Expression Of NADPH Oxidase Subunits Research Articles

P23 Saikosaponin a ameliorate learning and memory impairment via anti-inflammation effect in an AD mouse model

The present study investigated the effects of saikosaponin a (SSA), a natural bioactive compound from Radix bupleuri, on memory deficit and neuroinflammation in the Aβ-treated C57BL/6 mouse model of AD. Different doses of SSA (4, 8, or 16 mg/kg) were intragastrically administered once a day after intracerebroventricular Aβ injection (2.5 μg/μl, 4 μl). 15 days after Aβ injection, behavioural experiments including the Morris water maze and novel object recognition (NOR) tests were performed. 21 days after the Aβ injection, the concentrations of ROS, IL-1β and TNF-α, as well as expression of NADPH oxidase subunits in hippocampal tissue were all assessed. Results from the Morris water maze test revealed that Aβ injection could remarkably increase the escape latency time while SSA could significantly decrease it. In the NOR test, SSA significantly increased the Preference Index (PI), which remarkably decreased in the Aβ injection group. ROS, IL-1β and TNF-α levels all increased after Aβ injection, while SSA significantly reduced the concentration of ROS, IL-1β and TNF-α at doses of 8 mg/kg and 16 mg/kg. Protein expression of NADPH oxidase subunits gp91phox and p47phox were significantly reduced by SSA at doses of 8 and 16 mg/kg. The results indicated that SSA could ameliorate learning and memory impairment induced by Aβ treatment and these effects were mediated through inhibition of pro-inflammatory mediators in hippocampal tissue. Furthermore, NADPH oxidase may be involved in this effect. SSA may be a potent therapeutic drug for the treatment of neurodegenerative diseases associated with neuroinflammation and cognitive impairment such as AD.

Resveratrol prevents angiotensin II-induced hypertrophy of vascular smooth muscle cells through the transactivation of growth factor receptors

We previously showed that augmented levels of endogenous angiotensin II (AngII) contribute to vascular smooth muscle cell (VSMC) hypertrophy through the transactivation of growth factor receptors in spontaneously hypertensive rats. Resveratrol (RV), a polyphenolic component of red wine, has also been shown to attenuate AngII-evoked VSMC hypertrophy; however, the molecular mechanism mediating this response is obscure. The present study was therefore undertaken to examine whether RV could prevent AngII-induced VSMC hypertrophy through the transactivation of growth factor receptor and associated signaling pathways. AngII treatment of VSMC enhanced the protein synthesis that was attenuated towards control levels by RV pretreatment as well as by the inhibitors of NADPH oxidase, c-Src, and growth factor receptors. Furthermore, RV pretreatment also inhibited enhanced levels of superoxide anion, NADPH oxidase activity, increased expression of NADPH oxidase subunits, and phosphorylation of c-Src, EGF-R, PDGE-R, ERK1/2, and AKT1/2. In conclusion, these results indicate that RV attenuates AngII-induced VSMC hypertrophy through the inhibition of enhanced oxidative stress and activation of c-Src, growth factor receptors, and MAPK/AKT signaling. We suggest that RV could be used as a therapeutic agent in the treatment of vascular complications associated with hypertension and hypertrophy.

Treadmill exercise produces neuroprotective effects in a murine model of Parkinson’s disease by regulating the TLR2/MyD88/NF-κB signaling pathway

Parkinson’s disease (PD) is characterized by progressive dopamine depletion and a loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Treadmill exercise is a promising non-pharmacological approach for reducing the risk of PD and other neuroinflammatory disorders, such as Alzheimer’s disease. The goal of this study was to investigate the effects of treadmill exercise on α-synuclein-induced neuroinflammation and neuronal cell death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Eight weeks of treadmill exercise improved motor deficits and reduced α-synuclein expression, a major causative factor of PD-like symptoms, in MPTP mice. Treadmill exercise also down-regulated the expression of toll-like receptor 2 and its associated downstream signaling molecules, including myeloid differentiation factor-88, tumor necrosis factor receptor-associated factor 6, and transforming growth factor-β-activated protein kinase 1. These effects were associated with reduced ionized calcium-binding adapter molecule 1 expression, decreased IκBα and nuclear transcription factor-κB phosphorylation, decreased tumor necrosis factor α and interleukin-1β expression, and decreased NADPH oxidase subunit expression in the SNpc and striatum. Additionally, it promoted the expression of tyrosine hydroxylase and the dopamine transporter, as well as plasma dopamine levels, in MPTP mice; these effects were associated with decreased caspase-3 expression and cleavage, as well as increased Bcl-2 expression in the SNpc. Taken together, our data suggest that treadmill exercise improves MPTP-associated motor deficits by exerting neuroprotective effects in the SNpc and striatum, supporting the notion that treadmill exercise is useful as a non-pharmacological tool for the management of PD.

Crosstalk between HDAC6 and Nox2-based NADPH oxidase mediates HIV-1 Tat-induced pro-inflammatory responses in astrocytes

Histone deacetylase 6 (HDAC6) likely is important in inflammatory diseases. However, how HDAC6 exerts its effect on inflammatory processes remains unclear. HIV-1 transactivator of transcription (Tat) activates NADPH oxidase resulting in generation of reactive oxygen species (ROS), leading to extensive neuro-inflammation in the central nervous system. We investigated the correlation of HDAC6 and NADPH oxidase in HIV-1 Tat-stimulated astrocytes. HDAC6 knockdown attenuated HIV-1 Tat-induced ROS generation and NADPH oxidase activation. HDAC6 knockdown suppressed HIV-1 Tat-induced expression of NADPH oxidase subunits, such as Nox2, p47phox, and p22phox. Specific inhibition of HDAC6 using tubastatin A suppressed HIV-1 Tat-induced ROS generation and activation of NADPH oxidase. N-acetyl cysteine, diphenyl iodonium, and apocynin suppressed HIV-1 Tat-induced expression of HDAC6 and the pro-inflammatory chemokines CCL2, CXCL8, and CXCL10. Nox2 knockdown attenuated HIV-1 Tat-induced HDAC6 expression and subsequent expression of chemokines. The collective results point to the potential crosstalk between HDAC6 and NADPH oxidase, which could be a combined therapeutic target for relief of HIV-1 Tat-mediated neuro-inflammation.

Garcinia mangostana pericarp extract protects against oxidative stress and cardiovascular remodeling via suppression of p47phox and iNOS in nitric oxide deficient rats

Nω-Nitro-l-arginine methyl ester (l-NAME)-induced hypertension and cardiovascular remodeling are associated with oxidative stress and inflammation. Garcinia mangostana Linn., has been reported to have antioxidant and anti-inflammatory properties. This study investigated whether G. mangostana pericarp extract (GME) could prevent l-NAME-induced hemodynamic alterations, cardiovascular remodeling, oxidative stress and inflammation in rats. Male Sprague-Dawley rats were given 40mg/kg/day of l-NAME in drinking water to induce hypertension, and were simultaneously treated with GME at a dose of 200mg/kg/day. Rats that received l-NAME for five weeks had high blood pressure, left ventricular hypertrophy and thickening of aortic wall. Vascular superoxide production, plasma malondialdehyde (MDA), and plasma tumor necrosis factor alpha (TNF-α) were significantly increased in l-NAME-hypertensive rats (p<0.05). This was consistent with up-regulation of the p47phox NADPH oxidase subunit and iNOS protein expression in aortic tissues (p<0.05). Low levels of plasma nitric oxide metabolites were observed in l-NAME hypertension. GME prevented the development of hypertension and cardiovascular remodeling induced by l-NAME with reduced oxidative stress and inflammation. These data suggest that GME had a protective effect against l-NAME-induced hypertension and cardiovascular remodeling via suppressing p47phox NADPH oxidase subunit and iNOS protein expression resulting in enhancing NO bioavailability.

Replicative senescence promotes prothrombotic responses in endothelial cells: Role of NADPH oxidase- and cyclooxygenase-derived oxidative stress

Endothelial senescence has been suggested to promote endothelial dysfunction in age-related vascular disorders. This study evaluated the prothrombotic properties of senescent endothelial cells (ECs) and the underlying mechanism. Serial passaging from passage (P)1 to P4 (replicative senescence) of porcine coronary artery ECs, or treatment of P1 ECs with the endothelial nitric oxide synthase (eNOS) inhibitor L-NAME (premature senescence) induced acquisition of markers of senescence including increased senescence-associated-β-galactosidase (SA-β-gal) activity and p53, p21, p16 expression. Approximately 55% of P3 cells were senescent with a high level oxidative stress, and decreased eNOS-derived nitric oxide (NO) formation associated with increased expression of NADPH oxidase subunits (gp91phox, p47phox), cyclooxygenase (COX)-2 but not COX-1, and a decreased eNOS expression leading to a reduced ability of ECs to inhibit platelet aggregation. P3 cells also presented increased expression and activity of tissue factor (TF), a key initiator of the coagulation cascade.Treatment of senesecent cells with a NADPH oxidase inhibitor (VAS-2870) or by a COX inhibitor (indomethacin) reduced oxidative stress, decreased TF activity and expression, and reduced the expression of gp91phox, p47phox and COX-2 and restored the ability of ECs to inhibit effectively platelet aggregation. Thus, replicative endothelial senescence promotes a prothrombotic response involving the down-regulation of the protective NO pathway and the upregulation of the NADPH oxidase- and COXs-dependent oxidative stress pathway promoting TF expression and activity.

Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

BackgroundAtmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. MethodsUltrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. ResultsIn rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100μg/cm2 induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10μg/cm2) and rotenone (2nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91phox, p47phox and p40phox); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47phox and p67phox translocation assembling active NADPH oxidase. ConclusionufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to oxidative damage to DA neurons. Our findings delineated the potential role of ultrafine particles alone and in combination with pesticide rotenone in the pathogenesis of PD.

Administration of bovine casein-derived peptide prevents cognitive decline in Alzheimer disease model mice.

There is a growing interest in identifying natural food ingredients that may serve to prevent dementia such as that due to Alzheimer disease (AD). Peptides derived from food proteins have been demonstrated to have various physiological activities such as a hypotensive action. Recent findings have indicated possible associations of hypertension with AD progression, and suggest that angiotensin converting enzyme (ACE) inhibitors with potential to pass through the blood brain barrier (BBB) may reduce the risk of AD. In this study, we investigated the effect of milk peptide (CH-3) on cognitive function in AD model mice. CH-3 contains a tripeptide (methionine-lysine-proline, MKP) that has been found to have a strong ACE inhibitory effect and the potential to pass through the BBB. Adult male ddY mice were used in this study, and an animal model of AD was induced by intracerebroventricular (ICV) injection of Aβ1–42. CH-3 (250 mg/kg/day) or MKP (0.5 mg/kg/day) was orally administered every day starting 2 days before ICV injection. At 3 weeks after ICV injection, cognitive function was evaluated by the Morris water maze test. Brain samples were obtained after behavioral testing, and expression of inflammatory cytokines and NADPH oxidase subunits was measured by real-time quantitative RT-PCR. ICV injection of Aβ1–42 significantly impaired cognitive function compared with that in PBS-injected mice. Daily administration of CH-3 markedly attenuated this Aβ1-42-induced cognitive decline. Aβ1–42 injection significantly enhanced the expression of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and p22phox in the mouse hippocampus compared with PBS injection, and showed a tendency to increase the expression of monocyte chemoattractant protein-1 (MCP-1), p47phox and gp91phox, whereas CH-3 treatment markedly reduced Aβ1-42-induced TNF-α, MCP-1, iNOS, p47phox and gp91phox expression. Finally, administration of MKP also attenuated Aβ1-42-induced cognitive impairment with an increase in cerebral blood flow. The present study demonstrated that repeated oral administration of CH-3 to AD model mice not only improved cognitive function but also suppressed the expression of inflammatory cytokines and production of oxidative stress, and suggests its therapeutic potential for preventing cognitive impairment in AD.

Abstract WP289: Interaction Between Stroke and Amyloid-β Deposition in Cognitive Function ~Possible Involvement of Antagonist Effect of AT 2 Receptor Activation~

Background and Aim: Stroke is known to be causally related with Alzheimer’s disease pathology and amyloid-β (Aβ) deposition has been suggested to induce cerebral amyloid angiopathy resulting in stroke-associated cognitive decline. Angiotensin (Ang) type 1 (AT 1 ) receptor activation impairs cognitive performance, whereas Ang II type 2 (AT 2 ) receptor stimulation has been reported to improve cognitive impairment. We examined the interaction between stroke and Aβ deposition in cognitive function, and the possible antagonist effect of AT 2 receptor in vascular smooth muscle cells (VSMC) on this cognitive decline. Methods: Adult (10-12 weeks old) male wild-type (WT) mice (C57BL/6J mice) or the mice with AT 2 receptor overexpression in VSMC (SMAT2) were used. Mice were subjected to intracerebroventricular (ICV) injection of Aβ1-40 following 15 minutes global brain ischemia operation. Three weeks after Aβ1-40 ICV injection, cognitive function of spatial learning memory was evaluated by the Morris water maze test. Brain samples were obtained after behavioral testing, and the expression of inflammatory cytokines and NADPH oxidase subunits were measured by real-time quantitative RT-PCR. Results: ICV injection of Aβ1-40 in WT mice showed impaired cognitive function. On the other hand, WT mice with transient global brain ischemia did not decline significantly cognitive function. In contrast, WT mice with Aβ1-40 ICV injection with global brain ischemia exhibited more marked cognitive impairment compared with control mice. These results suggested that transient brain ischemia and amyloid-β deposition exerted at least additive effects on cognitive impairment. This cognitive decline was accompanied with increased expressions of inflammatory cytokines such as monocyte chemoattractant protein-1 and NADPH oxidase subunits including p22 phox . On the other hand, SMAT 2 mice did not show cognitive impairment by global brain ischemia with/without Aβ1-40 ICV injection. Conclusion: Brain ischemia and amyloid-β deposition induced additive or synergistic effect on cognitive impairment. AT 2 receptor activation in VSMC could play an inhibitory role in the cognitive decline induced by brain ischemia and amyloid-β deposition.

Abstract TP95: Exercise Too Soon After Stroke Exacerbates Hyperglycolysis-mediated Brain Damage

Introduction: Although physical exercise has emerged as a potential therapeutic modality for functional deficits following ischemic stroke, the extent of this effect appears to be contingent upon the time of exercise initiation. In the present study, we assessed how exercise timing affected brain damage through hyperglycolysis associated NADPH oxidase (NOX) activation. Methods: Using an intraluminal filament, adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h and assigned to one non-exercise and three exercise groups. Exercise on Rota-rod was initiated for 30 min at 6 h (considered very early), at 24 h (early), and at day 3 (relatively late) after reperfusion. Lactate production was measured 30 min after exercise completion, and NOX activity and protein expression of NOX subunits (p47 phox , gp91 phox , p22 phox and p67 phox ) and glucose transporter 1 and 3 (GLUT1 and 3) were measured at 3 and 24 h after exercise. Apoptotic cell death was determined at 24 h after exercise. Results: Lactate production and Glut-1 and Glut-3 expression were increased after very early exercise (6 h), but not after late exercise (3days), suggesting hyperglycolysis. NOX activity was increased with the initiation of exercise at 6 h (P&lt;0.05), but not 24 h or 3 days, following stroke. Early (6 and 24 h), but not late (3 days), post-stroke exercise was associated with increased (P&lt;0.05) expression of the NOX protein subunit p47 phox , gp91 phox and p67 phox . This may have led to enhanced apoptosis after early exercise in ischemic rats. Conclusion: Hyperglycolysis and NOX activation was associated with an elevation in apoptotic cell death after very early exercise, while the detrimental effect of exercise on stroke recovery began to decrease when exercise was initiated 24 h after reperfusion.

Long-Term Effects of Maternal Deprivation on Redox Regulation in Rat Brain: Involvement of NADPH Oxidase.

Maternal deprivation (MD) causes perinatal stress, with subsequent behavioral changes which resemble the symptoms of schizophrenia. The NADPH oxidase is one of the major generators of reactive oxygen species, known to play a role in stress response in different tissues. The aim of this study was to elucidate the long-term effects of MD on the expression of NADPH oxidase subunits (gp91phox, p22phox, p67phox, p47phox, and p40phox). Activities of cytochrome C oxidase and respiratory chain Complex I, as well as the oxidative stress parameters using appropriate spectrophotometric techniques were analyzed. Nine-day-old Wistar rats were exposed to a 24 h maternal deprivation and sacrificed at young adult age. The structures affected by perinatal stress, cortex, hippocampus, thalamus, and caudate nuclei were investigated. The most prominent findings were increased expressions of gp91phox in the cortex and hippocampus, increased expression of p22phox and p40phox, and decreased expression of gp91phox, p22phox, and p47phox in the caudate nuclei. Complex I activity was increased in all structures except cortex. Content of reduced glutathione was decreased in all sections while region-specific changes of other oxidative stress parameters were found. Our results indicate the presence of long-term redox alterations in MD rats.

The effects of urotensin II on migration and invasion are mediated by NADPH oxidase-derived reactive oxygen species through the c-Jun N-terminal kinase pathway in human hepatoma cells

AimsUrotensin II (UII) is a vasoactive neuropeptide involved in migration and invasion in various cell types. However, the effects of UII on human hepatoma cells still remain unclear. The aim of this study was to investigate the role and mechanism of UII on migration and invasion in human hepatoma cells. MethodsMigration was measured by wound healing assays and a Transwell® methodology, and invasion was analyzed using Matrigel® invasion chambers. Reactive oxygen species (ROS) levels were detected using a 2′, 7′-dichlorofluorescein diacetate probe, and flow cytometry, and protein expression levels were evaluated by western blotting. Cell proliferation and actin polymerization were examined using cell proliferation reagent WST-1 and F-actin immunohistochemistry staining. ResultsExposure to UII promoted migration and invasion in hepatoma cells compared with that in cells without UII. UII also increased matrix metalloproteinase-2 (MMP2) expression in a time-independent manner. Furthermore, UII markedly enhanced ROS generation and NADPH oxidase subunit expression, and consequently facilitated the phosphorylation of c-Jun N-terminal kinase (JNK). The UT antagonist urantide or the antioxidant/NADPH oxidase inhibitor apocynin decreased UII-induced ROS production. JNK phosphorylation, migration, invasion, and MMP9/2 expression were also reversed by pretreatment with apocynin. Urantide and JNK inhibitor SP600125 abrogated migration, invasion, or MMP9/2 expression in response to UII. UII induced actin polymerization and fascin protein expression, and could be reversed by apocynin and SP600125. ConclusionsExogenous UII induced migration and invasion in hepatoma cells that mainly involved NADPH oxidase-derived ROS through JNK activation. UT played an additional role in regulating hepatoma cells migration and invasion. Thus, our data suggested an important effect of UII in hepatocellular carcinoma metastasis.

Genetic and Pharmacologic Inhibition of the Chemokine Receptor CXCR2 Prevents Experimental Hypertension and Vascular Dysfunction.

The recruitment of leukocytes to the vascular wall is a key step in hypertension development. Chemokine receptor CXCR2 mediates inflammatory cell chemotaxis in several diseases. However, the role of CXCR2 in hypertension development and the underlying mechanisms remain unknown. Angiotensin II (490 ng·kg-1·min-1) or deoxycorticosterone acetate (DOCA) salt-induced mouse hypertensive models in genetic ablation, pharmacologic inhibition of CXCR2, and adoptive bone marrow transfer mice were used to determine the role of CXCR2 in hypertension (measured by radiotelemetry and tail-cuff system), inflammation (verified by flow cytometry and quantitative real-time polymerase chain reaction [PCR] analysis), vascular remodeling (studied by haematoxylin and eosin and Masson's trichrome staining), vascular dysfunction (assessed by aortic ring), and oxidative stress (indicated by nicotinamide adenine dinucleotide phosphate [NADPH] oxidase activity, dihydroethidium staining, and quantitative real-time PCR analysis). Moreover, the blood CXCR2+ cells in normotensive controls and hypertension patients were analyzed by flow cytometry. Angiotensin II significantly upregulated the expression of CXCR2 mRNA and protein and increased the number of CD45+ CXCR2+ cells in mouse aorta (n=8 per group). Selective CXCR2 knockout (CXCR2-/-) or pharmacological inhibition of CXCR2 markedly reduced angiotensin II- or DOCA-salt-induced blood pressure elevation, aortic thickness and collagen deposition, accumulation of proinflammatory cells into the vascular wall, and expression of cytokines (n=8 per group). CXCR2 inhibition also ameliorated angiotensin II-induced vascular dysfunction and reduced vascular superoxide formation, NADPH activity, and expression of NADPH oxidase subunits (n=6 per group). Bone marrow reconstitution of wild-type mice with CXCR2-/- bone marrow cells also significantly abolished angiotensin II-induced responses (n=6 per group). It is important to note that CXCR2 blockade reversed established hypertension induced by angiotensin II or DOCA-salt challenge (n=10 per group). Furthermore, we demonstrated that CXCR2+ proinflammatory cells were higher in hypertensive patients (n=30) compared with normotensive individuals (n=20). Infiltration of CXCR2+ cells plays a pathogenic role in arterial hypertension and vascular dysfunction. Inhibition of CXCR2 pathway may represent a novel therapeutic approach to treat hypertension.

Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats

We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilin-negative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats.

Nitrite-mediated reduction of macrophage NADPH oxidase activity is dependent on xanthine oxidoreductase-derived nitric oxide but independent of S-nitrosation

BackgroundInorganic nitrite has shown beneficial effects in cardiovascular and metabolic diseases partly via attenuation of NADPH-oxidase (NOX)-mediated oxidative stress. However, the exact mechanisms are still unclear. Here we investigated the role of S-nitrosation or altered expression of NOX subunits, and the role of xanthine oxidoreductase (XOR) in nitrite-derived nitric oxide (NO) production. MethodsMouse macrophages were activated with LPS in the presence or absence of nitrite. NOX activity was measured by lucigenin-dependent chemiluminescence. Gene and protein expression of NOX2 subunits and XOR were investigated using qPCR and Western Blot. S-nitrosation of Nox2 and p22phox was studied with a Biotin Switch assay. Uric acid levels in cell culture medium were analyzed as a measure of XOR activity, and NO production was assessed by DAF-FM fluorescence. ResultsNOX activity in activated macrophages was significantly reduced by nitrite. Reduced NOX activity was not attributed to decreased NOX gene expression. However, protein levels of p47phox and p67phox subunits were reduced by nitrite in activated macrophages. Protein expression of Nox2 and p22phox was not influenced by this treatment and neither was their S-nitrosation status. Increased uric acid levels after nitrite and diminished NO production during XOR-inhibition with febuxostat suggest that XOR is more active during nitrite-treatment of activated macrophages and plays an important role in the bioactivation of nitrite. ConclusionsOur findings contribute to the mechanistic understanding about the therapeutic effects associated with nitrite supplementation in many diseases. We show that nitrite-mediated inhibition of NOX activity cannot be explained by S-nitrosation of the NOX enzyme, but that changes in NOX2 expression and XOR function may contribute.

Intermittent local periodontal inflammation causes endothelial dysfunction of the systemic artery via increased levels of hydrogen peroxide concomitantly with overexpression of superoxide dismutase

BackgroundThe present study was designed to examine whether the intermittent local periodontal inflammation induces endothelial dysfunction of the systemic artery caused by oxidative stress and if increased levels of hydrogen peroxide coexisted with overexpression of superoxide dismutase (SOD) as well as NADPH oxidase contribute to the oxidative stress. MethodsThe rats in lipopolysaccharides (LPS) group received 1500μg LPS injection to bilateral gingiva of the lower jaw a week interval from eight- to eleven-week-old. Isolated mandibles or aortas were subjected to the evaluation of histopathological changes, isometric force recordings, reactive oxygen species using 2′,7′-dichlorofluorescin diacetate (10−5mol/L) and protein expression of NADPH oxidase subunits and SOD, respectively. ResultsMandible sections demonstrated the periodontal inflammation only in the LPS group at three days, but not seven days, after the LSP injection. Acetylcholine (10−9 to 10−5mol/L)-induced relaxation was reduced only in aortas from the LPS group. Gp91ds-tat and PEG-catalase restored the impaired dilation in arteries from the LPS group. Levels of reactive oxygen species were enhanced in aortas from the LPS group, whereas the increment was abolished by the treatment with gp91-ds-tat or PEG-catalase. Expression of a NADPH oxidase subunit p47phox and CuZn-SOD increased in the LPS group. ConclusionsThe intermittent local periodontal inflammation induces systemic endothelial dysfunction caused by overproduction of reactive oxygen species in the systemic artery of rats and that overexpression of CuZn-SOD as well as a NADPH oxidase cytosolic subunit contributes to increased levels of hydrogen peroxide in blood vessels of this animal model.

Diminished COX-2/PGE2-Mediated Antiviral Response Due to Impaired NOX/MAPK Signaling in G6PD-Knockdown Lung Epithelial Cells.

Glucose-6-phosphate dehydrogenase (G6PD) provides the reducing agent NADPH to meet the cellular needs for reductive biosynthesis and the maintenance of redox homeostasis. G6PD-deficient cells experience a high level of oxidative stress and an increased susceptibility to viral infections. Cyclooxygenase-2 (COX-2) is a key mediator in the regulation of viral replication and inflammatory response. In the current study, the role of G6PD on the inflammatory response was determined in both scramble control and G6PD-knockdown (G6PD-kd) A549 cells upon tumor necrosis factor-α (TNF-α) stimulation. A decreased expression pattern of induced COX-2 and reduced production of downstream PGE2 occurred upon TNF-α stimulation in G6PD-kd A549 cells compared with scramble control A549 cells. TNF-α-induced antiviral activity revealed that decreased COX-2 expression enhanced the susceptibility to coronavirus 229E infection in G6PD-kd A549 cells and was a result of the decreased phosphorylation levels of MAPK (p38 and ERK1/2) and NF-κB. The impaired inflammatory response in G6PD-kd A549 cells was found to be mediated through NADPH oxidase (NOX) signaling as elucidated by cell pretreatment with a NOX2-siRNA or NOX inhibitor, diphenyleneiodonium chloride (DPI). In addition, NOX activity with TNF-α treatment in G6PD-kd A549 cells was not up-regulated and was coupled with a decrease in NOX subunit expression at the transcriptional level, implying that TNF-α-mediated NOX signaling requires the participation of G6PD. Together, these data suggest that G6PD deficiency affects the cellular inflammatory response and the decreased TNF-α-mediated antiviral response in G6PD-kd A549 cells is a result of dysregulated NOX/MAPK/NF-κB/COX-2 signaling.

Effects of candesartan cilexetil and amlodipine orotate on receptor for advanced glycation end products expression in the aortic wall of Otsuka Long-Evans Tokushima Fatty (OETFF) type 2 diabetic rats.

The receptor for advanced glycation end products (RAGE) plays a key role in the development of vascular inflammation and acceleration of atherosclerosis in type 2 diabetes. We investigated the effect of candesartan cilexetil (CDRT) and amlodipine orotate (AMDP) on the expression of RAGE in the aortic walls of Otsuka Long-Evans Tokushima Fatty (OLETF) rats and AGE-treated endothelial cells. Twenty five-week-old OLETF rats were randomized to 8week treatments consisting of CDRT (n=8), AMDP (n=8) or saline (control, n=8). Immunohistochemical and dihydroethidine staining revealed reduced RAGE and reactive oxygen species (ROS) signals in rats treated with CDRT or AMDP compared with control rats. Both CDRT and AMDP suppressed the expression of p22phox and p47phox NADPH oxidase subunits. However, only CDRT significantly reduced expression of phosphorylated extracellular signal regulated kinase (pERK)1/2 in the aortic wall of OLETF rats. In addition, both drugs reduced RAGE expression and total and mitochondrial ROS production in the AGE-treated endothelial cells. Both ARBs and CCBs reduced RAGE expression in the aortic walls of OLETF rats, which was attributed to decreased ROS production through inhibition of NADPH oxidase. In addition, only CDRT reduced aortic expression of RAGE via suppression of the ERK1/2 pathway unlike AMDP.

Curcumin Protects Neonatal Rat Cardiomyocytes against High Glucose-Induced Apoptosis via PI3K/Akt Signalling Pathway.

The function of curcumin on NADPH oxidase-related ROS production and cardiac apoptosis, together with the modulation of protein signalling pathways, was investigated in cardiomyocytes. Primary cultures of neonatal rat cardiomyocytes were exposed to 30 mmol/L high glucose with or without curcumin. Cell viability, apoptosis, superoxide formation, the expression of NADPH oxidase subunits, and potential regulatory molecules, Akt and GSK-3β, were assessed in cardiomyocytes. Cardiomyocytes exposure to high glucose led to an increase in both cell apoptosis and intracellular ROS levels, which were strongly prevented by curcumin treatment (10 μM). In addition, treatment with curcumin remarkably suppressed the increased activity of Rac1, as well as the enhanced expression of gp91phox and p47phox induced by high glucose. Lipid peroxidation and SOD were reversed in the presence of curcumin. Furthermore, curcumin treatment markedly inhibited the reduced Bcl-2/Bax ratio elicited by high glucose exposure. Moreover, curcumin significantly increased Akt and GSK-3β phosphorylation in cardiomyocytes treated with high glucose. In addition, LY294002 blocked the effects of curcumin on cardiomyocytes exposure to high glucose. In conclusion, these results demonstrated that curcumin attenuated high glucose-induced cardiomyocyte apoptosis by inhibiting NADPH-mediated oxidative stress and this protective effect is most likely mediated by PI3K/Akt-related signalling pathway.

Urotensin II-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in HepG2 cells.

To investigated the effects of urotensin II (UII) on hepatic insulin resistance in HepG2 cells and the potential mechanisms involved. Human hepatoma HepG2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucose-oxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species (ROS) levels were detected with a multimode reader using a 2',7'-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase (JNK), insulin signal essential molecules such as insulin receptor substrate -1 (IRS-1), protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), and glucose transporter-2 (Glut 2), and NADPH oxidase subunits such as gp91(phox), p67(phox), p47(phox), p40(phox), and p22(phox) were evaluated by Western blot. Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption (P < 0.05) and glycogen content (P < 0.01) in HepG2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression (P < 0.01) and phosphorylation of IRS-1 (P < 0.05), associated with down-regulation of Akt (P < 0.05) and GSK-3β (P < 0.05) phosphorylation levels, and the expression of Glut 2 (P < 0.001), indicating an insulin-resistance state in HepG2 cells. Furthermore, UII enhanced the phosphorylation of JNK (P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1 (P < 0.001), phosphorylation of IRS-1 (P < 0.001) and GSK-3β (P < 0.05), and glycogen synthesis (P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation (P < 0.05) and NADPH oxidase subunit expression (P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production (P < 0.05), JNK phosphorylation (P < 0.05), and insulin resistance (P < 0.05) in HepG2 cells. UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG2 cells.