor crystal structure and revealed possible binding modes of LASSBio-897 at the orthosteric and allosteric sites. These findings highlight LASSBio-897 as a lead compound in drug development for silicosis, emphasizing the role of the A2A receptor as its putative site of action.

15-Deoxy-Delta-12,14-Prostaglandin J2 Inhibits Lung Inflammation and Remodeling in Distinct Murine Models of Asthma.

15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) has been described as an anti-inflammatory lipid mediator in several in vitro and in vivo studies, but its effect on allergic pulmonary inflammation remains elusive. The aim of this study was to investigate the therapeutic potential of 15d-PGJ2 based on distinct murine models of allergic asthma triggered by either ovalbumin (OVA) or house dust mite extract (HDM). Characteristics of lung inflammation, airway hyper-reactivity (AHR), mucus exacerbation, and lung remodeling in sensitized A/J mice treated or not with 15d-PGJ2 were assessed. 15d-PGJ2 treatments were carried out systemically or topically given via subcutaneous injection or intranasal instillation, respectively. Analyses were carried out 24 h after the last allergen provocation. Irrespective of the route of administration, 15d-PGJ2 significantly inhibited the peribronchial accumulation of eosinophils and neutrophils, subepithelial fibrosis and also mucus exacerbation caused by either OVA or HDM challenge. The protective effect of 15d-PGJ2 occurred in parallel with inhibition of allergen-induced AHR and lung tissue production of pro-inflammatory cytokines, such as interleukin (IL)-5, IL-13, IL-17, and TNF-α. Finally, 15d-PGJ2 was found effective in inhibiting NF-κB phosphorylation upon HDM challenge as measured by Western blotting. In conclusion, our findings suggest that 15d-PGJ2 can reduce crucial features of asthma, including AHR, lung inflammation, and remodeling in distinct murine models of the disease. These effects are associated with a decrease in lung tissue generation of pro-inflammatory cytokines by a mechanism related to downregulation of NF-κB phosphorylation.

Glucagon and glucagon-like peptide-1 as novel anti-inflammatory and immunomodulatory compounds.

Glucagon and glucagon-like peptide-1 (GLP-1) are polypeptide hormones that are produced by pancreatic α-cells and the intestine, respectively, whose main function is to control glucose homeostasis. The glucagon and GLP-1 levels are imbalanced in diabetes. Furthermore, type 1 diabetic patients and animals present with a diminished inflammatory response, which is related to some morbidities of diabetes, such as a higher incidence of infectious diseases, including sepsis. The focus of this review is to briefly summarize the state of the art concerning the effects of glucagon and GLP-1 on the inflammatory response. Here, we propose that glucagon and GLP-1 have anti-inflammatory properties, making them possible prototypes for the design and synthesis of new compounds to treat inflammatory diseases. In addition, glucagon, GLP-1 or their analogues or new derivatives may not only be important for managing inflammatory diseases but may also have the therapeutic potential to prevent, cure or ameliorate diabetes in patients by counteracting the deleterious effects of pro-inflammatory cytokines on the function and viability of pancreatic ß-cells. In addition, GLP-1, its analogues or drugs that inhibit GLP-1 metabolism may have a doubly beneficial effect in diabetic patients by inhibiting the inflammatory response and reducing glycaemia.

Antioxidant Treatment Induces Hyperactivation of the HPA Axis by Upregulating ACTH Receptor in the Adrenal and Downregulating Glucocorticoid Receptors in the Pituitary.

Glucocorticoid (GC) production is physiologically regulated through a negative feedback loop mediated by the GC, which appear disrupted in several pathological conditions. The inability to perform negative feedback of the hypothalamus-pituitary-adrenal (HPA) axis in several diseases is associated with an overproduction of reactive oxygen species (ROS); however, nothing is known about the effects of ROS on the functionality of the HPA axis during homeostasis. This study analyzed the putative impact of antioxidants on the HPA axis activity and GC-mediated negative feedback upon the HPA cascade. Male Wistar rats were orally treated with N-acetylcysteine (NAC) or vitamin E for 18 consecutive days. NAC-treated rats were then subjected to a daily treatment with dexamethasone, which covered the last 5 days of the antioxidant therapy. We found that NAC and vitamin E induced an increase in plasma corticosterone levels. NAC intensified MC2R and StAR expressions in the adrenal and reduced GR and MR expressions in the pituitary. NAC also prevented the dexamethasone-induced reduction in plasma corticosterone levels. Furthermore, NAC decreased HO-1 and Nrf2 expression in the pituitary. These findings show that antioxidants induce hyperactivity of the HPA axis via upregulation of MC2R expression in the adrenal and downregulation of GR and MR in the pituitary.

Activation of PPAR-γ reduces HPA axis activity in diabetic rats by up-regulating PI3K expression.

Increased hypothalamus-pituitary-adrenal axis (HPA) activity in diabetes is strongly associated with several morbidities noted in patients with the disease. We previously demonstrated that hyperactivity of HPA axis under diabetic conditions is associated with up-regulation of adrenocorticotrophic hormone (ACTH) receptors (MC2R) in adrenal and down-regulation of glucocorticoid receptors (GR and MR) in pituitary. This study investigates the role of peroxisome proliferator-activated receptor (PPAR)-γ in HPA axis hyperactivity in diabetic rats. Diabetes was induced by intravenous injection of alloxan into fasted rats. The PPAR-γ agonist rosiglitazone and/or PI3K inhibitor wortmannin were administered daily for 18 consecutive days, starting 3days after diabetes induction. Plasma ACTH and corticosterone were evaluated by radioimmunoassay, while intensities of MC2R, proopiomelanocortin (POMC), GR, MR, PI3K p110α and PPAR-γ were assessed using immunohistochemistry. Rosiglitazone treatment inhibited adrenal hypertrophy and hypercorticoidism observed in diabetic rats. Rosiglitazone also significantly reversed the diabetes-induced increase in the MC2R expression in adrenal cortex. We noted that rosiglitazone reduced the number of corticotroph cells and inhibited both anterior pituitary POMC expression and plasma ACTH levels. Furthermore, rosiglitazone treatment was unable to restore the reduced expression of GR and MR in the anterior pituitary of diabetic rats. Rosiglitazone increased the number of PPAR-γ+ cells and expression of PI3K p110α in both anterior pituitary and adrenal cortex of diabetic rats. In addition, wortmannin blocked the ability of rosiglitazone to restore corticotroph cell numbers, adrenal hypertrophy and plasma corticosterone levels in diabetic rats. In conclusion, our findings revealed that rosiglitazone down-regulates HPA axis hyperactivity in diabetic rats via a mechanism dependent on PI3K activation in pituitary and adrenal glands.

Glucagon induces airway smooth muscle relaxation by nitric oxide and prostaglandin E2.

Glucagon is a hyperglycemic pancreatic hormone that has been shown to provide a beneficial effect against asthmatic bronchospasm. We investigated the role of this hormone on airway smooth muscle contraction and lung inflammation using both in vitro and in vivo approaches. The action of glucagon on mouse cholinergic tracheal contraction was studied in a conventional organ bath system, and its effect on airway obstruction was also investigated using the whole-body pletysmographic technique in mice. We also tested the effect of glucagon on lipopolysaccharide (LPS)-induced airway hyperreactivity (AHR) and inflammation. The expression of glucagon receptor (GcgR), CREB, phospho-CREB, nitric oxide synthase (NOS)-3, pNOS-3 and cyclooxygenase (COX)-1 was evaluated by western blot, while prostaglandin E2 (PGE2) and tumour necrosis factor-α were quantified by enzyme-linked immunoassay and ELISA respectively. Glucagon partially inhibited carbachol-induced tracheal contraction in a mechanism clearly sensitive to des-His1-[Glu9]-glucagon amide, a GcgR antagonist. Remarkably, GcgR was more expressed in the lung and trachea with intact epithelium than in the epithelium-denuded trachea. In addition, the glucagon-mediated impairment of carbachol-induced contraction was prevented by either removing epithelial cells or blocking NOS (L-NAME), COX (indomethacin) or COX-1 (SC-560). In contrast, inhibitors of either heme oxygenase or COX-2 were inactive. Intranasal instillation of glucagon inhibited methacholine-induced airway obstruction by a mechanism sensitive to pretreatment with L-NAME, indomethacin and SC-560. Glucagon induced CREB and NOS-3 phosphorylation and increased PGE2 levels in the lung tissue without altering COX-1 expression. Glucagon also inhibited LPS-induced AHR and bronchoalveolar inflammation. These findings suggest that glucagon possesses airway-relaxing properties that are mediated by epithelium-NOS-3-NO- and COX-1-PGE2-dependent mechanisms.

Mangiferin prevents guinea pig tracheal contraction via activation of the nitric oxide-cyclic GMP pathway.

Previous studies have described the antispasmodic effect of mangiferin, a natural glucoside xanthone (2-C-ß-Dgluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) that is present in mango trees and other plants, but its mechanism of action remains unknown. The aim of this study was to examine the potential contribution of the nitric oxide-cyclic GMP pathway to the antispasmodic effect of mangiferin on isolated tracheal rings preparations. The functional effect of mangiferin on allergic and non-allergic contraction of guinea pig tracheal rings was assessed in conventional organ baths. Cultured tracheal rings were exposed to mangiferin or vehicle, and nitric oxide synthase (NOS) 3 and cyclic GMP (cGMP) levels were quantified using western blotting and enzyme immunoassays, respectively. Mangiferin (0.1-10 µM) inhibited tracheal contractions induced by distinct stimuli, such as allergen, histamine, 5-hydroxytryptamine or carbachol, in a concentration-dependent manner. Mangiferin also caused marked relaxation of tracheal rings that were precontracted by carbachol, suggesting that it has both anti-contraction and relaxant properties that are prevented by removing the epithelium. The effect of mangiferin was inhibited by the nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) (100 µM), and the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM), but not the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (SQ22536) (100 µM). The antispasmodic effect of mangiferin was also sensitive to K⁺ channel blockers, such as tetraethylammonium (TEA), glibenclamide and apamin. Furthermore, mangiferin inhibited Ca²âº-induced contractions in K⁺ (60 mM)-depolarised tracheal rings preparations. In addition, mangiferin increased NOS3 protein levels and cGMP intracellular levels in cultured tracheal rings. Finally, mangiferin-induced increase in cGMP levels was abrogated by co-incubation with either ODQ or L-NAME. These data suggest that the antispasmodic effect of mangiferin is mediated by epithelium-nitric oxide- and cGMP-dependent mechanisms.

Activation of PPARγ by restores mast cell numbers and reactivity in alloxan-diabetic rats by reducing the systemic glucocorticoid levels.

Mast cell function and survival have been shown to be down-regulated under diabetic conditions. This study investigates the role of the peroxisome proliferator-activated receptor (PPAR)-γ in reducing mast cell number and reactivity in diabetic rats. The effect of rosiglitazone on mast cell apoptosis was also evaluated. Diabetes was induced by intravenous injection of alloxan into fasted rats and PPARγ agonist rosiglitazone and/or specific antagonist 2-chloro-5-nitrobenzanilide (GW9662) were administered 3 day after diabetes induction, once daily for 18 consecutive days. Mast cell apoptosis and plasma corticosterone levels were evaluated by TUNEL and radioimmunoassay, respectively. Treatment with rosiglitazone restored mast cell numbers in the pleural cavity and mesenteric tissue of diabetic rats. Rosiglitazone also significantly reversed the diabetes-induced reduction of histamine release by mast cells, as measured by fluorescence, following activation with the antigen in vitro. Increased apoptosis in mast cells from diabetic rats were inhibited by rosiglitazone. Moreover, we noted that the increase in plasma corticosterone levels in diabetic rats was inhibited by rosiglitazone. In addition, GW9662 blocked the ability of rosiglitazone to restore baseline numbers of mast cells and plasma corticosterone in diabetic rats. In conclusion, our findings showed that rosiglitazone restored the number and reactivity of mast cells in diabetic rats, accompanied with a suppression of apoptosis, in parallel with impairment of diabetes hypercorticolism, indicating that PPARγ has an important role in these phenomena.

Inhibition of advanced glycation end products by aminoguanidine restores mast cell numbers and reactivity in alloxan-diabetic rats.

Mast cell number and reactivity have been shown to be down-regulated under diabetic conditions. This study was undertaken in order to investigate the role of the advanced glycation end products in the reduction of mast cell number and reactivity in diabetic rats. The effect of aminoguanidine on mast cell apoptosis was also evaluated. Diabetes was induced by intravenous injection of alloxan into fasted rats and aminoguanidine was administered after 3 days of diabetes induction, once daily for 18 consecutive days. Mast cell apoptosis and levels of Bax, a pro-apoptotic member of Bcl-2 family, were evaluated by TUNEL and western blot, respectively. Diabetes led to increased levels of fructosamine and AGEs in the plasma, an effect prevented by aminoguanidine. Treatment with aminoguanidine restored mast cell numbers in the pleural cavity and in mesenteric tissue of diabetic rats. Aminoguanidine also significantly reversed the diabetes-induced reduction in histamine release, as measured by fluorescence, following activation with substance P or antigen in vitro. Increased apoptosis and levels of Bax in mast cells from diabetic rats were inhibited by aminoguanidine. In conclusion, our findings showed that aminoguanidine restored the number and reactivity of mast cells in diabetic rats, accompanied by suppression of apoptosis, evidencing that advanced glycation end product formation has a critical role in mast cell behavior of diabetic rats.

Down-regulation of allergic responses in conditions of experimental diabetes: a role for glucocorticoids?

The prevalence of atopic diseases and diabetes is increasing worldwide, though the co-occurrence of both diseases in the same individual is less frequent than predicted. Previously published studies suggest that the Th1/Th2 concept could explain the inverse relationship between allergic diseases and type 1 diabetes. However, down-regulation of the IgE-mast cell system can also markedly contribute to the lack of responsiveness to local and systemic allergen challenges in diabetic conditions. Moreover, dysregulation of the hypothalamic-pituitary-adrenocortical axis and elevated endogenous glucocorticoid levels play a pertinent role in some of the pathological-related processes associated with poorly controlled or uncontrolled diabetes.