We previously demonstrated that the lentivirus lytic peptide 1 (LLP-1) corresponding to the carboxyl terminus of HIV-1 gp41 induced cell death in human neuronal cells. Present study was conducted to further elucidate the pathogenic mechanisms involved in HIV-1 gp41-induced neurodegeneration in AIDS patients with cognitive deficits. The effect of LLP-1 on activation of calpain-1, a calcium-activated cysteine protease, which has been implicated in neuronal degeneration and death, was monitored by the proteolysis of spectrin in rat organotypic hippocampal slice cultures. Protease specific spectrin breakdown products revealed that LLP-1 generated~150/145-kDa fragments characteristic of calpain-1 activation in hippocampus undergoing cell death as evidenced by LDH release. This spectrin cleavage pattern was further confirmed by in vitro calpain-1 proteolysis. Futhermore, calpectin and MDL28170, inhibitors of calpain activity, blocked calpain-1-mediated spectrin cleavage. Spectrin cleavage likely occurred in the absence of overt synaptic loss, as suggested by the preserved levels of synaptophysin. Among pharmacological agents tested, apocynin, NADPH oxidase inhibitor, ameliorated the LLP-1-induced spectrin. Given the role of spectrin essential for synapse stabilization, LLP-1-induced spectrin cleavage as occurs with the activation of calpain-1 may be an important effector in LLP-1mediated cell injury in hippocampus, which is primarily linked to cognitive dysfunction.
Animals ; Calpain ; Cell Death ; Cysteine Proteases ; Hippocampus ; HIV* ; HIV-1* ; Humans* ; Lentivirus* ; NADPH Oxidase ; Neurons ; Protein Structure, Tertiary* ; Proteolysis ; Rats* ; Spectrin* ; Synapses ; Synaptophysin

Atherosclerosis is considered as a chronic inflammatory process. However, the nature of the oxidant signaling that regulates monocyte adhesion and its underlying mechanism is poorly understood. We investigated the role of reactive oxygen species on the vascular cell adhesion molecule-1 (VCAM-1) and monocyte adhesion in the cultured endothelial cells. TNF-alpha at a range of 1~30 ng/ml induced VCAM-1 expression dose-dependently. BCECF-AM-labeled U937 cells firmly adhered on the surface of endothelial cells when the endothelial cells were incubated with TNF-alpha (15 ng/ml). Ten micromol/L of SB203580, an inhibitor of p38 MAPK, significantly reduced TNF-alpha-induced VCAM-1 expression, compared to the JNK inhibitor (40micromol/L of SP60015) or ERK inhibitor (40micrommol/L of U0126). Also, SB203580 significantly inhibited TNF-alpha-induced monocyte adhesion in HUVEC. Superoxide production was minimal in the basal condition, however, treatment of TNF-alpha induced superoxide production in the dihydroethidine-loaded endothelial cells. Diphenyleneiodonium (DPI, 10micromol/L), an inhibitor of NADPH oxidase, and rotenone (1micromol/L), an inhibitor of mitochondrial complex I inhibited TNF-alpha-induced superoxide production, VCAM-1 expression and monocyte adhesion in the endothelial cells. Taken together, our data suggest that NADPH oxidase and mitochondrial ROS were involved in TNF-alpha-induced VCAM-1 and monocyte adhesion in the endothelial cells.
Atherosclerosis ; Endothelial Cells* ; Monocytes* ; NADP* ; NADPH Oxidase* ; p38 Mitogen-Activated Protein Kinases ; Reactive Oxygen Species ; Rotenone ; Superoxides ; Tumor Necrosis Factor-alpha ; U937 Cells ; Vascular Cell Adhesion Molecule-1*

OBJECTIVETo investigate the effect of losartan in regulating oxidative stress and the underlying mechanism in mice with ventilator-induced lung injury.

METHODSThirty-six male C57 mice were randomly divided into control group, losartan treatment group, mechanical ventilation model group, and ventilation plus losartan treatment group. After the corresponding treatments, the lung injuries in each group were examined and the expressions of caveolin-1 and NOX4 in the lung tissues were detected.

RESULTSThe mean Smith score of lung injury was significantly higher in mechanical ventilation model group (3.3) than in the control group (0.4), and losartan treatment group (0.3); the mean score was significantly lowered in ventilation plus losartan treatment group (2.3) compared with that in the model group (P<0.05). The expressions of caveolin-1 and NOX4 were significantly higher in the model group than in the control and losartan treatment groups (P<0.05) but was obviously lowered after losartan treatment (P<0.05). Co-expression of caveolin-1 and NOX4 in the lungs was observed in the model group, and was significantly decreased after losartan treatment.

CONCLUSIONLosartan can alleviate ventilator-induced lung injury in mice and inhibit the expression of caveolin-1 and NOX4 and their interaction in the lungs.

Animals ; Caveolin 1 ; metabolism ; Losartan ; pharmacology ; Lung ; metabolism ; physiopathology ; Male ; Mice ; Mice, Inbred C57BL ; NADPH Oxidase 4 ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Respiration, Artificial ; Ventilator-Induced Lung Injury ; drug therapy ; metabolism

Objective To verify the expressions of genes associated with colorectal cancer with hyperglycemia and evaluate their diagnostic values.Methods Tumor tissues,distal normal intestinal mucosa,and peripheral blood samples were harvested from 109 colorectal cancer patients and peripheral blood samples from 30 diabetes patients and 30 healthy volunteers. The mRNA expressions of glucose regulated protein 78 (GRP78),NADPH oxidase-1 (NOX1),carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5),heat shock protein 60 (HSP60),and histone deacetylase 1(HDAC1) were detected by real-time quantitative polymerase chain reaction. The correlation between the gene expressions and clinicopathological parameters in colorectal cancer patients were analyzed using Pearson's correlation analysis. Diagnostic test accuracy evaluation was used to calculate the sensitivity,specificity,accuracy,predictability,Youden index,and likelihood ratio of serum gene expressions in colorectal cancer patients,and the receiver operating characteristic (ROC) curves were drawn. The area under the ROC curve was calculated to evaluate the diagnostic efficiency of the combined detection of multiple genes.Results The mRNA levels of GRP78 (P=0.001),NOX1 (P=0.022),CEACAM5 (P=0.000),HSP60 (P=0.044),and HDAC1 (P=0.047) were positively correlated with the fasting blood glucose level. The mRNA expressions of NOX1 (P=0.000,P=0.008) and HDAC1 (P=0.000,P=0.037) in tissues and serum were significantly higher in colorectal cancer patients than in patients with normal blood glucose levels. The NOX1 mRNA expression was positively correlated with the diameter of colorectal cancer (P=0.013),and the HDAC1 mRNA expression was significantly correlated with the tumor site (P=0.049),depth of primary tumor invasion (P=0.025),and TNM stage (P=0.042). The areas under the ROC curves of NOX1,CEACAM5,and HDAC1 were 0.931,0.852,and 0.860 respectively (all P=0.000). The specificity,accuracy,and negative predictive value of NOX1,HDAC1 mRNA expression in colorectal cancer patients with hyperglycemia were all above 90%. The diagnostic sensitivity and specificity of the combined detection of NOX1,CEACAM5,and HDAC1 were 98.82% and 99.93%,respectively.Conclusion Combined detection of genes associated with colorectal cancer accompanied by hyperglycemia can improve the diagnostic efficiency of early screening.
Biomarkers, Tumor ; genetics ; Carcinoembryonic Antigen ; genetics ; Case-Control Studies ; Colorectal Neoplasms ; complications ; diagnosis ; genetics ; Diabetes Mellitus ; genetics ; GPI-Linked Proteins ; genetics ; Heat-Shock Proteins ; genetics ; Histone Deacetylase 1 ; genetics ; Humans ; Hyperglycemia ; complications ; diagnosis ; genetics ; NADPH Oxidase 1 ; genetics ; ROC Curve

OBJECTIVETo screen the regulatory proteins involved in Nox1 promoter activation in a cell model of inflammation and oxidative stress.

METHODSA cell model of inflammation and oxidative stress was established by stimulating A549 cells with tumor necrosis factor-α (TNF-α). The differential proteins binding to Nox1 promoter were screened by DNA pull-down and the binding proteins were separated by 2D electrophoresis and selected according to the their differential expression levels (with over 1.5-fold changes relative to the control level). The screened proteins were finally identified by MALDI-TOF/TOF-MS.

RESULTSSeven differentially expressed protein spots (all upregulated in the cell model) were obtained, among which GLE1, DDX19A, KRT1 and KRT10 were identified by mass spectrometry.

CONCLUSIONGLE1, DDX19A, KRT1 and KRT10 participate in the activation of Nox1 promoter in TNF-α-induced A549 cells, and this result provides new insights into the biological roles of the regulatory proteins of Nox1 promoter in inflammation and oxidative stress.

Cell Line, Tumor ; DEAD-box RNA Helicases ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Inflammation ; Keratin-1 ; metabolism ; Keratin-10 ; metabolism ; Mass Spectrometry ; NADPH Oxidase 1 ; NADPH Oxidases ; genetics ; metabolism ; Nucleocytoplasmic Transport Proteins ; metabolism ; Oxidative Stress ; Promoter Regions, Genetic ; Tumor Necrosis Factor-alpha ; adverse effects

Oxidative stress has been paid increasing attention to as an important causative factor for diabetic vascular complications. Among possible various sources, accumulating evidence has indicated that NAD(P)H oxidase may be the most important source for reactive oxygen species production in diabetic vascular tissues. The mechanisms underlying activation and up-regulation of NAD(P)H oxidase has been supposed to be mediated by high glucose-induced protein kinase C (PKC) activation. In this review article, activation of local renin-angiotensin II system induced by chymase activation is also shown to amplify such a PKC-dependent activation of NAD(P)H oxidase. Additionally, human evidence showing the beneficial effect of antioxidants on diabetic vascular complications. Bilirubin has been recognized as a strong endogenous antioxidant. Here markedly lower prevalence of vascular complications is shown in diabetic patients with Gilbert syndrome, a congenital hyperbilirubinemia, as well as reduced markers of oxidative stress and inflammation. Lastly, statin, angiotensin II receptor blocker, chymase inhibitor, bilirubin and biliverdin, PKC beta isoform inhibitor, and glucagon-like peptide-1 analog, are shown to serve as antioxidants and have some beneficial effect on diabetic vascular complications, via inhibiting PKC-NAD(P)H oxidase activation, supporting the notion that this mechanism may be an effective therapeutic target for preventing diabetic vascular complications.
Angiotensin II ; Antioxidants ; Bilirubin ; Biliverdine ; Chymases ; Diabetic Angiopathies ; Gilbert Disease ; Glucagon-Like Peptide 1 ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; Hyperbilirubinemia ; Inflammation ; NADPH Oxidase ; Oxidative Stress ; Oxidoreductases ; Prevalence ; Protein Kinase C ; Reactive Oxygen Species ; Receptors, Angiotensin ; Up-Regulation

Rheumatoid arthritis (RA) is an autoimmune disease that starts with decreased tolerance to modified self-antigens and eventually leads to synovitis and destruction of bone and cartilage. Age is a risk factor for developing RA. Major changes in the immune system come with age due to chronic oxidative stress on the deoxyribonucleic acid (DNA) damage pathway, somatic mutation, modifications of auto-antigens, T cell tolerance and activation of fibroblast-like synoviocytes (FLS). Reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate oxidase 2 (NADPH oxidase 2) suppress T cell receptor signaling. Sirtuin 1 (SIRT1) is a critical immune suppressor of T cell activation and a key regulator of oxidative stress. When oxidative stress reduces activity of SIRT1, the breakdown of tolerance to modified self-antigens is expected. Generation of ROS can be perpetuated by enhanced DNA damage and dysfunctional mitochondria in a feedback loop during the development of RA. Through major T cell loss and selective proliferation of peripheral T cells, pro-inflammatory T cell pools with abnormal features are established in the T cell compartment. Hypoxic and inflammatory condition in synovium perpetuates ROS generation, which leads to the activation of FLS. In both T cell and synovium compartment, oxidative stress reshapes the immune system into the development of pre-clinical RA.
Arthritis, Rheumatoid* ; Autoantigens ; Autoimmune Diseases ; Cartilage ; DNA ; DNA Damage ; Immune System ; Mitochondria ; NADP ; NADPH Oxidase ; Oxidative Stress ; Oxidoreductases ; Reactive Oxygen Species* ; Receptors, Antigen, T-Cell ; Risk Factors ; Sirtuin 1 ; Synovial Membrane ; Synovitis ; T-Lymphocytes

BACKGROUND: Based on the assertion that apocynin diminishes acute lung injury (ALI) by inhibition of NADPH oxidase, the effect of apocynin was tested in interleukin-1alpha (IL-1)-induced ALI in rats. METHODS: IL-1 was insufflated into the trachea of Sprague-Dawley rats to induce ALI, and apocynin (8 mg/kg) was given intravenously for inhibition of NADPH oxidase. In addition, we determined whether apocynin inhibited generation of superoxide anions from isolated human neutrophils. Five hours after IL-1 instillation, lung injury parameters, expression of cytosolic phospholipase A2 (cPLA2) by cells from bronchoalveolar lavage (BAL), an index of oxidative stress in lung tissues (gamma-glutamyltranspeptidase, activity), and ultrastructure of alveolar type II (AT II) cells were evaluated. RESULTS: Apocynin decreased the generation of free radicals from phorbol myristate (PMA)-activated neutrophils in vitro, but did not ameliorate ALI. IL-1 induced enhancement of the expression of cPLA2 on neutrophils was not altered by apocynin. CONCLUSION: Apocynin induced suppression of the generation of superoxide anions from neutrophils by inhibition of NADPH oxidase does not attenuate IL-1-induced ALI in rats.
Acetophenones ; Acute Lung Injury ; Animals ; Bronchoalveolar Lavage ; Cytosol ; Free Radicals ; Humans ; Interleukin-1 ; Interleukin-1alpha ; Lung ; Lung Injury ; Myristic Acid ; NADPH Oxidase ; Neutrophils ; Oxidative Stress ; Phorbols ; Phospholipases A2 ; Rats ; Rats, Sprague-Dawley ; Superoxides ; Trachea

The effects of capsaicin (CAP), a transient receptor potential vanilloid subtype 1 (TRPV1) agonist, were determined on nigrostriatal dopamine (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). The results showed that TRPV1 activation by CAP rescued nigrostriatal DA neurons, enhanced striatal DA functions and improved behavioral recovery in MPTP-treated mice. CAP neuroprotection was associated with reduced expression of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1β) and reactive oxygen species/reactive nitrogen species from activated microglia-derived NADPH oxidase, inducible nitric oxide synthase or reactive astrocyte-derived myeloidperoxidase. These beneficial effects of CAP were reversed by treatment with the TRPV1 antagonists capsazepine and iodo-resiniferatoxin, indicating TRPV1 involvement. This study demonstrates that TRPV1 activation by CAP protects nigrostriatal DA neurons via inhibition of glial activation-mediated oxidative stress and neuroinflammation in the MPTP mouse model of PD. These results suggest that CAP and its analogs may be beneficial therapeutic agents for the treatment of PD and other neurodegenerative disorders that are associated with neuroinflammation and glial activation-derived oxidative damage.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine* ; Animals ; Capsaicin* ; Cytokines ; Dopamine* ; Dopaminergic Neurons* ; Mice ; NADPH Oxidase ; Necrosis ; Neurodegenerative Diseases ; Neurons ; Neuroprotection ; Nitric Oxide Synthase Type II ; Nitrogen ; Oxidative Stress* ; Oxygen ; Parkinson Disease*

OBJECTIVETo evaluate the effect of palmitic acid (PA) on oxidative stress and activation of inflammasomes in hepatocytes.

METHODSTo test the dose-dependent effect of PA on normal murine hepatocytes AML12, the cells were treated with 0, 0.15, 0.25 and 0.4 mmol/L of palmitic acid (PA). The cells were also divided into blank control group, 0.25 mmol/L PA group and 0.25 mmol/L PA+N-acetylcysteine (NAC) group to examine the effect of reactive oxygen species (ROS) on the activation of inflammasomes. After 24 h of treatment, lipid accumulation, total ROS, mitochondrial ROS, expression and localization of NOX4, and expressions of inflammasomes and IL-1β were detected in the hepatocytes.

RESULTSCompared with the control cells, PA treatment of the cells significantly increased cytoplasmic lipid accumulation, concentrations of total ROS (12 463.09±2.72 vs 6691.23±2.45, P=0.00) and mitochondrial ROS (64.98±0.94 vs 45.04±0.92, P=0.00), and the expressions of NOX4, NLRP3, ASC, caspase-1, and IL-1β (1603.52±1.32 vs 2629.33±2.57, P=0.00). The mitochondria and NOX4 were found to be co-localized in the cytoplasm. NAC obviously reduced cellular ROS level stimulated by PA (7782.15±2.87 vs 5445.6±1.17, P=0.00) and suppressed the expressions of NLRP3, ASC and caspase-1.

CONCLUSIONPA treatment can stimulate lipid accumulation in hepatocytes and induce oxidative stress through NOX4 and mitochondria pathway to activate inflammasomes and stimulate the secretion of IL-1β.

Acetylcysteine ; pharmacology ; Animals ; Carrier Proteins ; metabolism ; Caspase 1 ; metabolism ; Cells, Cultured ; Hepatocytes ; drug effects ; metabolism ; Inflammasomes ; drug effects ; metabolism ; Interleukin-1beta ; metabolism ; Mice ; Mitochondria ; drug effects ; NADPH Oxidase 4 ; NADPH Oxidases ; metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein ; Oxidative Stress ; Palmitic Acid ; pharmacology ; Reactive Oxygen Species ; metabolism