Contribution of anaphylatoxin C5a to late airway responses after repeated exposure of antigen to allergic rats

We attempted to elucidate the contribution of complement to allergic asthma. Rat sensitized to OVA received repeated intratracheal exposures to OVA for up to 3 consecutive days, and pulmonary resistance was then estimated for up to 6 h after the last exposure. Whereas the immediate airway response (IAR) in terms of R(L) tended to decrease in proportion to the number of OVA exposures, late airway response (LAR) became prominent only after three. Although premedication with two kinds of complement inhibitors, soluble complement receptor type 1 (sCR1) or nafamostat mesylate, resulted in inhibition of the IAR after either a single or a double exposure, the LAR was inhibited after the triple. Premedication with a C5a receptor antagonist (C5aRA) before every exposure to OVA also inhibited the LAR after three. Repeated OVA exposure resulted in eosinophil and neutrophil infiltration into the bronchial submucosa which was suppressed by premedication with sCR1 or C5aRA. Up-regulation of C5aR mRNA was shown in lungs after triple OVA exposure, but almost no up-regulation of C3aR. Pretreatment with sCR1 or C5aRA suppressed the up-regulation of C5aR expression as well as cytokine messages in the lungs. The suppression of LAR by pretreatment with sCR1 was reversed by intratracheal instillation of rat C5a desArg the action of which was inhibited by C5aRA. In contrast, rat C3a desArg or cytokine-induced neutrophil chemoattractant-1 induced cellular infiltration into the bronchial submucosa by costimulation with OVA, but these had no influence on the LAR. These differences might be explained by the fact that costimulation with OVA and C5a synergistically potentiated IAR, whereas that with OVA and either C3a or cytokine-induced neutrophil chemoattractant-1 did not. C5a generated by Ag-Ab complexes helps in the production of cytokines and contributes to the LAR after repeated exposure to Ag.     

Phosphatidylinositol 3-kinase inhibitor suppresses inducible nitric oxide synthase expression in bronchiole epithelial cells in asthmatic rats

Inducible nitric oxide synthase (iNOS) is known to produce nitric oxide (NO), which is a main contributor to asthmatic airway inflammation. Recent studies have shown that phosphatidylinositol 3-kinase (PI3K) is ubiquitously expressed in airway epithelial cells and its inhibition could relieve airway inflammation and hyperresponsiveness. This study aimed to explore the interaction of PI3K and NO signaling in allergic asthma. We investigated the effects of PI3K inhibitor wortmannin on iNOS expression in bronchiole epithelial cells and NO, IL-4 and IFN-γ levels in lung tissues of asthmatic rat model, which was prepared by 10% OVA solution sensitization and 1% OVA aerosol challenge. Our results showed that the ratio of eosinophils to total cells in BALF, PI3K activity, NO and IL-4 levels in lung tissues was increased after OVA sensitization and challenge, but then was attenuated by the administration of wortmannin. In contrast, IFN-γ level in lung tissues was decreased after OVA sensitization and challenge and increased after the administration of wortmannin. The expression of iNOS protein in bronchiole epithelial cells, iNOS mRNA level and iNOS activity in lung tissues was markedly upregulated after OVA sensitization and challenge, but the upregulation was significantly antagonized by wortmannin. Taken together, these data provide evidence that PI3K functions upstream to modulate iNOS/NO signaling, which then promotes the development of airway inflammation in asthmatic animal model. PI3K inhibitor wortmannin could lead to reduced iNOS expression and NO production, therefore inhibiting airway inflammatory responses.     

Heme oxygenase attenuates allergen-induced airway inflammation and hyperreactivity in guinea pigs

Heme oxygenase (HO), the heme-degrading enzyme, has shown anti-inflammatory effects in several models of pulmonary diseases. HO is induced in airways during asthma; however, its functional role is unclear. Therefore, we evaluated the role of HO on airway inflammation [evaluated by bronchoalveolar lavage (BAL) cellularity and BAL levels of eotaxin, PGE(2), and proteins], mucus secretion (evaluated by analysis of MUC5AC gene expression and periodic acid-Schiff staining), oxidative stress (evaluated by quantification of 4-hydroxynonenal adducts and carbonylated protein levels in lung homogenates), and airway responsiveness to histamine in ovalbumin (OVA)-sensitized and multiple aerosol OVA or saline-challenged guinea pigs (6 challenges, once daily, OVA group and control group, respectively). Airway inflammation, mucus secretion, oxidative stress, and responsiveness were significantly increased in the OVA group compared with the control group. HO upregulation by repeated administrations of hemin (50 mg/kg i.p.) significantly decreased airway responsiveness in control animals and airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These effects were reversed by the concomitant administration of the HO inhibitor tin protoporphyrin-IX (50 micromol/kg i.p.). Repeated administrations of tin protoporphyrin-IX alone significantly increased airway responsiveness in control animals but did not modify airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These results suggest that upregulation of the HO pathway has a significant protective effect against airway inflammation, mucus hypersecretion, oxidative stress, and hyperresponsiveness in a model of allergic asthma in guinea pigs.     

Inhibitors of inducible nitric oxide (NO) synthase are more effective than an NO donor in reducing carbon-tetrachloride induced acute liver injury

The exact functional role of nitric oxide (NO) in liver injury is currently a source of controversy. NO is enzymatically synthesized by nitric oxide synthase (NOS). In this study, we assessed the role of inducible NOS (iNOS) in carbon tetrachloride (CCl4)-induced acute liver injury using inhibitors of iNOS, and an NO donor. Adult ICR mice were injected with CCl4 with or without the iNOS inhibitors (5-methylisothiourea hemisulfate [SMT] and l-N6-(1-iminoethyl)-lysine [L-NIL]) and an NO donor (Sodium Nitroprusside [SNP]). Blood and liver tissues were collected for analysis. Immunohistochemistry (IHC), serum alanine aminotransferase (ALT), serum total 8-isoprostane analysis, RT-PCR, Western Blotting (WB) and EMSA were done. Our results showed increased levels of ALT, necrosis, total 8-isoprostane and nitrotyrosine after CCl4 administration. iNOS inhibitors and SNP abrogated these effects but the effect was more pronounced with SMT and L-NIL. RT-PCR, WB and IHC in CCl4-treated mice demonstrated upregulation of TNF-alpha, iNOS, and COX-2. The administration of iNOS inhibitors with CCl4 diminished the expression of these proinflammatory mediators. NF-kappaB was also upregulated in CCl4-treated mice and was reversed in mice pretreated with iNOS inhibitors. SNP pretreated mice also showed a lower expression of COX-2 when compared with CCl4 treated mice but TNF-alpha, iNOS and NF-kappaB activity were unaffected. We propose that a high level of nitric oxide is associated with CCl4-induced acute liver injury and the liver injury can be ameliorated by decreasing the NO level with iNOS inhibitors and an NO donor with the former more effective in reducing CCl4-induced liver injury.     

TLR-7 agonist attenuates airway reactivity and inflammation through Nrf2-mediated antioxidant protection in a murine model of allergic asthma

Toll-like receptors (TLRs) through innate immune system recognize pathogen associated molecular patterns and play an important role in host defense against bacteria, fungi and viruses. TLR-7 is responsible for sensing single stranded nucleic acids of viruses but its activation has been shown to be protective in mouse models of asthma. The NADPH oxidase (NOX) enzymes family mainly produces reactive oxygen species (ROS) in the lung and is involved in regulation of airway inflammation in response to TLRs activation. However, NOX-4 mediated signaling in response to TLR-7 activation in a mouse model of allergic asthma has not been explored previously. Therefore, this study investigated the role TLR-7 activation and downstream oxidant–antioxidant signaling in a murine model of asthma. Mice were sensitized with ovalbumin (OVA) intraperitoneally and treated with TLR-7 agonist, resiquimod (RSQ) intranasally before each OVA challenge from days 14 to 16. Mice were then assessed for airway reactivity, inflammation, and NOX-4 and nuclear factor E2-related factor 2 (Nrf2) related signaling [inducible nitric oxide synthase (iNOS), nitrotyrosine, lipid peroxides and copper/zinc superoxide dismutase (Cu/Zn SOD)]. Treatment with RSQ reduced allergen induced airway reactivity and inflammation. This was paralleled by a decrease in ROS which was due to induction of Nrf2 and Cu/Zn SOD in RSQ treated group. Inhibition of MyD88 reversed RSQ-mediated protective effects on airway reactivity/inflammation due to reduction in Nrf2 signaling. SOD inhibition produced effects similar to MyD88 inhibition. The current study suggests that TLR-7 agonist is beneficial and may be developed into a therapeutic option in allergic asthma.     

Curcumin attenuates ovalbumin-induced airway inflammation by regulating nitric oxide

Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that curcumin contributes to anti-inflammatory activity in the murine asthma model and lung epithelial cell A549 through suppression of nitric oxide (NO). To address this problem, curcumin was injected into the peritoneum of ovalbumin (OVA)-sensitized mice before the last allergen challenge. OVA challenge resulted in activation of the production of inducible nitric oxide (iNOS) in lung tissue, inflammatory cytokines, recruitment of eosinophils to lung airways, and airway hyper-responsiveness to inhaled methacholine. These effects of ovalbumin challenge were all inhibited by pretreatment of mice with curcumin. Furthermore, supplementation with curcumin in the A549 human airway epithelial cells decreased iNOS and NO production induced by IFN-γ. These findings show that curcumin may be useful as an adjuvant therapy for airway inflammation through suppression of iNOS and NO.     

Gene transfer of inducible nitric oxide synthase complementary DNA regresses the fibrotic plaque in an animal model of Peyronie's disease

The goal of the present study was to investigate the antifibrotic role of inducible nitric oxide synthase (iNOS) in Peyronie's disease (PD) by determining whether a plasmid expressing iNOS (piNOS) injected into a PD-like plaque can induce regression of the plaque. A PD-like plaque was induced with fibrin in the penile tunica albuginea of mice and then injected with a luciferase-expressing plasmid (pLuc), either alone or with piNOS, following luciferase expression in vivo by bioluminescence imaging. Rats were treated with either piNOS, an empty control plasmid (pC), or saline. Other groups were treated with pC or piNOS, in the absence of fibrin. Tissue sections were stained for collagen, transforming growth factor (TGF) beta1, and plasminogen-activator inhibitor (PAI-1) as profibrotic factors; copper-zinc superoxide dismutase (CuZn SOD) as scavenger of reactive oxygen species (ROS); and nitrotyrosine to detect nitric oxide reaction with ROS. Quantitative image analysis was applied. Both iNOS and xanthine oxido-reductase (XOR; oxidative stress) were estimated by Western blot analysis. Luciferase reporter expression was restricted to the penis, peaked at 3 days after injection, but continued for at least 3 wk. In rats receiving piNOS, iNOS expression also peaked at 3 days, but expression decreased at the end of treatment, when a considerable reduction of plaque size occurred. Protein nitrotyrosine, XOR, and CuZn SOD increased, and TGFbeta1 and PAI-1 decreased. The piNOS gene transfer regressed the PD plaque and expression of profibrotic factors, supporting the view that endogenous iNOS induction in PD is defense mechanism by the tissue against fibrosis.     

Ebselen suppresses late airway responses and airway inflammation in guinea pigs

Although ebselen, a seleno-organic compound, inhibits inflammation in various animal models, its efficacy as an anti-asthma drug remains to be clarified. In this study, we investigated the inhibitory effect of ebselen on a guinea pig asthma model. Ebselen was orally administered at dosages of 1-20 mg/kg 2 h before an ovalbumin (OA) challenge, and then airway responses, airway inflammation, the generation of superoxide, H(2)O(2), and nitrotyrosine, and the induction of inducible nitric oxide synthase (iNOS) were evaluated. Sensitized animals challenged with OA aerosol showed dual airflow limitations, i.e., immediate and late airway responses (IAR and LAR). Ebselen significantly inhibited LAR at dosages greater than 10 mg/kg, but did not inhibit IAR at any dosage. Bronchoalveolar lavage (BAL) examination showed that airway inflammation was significantly suppressed by ebselen at 10 mg/kg. The generation of superoxide and H(2)O(2) occurred on endothelial cells of LAR bronchi, and was inhibited by 10 mg/kg of ebselen. Superoxide generation was inhibited by diphenyleneiodonium chloride (DPI), a NAD(P)H oxidase inhibitor, but not by allopurinol, a xanthine oxidase inhibitor. Immunoreactivities for iNOS and nitrotyrosine were also observed on endothelial cells of LAR bronchi and were abolished in ebselen-treated animals. The present findings suggest that ebselen can be applied as a new therapeutic agent for asthma. The possible mechanisms by which ebselen inhibits LAR likely involve suppression of oxidant formation and iNOS induction in endothelial cells.     

Inhalation of sphingosine kinase inhibitor attenuates airway inflammation in asthmatic mouse model

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.     

姜黄素对脂多糖激活的小胶质细胞iNOS 表达的抑制及抗氧化作用

用姜黄素预处理小胶质细胞株BV,1 h后加用脂多糖(200 ng/ml)进行刺激,通过MTT检测细胞活性;硝酸还原酶法检测细胞上清液中一氧化氮(NO)的含量;Western 印迹、免疫细胞化学染色检测细胞活化后形态及诱导型一氧化氮合酶(iNOS)蛋白的表达;瞬时转染和荧光素酶报告基因鉴定iNOS和NF-κB基因表达活性;SOD和GSH-Px检测姜黄素的抗氧化能力.结果证明,脂多糖可促使小胶质细胞活化,使iNOS和NF-κB基因表达活性显著增强;iNOS蛋白表达明显上调,NO释放增多;细胞内SOD和GSH-Px活性明显下降.而姜黄素(10 μmol/L)可以显著抑制活化后小胶质细胞NO的产生、iNOS蛋白的表达及iNOS-Luc、NF-κB-Luc的表达活性,其机制可能通过NF-κB的信号转导途径抑制iNOS的表达.姜黄素可通过提高细胞内SOD和GSH-Px的活性发挥抗氧化作用.     

Nitric oxide confers cisplatin resistance in human lung cancer cells through upregulation of aldo-keto reductase 1B10 and proteasome

Abstract

In this study, we show that exposure of human lung cancer A549 cells to cisplatin (cis-diamminedichloroplatinum, CDDP) promotes production of nitric oxide (NO) through generation of reactive oxygen species (ROS) and resulting upregulation of inducible NO synthase (iNOS). The incubation of the cells with a NO donor, diethylenetriamine NONOate, not only reduced the CDDP-induced cell death and apoptotic alterations (induction of CCAAT-enhancer-binding protein homologous protein and caspase-3 activation), but also elevated proteolytic activity of 26S proteasome, suggesting that the activation of proteasome function contributes to the reduction of CDDP sensitivity by NO. Monitoring expression levels of six aldo-keto reductases (AKRs) (1A1, 1B1, 1B10, 1C1, 1C2, and 1C3) during the treatment with the NO donor and subsequent CDDP sensitivity test using the specific inhibitors also proposed that upregulation of AKR1B10 by NO is a key process for acquiring the CDDP resistance in A549 cells. Treatment with CDDP and NO increased amounts of nitrotyrosine protein adducts, indicative of peroxynitrite formation, and promoted the induction of AKR1B10, inferring a relationship between peroxynitrite formation and the enzyme upregulation in the cells. The treatment with CDDP or a ROS-related lipid aldehyde, 4-hydroxy-2-nonenal, facilitated the iNOS upregulation, which was restored by increasing the AKR1B10 expression. In contrast, the facilitation of NO production by CDDP treatment was hardly observed in AKR1B10-overexpressing A549 cells and established CDDP-resistant cancer cells (A549, LoVo, and PC3). Collectively, these results suggest the NO functions as a key regulator controlling AKR1B10 expression and 26S proteasome function leading to gain of the CDDP resistance.     

Adenovirus infection increases iNOS and peroxynitrite production in the lung

Host inflammatory and immune responses limit viral gene expression after administration of replication-deficient adenoviruses to the lung. The current study asks whether inducible nitric oxide synthase (iNOS) expression and peroxynitrite generation accompanied the inflammatory response following intratracheal administration of adenovirus. Pulmonary iNOS mRNA and protein were increased 2, 7, and 14 days following administration of 2 x 10(9) plaque-forming units of recombinant adenovirus (Av1Luc1) to BALB/c mice. Adenovirus infection was associated with a marked increase in nitrotyrosine staining. Intense nitrotyrosine staining was observed in alveolar macrophages, respiratory epithelial cells, conducting airways, and alveolar spaces 2 days postinfection. Two weeks after exposure to adenovirus, nitrotyrosine staining was detected within alveolar macrophages, suggesting adenovirus enhanced the nitration of proteins that were subsequently taken up by alveolar macrophages. Western blot analysis using anti-nitrotyrosine antibody did not demonstrate accumulation of nitrated surfactant protein A (SP-A), although a small fraction of aggregated SP-A comigrated with a nitrotyrosine-positive protein. iNOS expression, peroxynitrite, and nitrotyrosine generation accompany and may contribute to inflammatory responses to adenovirus in the lung.     

Endogenous hydrogen sulfide reduces airway inflammation and remodeling in a rat model of asthma

Endogenous hydrogen sulfide (H₂S) is hypothesized to have an important role in systemic inflammation. We investigated if endogenous H₂S may be a crucial mediator in airway inflammation and airway remodeling in a rat model of asthma and if endogenous H₂S may exert its anti-inflammatory effect by inhibiting inducible nitric oxide synthase (iNOS)/NO pathway. Cystathionine-γ-lyase (CSE; a H₂S-synthesizing enzyme) was mainly expressed in airway and vascular smooth muscle cells in rat lung tissue. Levels of endogenous H₂S was decreased in pulmonary tissue in ovalbumin (OVA)-treated rats. Exogenous administration of NaHS alleviated airway inflammation and airway remodeling: peak expiratory flow (PEF) increased, goblet cell hyperplasia and collagen deposition score decreased, with decreased total cells recovered from bronchoalveolar fluid (BALF) and influx of eosinophils and neutrophils. The H₂S levels of serum and lung tissue were positively correlated with PEF and negatively correlated with the level of eosinophils and neutrophils in BALF, score of lung pathology. NaHS treatment significantly attenuated pulmonary iNOS activation in OVA-treated rats. These results suggest that the CSE/H₂S pathway plays an anti-inflammatory and anti-remodeling part in asthma pathogenesis and could be a novel target in prevention and treatment of asthma.     

Cannabidiol attenuates high glucose-induced endothelial cell inflammatory response and barrier disruption

A nonpsychoactive cannabinoid cannabidiol (CBD) has been shown to exert potent anti-inflammatory and antioxidant effects and has recently been reported to lower the incidence of diabetes in nonobese diabetic mice and to preserve the blood-retinal barrier in experimental diabetes. In this study we have investigated the effects of CBD on high glucose (HG)-induced, mitochondrial superoxide generation, NF-kappaB activation, nitrotyrosine formation, inducible nitric oxide synthase (iNOS) and adhesion molecules ICAM-1 and VCAM-1 expression, monocyte-endothelial adhesion, transendothelial migration of monocytes, and disruption of endothelial barrier function in human coronary artery endothelial cells (HCAECs). HG markedly increased mitochondrial superoxide generation (measured by flow cytometry using MitoSOX), NF-kappaB activation, nitrotyrosine formation, upregulation of iNOS and adhesion molecules ICAM-1 and VCAM-1, transendothelial migration of monocytes, and monocyte-endothelial adhesion in HCAECs. HG also decreased endothelial barrier function measured by increased permeability and diminished expression of vascular endothelial cadherin in HCAECs. Remarkably, all the above mentioned effects of HG were attenuated by CBD pretreatment. Since a disruption of the endothelial function and integrity by HG is a crucial early event underlying the development of various diabetic complications, our results suggest that CBD, which has recently been approved for the treatment of inflammation, pain, and spasticity associated with multiple sclerosis in humans, may have significant therapeutic benefits against diabetic complications and atherosclerosis.     

Immunohistochemical Artifact for Nitrotyrosine in Eosinophils or Eosinophil Containing Tissue

Immunohistochemical artifacts for nitrotyrosine were investigated in eosinophils with regard to fixatives. Immunoreactivity for nitrotyrosine was revealed in separated eosinophils and in gastric mucosa fixed with periodate, lysine-paraformaldehyde (PLP). The increase in immunoreactivity by PLP was due to periodate itself, a component of PLP. Nitrotyrosine formed by peroxidase using NO 2 &#109 and H 2 O 2 or by peroxynitrite was not completely inhibited by 100 mM dithionite but the immunoreactivity for nitrotyrosine antibodies by PLP was completely inhibited by 5.7 mM dithionite. Although untreated eosinophils or ovalbumin (OVA) did not show protein tyrosine nitration in a standard Western blot, the treatment of the blotted membrane with PLP increased the reactivities of proteins from eosinophils with anti-nitrotyrosine antibodies. The increase in immunoreactivity of OVA with anti-nitrotyrosine antibodies by PLP did not change with pre-treatment with dithionite but was abolished by treatment with dithionite after PLP fixation. In HPLC assays, periodate did not generate nitrotyrosine from l -tyrosine and aminotyrosine. These results suggest that the treatment of eosinophils or eosinophil-containing tissues with PLP fixative augments the immunoreactivity of nitrotyrosine antibodies with eosinophils due to the formation of epitopes similar to nitrotyrosine by an oxidation reaction of periodate, which evokes an artifact in nitrotyrosine immunohistochemistry.     

Splenic dendritic cells induced by oral antigen administration are important for the transfer of oral tolerance in an experimental model of asthma

Peripheral tolerance can be induced after the feeding of Ag, which is referred to as oral tolerance. We demonstrated in this study that the oral administration of OVA induced tolerance in an experimental model of asthma in mice, and investigated which cells function as the regulatory cells in the transfer of this oral tolerance. In OVA-fed mice, the percentage of eosinophils in bronchoalveolar lavage fluid, serum IgE levels, airway hyperresponsiveness, and mRNA levels of IL-13 and eotaxin were significantly lower than found in nonfed mice. Histological examination of lung tissue showed a suppression of the accumulation of inflammatory cells in the peribronchial area of OVA-fed mice. Feeding after the first immunization or between the first and the second immunization suppressed these findings, whereas feeding just before the airway Ag challenge did not. The suppression of disease in OVA-fed mice was successfully transferred by injection of whole spleen cells of OVA-fed mice. When CD11c+ dendritic cells (DCs) were removed from splenocytes, this transfer of suppression was completely abolished. The injection of splenic DCs purified from OVA-fed mice alone transferred the suppression, whereas the injection of splenic DCs from naive mice that were cocultured with OVA in vitro did not. These data suggest that not only CD4+ T cells, but also CD11c+ DCs induced by Ag feeding are important for the active transfer of oral tolerance in this murine experimental model of asthma.