Inhibition of LPS-induced cyclooxygenase 2 and nitric oxide production by transduced PEP-1-PTEN fusion protein in Raw 264.7 macrophage cells

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor. Although it is well known to have various physiological roles in cancer, its inhibitory effect on inflammation remains poorly understood. In the present study, a human PTEN gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-PTEN fusion protein. The expressed and purified PEP-1-PTEN fusion protein were transduced efficiently into macrophage Raw 264.7 cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-PTEN protein was stable for 24 h. Transduced PEP-1-PTEN fusion protein inhibited the LPS-induced cyclooxygenase 2 (COX-2) and iNOS expression levels in a dose-dependent manner. Furthermore, transduced PEP-1-PTEN fusion protein inhibited the activation of NF-κB induced by LPS. These results suggest that the PEP-1-PTEN fusion protein can be used in protein therapy for inflammatory disorders.     

Effect of sildenafil citrate on interleukin-1beta-induced nitric oxide synthesis and iNOS expression in SW982 cells

The purpose of this study was to identify the effect of sildenafil citrate on IL-1β-induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1β stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1β-induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1β treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1β-induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1β-induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.     

Role of inducible nitric oxide synthase on the development of virus-associated asthma exacerbation which is dependent on Th1 and Th17 cell responses

Asthma is characterized by airway inflammation induced by immune dysfunction to inhaled antigens. Although respiratory viral infections are the most common cause of asthma exacerbation, immunologic mechanisms underlying virus-associated asthma exacerbation are controversial. Clinical evidence indicates that nitric oxide (NO) levels in exhaled air are increased in exacerbated asthma patients compared to stable patients. Here, we evaluated the immunologic mechanisms and the role of NO synthases (NOSs) in the development of virus-associated asthma exacerbation. A murine model of virus-associated asthma exacerbation was established using intranasal challenge with ovalbumin (OVA) plus dsRNA for 4 weeks in mice sensitized with OVA plus dsRNA. Lung infiltration of inflammatory cells, especially neutrophils, was increased by repeated challenge with OVA plus dsRNA, as compared to OVA alone. The neutrophilic inflammation enhanced by dsRNA was partly abolished in the absence of IFN-gamma or IL-17 gene expression, whereas unaffected in the absence of IL-13. In terms of the roles of NOSs, dsRNA-enhanced neutrophilic inflammation was significantly decreased in inducible NOS (iNOS)-deficient mice compared to wild type controls; in addition, this phenotype was inhibited by treatment with a non-specific NOS inhibitor (L-NAME) or an specific inhibitor (1400 W), but not with a specific endothelial NOS inhibitor (AP-CAV peptide). Taken together, these findings suggest that iNOS pathway is important in the development of virus-associated exacerbation of neutrophilic inflammation, which is dependent on both Th1 and Th17 cell responses.     

Over-expression of extracellular superoxide dismutase in mouse synovial tissue attenuates the inflammatory arthritis

Oxidative stress such as reactive oxygen species (ROS) within the inflamed joint have been indicated as being involved as inflammatory mediators in the induction of arthritis. Correlations between extracellular- superoxide dismutase (EC-SOD) and inflammatory arthritis have been shown in several animal models of RA. However, there is a question whether the over-expression of EC-SOD on arthritic joint also could suppress the progression of disease or not. In the present study, the effect on the synovial tissue of experimental arthritis was investigated using EC-SOD over-expressing transgenic mice. The over-expression of EC- SOD in joint tissue was confirmed by RT-PCR and immunohistochemistry. The degree of the inflammation in EC-SOD transgenic mice was suppressed in the collagen-induced arthritis model. In a cytokine assay, the production of pro-inflammatory cytokines such as, IL-1β, TNFα, and matrix metalloproteinases (MMPs) was decreased in fibroblast-like synoviocyte (FLS) but not in peripheral blood. Histological examination also showed repressed cartilage destruction and bone in EC-SOD transgenic mice. In conclusion, these data suggest that the over-expression of EC-SOD in FLS contributes to the activation of FLS and protection from joint destruction by depressing the production of the pro-inflammatory cytokines and MMPs. These results provide EC-SOD transgenic mice with a useful animal model for inflammatory arthritis research.     

Regulation of pro-inflammatory responses by lipoxygenases via intracellular reactive oxygen species in vitro and in vivo

Reactive oxygen species (ROS) performs a pivotal function as a signaling mediator in receptor-mediated signaling. However, the sources of ROS in this signaling have yet to be determined, but may include lipoxygenases (LOXs) and NADPH oxidase. The stimulation of lymphoid cells with TNF-alpha, IL-1beta, and LPS resulted in significant ROS production and NF-kappaB activation. Intriguingly, these responses were markedly abolished via treatment with the LOXs inhibitor nordihydroguaiaretic acid (NDGA). We further examined in vivo anti-inflammatory effects of NDGA in allergic airway inflammation. Both intraperitoneal and intravenous NDGA administration attenuated ovalbumin (OVA)-induced influx into the lungs of total leukocytes, as well as IL-4, IL-5, IL-13, and TNF-alpha levels. NDGA also significantly reduced serum levels of OVA-specific IgE and suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. The results of our histological studies and flow cytometric analyses showed that NDGA inhibits OVA-induced lung inflammation and the infiltration of CD11b+ macrophages into the lung. Collectively, our findings indicate that LOXs performs an essential function in pro-inflammatory signaling via the regulation of ROS regulation, and also that the inhibition of LOXs activity may have therapeutic potential with regard to the treatment of allergic airway inflammation.     

Syringaresinol causes vasorelaxation by elevating nitric oxide production through the phosphorylation and dimerization of endothelial nitric oxide synthase

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS(-/-) mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor N(G)-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca(2+) levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca(2+) levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol- treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca(2+) chelator, calmodulin antagonist, and CaMKKβ siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca(2+)-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca(2+)/CaMKKβ-dependent eNOS phosphorylation and Ca(2+)-dependent eNOS dimerization.     

TNF-alpha-induced up-regulation of intercellular adhesion molecule-1 is regulated by a Rac-ROS-dependent cascade in human airway epithelial cells

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the lung airway epithelium is associated with the epithelium-leukocyte interaction, critical for the pathogenesis of various lung airway inflammatory diseases such as asthma. However, little is known about how ICAM-1 is up-regulated in human airway epithelial cells. In this study, we show that tumor TNF-alpha induces monocyte adhesion to A549 human lung airway epithelium and also up-regulation of ICAM-1 expression. These effects were significantly diminished by pre-treatment with diphenyliodonium (DPI), an inhibitor of NADPH oxidase-like flavoenzyme. In addition, the level of reactive oxygen species (ROS) was increased in response to TNF-alpha in A549 cells, suggesting a potential role of ROS in the TNF-alpha-induced signaling to ICAM-1 expression and monocyte adhesion to airway epithelium. Further, we found out that expression of RacN17, a dominant negative mutant of Rac1, suppressed TNF-alpha-induced ROS generation, ICAM-1 expression, and monocyte adhesion to airway epithelium. These findings suggest that Rac1 lies upstream of ROS generation in the TNF-alpha-induced signaling to ICAM-1 expression in airway epithelium. Finally, pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, reduced TNF-alpha-induced ICAM-1 expression and both DPI and RacN17 significantly diminished NF-kappaB activation in response to TNF-alpha. Together, we propose that Rac1-ROS-linked cascade mediate TNF-alpha-induced ICAM-1 up-regulation in the airway epithelium via NF-kappaB-dependent manner.     

Rutin inhibits osteoclast formation by decreasing reactive oxygen species and TNF-alpha by inhibiting activation of NF-kappaB

Rutin, a glycoside of flavonol, inhibits osteoclast formation induced by receptor activator of NF-kappaB ligand (RANKL) in bone marrow-derived macrophages. It reduces reactive oxygen species produced by RANKL and its inhibitory effect results from reduced levels of TNF-alpha. Rutin also lowers NF-kappaB activation in response to RANKL.     

Genistein activates endothelial nitric oxide synthase in broiler pulmonary arterial endothelial cells by an Akt-dependent mechanism

Deregulation of endothelial nitric oxide synthase (eNOS) plays an important role in the development of multiple cardiovascular diseases. Our recent study demonstrated that genistein supplementation attenuates pulmonary arterial hypertension in broilers by restoration of endothelial function. In this study, we investigated the molecular mechanism by using broiler pulmonary arterial endothelial cells (PAECs). Our results showed that genistein stimulated a rapid phosphorylation of eNOS at Ser(1179) which was associated with activation of eNOS/NO axis. Further study indicated that the activation of eNOS was not mediated through estrogen receptors or tyrosine kinase inhibition, but via a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent signaling pathway, as the eNOS activity and related NO release were largely abolished by pharmacological inhibitors of PI3K or Akt. Thus, our findings revealed a critical function of Akt in mediating genistein-stimulated eNOS activity in PAECs, partially accounting for the beneficial effects of genistein on the development of cardiovascular diseases observed in animal models.     

Potential role of HMG CoA reductase inhibitor on oxidative stress induced by advanced glycation endproducts in vascular smooth muscle cells of diabetic vasculopathy

Advanced glycation endproducts (AGEs)-induced vascular smooth muscle cell (VSMCs) proliferation and formation of reactive oxygen species (ROS) are emerging as one of the important mechanisms of diabetic vasculopathy but little is known about the antioxidative action of HMG CoA reductase inhibitor (statin) on AGEs. We hypothesized that statin might reduce AGEs-induced intracellular ROS of VSMCs and analyzed the possible mechanism of action of statin in AGEs-induced cellular signaling. Aortic smooth muscle cell of Sprague-Dawley rat (RASMC) culture was done using the different levels of AGEs stimulation in the presence or absence of statin. The proliferation of RASMC, ROS formation and cellular signaling was evaluated and neointimal formation after balloon injury in diabetic rats was analyzed. Increasing concentration of AGEs stimulation was associated with increased RASMC proliferation and increased ROS formation and they were decreased with statin in a dose-dependent manner. Increased NF-κB p65, phosphorylated ERK, phosphorylated p38 MAPK, cyclooxygenase-2, and c-jun by AGEs stimulation were noted and their expression was inhibited by statin. Neointimal formation after balloon injury was much thicker in diabetic rats than the sham-treated group but less neointimal growth was observed in those treated with statin after balloon injury. Increased ROS formation, subsequent activation of MAPK system and increased VSMC proliferation may be possible mechanisms of diabetic vasculopathy induced by AGEs and statin may play a key role in the treatment of AGEs-induced diabetic atherosclerosis.     

Accumulation of argpyrimidine, a methylglyoxal-derived advanced glycation end product, increases apoptosis of lens epithelial cells both in vitro and in vivo

The formation of advanced glycation end products (AGEs) has been considered to be a potential causative factor of injury to lens epithelial cells (LECs). Damage of LECs is believed to contribute to cataract formation. The purpose of this study was to investigate the cytotoxic effect of AGEs on LECs both in vitro and in vivo. We examined the accumulation of argpyrimidine, a methylglyoxal-derived AGE, and the expression of apoptosis-related molecules including nuclear factor- kappaB (NF-κB), Bax, and Bcl-2 in the human LEC line HLE-B3 and in cataractous lenses of Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes. In cataractous lenses from twenty-oneweek- old ZDF rats, LEC apoptosis was markedly increased, and the accumulation of argpyrimidine as well as subsequent activation of NF-κB in LECs were significantly enhanced. The ratio of Bax to Bcl-2 protein levels was also increased. In addition, the accumulation of argpyrimidine triggered apoptosis in methylglyoxal- treated HLE-B3 cells. However, the presence of pyridoxamine (an AGEs inhibitor) and pyrrolidine dithiocarbamate (a NF-κB inhibitor) prevented apoptosis in HLE-B3 cells through the inhibition of argpyrimidine formation and the blockage of NF-κB nuclear translocalization, respectively. These results suggest that the cellular accumulation of argpyrimidine in LECs is NF-κB-dependent and pro-apoptotic.     

Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min. Precision in terms of relative standard deviation (n = 5) is of 3–5% and detection limits is 0.21 ng Cu g⁻¹.     

β-Carotene Inhibits Expression of Matrix Metalloproteinase-10 and Invasion in Helicobacter pylori-Infected Gastric Epithelial Cells

Matrix metalloproteinases (MMPs), key molecules of cancer invasion and metastasis, degrade the extracellular matrix and cell–cell adhesion molecules. MMP-10 plays a crucial role in Helicobacter pylori-induced cell-invasion. The mitogen-activated protein kinase (MAPK) signaling pathway, which activates activator protein-1 (AP-1), is known to mediate MMP expression. Infection with H. pylori, a Gram-negative bacterium, is associated with gastric cancer development. A toxic factor induced by H. pylori infection is reactive oxygen species (ROS), which activate MAPK signaling in gastric epithelial cells. Peroxisome proliferator-activated receptor γ (PPAR-γ) mediates the expression of antioxidant enzymes including catalase. β-Carotene, a red-orange pigment, exerts antioxidant and anti-inflammatory properties. We aimed to investigate whether β-carotene inhibits H. pylori-induced MMP expression and cell invasion in gastric epithelial AGS (gastric adenocarcinoma) cells. We found that H. pylori induced MMP-10 expression and increased cell invasion via the activation of MAPKs and AP-1 in gastric epithelial cells. Specific inhibitors of MAPKs suppressed H. pylori-induced MMP-10 expression, suggesting that H. pylori induces MMP-10 expression through MAPKs. β-Carotene inhibited the H. pylori-induced activation of MAPKs and AP-1, expression of MMP-10, and cell invasion. Additionally, it promoted the expression of PPAR-γ and catalase, which reduced ROS levels in H. pylori-infected cells. In conclusion, β-carotene exerts an inhibitory effect on MAPK-mediated MMP-10 expression and cell invasion by increasing PPAR-γ-mediated catalase expression and reducing ROS levels in H. pylori-infected gastric epithelial cells.     

Electrochemistry and chemiluminescence techniques compared in the detection of NADPH oxidase activity in phagocyte cells

Several methodologies have been used in clinical chemistry for real-time assessment of NADPH oxidase primary product superoxide anion which dismutases to hydrogen peroxide. Among these methodologies, isoluminol chemiluminescence (CL) is considered to be one of the more sensitive and reliable techniques for the assessment of NADPH oxidase activity in neutrophils. The electrochemical technique was recently designed and also applied for real-time detection of NADPH oxidase activity in neutrophils but its reliability and sensitivity has not been investigated so far. In this study, isoluminol CL and electrochemical techniques were investigated and compared by monitoring the generation of superoxide and hydrogen peroxide in both PLB 985 cell line differentiated into neutrophil-like cells and human neutrophils. The electrochemical technique was shown to be as sensitive as that of CL and able to detect the reactive oxygen species (ROS) release of as low as 500 cells. Thus, the electrochemical technique could be used as an alternative to optical techniques for the evaluation of extracellular ROS in phagocyte cells.