Eugenol Inhibits Neutrophils Myeloperoxidase In Vitro and Attenuates LPS-Induced Lung Inflammation in Mice.

Eugenol (Eug) is a polyphenol extracted from the essential oil of Syzygium aromaticum (L.) Merr. and Perry (Myrtaceae). The health benefits of eugenol in human diseases were proved in several studies. This work aims to evaluate the effect of eugenol on lung inflammatory disorders. For this, using human neutrophils, the antioxidant activity of eugenol was investigated in vitro. Furthermore, a model of LPS-induced lung injury in mice was used to study the anti-inflammatory effect of eugenol in vivo. Results showed that eugenol inhibits luminol-amplified chemiluminescence of resting neutrophils and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLF) peptide or phorbol myristate acetate (PMA). This effect was dose dependent and was significant from a low concentration of 0.1 µg/mL. Furthermore, eugenol inhibited myeloperoxidase (MPO) activity without affecting its degranulation. Eugenol has no scavenging effect on hydrogen peroxide (H2O2) and superoxide anion (O2-). Pretreatment of mice with eugenol prior to the administration of intra-tracheal LPS significantly reduced neutrophil accumulation in the bronchoalveolar lavage fluid (BALF) and decreased total proteins concentration. Moreover, eugenol clearly inhibited the activity of matrix metalloproteinases MMP-2 (21%) and MMP-9 (28%), stimulated by LPS administration. These results suggest that the anti-inflammatory effect of eugenol against the LPS-induced lung inflammation could be exerted via inhibiting myeloperoxidase and metalloproteinases activity. Thus, eugenol could be a promising molecule for the treatment of lung inflammatory diseases.

Levan from a new isolated Bacillus subtilis AF17: Purification, structural analysis and antioxidant activities.

A strain of Bacillus subtilis AF 17 with high exopolysaccharide (EPS) production ability was isolated and identified based on morphological and physiological characteristics and phylogenetic analysis of 16S rDNA sequences. EPS was isolated from the strain fermentation broth by alcohol precipitation and gel-filtration chromatography. Its structural characteristics were investigated and elucidated by methylation analysis, gas chromatography mass spectrometry and nuclear magnetic resonance spectroscopy. Based on the obtained data, the EPS was found to be a levan containing a backbone of 6-substituted ß-fructoses, with a low grade of branching at position 1 (linear/branched ratio 20:1). Levan showed a molecular weight of about 20 MDa. The antioxidant activity of this biopolymer was studied and revealed that levan showed an interesting 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity (IC50 levan = 1.42 mg/mL), reducing power, and also a strong total antioxidant activity. Overall, the results suggest that levan is a promising source of natural antioxidants and can be used as additive in food and pharmaceutical preparations.

Eugenol prevents fMLF-induced superoxide anion production in human neutrophils by inhibiting ERK1/2 signaling pathway and p47phox phosphorylation.

Eugenol is a polyphenol extracted from Syzygium aromaticum essential oil. It is known to have anti-inflammatory and chemoprotective properties as well as a potent anti-oxidant activity due the presence of its phenolic group. In this study, we examined the effects of eugenol on neutrophil superoxide production, a key process involved in innate immunity and inflammation. Superoxide anion generationin human neutrophils was measured by cytochrome c reduction assay. Western blotting was used to analyze the phosphorylation of, p47phox, MAPKinases (p38 and ERK1/2), MEK1/2 and Raf, key proteins involved in the activation of NADPH oxidase. Pretreatment of neutrophils by increasing concentrations (2.5 µg/mL-20 µg/mL) of eugenol for 30 min, inhibited significantly (p < 0.001) superoxide anion generation induced by the chemotactic peptide formyl-Met-Leu-Phe (fMLF) with an IC50 of 5 µg/mL. Phorbolmyristate acetate (PMA)-stimulated O2- production was affected only at the highest eugenol concentration (20 µg/mL). Results showed that eugenol decreased the phosphorylation of p47phox onSer-345 and Ser-328, the translocation of p47phox to the membranesand the phosphorylation of Raf, MEK1/2 and ERK1/2 proteins. Taken together, our results suggest that eugenol inhibits the generation of superoxide anion by neutrophils via the inhibition of Raf/MEK/ERK1/2/p47phox-phosphorylation pathway.

Phosphorylation of NADPH oxidase activator 1 (NOXA1) on serine 282 by MAP kinases and on serine 172 by protein kinase C and protein kinase A prevents NOX1 hyperactivation.

NADPH oxidase activator 1 (NOXA1) together with NADPH oxidase organizer 1 (NOXO1) are key regulatory subunits of the NADPH oxidase NOX1. NOX1 is expressed mainly in colon epithelial cells and could be involved in mucosal innate immunity by producing reactive oxygen species (ROS). Contrary to its phagocyte counterpart NOX2, the mechanisms involved in NOX1 activation and regulation remain unclear. Here we report that NOX1 activity is regulated through MAP kinase (MAPK), protein kinase C (PKC), and protein kinase A (PKA)-dependent phosphorylation of NOXA1. We identified Ser-282 as target of MAPK and Ser-172 as target of PKC and PKA in vitro and in a transfected human embryonic kidney 293 (HEK293) cell model using site directed mutagenesis and phosphopeptide mapping analysis. In HEK293 cells, phosphorylation of these sites occurred at a basal level and down-regulated constitutive NOX1 activity. Indeed, S172A and S282A single mutants of NOXA1 significantly up-regulated constitutive NOX1-derived ROS production, and S172A/S282A double mutant further increased it, as compared to wild-type NOXA1. Furthermore, phosphorylation of NOXA1 on Ser-282 and Ser-172 decreased its binding to NOX1 and Rac1. These results demonstrated a critical role of NOXA1 phosphorylation on Ser-282 and Ser-172 in preventing NOX1 hyperactivation through the decrease of NOXA1 interaction to NOX1 and Rac1.

Heme oxygenase-1 prevents airway mucus hypersecretion induced by cigarette smoke in rodents and humans.

We investigated the role of heme oxygenase-1 (HO-1), a powerful anti-inflammatory and anti-oxidant enzyme, in modulating cigarette smoke (CS)-induced mucus secretion. In both rats and mice, 5-day CS exposure increased HO-1 expression and activity, mucus secretion, MUCIN 5AC (MUC5AC) gene and protein expression, and local inflammation, along with up-regulation of dual oxidase 1 gene expression and both the activity and phosphorylation of the epidermal growth factor receptor, which is involved in MUC5AC induction. Pharmacological induction of HO-1 prevented these actions and inhibition of HO-1 expression by a specific siRNA potentiated them. In French participants to the European Community Respiratory Health Survey II (n = 210, 30 to 53 years of age, 50% males) exposed to CS, a significant increase in the percentage of participants with chronic sputum was observed in those harboring at least one allele with a long (GT)(n) in the HO-1 promoter gene (>33 repeats), which is associated with a low level of HO-1 protein expression, compared with those with a short number of (GT)n repeats (21.7% versus 8.6%, P = 0.047). No such results were observed in those who had never smoked (n = 297). We conclude that HO-1 has a significant protective effect against airway mucus hypersecretion in animals and humans exposed to CS.

Biological effects of particles from the paris subway system.

Particulate matter (PM) from atmospheric pollution can easily deposit in the lungs and induce recruitment of inflammatory cells, a source of inflammatory cytokines, oxidants, and matrix metalloproteases (MMPs), which are important players in lung structural homeostasis. In many large cities, the subway system is a potent source of PM emission, but little is known about the biological effects of PM from this source. We performed a comprehensive study to evaluate the biological effects of PM sampled at two sites (RER and Metro) in the Paris subway system. Murine macrophages (RAW 264.7) and C57Bl/6 mice, respectively, were exposed to 0.01-10 microg/cm2 and 5-100 microg/mouse subway PM or reference materials [carbon black (CB), titanium dioxide (TiO2), or diesel exhaust particles (DEPs)]. We analyzed cell viability, production of cellular and lung proinflammatory cytokines [tumor necrosis factor alpha (TNFalpha), macrophage inflammatory protein (MIP-2), KC (the murin analog of interleukin-8), and granulocyte macrophage-colony stimulating factor (GM-CSF)], and mRNA or protein expression of MMP-2, -9, and -12 and heme oxygenase-1 (HO-1). Deferoxamine and polymixin B were used to evaluate the roles of iron and endotoxin, respectively. Noncytotoxic concentrations of subway PM (but not CB, TiO2, or DEPs) induced a time- and dose-dependent increase in TNFalpha and MIP-2 production by RAW 264.7 cells, in a manner involving, at least in part, PM iron content (34% inhibition of TNF production 8 h after stimulation of RAW 264.7 cells with 10 microg/cm2 RER particles pretreated with deferoxamine). Similar increased cytokine production was transiently observed in vivo in mice and was accompanied by an increased neutrophil cellularity of bronchoalveolar lavage (84.83+/-0.98% of polymorphonuclear neutrophils for RER-treated mice after 24 h vs 7.33+/-0.99% for vehicle-treated animals). Subway PM induced an increased expression of MMP-12 and HO-1 both in vitro and in vivo. PM from the Paris subway system has transient biological effects. Further studies are needed to better understand the pathophysiological implications of these findings.

Keratinocyte growth factor protects against elastase-induced pulmonary emphysema in mice.

Pulmonary emphysema is characterized by persistent inflammation and progressive alveolar destruction. The keratinocyte growth factor (KGF) favorably influences alveolar maintenance and repair and possesses anti-inflammatory properties. We aimed to determine whether exogenous KGF prevented or corrected elastase-induced pulmonary emphysema in vivo. Treatment with 5 mg x kg(-1) x day(-1) KGF before elastase instillation prevented pulmonary emphysema. This effect was associated with 1) a sharp reduction in bronchoalveolar lavage fluid total protein and inflammatory cell recruitment, 2) a reduction in the pulmonary expression of the chemokines CCL2 (or monocyte chemoattractant protein-1) and CXCL2 (or macrophage inflammatory protein-2alpha) and of the adhesion molecules ICAM-1 and VCAM-1, 3) a reduction in matrix metalloproteinase (MMP)-2 and MMP-9 activity at day 3, and 4) a major reduction in DNA damage detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) in alveolar cells at day 7. Treatment with KGF after elastase instillation had no effect on elastase-induced emphysema despite the conserved expression of the KGF receptor in the lungs of elastase-instilled animals as determined by immunohistochemistry. In vitro, KGF abolished the elastase-induced increase in CCL2, CXCL2, and ICAM-1 mRNA in the MLE-12 murine alveolar epithelial cell line. We conclude that KGF pretreatment protected against elastase-induced pulmonary inflammation, activation of MMPs, alveolar cell DNA damage, and subsequent emphysema in mice.

Diesel exhaust particles induce matrix metalloprotease-1 in human lung epithelial cells via a NADP(H) oxidase/NOX4 redox-dependent mechanism.

Chronic exposure to particulate air pollution is associated with lung function impairment. To determine the molecular mechanism(s) of this phenomenon, we investigated, in an alveolar human epithelial cell line (A549), whether diesel exhaust particles (DEPs), a main component of particulate air pollution, modulates the expression and activity of the matrix metalloprotease (MMP)-1, a collagenase involved in alveolar wall degradation. Interaction of DEPs with cigarette smoke, which also produces structural and functional lung alterations, was also investigated. A noncytotoxic concentration of DEPs induced an increase in MMP-1 mRNA and protein expression and activity in A549 cells without modifying the expression of the MMP inhibitors TIMP-1 and -2. This effect was not potentiated when cells were coexposed to noncytotoxic concentrations of cigarette smoke condensate. DEP-induced MMP-1 was associated with increased ERK 1/2 phosphorylation and upregulation of expression and activity of the NADPH oxidase analog NOX4. Cell transfection with a NOX4 small interfering RNA prevented these phenomena, showing the critical role of a NOX4 ERK 1/2 pathway in DEP-induced MMP-1 expression and activity. Similar results to those observed in A549 cells were obtained in another human lung epithelial cell line, NCI-H292. Furthermore, experiments in mice intratracheally instilled with DEPs confirmed the in vitro findings, showing the induction of NOX4 and MMP-1 protein expression in alveolar epithelial cells. We conclude that alveolar alterations secondary to MMP-1 induction could explain lung function impairment associated with exposure to particulate pollution.