Role of nitric oxide in pathological responses of the lung to exposure to environmental/occupational agents

Conflicting evidence exists as to whether nitric oxide expresses damaging/inflammatory or antioxidant/anti-inflammatory properties. Data presented in this review indicate that in vitro or in vivo exposure to selected environmental or occupational agents, such as asbestos, silica, ozone or lipopolysaccharide, can result in up-regulation of inducible nitric oxide synthase by alveolar macrophages and pulmonary epithelial cells. In the case of silica exposure, evidence consistently supports a damaging/inflammatory role of nitric oxide and/or peroxynitrite in the pathogenesis of lung disease. Although conflicting data have been reported, the majority of published studies suggest that nitric oxide plays a damaging role in pulmonary injury resulting from exposure to ozone or asbestos. In contrast, most information supports an anti-inflammatory role of nitric oxide following exposure to lipopolysaccharide. Further investigation is required to elucidate fully the mechanisms involved in determining the role of nitric oxide in the initiation and progression of various pulmonary diseases.     

Role of Nitric Oxide in the Progression of Pneumoconiosis

Conflicting evidence has been reported as to whether nitric oxide (NO) possesses anti-inflammatory or inflammatory properties. Data are presented indicating that in vitro or in vivo exposure to selected occupational dusts, i.e., crystalline silica, organic dust contaminated with endotoxin, or asbestos, results in upregulation of inducible nitric oxide synthase (iNOS) and the production of NO by alveolar macrophages and pulmonary epithelial cells. Nitric oxide production is associated temporally and anatomically with pulmonary damage, inflammation, and disease progression in response to occupational dusts. Blockage of inducible nitric oxide synthase by administration of NOS inhibitors or in iNOS knockout mice decreases the magnitude of injury and inflammation following in vivo exposure to silica, endotoxin, or asbestos. Therefore, NO may play an important role in the initiation and progression of pneumoconiosis.     

Anti-inflammatory phenolics isolated from Juniperus rigida leaves and twigs in lipopolysaccharide-stimulated RAW264.7 macrophage cells

Inflammation is an essential host defense system particularly in response to infection and injury; however, excessive or undesirable inflammatory responses contribute to acute and chronic human diseases. A high-throughput screening effort searching for anti-inflammatory compounds from medicinal plants deduced that the methanolic extract of Juniperus rigida S. et L. (Cupressaceae) inhibited significantly nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Activity-guided fractionation and isolation yielded 13 phenolic compounds, including one new phenylpropanoid glycosides, 3,4-dimethoxycinnamyl 9-O-β-D-glucopyranoside (1). Among the isolated compounds, phenylpropanoid glycosides with p-hydroxy group (2, 4) and massoniaside A (7), (+)-catechin (10), amentoflavone (11) effectively inhibited LPS-induced NO production in RAW264.7 cells.     

Anti-Inflammatory Activities of Methanol Extract of Mastixia arborea C.B. Clarke as to Mouse Macrophage and Paw Edema

The biological activity of Mastixia arborea (MA) relates to inflammation, but the underlying mechanisms are largely unknown. We confirmed the anti-inflammatory effects of a methanol extract of MA extract on lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells and carrageenan-induced mice paw edema. The MA extract significantly inhibited nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin-1β (IL-1β), and IL-6 production in LPS-stimulated RAW264.7 cells. In vitro expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was suppressed by the extract. The extract attenuated acute inflammatory responses in carrageenan-induced mice paw edema. A mechanism study indicated that translocation of the NF-κB (p65) subunit into the nucleus and phosphorylation of ERK and JNK were inhibited by the extract. These results indicate that the extract is an effective suppressor of the inflammatory response, blocking the phosphorylation of ERK and JNK and the translocation of NF-κB in macrophages, thereby producing an anti-inflammatory effect in vivo.     

Synthesis of some new amide-linked bipyrazoles and their evaluation as anti-inflammatory and analgesic agents

Four series of new bipyrazoles comprising the N-phenylpyrazole scaffold linked to polysubstituted pyrazoles or to antipyrine moiety through different amide linkages were synthesized. The synthesized compounds were evaluated for their anti-inflammatory and analgesic activities. In vitro COX-1/COX-2 inhibition study revealed that compound 16b possessed the lowest IC₅₀ value against both COX-1 and COX-2. Moreover, the effect of the most promising compounds on inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) protein expression in lipopolysaccharide (LPS)-activated rat monocytes was also investigated. The results revealed that some of the synthesized compounds showed anti-inflammatory and/or analgesic activity with less ulcerogenic potential than the reference drug diclofenac sodium and are well tolerated by experimental animals. Moreover, they significantly inhibited iNOS and COX-2 protein expression induced by LPS stimulation. Compounds 16b and 18 were proved to display anti-inflammatory activity superior to diclofenac sodium and analgesic activity equivalent to it with minimal ulcerogenic potential.     

Quercetin 3,7-O-dimethyl ether from Siegesbeckia pubescens suppress the production of inflammatory mediators in lipopolysaccharide-induced macrophages and colon epithelial cells

Siegesbeckia pubescens (Compositae) is an annual herb indigenous to Korean mountainous regions. Recent reports have been issued on some compounds derived from S. pubescens for its anti-inflammatory activity or mode of action. The quercetin 3,7-O-dimethyl ether (QDE) isolated from the herbs of S. pubescens suppressed the lipopolysaccharide (LPS)-induced nitric oxide and inducible nitric oxide synthase (iNOS) protein production in mouse macrophages. QDE downregulated pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β, tumor necrosis factor -α levels in LPS-stimulated macrophages. Also, QDE decreased the expression of LPS-induced iNOS and cyclooxygenase-2 (COX-2) protein and the production of IL-8 in LPS-induced HT-29 cells. Macrophages and colon epithelial cells are important for regulating the colon immune systems, thus QDE may regulate inflammatory colon disease via LPS-induced inflammation in macrophages and colon epithelial cells. QDE, anti-inflammatory constituent of S. pubescens herbs, can be expected to be a potential candidate for therapeutics against inflammatory bowel disease.     

Changes in the expression of NO synthase isoforms after ozone: the effects of allergen exposure

BackgroundThe functional role of nitric oxide (NO) and various nitric oxide synthase (NOS) isoforms in asthma remains unclear.ObjectiveThis study investigated the effects of ozone and ovalbumin (OVA) exposure on NOS isoforms.MethodsThe expression of inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) in lung tissue was measured. Enhanced pause (P_enh) was measured as a marker of airway obstruction. Nitrate and nitrite in bronchoalveolar lavage (BAL) fluid were measured using a modified Griess reaction.ResultsThe nitrate concentration in BAL fluid from the OVA-sensitized/ozone-exposed/OVA-challenged group was greater than that of the OVA-sensitized/saline-challenged group. Methacholine-induced P_enh was increased in the OVA-sensitized/ozone-exposed/OVA-challenged group, with a shift in the dose-response curve to the left, compared with the OVA-sensitized/saline-challenged group. The levels of nNOS and eNOS were increased significantly in the OVA-sensitized/ozone-exposed/OVA-challenged group and the iNOS levels were reduced compared with the OVA-sensitized/saline-challenged group.ConclusionIn mice, ozone is associated with increases in lung eNOS and nNOS, and decreases in iNOS. None of these enzymes are further affected by allergens, suggesting that the NOS isoforms play different roles in airway inflammation after ozone exposure.     

Synthesis and bio-evaluation of human macrophage migration inhibitory factor inhibitor to develop anti-inflammatory agent

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is involved in the development of an array of inflammatory disorders including rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis and sepsis. The synthesis of MIF-inhibitor is a rationale approach to develop novel anti-inflammatory agent to treat multitude of inflammatory diseases. In this work, we have synthesized and evaluated MIF-inhibitory activity of a series of small molecules containing isoxazoline skeleton. Mode of binding of this inhibitor to human MIF (huMIF) was determined by docking studies. The synthesized molecules inhibit tautomerase activity of huMIF. The anti-inflammatory activity of the most active inhibitor, 4-((3-(4-hydroxy-3-methoxyphenyl)-4, 5-dihydroisoxazol-5-yl) methoxy) benzaldehyde (4b) was evaluated against huMIF-induced inflammation in a cellular model (RAW 264.7 cell). Compound 4b significantly inhibits huMIF-mediated NF-κB translocation to the nucleus, up-regulation of inducible nitric oxide synthase and nitric oxide production in RAW 264.7 cell which are the markers for inflammation. The compound 4b is not cytotoxic as evident from cell viability assay. Hence, the compound 4b has potential to be a novel anti-inflammatory agent.     

The spin trap 5,5-dimethyl-1-pyrroline N-oxide inhibits lipopolysaccharide-induced inflammatory response in RAW 264.7 cells

AimExposure of macrophages to lipopolysaccharide (LPS) induces oxidative and inflammatory stresses, which cause cell damage. Antioxidant and anti-inflammatory properties have been attributed to the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), commonly used in free radical analysis, but these aspects of DMPO have been little explored. In this study, we sought to establish the anti-inflammatory activity of DMPO, presumably by removing free radicals which otherwise help activate inflammatory response and damage cells.Main methodsRAW 264.7 macrophages were treated with LPS and/or DMPO for different time points, cell damage, production of inflammatory mediators, inducible nitric oxide synthase (iNOS) expression, NF-κB p65 activation, phosphorylation of MAPKs and Akt, and intracellular reactive oxygen species (ROS) were determined.Key findingsAfter cells were treated with LPS and/or DMPO for 24 h, DMPO reduced the LPS-induced inflammatory response as indicated by downregulated iNOS expression and production of inflammatory mediators. Accordingly, DMPO protected cells from LPS-induced cytotoxicity. In order to understand the mechanistic basis of these DMPO effects, the NF-κB p65 activation and the phosphorylation of MAPKs and Akt were examined. We found, by assaying cells treated with LPS and/or DMPO for 15–60 min, that DMPO inhibited the phosphorylation of MAPKs, Akt, and IκBα, and reduced the NF-κB p65 translocation. Furthermore, we demonstrated that DMPO inhibited LPS-induced ROS production.SignificanceDMPO showed the anti-inflammatory activity and attenuated LPS-induced cell damage, most likely by reducing ROS production and thus preventing the subsequent inflammatory activation and damage.     

Effects of globin digest and its active ingredient Trp-Thr-Gln-Arg on galactosamine/lipopolysaccharide-induced liver injury in ICR mice

AimsWe investigated the effects of globin digest (GD) and its active ingredient Trp-Thr-Gln-Arg (WTQR) on galactosamine/lipopolysaccharide (GalN/LPS)-induced liver injury in imprinting control region (ICR) mice.Main methodsThe effects of WTQR and GD on the liver injury were examined by measuring the survival rate, serum aminotransferase activities, hepatic components, antioxidant enzyme activities, histopathological analysis, serum levels and hepatic gene expression of tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2), and nitric oxide (NO) or inducible nitric oxide synthase (iNOS), and nuclear factor-kappa B (NF-κB) p65 content in GalN/LPS-treated ICR mice. RAW264 mouse macrophages were used to confirm the anti-inflammatory effects of WTQR and GD on the macrophages.Key findingsWTQR and GD increased the survival rate, suppressed the serum aminotransferase activities, serum levels and hepatic gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in GalN/LPS-treated mice; decreased the oxidized glutathione content, increased the superoxide dismutase activity, and decreased the histopathological grade values of the hepatocyte necrosis and lobular inflammation in GalN/LPS-injured liver; and suppressed the release levels and gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in LPS-stimulated RAW264 macrophages. WTQR and GD may improve the antioxidant defense system and inflammatory status in GalN/LPS-injured liver.SignificanceThese findings indicate that WTQR and GD have hepatoprotective effects on GalN/LPS-induced liver injury in ICR mice.     

Effect of inhaled crystalline silica in a rat model: Time course of pulmonary reactions

Numerous investigations have been conducted to elucidate mechanisms involved in the initiation and progression of silicosis. However, most of these studies involved bolus exposure of rats to silica, i.e. intratracheal instillation or a short duration inhalation exposure to a high dose of silica. Therefore, the question of pulmonary overload has been an issue in these studies. The objective of the current investigation was to monitor the time course of pulmonary reactions of rats exposed by inhalation to a non-overload level of crystalline silica. To accomplish this, rats were exposed to 15 mg/m³ silica, 6 h/day, 5 days/week for up to 116 days of exposure. At various times (5–116 days exposure), animals were sacrificed and silica lung burden, lung damage, inflammation, NF-B activation, reactive oxygen species and nitric oxide production, cytokine production, alveolar type II epithelial cell activity, and fibrosis were monitored. Activation of NF-B/DNA binding in BAL cells was evident after 5 days of silica inhalation and increased linearly with continued exposure. Parameters of pulmonary damage, inflammation and alveolar type II epithelial cell activity rapidly increased to a significantly elevated but stable new level through the first 41 days of exposure and increased at a steep rate thereafter. Pulmonary fibrosis was measurable only after this explosive rise in lung damage and inflammation, as was the steep increase in TNF- and IL-1 production from BAL cells and the dramatic rise in lavageable alveolar macrophages. Indicators of oxidant stress and pulmonary production of nitric oxide exhibited a time course which was similar to that for lung damage and inflammation with the steep rise correlating with initiation of pulmonary fibrosis. Staining for iNOS and nitrotyrosine was localized in granulomatous regions of the lung and bronchial associated lymphoid tissue. Therefore, these data demonstrate that the generation of oxidants and nitric oxide, in particular, is temporally and anatomically associated with the development of lung damage, inflammation, granulomas and fibrosis. This suggests an important role for nitric oxide in the initiation of silicosis.     

Evaluation of analgesic and anti-inflammatory activities and molecular docking analysis of steroidal lactones from Datura stramonium L.

BackgroundDatura stramonium L. is widely used across the world for its therapeutic potential to treat inflammatory disorders. The current work was designed to isolate and identify steroidal lactones from D. stramonium leaves and evaluate their anti-inflammatory and analgesic properties.MethodsSeveral compounds were isolated from D. stramonium leaves and characterized by nuclear magnetic resonance and high-resonance electron spray ionization mass spectrometry techniques. Further, anti-inflammatory properties of these compounds were evaluated by in vitro assays, such as release of NO and pro-inflammatory cytokines by lipopolysaccharide (LPS)-activated J774A.1 macrophages. Using in vivo models, anti-inflammatory and analgesic effects were examined by mouse tail-flick, carrageenan-induced inflammation in rat paw model, vascular permeability in rats, and acetic acid-induced writhing in mice. The docking studies were performed for assessing the binding efficiency of the test compounds with cyclooxygenase-1 (COX-1) and COX-2, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), inducible nitric oxide synthases (iNOS) and nuclear factor-κB (NF-κB).ResultsThree lactones were isolated and confirmed as daturalactone (D1), 12-deoxywithastramonolide (D23), and daturilin (D27). Further, the isolated compounds showed nitric oxide inhibition and pro-inflammatory cytokines released by LPS-activated J774A.1 macrophages. The in vivo results suggest that D1, D23 and D27 (20 mg/kg) were able to reduce the pain and inflammation in various animal models. The docking analysis showed that these three compounds actively bind with COX-1, COX-2, LOX-1, NF-κB, and iNOS, validating the anti-inflammatory effects of the lactones.ConclusionThese findings demonstrate substantial anti-inflammatory and analgesic properties of D. stramonium-derived lactones and their potential as anti-inflammatory agents to treat chronic inflammatory ailments.     

Functional properties of anti-inflammatory substances from quercetin-treated Bifidobacterium adolescentis

Abstract

The genus Bifidobacterium is well known to have beneficial health effects. We discovered that quercetin and related polyphenols enhanced the secretion of anti-inflammatory substances by Bifidobacterium adolescentis. This study investigated characteristics of the anti-inflammatory substances secreted by B. adolescentis. The culture supernatant of B. adolescentis with quercetin reduced the levels of inflammatory mediators in activated macrophages. Spontaneous quercetin degradant failed to increase anti-inflammatory activity, while the enhancement of anti-inflammatory activity by quercetin was sustained after washout of quercetin. Physicochemical treatment of the culture supernatant indicated that its bioactive substances may be heat-stable, non-phenolic, and acidic biomolecules with molecular weights less than 3 kDa. Acetate and lactate have little or no effect on nitric oxide production. Taken together, the anti-inflammatory substances secreted by B. adolescentis may be small molecules but not short chain fatty acids. In agreement with these findings, stearic acid was tentatively identified as a bioactive candidate compound.     

Anthocyanins isolated from Hibiscus syriacus L. attenuate lipopolysaccharide-induced inflammation and endotoxic shock by inhibiting the TLR4/MD2-mediated NF-κB signaling pathway

BackgroundHibiscus syriacus L. has been used as a medicinal plant in many Asian countries. However, anti-inflammatory activity of H. syriacus L. remains unknown.PurposeThis study was aimed to investigating the anti-inflammatory effect of anthocyanin fractions from the H. syriacus L. variety Pulsae (PS) on the lipopolysaccharide (LPS)-induced inflammation and endotoxic shock.Study design and methodsMTT assay and flow cytometry analysis were performed to determine cytotoxicity of PS. RT-PCR, western blotting, and ELISA were conducted to evaluate the expression of proinflammatory mediators and cytokines. Molecular docking study predicted the binding scores and sites of PS to TLR4/MD2 complex. Immunohistochemical assay was conducted to evaluate the binding capability of PS to TLR4/MD2 and nuclear translocation of NF-κB p65. A zebrafish endotoxic shock model was used to evaluate anti-inflammatory activity of PS in vivo.ResultsPS suppressed LPS-induced nitric oxide and prostaglandin E₂ secretion concomitant with the downregulation of inducible nitric oxide synthase and cyclooxygenase-2 expression. Furthermore, PS inhibited the production of proinflammatory cytokines such as TNF-α, IL-6, and IL-12 in LPS-stimulated RAW 264.7 macrophages. Additionally, molecular docking data showed that PS mostly fit into the hydrophobic pocket of MD2 and bound to TLR4. In particular, apigenin-7-O-glucoside powerfully bound to MD2 and TLR4 via hydrogen bonding. Additionally, immunohistochemistry assay revealed that PS inhibited LPS-induced TLR4 dimerization or expression on the cell surface, which consequently decreased MyD88 recruitment and IRAK4 phosphorylation, resulting in the inhibition of NF-κB activity. PS also attenuated LPS-mediated mortality and abnormality in zebrafish larvae and diminished the recruitment of neutrophils and macrophages at the inflammatory site accompanied by the low levels of proinflammatory mediators and cytokines.ConclusionPS might be a novel immunomodulator for the effective treatment of LPS-mediated inflammatory diseases.     

Susceptibility to ozone-induced acute lung injury in iNOS-deficient mice

Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO]NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide.     

The Role of the Receptor for Advanced Glycation End-Products in a Murine Model of Silicosis

Background

The role of the receptor for advanced glycation end-products (RAGE) has been shown to differ in two different mouse models of asbestos and bleomycin induced pulmonary fibrosis. RAGE knockout (KO) mice get worse fibrosis when challenged with asbestos, whereas in the bleomycin model they are largely protected against fibrosis. In the current study the role of RAGE in a mouse model of silica induced pulmonary fibrosis was investigated.

Methodology/Principal Findings

Wild type (WT) and RAGE KO mice received a single intratracheal (i.t.) instillation of silica in saline or saline alone as vehicle control. Fourteen days after treatment mice were subjected to a lung mechanistic study and the lungs were lavaged and inflammatory cells, protein and TGF-β levels in lavage fluid determined. Lungs were subsequently either fixed for histology or excised for biochemical assessment of fibrosis and determination of RAGE protein- and mRNA levels. There was no difference in the inflammatory response or degree of fibrosis (hydroxyproline levels) in the lungs between WT and RAGE KO mice after silica injury. However, histologically the fibrotic lesions in the RAGE KO mice had a more diffuse alveolar septal fibrosis compared to the nodular fibrosis in WT mice. Furthermore, RAGE KO mice had a significantly higher histologic score, a measure of affected areas of the lung, compared to WT silica treated mice. A lung mechanistic study revealed a significant decrease in lung function after silica compared to control, but no difference between WT and RAGE KO. While a dose response study showed similar degrees of fibrosis after silica treatment in the two strains, the RAGE KO mice had some differences in the inflammatory response compared to WT mice.

Conclusions/Significance

Aside from the difference in the fibrotic pattern, these studies showed no indicators of RAGE having an effect on the severity of pulmonary fibrosis following silica injury.     

Effect of inhaled crystalline silica in a rat model: time course of pulmonary reactions

Numerous investigations have been conducted to elucidate mechanisms involved in the initiation and progression of silicosis. However, most of these studies involved bolus exposure of rats to silica, i.e. intratracheal instillation or a short duration inhalation exposure to a high dose of silica. Therefore, the question of pulmonary overload has been an issue in these studies. The objective of the current investigation was to monitor the time course of pulmonary reactions of rats exposed by inhalation to a non-overload level of crystalline silica. To accomplish this, rats were exposed to 15 mg/m3 silica, 6 h/day, 5 days/week for up to 116 days of exposure. At various times (5-116 days exposure), animals were sacrificed and silica lung burden, lung damage, inflammation, NF-KB activation, reactive oxygen species and nitric oxide production, cytokine production, alveolar type II epithelial cell activity, and fibrosis were monitored. Activation of NF-KB/DNA binding in BAL cells was evident after 5 days of silica inhalation and increased linearly with continued exposure. Parameters of pulmonary damage, inflammation and alveolar type II epithelial cell activity rapidly increased to a significantly elevated but stable new level through the first 41 days of exposure and increased at a steep rate thereafter. Pulmonary fibrosis was measurable only after this explosive rise in lung damage and inflammation, as was the steep increase in TNF-alpha and IL-1 production from BAL cells and the dramatic rise in lavageable alveolar macrophages. Indicators of oxidant stress and pulmonary production of nitric oxide exhibited a time course which was similar to that for lung damage and inflammation with the steep rise correlating with initiation of pulmonary fibrosis. Staining for iNOS and nitrotyrosine was localized in granulomatous regions of the lung and bronchial associated lymphoid tissue. Therefore, these data demonstrate that the generation of oxidants and nitric oxide, in particular, is temporally and anatomically associated with the development of lung damage, inflammation, granulomas and fibrosis. This suggests an important role for nitric oxide in the initiation of silicosis.