Protein Levels Of NADPH Oxidase Research Articles

Acylated Ghrelin Attenuates l-Thyroxin-induced Cardiac Damage in Rats by Antioxidant and Anti-inflammatory Effects and Downregulating Components of the Cardiac Renin-angiotensin System.

This study investigated the protective effect of acylated ghrelin (AG) against l-thyroxin (l-Thy)-induced cardiac damage in rats and examined possible mechanisms. Male rats were divided into five intervention groups of 12 rats/group: control, control + AG, l-Thy, l-Thy + AG, and l-Thy + AG + [D-Lys3]-GHRP-6 (AG antagonist). l-Thy significantly reduced the levels of AG and des-acyl ghrelin and the AG to des-acyl ghrelin ratio. Administration of AG to l-Thy-treated rats reduced cardiac weights and levels of reactive oxygen species and preserved the function and structure of the left ventricle. In addition, AG also reduced the protein levels of cleaved caspase-3 and cytochrome c and prevented mitochondrial permeability transition pore opening. In the left ventricle of both control + AG-treated and l-Thy + AG-treated rats, AG significantly increased left ventricular levels of manganese superoxide dismutase (SOD2), total glutathione (GSH), and Bcl2. It also reduced the levels of malondialdehyde, tumor necrosis factor-α (TNF-α), interleukin-6, and Bax and the nuclear activity of nuclear factor-kappa B. Concomitantly, in both treated groups, AG reduced the mRNA and protein levels of NADPH oxidase 1, angiotensin (Ang) II type 1 receptor, and Ang-converting enzyme 2. All the beneficial effects of AG in l-Thy-treated rats were prevented by the coadministration of [D-Lys3]-GHRP-6, a selective growth hormone secretagogue receptor subtype 1a antagonist. In conclusion, AG protects against hyperthyroidism-induced cardiac hypertrophy and damage, which is mainly due to its antioxidant and anti-inflammatory potentials and requires the activation of GHS-R1a.

Inhibition of NLRP3 inflammasome by glibenclamide attenuated dopaminergic neurodegeneration and motor deficits in paraquat and maneb-induced mouse Parkinson’s disease model

Pesticides exposure can lead to damage of dopaminergic neurons, which are associated with increased risk of Parkinson’s disease (PD). However, the etiology of PD remains poorly understood and no therapeutic strategy is available. Previous studies suggested the involvement of NLRP3 inflammasome in the onset of PD. This study was designed to investigate whether glibenclamide, an inhibitor of NLRP3 inflammasome, could offer a reliable protective strategy for PD in a mouse PD model induced by paraquat and maneb. We found that glibenclamide exerted potent neuroprotection against paraquat and maneb-induced upregulation of α-synuclein, dopaminergic neurodegeneration and motor impairment in brain of mice. Mechanistically, glibenclamide treatment blocked NLRP3 inflammasome activation evidenced by reduced expressions of NLRP3, activated caspase-1 and mature interleukin-1β in glibenclamide co-treated mice compared with those in paraquat and maneb group mice. Furthermore, glibenclamide treatment mitigated paraquat and maneb-induced microglial M1 proinflammatory response and nuclear factor-κB activation in mice. Finally, the increased superoxide production, lipid peroxidation, protein levels of NADPH oxidase 2 (NOX2) and inducible nitric oxide synthase (iNOS) induced by paraquat and maneb were all attenuated by glibenclamide. Overall, our findings demonstrated that glibenclamide protected dopaminergic neurons in a mouse PD model induced by combined exposures of paraquat and maneb through suppression of NLRP3 inflammasome activation, microglial M1 polarization and oxidative stress.

Overexpression of heat shock protein 70 inhibits epithelial-mesenchymal transition and cell migration induced by transforming growth factor-β in A549 cells.

Heat shock protein 70 (HSP70) is a key member of the HSP family that contributes to a pre-cancerous environment; however, its role in lung cancer remains poorly understood. The present study used geranylgeranylacetone (GGA) to induce HSP70 expression, and transforming growth factor-β (TGF-β) was used to construct an epithelial-mesenchymal transition (EMT) model by stimulating A549 cells in vitro. Western Blot was performed to detect protein levels of NADPH oxidase 4 (NOX4) and the EMT-associated proteins E-cadherin and vimentin both before and after HSP70 expression. Cell morphological changes were observed, and the effect of HSP70 on cell migration ability was detected via the wound healing. The results demonstrated that GGA at 50 and 200 μmol/L could significantly induce HSP70 expression in A549 cells (P < 0.05). Furthermore, HSP70 induced by 200 μmol/L GGA significantly inhibited the changes of E-cadherin, vimentin, and cell morphology induced by TGF-β (P < 0.05), while HSP70 induced by 50 μmol/L GGA did not. The results of the wound healing assay indicated that 200 μmol/L GGA significantly inhibited A549 cell migration induced by TGF-β. Taken together, the results of the present study demonstrated that overexpression of HSP70 inhibited the TGF-β induced EMT process and changed the cell morphology and migratory ability induced by TGF-β in A549 cells.

Age-related insult of cochlear ribbon synapses: An early-onset contributor to D-galactose-induced aging in mice

Presbycusis results from age-related degeneration of the auditory system. D-galactose (D-gal)-induced aging is an ideal and commonly used animal model in aging research. Previous studies demonstrate that administration of D-gal can activate mitochondria-dependent apoptosis in the cochlear stria vascularis. However, D-gal-induced changes to cochlear inner (IHCs) and outer (OHCs) hair cells, spiral ganglion cells (SGCs), and ribbon synapses connecting IHCs and SGCs have not been systematically reported. The current study investigated changes in the numbers of hair cells, SGCs, and ribbon synapses in the mouse model of aging. We found that in comparison to control mice, the numbers of ribbon synapses and their nerve fibers were significantly decreased in D-gal-treated mice, whereas the numbers of OHCs, IHCs, and SGCs were almost unchanged. Moreover, hair cell stereocilia were also not obviously influenced by D-gal administration. Although D-gal-induced aging did not significantly shift the auditory brainstem response (ABR) thresholds in the 8, 16, and 32 kHz frequency bands, the amplitude and latency of the ABR wave I, reflecting ribbon synapse functions, were abnormal in D-gal-treated mice compared to control mice. We also found that 8-hydroxy-2-deoxyguanosine, a marker of oxidative DNA damage, was significantly increased in mitochondria of cochleae from mice exposed to D-gal-induced aging in comparison to control mice. Moreover, D-gal administration increased the levels of H2O2 and mitochondrial 3860-bp common deletion, and decreased superoxide dismutase activity and ATP production in the cochlea. Furthermore, compared with control mice, the protein levels of NADPH oxidase 2 and uncoupling protein 2 were significantly increased in the cochlea of D-gal-treated mice. Taken together, these findings support that the cochlear ribbon synapse is the primary insult site in the early stage of presbycusis, and mitochondrial oxidative damage and subsequent dysfunctions might be responsible for this insult.

Magnesium lithospermate B prevents phenotypic transformation of pulmonary arteries in rats with hypoxic pulmonary hypertension through suppression of NADPH oxidase

Magnesium lithospermate B (MLB) shows multiple biological activities including anti-oxidation and anti-proliferation in various diseases. However, the function of MLB in pulmonary arterial hypertension (PAH) is still unknown. This study aims to investigate the effect of MLB on hypoxia-induced phenotypic transformation of pulmonary arterial smooth muscle cells (PASMCs) and the underlying mechanisms. SD rats (or PASMCs) were exposed to 10% O2 for 3 weeks (or 3% O2 for 48 h) along with MLB or NADPH oxidase （NOX） inhibitor intervention. The effects of MLB on hemodynamics, pulmonary vascular remodeling and phenotypic transformation of PASMCs were observed first. Then, its effects on the protein levels of NOX (NOX2 and NOX4), ERK and p-ERK were examined. The results showed that MLB prevented the elevation in right ventricular systolic pressure and the increase in ratio of wall thickness to vessel external diameter of pulmonary arteries in PAH rats, and attenuated phenotypic transformation of PASMCs (decrease in α-smooth muscle actin while increase in osteopontin), accompanied by downregulation of NOX (NOX2 and NOX4) protein levels, decrease of ROS and H2O2 production, and suppression of the phosphorylation of ERK. NOX inhibitor (VAS2870) achieved similar results to that of MLB did in the hypoxia-treated PASMCs. Based on the observations, we conclude that MLB is able to prevent phenotypic transformation of pulmonary arteries in hypoxic PAH rats through suppression of NOX/ROS/ERK pathway, and MLB might have the potentials in PAH therapy.

Angiotensin (1-7) inhibits arecoline-induced migration and collagen synthesis in human oral myofibroblasts via inhibiting NLRP3 inflammasome activation.

Arecoline induces oral submucous fibrosis (OSF) via promoting the reactive oxygen species (ROS). Angiotensin (1-7) (Ang-(1-7))protects against fibrosis by counteracting angiotensin II (Ang-II) via the Mas receptor. However, the effects of Ang-(1-7) on OSF remain unknown. NOD-like receptors (NLRs) family pyrin domain containing 3 (NLRP3) inflammasome is identified as the novel mechanism of fibrosis. Whereas the effects of arecoline on NLRP3 inflammasome remain unclear. We aimed to explore the effect of Ang-(1-7) on NLRP3 inflammasome in human oral myofibroblasts. In vivo, activation of NLRP3 inflammasomes with an increase of Ang-II type 1 receptor (AT1R) protein level and ROS production in human oral fibrosis tissues. Ang-(1-7) improved arecoline-induced rats OSF, reduced protein levels of NADPH oxidase 4 (NOX4) and the NLRP3 inflammasome. In vitro, arecoline increased ROS along with upregulation of the angiotensin-converting enzyme (ACE)/Ang-II/AT1R axis and NLRP3 inflammasome/interleukin-1β axis in human oral myofibroblasts, which were reduced by NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine, and NOX4 small interfering RNA (siRNA). Furthermore, arecoline induced collagen synthesis or migration via the Smad or RhoA-ROCK pathway respectively, which could be inhibited by NLRP3 siRNA or caspase-1 blocker VX-765. Ang-(1-7) shifted the balance of RAS toward the ACE2/Ang-(1-7)/Mas axis, inhibited arecoline-induced ROS and NLRP3 inflammasome activation, leading to attenuation of migration or collagen synthesis. In summary, Ang-(1-7) attenuates arecoline-induced migration and collagen synthesis via inhibiting NLRP3 inflammasome in human oral myofibroblasts.

Regional Heterogeneity of Cerebral Microvessels and Brain Susceptibility to Oxidative Stress

The hippocampus is one of the earliest and most affected regions in Alzheimer’s disease (AD), followed by the cortex while the cerebellum is largely spared. Importantly, endothelial dysfunction is a common feature of cerebral blood vessels in AD. In this study, we sought to determine if regional heterogeneity of cerebral microvessels might help explain the susceptibility of the hippocampus and cortex as compared to the cerebellum. We isolated microvessels from wild type mice from the cerebellum, cortex, and hippocampus to characterize their vascular phenotype. Superoxide anion was significantly higher in microvessels isolated from the cortex and hippocampus as compared to the cerebellum. Importantly, protein levels of NADPH oxidase (NOX)-2 and NOX-4 were significantly higher in the cortical and hippocampal microvessels as compared to microvessels from the cerebellum. In addition, expression of manganese superoxide dismutase protein was significantly lower in microvessels from the cortex and hippocampus as compared to cerebellum while other antioxidant enzymes were unchanged. There was no difference in eNOS protein expression between the microvessels of the three brain regions; however, bioavailability of tetrahydrobiopterin (BH4), an essential cofactor for eNOS activity, was significantly reduced in microvessels from the hippocampus and cortex as compared to the cerebellum. Higher levels of superoxide and reduced tetrahydrobiopterin bioavailability may help explain the vulnerability of the hippocampus and cortical microvessels to oxidative stress and development of endothelial dysfunction.

Role of NADPH oxidase-4 in saturated fatty acid-induced insulin resistance in SK-Hep-1 cells

We aimed to develop a cell culture model of type 2 diabetes by treating SK-Hep-1 cells with four free fatty acids [i.e., palmitic acid, stearic acid (SA), linoleic acid and oleic acid]. The results showed that Akt phosphorylation was increased in SK-Hep-1 cells treated with insulin in a time- and concentration-dependent manner, which was inhibited by saturated fatty acids, but not by unsaturated fatty acids. Moreover, protein levels of NADPH oxidase (NOX) 4 but not NOX2 were increased following SA treatment and, consequently, increased reactive oxygen species production and decreased cellular glutathione were observed. Apocynin, a NOX4 inhibitor, restored the SA-induced inhibition of Akt phosphorylation, suggesting the role of NOX4 in insulin resistance induced by SA. Neither phosphorylation level nor protein level of the stress signaling kinases, such as c-Jun N-terminal kinase or p38 mitogen activated protein kinase, was changed by SA treatment. Although binding immunoglobulin protein, a marker of endoplasmic reticulum stress, was transiently increased in SKHep-1 cells treated with SA, 4-phenyl butyric acid, a chemical chaperone, had no effect on the insulin-mediated Akt phosphorylation inhibited by SA. The present study provides a useful model for screening anti-insulin resistance drugs and finding new drug targets for treatment of diabetes.

NADPH oxidase-dependent oxidative stress and mitochondrial damage in hippocampus of D-galactose-induced aging rats

Mitochondrial DNA (mtDNA) common deletion (CD) plays a significant role in aging and age-related diseases. In this study, we used D-galactose (D-gal) to generate an animal model of aging and the involvement and causative mechanisms of mitochondrial damage in such a model were investigated. Twenty 5-week-old male Sprague-Dawley rats were randomly divided into two groups: D-gal group (n=10) and control group (n=10). The quantity of the mtDNA CD in the hippocampus was determined using a TaqMan real-time PCR assay. Transmission electron microscopy was used to observe the mitochondrial ultrastructure in the hippocampus. Western blot was used to detect the protein levels of NADPH oxidase (NOX) and uncoupling protein 2 (UCP2). We found that the level of mtDNA CD was significantly higher in the hippocampus of D-gal-induced aging rats than in control rats. In comparison with the control group, the mitochondrial ultrastructure in the hippocampus of D-gal-treated rats was damaged, and the protein levels of NOX and UCP2 were significantly increased in the hippocampus of D-gal-induced aging rats. This study demonstrated that the levels of mtDNA CD and NOX protein expression were significantly increased in the hippocampus of D-gal-induced aging rats. These findings indicate that NOX-dependent reactive oxygen species generation may contribute to D-gal-induced mitochondrial damage.

PKCβ inhibition with ruboxistaurin reduces oxidative stress and attenuates left ventricular hypertrophy and dysfuntion in rats with streptozotocin-induced diabetes

Oxidative stress plays critical roles in the development of diabetic cardiovascular complications, including myocardial hypertrophy. The β isoform of PKC (protein kinase C) is preferentially overexpressed in the myocardium of diabetic subjects accompanied with increased activation of the pro-oxidant enzyme NADPH oxidase, which may exacerbate oxidative stress. We hypothesized that myocardial PKCβ is a major upstream mediator of oxidative stress in diabetes and that PKCβ inhibition can attenuate myocardial hypertrophy and dysfunction. Control or streptozotocin-induced diabetic rats were treated with the selective PKCβ inhibitor RBX (ruboxistaurin; 1mg/kg of body weight per day) or the antioxidant NAC (N-acetylcysteine) for 4weeks. LV (left ventricular) dimensions and functions were detected by echocardiography. 15-F2t-isoprostane (a specific index of oxidative stress) and myocardial activities of superoxide dismutase as well as protein levels of NADPH oxidase were assessed by immunoassay or Western blotting. Echocardiography revealed that the LV mass/body weight ratio was significantly increased in diabetic rats (P<0.01 compared with the control group) in parallel with the impaired LV relaxation. A significant increase in cardiomyocyte cross-sectional area was observed in diabetic rats accompanied by an increased production of O2- (superoxide anion) and 15-F2t-isoprostane (all P<0.05 compared with the control group). RBX normalized these changes with concomitant inhibition of PKCβ2 activation and prevention of NADPH oxidase subunit p67phox membrane translocation and p22phox overexpression. The effects of RBX were comparable with that of NAC, except that NAC was inferior to RBX in attenuating cardiac dysfunction. It is concluded that RBX can ameliorate myocardial hypertrophy and dysfunction in diabetes, which may represent a novel therapy in the prevention of diabetic cardiovascular complications.