Article

N-acetylcysteine exerts therapeutic action in a rat model of allergic rhinitis

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Abstract

Background: The pathophysiologic mechanism of allergy is dependent on the action of many redox-sensitive proinflammatory mediators. However, even though redox disturbances are believed to be a hallmark of inflammation, little is known of the effect of redox imbalance to the pathophysiology of allergic rhinitis. We thus opted to investigate the relation of oxidative stress and allergic rhinitis, through the utilization of a potent antioxidant substance (N-acetylcysteine [NAC]) in a rat model of allergic rhinitis and the evaluation of its action on specific markers of inflammation. Methods: NAC (50 mg/kg and 250 mg/kg) was intraperitoneally administered to ovalbumin (OVA)-sensitized rats prior to intranasal challenge with OVA. Mucosal congregation of inflammatory cells (eosinophils and mast cells), mucosal expression of redox-sensitive enzymes (inducible nitric oxide synthase [iNOS] and cyclooxygenase 2 [COX-2]), and the blood levels of a key proinflammatory mediator (tumor necrosis factor-α [TNF-α]) were evaluated. Results: Intranasal OVA challenges lead to mucosal inflammation, induction of the mucosal expression of iNOS and COX-2 and elevation of TNF-α blood levels. NAC significantly inhibited accumulation of inflammatory cells and downregulated iNOS expression and TNF-α serum levels. The role of COX-2 appeared to be 2-fold and its expression was divergently modulated by NAC. Conclusion: Our findings suggest that redox balance is involved in the pathophysiology of allergic rhinitis in rats and that NAC can potentially suppress the allergen-induced nasal inflammatory cascade. The investigation of the role of oxidative stress in atopy could help in the evaluation of the therapeutic potential of antioxidant substances in allergic diseases.

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... After a 10-min adaptation period, rats were observed over 10 min and nasal symptoms, including sneezing, itching, and rhinorrhea, were rated on a 0-3 point scale (Fig. 1a) [13]. Allergic rhinitis scores were recorded on days 15,16,18,20,21, and 22 (Fig. 2). The allergic rhinitis model was considered to be successful when the total score exceeded five points. ...
... Several allergens, such as ovalbumin, pollen from Cryptomeria japonica (Sugi tree), toluene 2-4-diisocyanate (TDI), Schistosoma mansoni egg antigen (SEA), and Ascaris suum extract antigen (ASA), have been used to develop animal models of allergic rhinitis [14][15][16][17]. For immunization with antigen, ovalbumin has been given with an adjuvant such as aluminum via an intraperitoneal route followed by an intranasal route at different doses and times, and increased inflammatory cells and eosinophils were observed in the nasal mucosa of subjects given ovalbumin in histopathological evaluation [18,19]. In our study, histopathological findings of allergic rhinitis, such as increased inflammation, eosinophil count, vasodilatation, dilatation of secretory ducts of serous glands, and edema, were detected in the control group receiving ovalbumin alone in histopathological evaluations. ...
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We aimed to investigate whether quercetin had a therapeutic effect in an experimental rat model of allergic rhinitis. The study was conducted with 35 rats, which were randomly assigned into 4 groups: group 1 (n = 5), sham group; group 2 (quercetin group, n = 10) received 80 mg/kg day quercetin; group 3 (steroid group, n = 10) received steroid (mometasone furoate); and group 4 (control group, n = 10), received ovalbumin alone. Rats were sensitized by administration of ovalbumin on alternate days over 14 days via an intraperitoneal route. On day 15, in addition to ovalbumin via an intranasal route, quercetin and steroid were given over 7 days to the corresponding groups. All rats were then sacrificed and nasal turbinates were evaluated histopathologically, and serum total IgE and ovalbumin (OVA)-specific IgE values were measured before and after treatment. A significant increase in OVA-specific IgE values was detected in all groups except sham group. A significant increase was detected in post-treatment total IgE levels in the control group, while no significant change was detected in the sham, quercetin, and intranasal steroid groups. On histopathological evaluation, it was observed that findings of allergic rhinitis were suppressed in the quercetin group when compared to the control group. In immunohistochemical evaluation, it was detected that COX-2 and VIP expressions were weaker in the quercetin group compared to the control group. Based on these findings, we conclude that quercetin was effective in allergic rhinitis induced by ovalbumin in rats both histopathologically and serologically.
... All guinea pigs were acclimated for seven days and divided into normal (n � 10) and model groups (n � 20) according to the random number table method. e model group was replicated into AR models using ovalbumin (OVA) (Sigma, USA) sensitization [8]. (1) Sensitization: each guinea pig was intraperitoneally injected with 1 ml suspension containing 0.5 mg/ml OVA + 30 mg/ml aluminum hydroxide (Sigma, USA), once every other day for seven times to make the guinea pig systemic sensitized; (2) challenge: on the 4th day after the completion of the sensitization, the guinea pigs were held up heads and challenged by dropping 50 μl of 2% OVA solution into each nostril, once every other day for five times; (3) modeling effect evaluation according to the concentration of serum IgE (guinea pig IgE ELISA Kit, Shanghai Enzymelinked, China) and National Allergic Rhinitis Diagnostic and Efficacy Evaluation Standards: within 30 minutes after each nasal challenge, the number of sneezes, nose scratching, and nasal discharge were observed and sneezing of 3∼9 times was scored as 1 point; 10∼14 times, 2 points; ≥15 times, 3 points; nose scratching of 2∼3 times was scored as 1 point; 4∼5 times, 2 points; ≥5 times, 3 points; runny nose to the nostrils was scored as 1 point, over the nostrils, 2 points; to all the faces, 3 points. ...
... AR: allergic rhinitis; STAT5: signal transduction and activator of transcription 5; TGF-β1: transforming growth factor-β1; XGND: Xingbi gel nasal drops. 8 Evidence-Based Complementary and Alternative Medicine point, suggesting that the two drugs interfere with AR fibroblasts in a similar regulatory pathway. e results showed that XGND could reduce Fyn and SCF expression and hinder FcεRI receptor cross-linking with treatment for 24 h, inhibit proliferation and degranulation of mast cells, and reduce the release of histamine and various inflammatory cytokines. ...
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Fyn-STAT5 is considered to be the frontier signaling pathway of IgE-mediated allergic reactions related to mast cell activation, but research on allergic rhinitis (AR) has been rarely reported. Xingbi gel nasal drops (XGND) are a compound preparation of traditional Chinese medicine, which has the exact therapeutic efficacy on AR. The current study aimed to observe the effects of XGND on Fyn-STAT5 pathway in AR guinea pig nasal mucosal fibroblasts in vitro and further illuminate the possible therapeutic mechanism of XGND on AR. The isolated and cultured nasal mucosa fibroblasts from AR guinea pigs were identified by immunocytochemical staining. Real-time PCR and western blot were performed to detect the mRNA and protein levels of the Fyn-STAT5 pathway and related cytokines in AR guinea pig nasal mucosal fibroblasts. The results indicated that XGND may interfere with the Fyn-STAT5 pathway by reducing the expression of Fyn and SCF and upregulating STAT5 and IL-10, thereby inhibiting proliferation and degranulation of mast cells, correcting Th1/Th2 immune imbalance, and then alleviating the immune response of AR fibroblasts. Our study revealed the possible regulatory mechanism of XGND in AR and laid an experimental foundation for improving the clinical efficacy of AR and enriching the clinical medication for AR.
... For these reasons, to confirm allergic inflammation in nasal epithelium, we decided to measure iNOS gene expression. This approach does not allow to verify structural changes in nasal epithelium upon HDM exposition, but it was shown previously (Guibas et al., 2013), that increased iNOS expression accompanied mucosal inflammation in histopathological staining thus confirming allergic inflammation in nasal epithelium in the rat model. ...
Article
Introduction Atopic asthma and allergic rhinitis are common chronic inflammatory diseases affecting lower airways and nasal mucosa, respectively. Several reports demonstrated frequent co-occurrence of these two diseases, however, the exact molecular mechanism has not been described. The present study aimed to investigate if small non-coding RNA might be responsible for the co-occurrence of asthma and allergic rhinitis in an animal model of allergic airway inflammation. Materials and methods As an in vivo model of allergic airway inflammation, we used Brown Norway rats exposed intranasally to house dust mite (HDM). Histological analysis, total IgE concentration, eosinophil counts and iNOS gene expression were determined to confirm inflammatory changes. Small RNA sequencing in the lung tissue and nasal epithelium was performed with TruSeq Small RNA Library Preparation Kit and analyzed using the BaseSpace tool. Validation of sequencing results was performed using qPCR. To assess the functional role of hsa-miR-223–3p, we transfected normal human bronchial epithelial (NHBE) cells with specific LNA-inhibitor and measured phosphorylated protein level of NF-kB with ELISA. Expression analysis of NF-kB pathway-related genes was performed using qPCR with SYBR Green and analyzed in DataAssist v3.01. Statistical analysis were done with STATISTICA version 13. Results We found 9 miRNA genes differentially expressed in the lungs of allergic rats. In nasal epithelium, only rno-miR-184 was upregulated in animals exposed to HDM. Validation with qPCR confirmed increased expression only for rno-miR-223–3p in the lungs from allergic rats. The expression of this miRNA was also increased in normal bronchial epithelial ALI cell culture stimulated with IL-13, but not in cells cultured in monolayer due to the low mRNA level of IL13RA1 and IL13RA2. Transfecting NHBE cells with hsa-miR-223–3p inhibitor increased the amount of phosphorylated NF-kB protein level and expression of MUC5AC, CCL24 and TSLP genes. Conclusions These findings suggest that miRNAs that regulate allergic inflammation in the lungs and nasal epithelium are specific for upper and lower airways. Furthermore, our study provides new insight on the role of hsa-miR-223–3p, that via targeting NF-kB signaling pathway, regulates the expression of MUC5AC, CCL24 and TSLP. Taken together, our study suggests that miR-223–3p is a regulator of allergic inflammation and could potentially be used to develop novel and targeted therapy for asthma.
... NAC has been shown to reduce the allergen-induced nasal inflammatory cascade in allergic rhinitis in animal models. [41]. Topical application of NAC prior to ragweed exposure resulted in attenuation of the late phase allergic response mediated nasal symptoms [42]. ...
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Objective: To review the clinical usefulness of N-acetylcysteine (NAC) as treatment or adjunctive therapy in a number of medical conditions. Use in Tylenol overdose, cystic fibrosis, and chronic obstructive lung disease has been well documented, but there is emerging evidence many other conditions would benefit from this safe, simple, and inexpensive intervention. Quality of Evidence. PubMed, several books, and conference proceedings were searched for articles on NAC and health conditions listed above reviewing supportive evidence. This study uses a traditional integrated review format, and clinically relevant information is assessed using the American Family Physician Evidence-Based Medicine Toolkit. A table summarizing the potential mechanisms of action for N-acetylcysteine in these conditions is presented. Main Message. N-acetylcysteine may be useful as an adjuvant in treating various medical conditions, especially chronic diseases. These conditions include polycystic ovary disease, male infertility, sleep apnea, acquired immune deficiency syndrome, influenza, parkinsonism, multiple sclerosis, peripheral neuropathy, stroke outcomes, diabetic neuropathy, Crohn's disease, ulcerative colitis, schizophrenia, bipolar illness, and obsessive compulsive disorder; it can also be useful as a chelator for heavy metals and nanoparticles. There are also a number of other conditions that may show benefit; however, the evidence is not as robust. Conclusion: The use of N-acetylcysteine should be considered in a number of conditions as our population ages and levels of glutathione drop. Supplementation may contribute to reducing morbidity and mortality in some chronic conditions as outlined in the article.
... 125 Nacetylcysteine maintains a potent antioxidant effect in in vitro studies 98 or in ovalbuminsensitized rats by downregulating tumor necrosis factor-alpha in recruited inflammatory cells. 126 Along these lines, intranasal steroids can exhibit an exogenous antioxidant regulatory role in seasonal AR by decreasing exhaled breath condensates of leukotriene B4 and 8-Isoprostane, although no effect was seen on exhaled carbon monoxide and nitrogen oxide. 106 In another study involving children with AR and asthma, no effect of topical nasal steroid therapy was noted on measured lipid peroxidation oxidative stress biomarkers and antioxidant enzymes. ...
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Air pollution causes significant morbidity and mortality in patients with inflammatory airway diseases (IAD) such as allergic rhinitis (AR), chronic rhinosinusitis (CRS), asthma, and chronic obstructive pulmonary disease (COPD). Oxidative stress in patients with IAD can induce eosinophilic inflammation in the airways, augment atopic allergic sensitization, and increase susceptibility to infection. We reviewed emerging data depicting the involvement of oxidative stress in IAD patients. We evaluated biomarkers, outcome measures and immunopathological alterations across the airway mucosal barrier following exposure, particularly when accentuated by an infectious insult.
... There was no significant difference between the three groups. Guibas et al. [42] showed that NAC significantly inhibited the accumulation of inflammatory cells and down regulated iNOS expression in a rat model of allergic rhinitis. The effect of NAC on NOS can be attributed mostly to the inhibitory effect of NAC on one of the events leading to iNOS protein expression [43]. ...
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Background: Antinociceptive anti-inflammatory drugs have many adverse effects. The goal of this investigation is to study the probable anti-inflammatory and analgesic effects of verapamil and N-acetylcysteine (NAC) in experimental rats. Methods: Adult male Wistar rats were randomly divided into 4 groups in the antinociceptive study, each containing 6 rats; the normal control group, which received saline (1 mL/kg); the diclofenac group, which received diclofenac sodium (5 mg/kg); the NAC group, which received NAC (125 mg/kg); and the verapamil group, which received verapamil (8 mg/kg). In the anti-inflammatory study, 5 groups were used, the 4 previous groups with the addition of an edema control group, received saline and were subjected to formalin test. Hot plate latency time was recorded for antinociceptive evaluation. Paw edema thickness and biochemical parameters were recorded for anti-inflammatory evaluation. Results: Administration of NAC showed significant prolongation of hot plate latency time at 1 hour when compared to the control group while verapamil showed a significant prolongation of hot plate latency time at 1 and 2 hours when compared to the control group and NAC group values. Administration of NAC and verapamil significantly decreased paw edema thickness at 2, 4, and 8 hours when compared to edema control values. Regarding biochemical markers, NAC and verapamil significantly decreased serum nitric oxide synthase, C-reactive protein, and cyclooxygenase- 2 levels compared to the edema control value. In accordance, a marked improvement of histopathological findings was observed with both drugs. Conclusions: NAC and verapamil have antinociceptive and anti-inflammatory effects comparable to diclofenac sodium.
... 8 Infiltration of the nasal mucosa with eosinophils and other inflammatory cells and pathological changes are characteristic features of an allergic reaction due to aller- gen-organism interaction. 7,[30][31][32] Histopathological examination is an objective analysis that can be used to determine anti-inflammatory activity of drugs. 31 In our study, histopathological findings of AR such as increased inflammation, mucosal edema, glandular hyperplasia, vascular dilatation, and eosinophil infiltra- tion in the lamina propria and loss of cilia, vacuoliza- tion, and dilatation in the pseudostratified columnar epithelium on the surface of the nasal mucosa were detected in the AR model group (Figure 4(b)). ...
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Background Mometasone furoate, one of the second generation intranasal corticosteroids, is currently used in suspension form due to its poor solubility. However, this is not favorable for nasal application because of the rapid elimination of the instilled drug from the nasal cavity by mucociliary clearance and delayed onset of action due to the slow dissolution of drug in suspension. Objective The aim of this study was to determine the antiallergic effects of mucoadhesive thermosensitive in situ gel containing mometasone furoate that we developed previously to prolong the contact between the drug and nasal mucosa and to prevent drainage of the formulation in an ovalbumin-induced rat model of allergic rhinitis. Methods An experimental allergic rhinitis model was developed in female Wistar albino rats by intraperitoneal injection of ovalbumin every 2 days for 14 days followed by its repeated intranasal instillation for 7 consecutive days. Intranasal instillation of ovalbumin was continued every other day for 14 days. Mometasone furoate in situ gel (5 μg/10 µl), mometasone furoate suspension (5 μg/10 µl), and physiological saline (10 µl) were administered into the bilateral nasal cavities from day 22 to day 35. Antiallergic effects were evaluated through histopathological evaluation, analysis of ovalbumin-specific serum immunoglobulin E, and a symptom score. Results Mometasone furoate in situ gel significantly decreased the nasal symptoms and ovalbumin-specific serum immunoglobulin E level as compared with mometasone furoate suspension and physiological saline. Additionally, inflammatory histological symptoms such as mucosal edema, vascular dilatation, eosinophil infiltration, and loss of cilia within the nasal mucosa of allergic rhinitis model rats were remarkably improved with the treatment of mometasone furoate in situ gel. Conclusion These results suggest that mometasone furoate in situ gel has a better therapeutic potential for the treatment of allergic rhinitis compared to mometasone furoate suspension.
... Accordingly, in a previous work of ours, we have used 10% EDTA solution for 15 days to decalcify nasal bone of 11 w.o. rats ( Guibas et al., 2013); although we were able to efficiently conduct the analyses in this work, tissue sectioning and mounting on glass slides was cumbersome. This provided the impetus for the present study in which we assessed the decalcifying efficiency and potential adverse effects of 20 and 40 days of EDTA. ...
Article
Introduction: Decalcification of osseous specimens is required for histological analysis; this however may cause tissue damage. In rodent models of allergic rhinitis (AR), epithelial histologic assessment necessitates prior decalcification of the nasal osseous structures. However, respiratory epithelium is highly susceptible to damage, and rat nasal architecture is elaborate and its sectioning is challenging. Nevertheless, decalcification is not standardized in experimental AR. We therefore undertook this task, in order to reduce experimental bias. Methods: Six-to-eight week-old Wistar rats underwent an AR protocol. Subsequently, nasal structures were decalcified in the following mediums: (i) formic acid 10% for 5 and 20 days; (ii) formic acid 15% for 5 and 15 days; (iii) Morse Solution for 5 and 20 days and (iv) EDTA for 20 and 40 days. Decalcification efficiency/speed was evaluated via radiographic analysis. Furthermore, specimens were stained with hematoxylin and eosin and assessed for preservation of epithelial features. Results: Specimens were appropriately decalcified in 5 days in the formic acid-based mediums and in 20 days in EDTA with minimal epithelial damage. EDTA for 40 days had no unacceptable adverse effects; conversely, 15 and/or 20 days in acid-based agents provided no extra benefit for decalcification and were detrimental to the epithelium. Conclusions: EDTA treatment for 20 days is appropriate for decalcification of nasal structures in rat models of allergic rhinitis; further incubation preserves epithelial integrity but is not required. When urgency is a factor, formic-acid-based decalcification for 5 days yields acceptable results.
Objective We investigate whether lycopene has a protective effect in an experimental rat model of allergic rhinitis. Methods Experimental animals (65 rats) were randomized to 7 groups (Sham-Control, Lycopene 10mg/kg/day, Lycopene 20mg/kg/day, Intranasal lycopene drops, Intranasal steroid, Corn oil, Allergic Rhinitis). Rats were sensitized by administering of ovalbumin intraperitoneally and intranasally. In addition to ovalbumin; lycopene, corn oil and steroids were given to the relevant groups. Nasal symptom scores of the rats were recorded throughout the study. At end of the study, after intracardiac blood sample collection, all rats were sacrified, and nasal tissues were examined histopathologically. Serum total immunoglobulin E (IgE) and ovalbumin (OVA) specific IgE were studied from all rats before and after the study. Results There was a statistically significant increase (p < 0.05) in OVA specific IgE values measured before and after the study in all groups except the sham group. In serum total IgE values; there was a statistically significant increase after treatment in allergic rhinitis, corn oil, lycopene 10mg and intranasal lycopene drops group, but other groups did not show any significant change. Histopathological study with hematoxylin-eosin staining and cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), vasoactive intestinal peptide (VIP) expression found that lycopene suppresses inflammation with both nasal administration and increased dose. Nasal symptom scores were observed to decrease significantly in all lycopene and steroid groups compared to allergic rihinits and corn groups. Conclusion It was determined that lycopene were effective in the treatment of allergic rhinitis, and this effect was found to be stronger with increasing doses of lycopene.
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Ginseng (the root of Panax ginseng Meyer) has been reported to have many biologic therapeutic effects, including anti-inflammatory properties, and ginsenosides are considered as one of the factors responsible for these therapeutic effects. To improve their therapeutic action, probiotic bacteria are used to ferment and chemically transform ginsenosides in red ginseng (RG). In this study, we aimed to investigate the beneficial effects of RG fermented by probiotic bacteria (FRG) against ovalbumin (OVA)-induced allergic rhinitis in a mouse model. We induced the mouse model via OVA inhalation; experimental results revealed increased immunoglobulin E (IgE) and interleukin (IL)-4 levels, leading to Th2-type cytokine response. The mice with induced allergy were then orally administered RG and FRG over 2 weeks, as a result of which, IL-4 and IgE levels in bronchoalveolar lavage fluid, nasal fluid, and serum were found to be ameliorated more effectively by FRG than by RG, suggesting that FRG has better immune regulatory effects than RG. FRG also downregulated immune cell levels, such as those of eosinophils and basophils, and significantly decreased the thickness of OVA-induced respiratory epithelium compared to RG. Collectively, the results showed that FRG treatment alleviates inflammation, thereby extending a protective effect to mice with OVA-induced inflammatory allergic rhinitis.
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Context: Prevalence of bronchial asthma massively increases worldwide, while the frequent therapies are still not sufficient. Polydatin, a naturally occurring glycoside, was known as to have anti-inflammatory and anti-oxidant effects. Objective: The current study aimed to investigate the possible protective effect of polydatin against experimental bronchial asthma in rats. Material and methods: Bronchial asthma was induced by ovalbumin (OVA) sensitization and challenge. Rats were randomly allocated into five groups; Group I (normal control group); Group II (asthma control group) received OVA; Group III (reference standard treatment group) received dexamethasone (1 mg/kg/day); Group IV (treatment group) received polydatin (200/mg/kg); and Group V (polydatin control group). The inflammatory biomarkers interleukin-4 (IL-4), IL-5, IL-13, tumor necrosis factor-alpha, interferon-gamma and absolute eosinophil count in bronchoalveolar lavage fluid (BALF), as well as serum immunoglobulin E were assessed, coupled with the oxido-nitrative stress biomarkers malondialdehyde and glutathione reduced levels and superoxide dismutase activity in the lung tissue, besides inducible nitric oxide synthase level in BALF. Western blot analysis of surfactant-D and immunohistochemical assay of urocortin (UCN) expression in the lung was performed. Results: Polydatin significantly reduced the inflammatory mediators and restored the normal values of oxidative and nitrosative stress biomarkers. It also significantly reduced the expression of surfactant-D and UCN as compared to asthma control. The histopathological study strongly augmented the biochemical results. Discussion and conclusions: Polydatin may be a promising protective agent against experimentally induced bronchial asthma. Modulation of SP-D and UCN expressions seems to mediate such protective effects.
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Eosinophils are oxidant-sensitive cells considered relevant in allergic inflammation. The present study aimed to examine the effects of the antioxidant N-acetyl-L-cysteine (NAC) on constitutive and cytokine-delayed apoptosis in human isolated eosinophils. Human eosinophils were purified from the blood of healthy donors by a magnetic separation system. Apoptosis and cellular glutathione were assessed by cytofluorometric analysis and nuclear factor (NF)-kappaB binding activity assessed by electrophoresis mobility shift assay. The rate of spontaneous apoptosis of human eosinophils after 24 h culture, as assessed by annexin-V-positive staining, was mean+/-sem 48.2+/-1.4%, n = 5. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng.mL(-1)) decreased apoptosis to 19.4+/-1.8%, n = 5. NAC (5 mM) inhibited spontaneous apoptosis (33.6+/-2.7%, n = 5) but augmented apoptosis in the presence of GM-CSF (30.9+/-1.5%, n = 5). NAC (5 mM) also increased the rate of apoptosis in the presence of tumour necrosis factor (TNF)-alpha (10 ng.mL(-1)) and interleukin-5 (5 ng.mL(-1)). NAC (5 mM) increased eosinophil glutathione content. The increase in eosinophil NF-kappaB binding activity induced by GM-CSF and TNF-alpha was suppressed by NAC. In conclusion, N-acetylcysteine modulates eosinophil apoptosis by inhibiting constitutive apoptosis but reversing the survival effect produced by inflammatory cytokines in human eosinophils.
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We have previously shown that tumor necrosis factor-a (TNF-α) reduces interleukin-4 (IL-4)-induced Fce RII expression in human monocytes. It has been shown that TNF-α activates nuclear transcriptional factors through the generation of reactive oxygen intermediates (ROIs), and antioxidant N-acetylcysteine (NAC) inhibits TNF-α-induced activation of nuclear transcriptional factors. Therefore, we hypothesized that TNF-α-dependent reduction of IL-4-induced Fce RII expression in monocytes might be mediated through the ROIs-activated mechanism. In the present study, to test our hypothesis, we examined the effect of NAC on TNF-α-dependent reduction of IL-4-induced FcɛRII expression in human monocytes. NAC attenuated TNF-α-dependent reduction of IL-4-induced Fcɛ RII expression by attenuating TNF-a-dependent reduction of Fcɛ RII mRNA expression. Similarly, the structurally unrelated antioxidant, pyrrolidine dithiocarbamate (PDTC), also effectively attenuated this reduction. These results indicate that an ROIs-activated and antioxidant-sensitive mechanism might be involved in TNF-α-dependent reduction of IL-4-induced FcɛRII expression in monocytes.
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Conventional rodent models of respiratory allergy that employ intraperitoneal sensitization to aeroallergen plus adjuvant, have offered greatly to our current knowledge of the pathophysiology of allergic airway diseases. Notwithstanding this significant contribution, non-adjuvant aided sensitization via respiratory presentation of the allergen, is more naturally relevant and more closely mimics the human exposure. Nevertheless, in the experimental setting, primary respiratory exposure to inert antigen is likely to lead to inhalation tolerance. Inasmuch as divergent and discrepant results are often reported in experimental models employing this method of sensitization, we set out to review the relative literature and identify and discuss factors that are liable to interfere in such protocols and modify the immune response, hence leading to variable outcomes. Protocol design features (including the use of anaesthesia, the nature and dosage of the antigen and the strain/age/sex and handling of the animals) as well as environmental factors (including airborne substances, viruses and lipopolysaccharide) have been identified as key modulators of the immune response that evolves, following primary airway exposure of laboratory rodents to aeroallergen. Delineation of the effect of those factors to induction or abrogation of inhalation tolerance can have important implications in the design of both improved experimental protocols of respiratory allergy and methods to intercept sensitization to inert aeroallergens in the clinical field.
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BACKGROUND The cyclooxygenase (COX) enzyme catalyzes the formation of prostaglandins, which can affect cell proliferation and alter the response of the immune system to malignant cells. The inducible form of COX, COX-2, has been shown to be important in carcinogenesis.METHODS The authors studied COX-1 and -2 expression in 20 tumors of the lung, colon, and breast (60 total) by using commercially available monoclonal and polyclonal antibodies on formalin fixed, paraffin embedded tissue. Our evaluation also included seven carcinoma-associated colonic adenomas and 10 mammary ductal carcinomas in situ (DCIS). Quantitation of immunoreactivity was accomplished using an immunohistochemical scoring system that approximates the use of image analysis-based systems.RESULTSNinety percent of lung tumors (squamous cell carcinomas and adenocarcinomas), 71% of colon adenocarcinomas and 56% of breast tumors (DCIS and infiltrating ductal and lobular carcinomas) expressed COX-2 at a moderate to strong level, which was significantly different from the negligible expression in distant nonneoplastic epithelium (controls; P < 0.0001). Poorly differentiated histologic features were correlated with low COX-2 expression overall, especially in colon carcinomas. Among breast carcinomas, DCIS was more likely to express COX-2 than invasive carcinomas. Adenomatous colonic epithelium showed moderate COX-2 expression, as did adjacent nonneoplastic epithelium. COX-1 immunoreactivity was essentially weak to moderate in all tissues evaluated.CONCLUSIONSCOX-2 expression is upregulated in well and moderately differentiated carcinomas of the lung, colon, and breast whereas COX-1 appears to be constitutively expressed at low levels. A possible COX-2 paracrine effect is suggested by moderate immunoreactivity in adjacent nonneoplastic epithelium. Cancer 2000;89:2637–45. © 2000 American Cancer Society.
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We have studied the effects of N-acetylcysteine which is thought to have antioxidant properties, on the susceptibility of low-density lipoprotein to oxidation and on whole-blood glutathione concentrations in six healthy volunteers. N-acetylcysteine was given orally in a dosage of at 1.2 g per day for 4 weeks, followed by 2.4 g per day for a further two weeks. The susceptibility of low-density lipoprotein toin vitro Cu2+-oxidation was determined by continuously measuring the formation of conjugated dienes. Whole-blood concentrations of reduced and oxidized glutathione were also determined. N-acetylcysteine had no effect on the susceptibility of LDL to oxidation. Concentrations of vitamin E in the serum and in low-density lipoprotein were not changed. Compared with controls the concentration of glutathione in N-acetylcysteine treated subjects was reduced (−48 %) and the concentration of oxidized glutathione was higher (+80%). The GSH/GSSG-ratio, a marker of oxidative stress was 83 % lower. The results do not support the supposed antioxidative action of N-acetylcysteine. It seems more likely that N-acetylcysteine acts as a pro-oxidant in the dosage used.
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The mucolytic drug N-acetyl cysteine has been shown to release histamine from cultured mouse mast cells and from human basophils. At neutral pH the release was moderate and non-cytotoxic. If the acidity of the drug was not neutralized, this histamine release was markedly potentiated, but was then associated with a reduction in the viability of the cells. However, the high level of release could not be reproduced by simply exposing the cells to an acidic medium. The results are discussed in terms of a possible mechanism for the adverse reactions sometimes observed during N-acetyl cysteine therapy.
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Nitric oxide (NO) is present in air derived from the nasal airways. However, the precise origin and physiological role of airway-derived NO are unknown. We report that NO in humans is produced by epithelial cells in the paranasal sinuses and is present in sinus air in very high concentrations, close to the highest permissible atmospheric pollution levels. In immunohistochemical and mRNA in situ hybridization studies we show that an NO synthase most closely resembling the inducible isoform is constitutively expressed apically in sinus epithelium. In contrast, only weak NO synthase activity was found in the epithelium of the nasal cavity. Our findings, together with the well-known bacteriostatic effects of NO, suggest a role for NO in the maintenance of sterility in the human paranasal sinuses.
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Reduced glutathione (GSH), at physiological concentrations, was found to markedly alter the profile of arachidonate metabolism by prostaglandin H2 synthase. In 1 mM GSH, the constitutive (COX-1) and the mitogen inducible (COX-2) isoforms metabolized arachidonate to 12-hydroxyheptadecatrienoic acid (12-HHT) (88% and 78% of total products, respectively). Prostanoid formation was consequently reduced to only 12% (COX-1) and 19% (COX-2) of the total metabolites. The GSH-dependent production of 12-HHT was regio- and enantioselective for the 12(S)-isomer. We propose that 12(S)-HHT is formed by a GSH-dependent enzymatic cleavage of the PGH2 8,9 and 11,12 carbon-carbon bonds based on the following: (a) nonsignificant GSH-dependent formation of 12(S)-HHT during chemical decomposition of synthetic PGH2, (b) the structural similarities between the asymmetric carbons at C(12) in 12-HHT and C(15) in PGH2, (c) the GSH concentration-dependent product/precursor relationship between 12-HHT and prostanoid production, and (d) aspirin inhibition of 12-HHT formation by both enzymes. Arachidonic acid oxidation by COX-1, and not by COX-2, was inhibited by the combined presence of GSH and liver cytosol. In contrast, metabolism by neither isoform was inhibited when the cytosol was obtained from selenium-depleted animals. This is consistent with a unique, selenium dependent, cytosolic GSH peroxidase that inhibits specifically prostanoid and 12(S)-HHT formation by COX-1. These results suggest an additional role for GSH and GSH peroxidase(s) in regulating prostanoid biosynthesis. Differences between the isoforms in their sensitivities to GSH peroxidase may reflect differences in their requirements for an "initiator hydroperoxide".
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The nasal mucosa plays an important role in defense of the lung against harmful agents. It has been suggested that this is partly mediated by the production of nitric oxide (NO). We have investigated the localization of the messenger ribonucleic acids (MRNAs) for human endothelial NO synthase (Type III NOS) and inducible NO synthase (Type II NOS) and the immunoreactivities of these enzymes in human nasal mucosa by immunohistochemistry, in situ hybridization, and reduced nicotinamide adenine diphosphate (NADPH) diaphorase histochemistry. Inferior nasal turbinates were obtained from 27 patients at the time of surgery for local disease. Strong immunostaining for Type III NOS was localized to vascular endothelium, surface epithelium, and submucosal glands in all subjects. Moderate immunostaining for Type II NOS was seen in surface epithelium; glandular, inflammatory, and vascular endothelial cells; and smooth-muscle cells in the specimens from patients with chronic rhinitis only. In situ hybridization showed expression of the mRNA for Type III NOS in similar sites to those shown by immunohistochemistry, whereas the mRNA for Type II NOS was predominantly localized to inflammatory cells. The sites of NOS expression were further confirmed by NADPH histochemical staining. These findings demonstrate the cellular expression of NOS in the human nasal mucosa and suggest a possible role for Types II and III NO synthase in the regulation of blood flow, nasal secretion, and ciliary movement in health and disease.
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We hypothesized that endotoxin injection in rats would stimulate in vivo nuclear factor-kappa B (NF-kappa B) activation in lung tissue and that antioxidant treatment before endotoxin injection would attenuate endotoxin-induced NF-kappa B activation, chemokine gene expression, and neutrophilic lung inflammation. We studied NF-kappa B activation in rat lung tissue following a single i.p. injection of endotoxin (6 mg/kg). After in vivo endotoxin treatment, lung NF-kappa B activation peaked at 2 h and temporally correlated with the expression of cytokine-induced neutrophil chemoattractant mRNA in lung tissue. Treatment with the antioxidant N-acetylcysteine (NAC) 1 h before endotoxin resulted in decreased lung NF-kappa B activation in a dose-dependent manner (from 200-1000 mg/kg) and diminished cytokine-induced neutrophil chemoattractant mRNA expression in lung tissue. Treatment with NAC significantly suppressed endotoxin-induced neutrophilic alveolitis. The average total lung lavage neutrophil count was 5.5 x 10(6) with endotoxin treatment vs 0.9 x 10(6) with NAC treatment before endotoxin. The NF-kappa B pathway represents an attractive therapeutic target for strategies to control neutrophilic inflammation and lung injury.
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The diversity of application of the thiol drug NAC in both the experimental setting, as a tool for the study of the mechanisms and consequences of oxidative stress, and the clinical setting, as a therapeutic agent, clearly reflects the central role played by the redox chemistries of the group XVI elements, oxygen and sulfur, in biology. As our understanding of such redox processes increases, particularly their roles in specific pathophysiological processes, new avenues will open for the use of NAC in the clinical setting. As a drug, NAC represents perhaps the ideal xenobiotic, capable of directly entering endogenous biochemical processes as a result of its own metabolism. Thus, it is hoped that the experience gained with this unique agent will help in future efforts to design antioxidants and chemoprotective principles which are able to more accurately utilize endogenous biochemical processes for cell- or tissue-specific therapy.
Article
Nitric oxide (NO) plays a major role in the regulation of vascular tone and in non-specific host defence. The epithelium in the paranasal sinuses was recently identified as the major site of NO production in the upper airways. To investigate NO status in allergic rhinitis, we compared the NO concentration in the nasal cavities of control subjects (n = 19) and in patients with allergic rhinitis (n = 36) with symptoms (WS, n = 17) or without symptoms (WOS, n = 19) on the day of the test. NO concentration was measured using a chemiluminescent analyser aspiring from each nasal cavity at a sampling flow rate of 0.7 L/min, before and 10 min after administration of a nasal vasoconstrictor. The mean NO concentration (+/- SE) in the control was 235 +/- 11 ppb and 225 +/- 9 ppb in the right and left nostrils respectively, and was decreased by 14% and 12% by the nasal vasoconstrictor (P < 0.001). The NO concentration in patients with allergic rhinitis was significantly higher in the right and left nostrils (382 +/- 20 ppb and 396 +/- 28 respectively, P < 0.0001 versus control). All WOS patients demonstrated normal or increased NO concentrations in both nostrils, whereas two WS patients showed decreased NO concentrations in the left nostril. Inhalation of a nasal vasoconstrictor increased NO concentration by 6% and 27% in the right and left nostrils respectively in WS patients. Nasal NO concentration is increased in patients with allergic rhinitis. Interestingly, patients without symptoms on the day of the test also showed a clear-cut increase in nasal NO production, which could reflect a permanent inflammation of the sinus mucosa.
Article
We have previously shown that tumor necrosis factor-alpha (TNF-alpha) reduces interleukin-4 (IL-4)-induced Fc epsilon RII expression in human monocytes. It has been shown that TNF-alpha activates nuclear transcriptional factors through the generation of reactive oxygen intermediates (ROIs), and antioxidant N-acetylcysteine (NAC) inhibits TNF-alpha-induced activation of nuclear transcriptional factors. Therefore, we hypothesized that TNF-alpha-dependent reduction of IL-4-induced Fc epsilon RII expression in monocytes might be mediated through the ROIs-activated mechanism. In the present study, to test our hypothesis, we examined the effect of NAC on TNF-alpha-dependent reduction of IL-4-induced Fc epsilon RII expression in human monocytes. NAC attenuated TNF-alpha-dependent reduction of IL-4-induced Fc epsilon RII expression by attenuating TNF-alpha-dependent reduction of Fc epsilon RII mRNA expression. Similarly, the structurally unrelated antioxidant, pyrrolidine dithiocarbamate (PDTC), also effectively attenuated this-reduction. These results indicate that an ROIs-activated and antioxidant-sensitive mechanism might be involved in TNF-alpha-dependent reduction of IL-4-induced Fc epsilon RII expression in monocytes.
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Vascular endothelial cells (ECs) are constantly subjected to flow-induced shear stress. Although the effects of shear stress on ECs are well known, the intracellular signal mechanisms remain largely unclear. Reactive oxygen species (ROS) have recently been suggested to act as intracellular second messengers. The potential role of ROS in shear-induced gene expression was examined in the present study by subjecting ECs to a shear force using a parallel-plate flow chamber system. ECs under shear flow increased their intracellular ROS as indicated by superoxide production. This superoxide production was maintained at an elevated level as shear flow remained. Sheared ECs, similar to TNF(alpha)-, PMA-, or H2O2-treated cells, increased their intercellular adhesion molecule-1 (ICAM-1) mRNA levels in a time-dependent manner. Pretreatment of ECs with an antioxidant, N-acetyl-cysteine (NAC) or catalase, inhibited this shear-induced or oxidant-induced ICAM-1 expression. ROS that were involved in the shear-induced ICAM-1 gene expression were further substantiated by functional analysis using a chimera containing the ICAM-1 promoter region (-850 bp) and the reporter gene luciferase. Shear-induced promoter activities were attenuated by pretreating sheared ECs with NAC and catalase. Flow cytometric analysis and monocytic adhesion assay confirmed the inhibitory effect of NAC and catalase on the shear-induced ICAM-1 expression on ECs. These results clearly demonstrate that shear flow to ECs can induce intracellular ROS generation that may result in an increase of ICAM-1 mRNA levels via transcriptional events. Our findings thus support the importance of intracellular ROS in modulating hemodynamically induced endothelial responses.
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The present study underscores the importance of N-acetyl cysteine (NAC), a potent antioxidant, in inhibiting the induction of NO production by lipopolysaccharides (LPS) and cytokines in peritoneal macrophages, C6 glial cells and primary astrocytes. LPS, interleukin-1 beta (IL-1beta), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) alone or in combinations induced the production of NO to different degrees. NAC when added 2 h earlier to the addition of these stimuli potentially blocked the increase in NO production in macrophages, astrocytes and C6 glial cells. The decrease in NO production by NAC was accompanied by a decrease in inducible nitric oxide synthase (iNOS) activity, in iNOS protein detected by immunoblot analysis with antibodies against iNOS, and in iNOS mRNA determined by reverse-transcriptase coupled polymerase chain reaction (RT-PCR). Time course studies show that inhibition was maximum when NAC was added 2 h prior to the addition of LPS and the degree of inhibition decreased progressively with the increase in time interval when NAC was added after the addition of LPS. In addition to NAC, another antioxidant pyrrolidine dithiocarbamate (PDTC) was also found to inhibit the induction of NO production effectively. Since activation of NF-kappaB is necessary for the induction of iNOS, we examined the effect of NAC on the activation of NF-kappaB. Inhibition of LPS-induced activation of NF-kappaB by NAC in rat peritoneal macrophages suggests that the inhibitory effect of NAC on the induction of iNOS is due to the inhibition of NF-kappaB. Besides NO, NAC also blocked the production of TNF-alpha in rat peritoneal macrophages activated with endotoxin. These results suggest that expression of iNOS and TNF-alpha in macrophages do involve oxygen radicals. The importance of these results in relation to controlling various harmful effects of cytokines released by activated macrophages and glial cells is discussed.
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Intraperitoneal administration of urate crystals to mice reduced subsequent macrophage conversion of arachidonic acid (AA) to prostaglandins (PGs) and 12-hydroxyeicosatetraenoic acid for up to 6 h. In contrast, levels of 12-hydroxyheptadecatrienoic acid (12-HHT) were markedly elevated. This metabolic profile was previously observed in vitro when recombinant cyclooxygenase (COX) enzymes were incubated with reduced glutathione (GSH). Analysis of peritoneal GSH levels revealed a fivefold elevation after urate crystal administration. The GSH synthesis inhibitor L-buthionine-[S,R]-sulfoximine partially reversed the urate crystal effect on both GSH elevation and PG synthesis. Moreover, addition of exogenous GSH to isolated peritoneal macrophages shifted AA metabolism from PGs to 12-HHT. Urate crystal administration reduced COX-1, but induced COX-2 expression in peritoneal cells. The reduction of COX-1 may contribute to the attenuation of PG synthesis after 1 and 2 h, but PG synthesis remained inhibited up to 6 h, when COX-2 levels were high. Overall, our results indicate that elevated GSH levels inhibit PG production in this model and provide in vivo evidence for the role of GSH in the regulation of PG biosynthesis.
Article
The genes encoding inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2, also known as prostaglandin-endoperoxide synthase-2) are induced in many types of cells in response to proinflammatory cytokines. We have previously shown that interleukin-1beta (IL) stimulates iNOS and COX-2 mRNA in cardiac myocytes. Because IL has been shown to activate mitogen-activated protein kinase (MAPK) signaling pathways in many different cells, we tested whether the p42/44 and p38 MAPK pathways were involved in IL stimulation of iNOS and COX-2, using a specific inhibitor of p42/44 activation, PD98059 (PD), and the p38 inhibitor SB205380 (SB). Nitrites were measured using the Griess reagent, prostaglandin PGE2 by an enzyme immunoassay, iNOS and COX-2 protein by Western blot analysis, and iNOS mRNA by Northern blot analysis. Tested separately, the p38 kinase and MAPK inhibitors partially reduced IL stimulation of nitrite, iNOS protein, and iNOS mRNA; used together, they completely abolished the effect of IL. SB and PD inhibited IL-stimulated COX-2 protein by 60% and 80%, respectively, and IL-stimulated COX-2 protein was totally prevented by the combination of inhibitors. PGE2 production was inhibited more than 99% by either drug alone, suggesting a posttranslational effect on enzyme activity. To test whether this posttranslational effect involved the cytosolic phospholipase A2 (cPLA2) isoform, Western blots were probed for cPLA2 protein. Results indicated that IL stimulated cPLA2 activity and synthesis, which was inhibited by SB but not PD. These data indicate that (1) IL induction of iNOS synthesis depends on both the p42/44 and p38 signaling pathways, acting primarily at the level of transcriptional regulation; and (2) IL regulation of COX-2 synthesis involves the p42/44 and p38 signaling pathways, with an additional level of regulation occurring posttranslationally, perhaps at the level of activation of the cPLA2 isoform, which may be involved in intracellular signaling, as well as regulation of arachidonic acid release for COX-2 activity.
Article
Nitric oxide (NO) plays an important role in the regulation of upper respiratory function. In the nasal cavity, the concentration of NO in the air in patients with untreated allergic rhinitis is higher than that in normal individuals. NO is produced by the action of NO synthase (NOS) using L-arginine as a substrate. To investigate the expression of NOS in human nasal mucosa, histochemical staining for NADPH diaphorase and immunohistochemical staining for NOS isoforms were carried out in nasal inferior turbinate mucosa from patients with nasal allergy. Those without nasal allergy served as controls. NADPH diaphorase histochemical study revealed that NOS was expressed in the nasal epithelium, submucosal glands, nerve fibres and the endothelium in specimens of both allergic and control groups. Immunoreactivity to endothelial NOS (eNOS) was localized to epithelial and endothelial cells in both allergic and control groups. In some specimens in both groups, nerve fibres around submucosal glands stained positively for eNOS. Immunoreactivity to eNOS, however, was slightly stronger in the epithelia of the allergic group than in those of the controls. Immunoreactivity to inducible NOS (iNOS) was localized to epithelial cells, endothelial cells, nasal glands and inflammatory cells. The staining of epithelial cells and inflammatory cells was more marked in the allergic group than the controls. These findings may suggest that the greater amounts of NO in the nasal air of patients with allergic rhinitis are mainly induced by iNOS activity.
Article
Since nitric oxide (NO) can be involved in multiple physiological and pathological functions, we evaluated its possible involvement and that of peroxynitrite in the pathogenesis of rhinitis. Inferior nasal turbinates were obtained from allergic rhinitis and nonallergic rhinitis patients during corrective nasal surgery. The expressions of the inducible form of nitric oxide synthase (iNOS) and the production of peroxynitrite and its metabolite 3-nitrotyrosine were examined by immunohistochemistry in consecutive tissue sections. Each section (or tissue compartment) was given a score of 0-4 according to the labeling intensity seen, with the highest number representing the highest labeling intensity. The results showed that iNOS expression was present mainly in the mucosal epithelium, vascular endothelium, and submucosal glands. A significant difference was only observed in the labeling scores of glandular tissues of the allergic group, which had a higher iNOS labeling score. We also found that sections with a higher iNOS level did not necessarily exhibit a higher 3-nitrotyrosine labeling intensity. These data suggest that iNOS-derived NO may have a role in the pathophysiology of rhinitis, especially the glandular function of allergic nasal mucosa. Moreover, our findings suggest that the production of peroxynitrite in rhinitis patients is not dependent on the level of iNOS alone.
Article
1. The effects of inhibitors of nitric oxide synthase (NOS) on the responsiveness of the human nasal airway were investigated, by measuring the nasal response to histamine and bradykinin. 2. Repeated intranasal administration of N(G)-nitro-L-arginine methyl ester (L-NAME) or N(G)-monomethyl-L-arginine (L-NMMA), 1 micromol per nostril every 30 min for 6 h, increased the nasal obstruction induced by histamine, 50 - 500 microg, and bradykinin, 200 microg per nostril. A single administration of L-NAME, 1 micromol per nostril did not induce hyperresponsiveness to histamine. 3. Pretreatment with L-arginine, 30 micromol, abolished the hyperresponsiveness to histamine caused by L-NAME, 1 micromol. Pretreatment with N(G)-nitro-D-arginine methyl ester (D-NAME), 1 micromol, did not induce hyperresponsiveness to histamine. 4. Repeated administration of L-NAME, 1 micromol, caused a significant reduction in the amount of nitric oxide measured in the nasal cavity. 5. Neither L-NMMA, 1 micromol, nor L-arginine, 30 micromol, altered the nasal hyperresponsiveness induced by platelet activating factor (PAF), 60 microg. PAF did not alter the levels of nitric oxide in the nasal cavity. 6. The results suggest that inhibition of nitric oxide synthase induces a hyperresponsiveness in the human nasal airway, and that this occurs by a mechanism different from that involved in PAF-induced hyperresponsiveness.
Article
We examined whether changes in intracellular reduced (GSH) or oxidized (GSSG) glutathione of human monocytes regulate lipopolysaccharide (LPS)-induced IL-12 production and defined the molecular mechanism that underlies glutathione redox regulation. Monocytes exposed to glutathione reduced form ethyl ester (GSH-OEt) or maleic acid diethyl ester (DEM) increased or decreased the intracellular GSH/GSSG ratio, respectively. LPS-induced IL-12 production and p38 mitogen-activated protein (MAP) kinase activation were enhanced by GSH-OEt but suppressed by DEM. Selective p38 inhibitors showed that p38 promoted GSH-OEt-enhanced IL-12 production. Furthermore, IFN-gamma priming increased the GSH/GSSG ratio and enhanced IL-12 production through p38, and DEM negated the priming effect of IFN-gamma on p38 activation and IL-12 production as well as on the GSH/GSSG ratio. These findings reveal that glutathione redox regulates LPS-induced IL-12 production from monocytes through p38 MAP kinase activation and that the priming effect of IFN-gamma on IL-12 production is partly a result of the glutathione redox balance.
Article
Oxidative stress appears to be relevant to asthma pathogenesis. Therefore, the effectiveness of the antioxidant N -acetylcysteine was examined on antigen-induced pulmonary responses in sensitized Brown-Norway rats. N -acetylcysteine (oral, 1 mmol kg(-1)per day for 7 days before challenge) did not reduce the immediate bronchospasm that followed aerosol antigen exposure but prevented airway hyperreactivity to 5-hydroxytryptamine at 24 h after antigen challenge, and reduced the eosinophils (from 0.178 +/- 0.038 in the absence to 0.064 +/- 0.020 x10(6)cells ml(-1)in the presence of N -acetylcysteine;P< 0.05), and Evans blue dye extravasation in bronchoalveolar lavage fluid. Taurine levels in bronchoalveolar lavage fluid from antigen-challenged rats were higher than control values but treatment with N -acetylcysteine failed to further increase these augmented levels. In conclusion, oral N -acetylcysteine showed beneficial effects in an in vivo model of experimental asthma, which confirm and extend the previous positive findings obtained in other models of lung injury.
Article
Glutathione (GSH) is the main intracellular thiol antioxidant, and as such participates in a number of cellular antitoxic and defensive functions. Nevertheless, non-antioxidant functions of GSH have also been described, e.g. in modulation of cell proliferation and immune response. Recent studies from our and other laboratories have provided evidence for a third functional aspect of GSH, i.e. the prooxidant roles played by molecular species originating during its catabolism by the membrane ectoenzyme gamma-glutamyl transpeptidase (GGT). The reduction of metal ions effected by GSH catabolites is capable to induce redox cycling processes leading to the production of reactive oxygen species (superoxide, hydrogen peroxide), as well as of other free radicals. Through the action of these reactive compounds, GSH catabolism can ultimately lead to oxidative modifications on a variety of molecular targets, involving oxidation and/or S-thiolation of protein thiol groups in the first place. Modulating effects of this kind have been observed on several important, redox-sensitive components of the signal transduction chains, such as cell surface receptors, protein phosphatase activities and transcription factors. Against this background, the prooxidant reactions induced by GSH catabolism appear to represent a novel, as yet unrecognized mechanism for modulation of cellular signal transduction.
Article
This study was conducted to clarify the effect of the n-butanol fraction from the anomalous fruits of Gleditsia sinensis LAM. (NBGS) on experimental allergic rhinitis. NBGS (100, 200, 400 mg/kg, p.o.) dose-dependently inhibited nasal symptoms (sneezing and nasal rubbing) and dye leakage induced by antigen challenge into the nasal cavity of actively sensitized rats. Significant effects were observed at doses of 200 and 400 mg/kg. NBGS (200, 400 mg/kg) also showed a clear inhibition of sneezing and an inhibitory tendency on nasal rubbing induced by histamine in normal rats. At 400 mg/kg, it significantly reduced dye leakage induced by histamine into the nasal cavity of rats. Terfenadine (10 mg/kg, p.o.), an antihistaminic drug, clearly inhibited the nasal symptoms and the amount of dye leakage induced by antigen or histamine. Furthermore, NBGS significantly reduced in vitro histamine release from rat peritoneal mast cells triggered by compound 48/80 at concentrations of 30 and 100 microg/ml. These results suggest that NBGS may be clinically effective in alleviating the nasal symptoms of allergic rhinitis, probably by inhibiting both histamine release from mast cells and nasal vascular permeability.
Article
Allergic rhinitis is an inflammation involving T(H)2-type cytokine production, with pathologic eosinophil infiltration in the nasal mucosa. Although TNF-alpha is thought to be a pro-inflammatory cytokine, the relationship between TNF-alpha and allergic rhinitis has not been clarified. The role of TNF-alpha in a murine model of ovalbumin (OVA)-sensitized allergic rhinitis was investigated by using mice deficient in the gene encoding TNF-alpha (TNF-alpha(-/-) mice). Both wild-type (TNF-alpha(+/+)) and TNF-alpha(-/-) mice were sensitized with OVA by means of intraperitoneal injection. They were then challenged with intranasal OVA, and various allergic responses were assessed. The production of OVA-specific IgE in the serum (P <.05) and the frequency of sneezes (P <.05) and nasal rubs (P <.05) decreased significantly in TNF-alpha(-/-) mice after OVA sensitization compared with that in TNF-alpha(+/+) mice (P <.05). The mRNA expression of IL-4, IL-10, and eotaxin in nasal mucosa in TNF-alpha(-/-) mice was also significantly suppressed compared with that in TNF-alpha(+/+) mice after OVA sensitization (P <.05). Furthermore, the expression of both endothelial-leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1 mRNA in the nasal mucosa was significantly suppressed (P <.05), although intercellular adhesion molecule 1 mRNA expression did not decrease significantly in TNF-alpha(-/-) mice compared with that in TNF-alpha(+/+) mice after OVA sensitization. In addition, the effect of TNF-alpha on endothelial-leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1 expression by means of Western blot analysis was compatible with the mRNA results. Pathologically, eosinophil infiltration in nasal mucosa was significantly restricted in TNF-alpha(-/-) mice compared with in TNF-alpha(+/+) mice after OVA sensitization (P <.05). TNF-alpha is necessary for antigen-specific IgE production and for the induction of T(H)2-type cytokines and chemokines. Furthermore, TNF-alpha might be important for the expression of adhesion molecules to recruit eosinophils to the allergic inflammatory site. We conclude that the lack of TNF-alpha inhibited the development of allergic rhinitis.
Article
This study examines whether endotoxin (LPS)-stimulated COX-2 is modulated by an interaction between mitogen activated protein kinases (MAPK) and intracellular glutathione. Bovine pulmonary artery endothelial cells (BPAEC) were pretreated for 30 min with the following prior to addition of 0.1 microg/ml endotoxin in 2% FBS in medium 199: 5 mM N-acetylcysteine (NAC) or 5 mM glutathione ethyl ester (GSE) (modulators of intracellular glutathione); 10 microM SB203580 or 25 microM PD98059 (inhibitors of p38 and p42/44 MAPKs, respectively). End-points included assessment of COX-1 and COX-2 gene expression by reverse transcription polymerase chain reaction (RT-PCR); COX-1, COX-2, p38, and p42/44 protein by Western analysis; and measurement of PGE2 and 6-keto-PGF1alpha releases by GC/MS. Both GSE and NAC resulted in significant exacerbation of the LPS-stimulated increase in COX-2 gene and protein expression and prostaglandin release, and suppressed the LPS-induced decrease in COX-1. LPS caused a biphasic activation of p42/44 MAPKs, an early increase peaking at 30 min and a second sustained phase, lasting up to 24 h; LPS also caused an early and sustained increase p38 MAPK activity. Pretreatment of cells with either GSE or NAC increased the early LPS-stimulated activation of p42/44 but had little effect on the sustained phase. Inhibition of either p38 or p42/44 MAPKs suppressed LPS-stimulated COX-2 gene and protein expression, and prostaglandin release (P<0.05) but had little effect on COX-1. We conclude that intracellular glutathione modulates LPS-stimulated COX-2 gene expression and prostaglandin synthesis in BPAEC via early activation of p42/44 MAPKs.
Article
Cyclooxygenase-2 (COX-2) is an inducible enzyme believed to be responsible for prostaglandin synthesis at site of inflammation. Recently, the activation of COX-2 expression may be one of the important pathogenesis of root-canal-sealers-induced periapical inflammation. However, little is known about whether chemical interaction can modulate the COX-2 expression and cytotoxicity induced by formaldehyde-containing-ZOE-based root canal sealers. The aim of this study was to investigate the effects of antioxidants such as catalase, superoxide dismutase (SOD), and N-acetyl-L-cysteine (NAC) on N2- and endomethasone-induced COX-2 mRNA gene and cytotoxicity in human osteoblastic cell line U2OS cells. Our data demonstrated that both formaldehyde-containing-ZOE-based root canal sealers were found to induce COX-2 mRNA gene expression in U2OS cells. The addition of glutathione (GSH) precursor NAC led to decrease the induction of COX-2 mRNA gene expression and cytotoxicity by both N2 and Endomethasone (p < 0.05). However, catalase and SOD lacked the ability to prevent cytotoxicity and COX-2 mRNA gene expression induced by N2 and Endomethasone (p > 0.05). The data presented here demonstrated that the activation of COX-2 mRNA gene expression may be one of the pathogenesis of formaldehyde-containing-ZOE-based root-canal-sealers-induced periapical inflammation. In addition, GSH depletion, but not the attack of oxygen free radicals, could be the mechanism for cytotoxicity and COX-2 mRNA gene expression induced by formaldehyde-containing-ZOE-based root canal sealers. NAC appears as a useful agent in protecting cell damage mediated by formaldehyde-containing-ZOE-based root canal sealers.
Article
This study examines the activity of the antioxidant N-acetylcysteine on bleomycin-induced pulmonary fibrosis in rats with emphasis on the early inflammatory phase. Rats receiving N-acetylcysteine (300 mg kg−1 day−1, intraperitoneal) had less augmented lung wet weight, and lower levels of proteins, lactate dehydrogenase, neutrophil and macrophage counts in bronchoalveolar lavage fluid and lung myeloperoxidase activity with a betterment of histological score at 3 days postbleomycin. A diminished lung GSH/GSSG ratio and augmented lipid hydroperoxides were observed 3 days postbleomycin. These changes were attenuated by N-acetylcysteine. Alveolar macrophages from bleomycin-exposed rats released augmented amounts of superoxide anion and nitric oxide. N-Acetylcysteine did not modify superoxide anion generation but reduced the increased production of nitric oxide. N-Acetylcysteine suppressed the bleomycin-induced increased activation of lung NF-κB (shift assay and immunohistochemistry), and decreased the augmented levels of the early inflammatory cytokines, tumour necrosis factor-α, interleukin-β, interleukin-6 and macrophage inflammatory protein-2 observed in bronchoalveolar lavage fluid at 1 and 3 days postbleomycin exposure. At 15 days postbleomycin, N-acetylcysteine decreased collagen deposition in bleomycin-exposed rats (hydroxyproline content: 6351±669 and 4626±288 μg per lung in drug vehicle- and N-acetylcysteine-treated rats, respectively; P<0.05). Semiquantitative histological assessment at this stage showed less collagen deposition in N-acetylcysteine-treated rats compared to those receiving bleomycin alone. These results indicate that N-acetylcysteine reduces the primary inflammatory events, thus preventing cellular damage and the subsequent development of pulmonary fibrosis in the bleomycin rat model. British Journal of Pharmacology (2003) 138, 1037–1048. doi:10.1038/sj.bjp.138-0705138
Article
TNF-alpha is a key molecule in obesity-related metabolic disturbances. This study was designed to determine whether N-acetylcysteine (NAC), an antioxidant, prevents the activation of nuclear factor-kappaB (NF-kappaB) by exogenously administered TNF-alpha in adipocytes, and whether such change affects the production of adipocytokines. The treatment of well-differentiated 3T3-L1 cells with 20 mM of NAC significantly increased the reduced glutathione concentration up to 150% of control. The treatment with 10 ng/ml of TNF-alpha decreased antioxidant enzyme levels such as CuZn-superoxide dismutase (SOD), MnSOD and catalase, and activated NF-kappaB in 3T3-L1 adipocytes. The activation of NF-kappaB was significantly prevented by the pretreatment with 20 mM of NAC. TNF-alpha (1-10 ng/ml) dose-dependently increased interleukin (IL)-6 and plasminogen activator inhibitor-1 (PAI-1) secretion from 3T3-L1 adipocytes, while decreased adiponectin secretion. NAC (5-20 mM) attenuated the TNF-alpha-induced changes in these adipocytokine secretions in a dose-dependent manner. The effect of TNF-alpha and NAC on the adipocytokine productions was exerted at the m-RNA level, judging from results of the real time RT-PCR analysis. The present study revealed that NAC inhibited the TNF-alpha-mediated activation of NF-kappaB and improved the adverse changes in the levels of IL-6, PAI-1 and adiponectin in 3T3-L1 adipocytes. NAC may have the potential to improve the obesity-related abnormal adipocytokine metabolism by attenuating the TNF-alpha-induced oxidant-antioxidant imbalance in adipocytes.
Article
This study was undertaken to investigate the involvement of cyclooxygenase-2 (COX-2) in allergic nasal inflammation in actively sensitized rats. An allergic rhinitis model was developed by the repeated topical application of antigen into the nasal cavities in the sensitized rats. The severity of allergic rhinitis was studied by measuring the nasal behavior, as well as electroencephalogram (EEG) activity by antigen challenge. The electrodes were implanted chronically into the bilateral olfactory bulb of the rats and the EEG was measured monopolarly with an electroencephalograph (EEG, Nohon Kohden, Japan). The intranasal application of antigen caused the increase of nasal allergic signs as well as an EEG spike in a dose-dependent fashion, and at a dose of 50 microg/site, it showed a significant effect. The responses induced by the antigen were evaluated with certain drugs, etodolac (a selective COX-2 inhibitor), indomethacin (a non-selective COX inhibitor), ramatroban (a thromboxane A2 receptor antagonist) and zafirlukast (a cys-leukotriene receptor antagonist). Etodolac showed the inhibition of nasal behavior and EEG spike in a dose-related fashion, and at doses of 3 and 10 mg/kg, it showed a significant effect. Moreover, ramatroban also caused the dose-related inhibition of nasal behavior and EEG spike induced by antigen. On the other hand, both indomethacin and zafirlukast had no effects on the responses induced by antigen, even at a higher dose. Therefore, it can be concluded that cyclooxygenase-2 actively participates in the allergic nasal inflammation in actively sensitized rats.
Article
To investigate protective effect of glutathione (GSH) against lipopolysaccharide (LPS)-induced inflammation and mortality in rats. Male Sprague-Dawley rats were injected intraperitoneally (i. p.) with GSH (40 mg/kg) 30 min prior to LPS injection (20 mg/kg). Animal survival rate and the production of nitric oxide in serum were measured. Morphology of lung was observed using hematoxylin and eosin staining. Western blotting was used to assess the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and the phosphorylation of p38 in rat peritoneal macrophages. GSH significantly decreased LPS-induced mortality, inhibited serum NO production and eliminated lung histological injury. Furthermore, GSH inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins and the phosphorylation of p38 in LPS-stimulated rat peritoneal macrophages. These results indicated that GSH suppressed LPS-induced systemic inflammatory response in rats and p38 MAPK signal pathway was involved in this process.
Article
Oxidative stress appears to be relevant in the pathogenesis of inflammation in allergic diseases like bronchial asthma. Eosinophils are oxidant-sensitive cells considered as key effectors in allergic inflammation. The aim of this work was to study the effects of the clinically used antioxidant N-acetyl-L-cysteine (NAC) on the functional responses of human-isolated eosinophils. Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of NAC were investigated on the generation of reactive oxygen species (chemiluminescence and flow cytometry), Ca(2+) signal (fluorimetry), intracellular glutathione (GSH; flow cytometry), p47(phox)-p67(phox) translocation (Western blot) and eosinophil cationic protein (ECP) release (radioimmunoassay). NAC (0.1-1 mm) inhibited the extracellular generation of oxygen species induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and eotaxin (in the presence of IL-5) with -logIC(50) values of 3.61+/-0.03 and 3.36+/-0.09, respectively. Also, the intracellular generation of hydrogen peroxide was virtually abolished by NAC (0.5-1 mm). NAC (1 mm) did not alter the fMLP-induced Ca(2+) signal but augmented the eosinophil content of reduced GSH and inhibited p47(phox)-p67(phox) translocation. NAC inhibited the release of ECP ( approximately 90% inhibition at 1 mm) from fMLP-activated eosinophils. Inhibition by NAC of human eosinophil functions in vitro is potentially useful in the treatment of allergic inflammation.
Involvement of cyclooxygenase-2 in allergic International Forum of 6 Effect of NAC on experimental allergic rhinitis nasal inflammation in rats
  • A Rahman
  • R Yatsuzuka
  • S Jiang
  • Y Ueda
  • Kamei
Rahman A, Yatsuzuka R, Jiang S, Ueda Y, Kamei C. Involvement of cyclooxygenase-2 in allergic International Forum of Allergy & Rhinology, Vol. 00, No. 0, Month 2013 6 Effect of NAC on experimental allergic rhinitis nasal inflammation in rats. Int Immunopharmacol. 2006;6:1736–1742.
Antioxidant and pro-oxidant effect of the thiolic compounds
  • Sagrist
  • Ml Garcí Ae
  • Africa M De
  • Mora
1. Sagrist a ML, Garcí AE, Africa De Madariaga M, Mora M. Antioxidant and pro-oxidant effect of the thiolic compounds N-acetyl-L-cysteine and