OBJECTIVETo investigate the inhibitory potential of aldosterone antagonist on NOX4 protein expression in hepatic fibrosis by using a rat model of carbon tetrachloride (CCl4)-induced hepatotoxicity.

METHODSTwenty-four male Wistar rats were randomly divided into three equal groups: fibrosis model group (receiving three subcutaneous injections per week of 2.5 ml/kg 40% CCl4); spironolactone (Sp)-treated fibrosis model group (receiving CCl4 regimen plus three injections per day of 20 mg/kg Sp in olive oil); negative-treatment fibrosis model group (receiving CCl4 regimen plus three injections per day of olive oil alone). Unmanipulated rats (receiving no CCl4 and no supplemental treatments) served as normal controls. After 4 weeks, liver histology was carried out to assess cytotoxicity (by hematoxylin-eosin staining), fibrosis (by Masson staining and METAVIR scoring), and NOX4 protein expression (by immunohistochemistry). In addition, in vitro analyses of immortalized rat hepatic stellate cells, HSC-T6, were performed to evaluate dose-response (10-9, 10-7 and 10-5 mol/L) and time-response (6, 12 and 24 h) of aldosterone agonist (Ald) and an aldosterone antagonist, eplerenone (EPLE). Effects on NOX4 protein expression were evaluated by western blotting.

RESULTSThe fibrosis model group showed significantly more fibrosis than the normal control group (16.060 +/- 0.300 vs. 2.471 +/- 0.160, P = 0.000]; however, the Sp-treated fibrosis model group showed significantly less CCl4-induced fibrosis (5.761 +/- 0.152 vs. model: 16.060 +/- 0.300, P = 0.000). The fibrosis model group also showed significantly higher NOX4 protein expression in liver tissues than the normal control group (7.231 +/- 0.211 vs. 1.350 +/- 0.252, P = 0.000), and the Sp-treated fibrosis model tissues showed significantly less CCl4-induced up-regulated NOX4 protein expression (4.270 +/- 0.242 vs. model: 7.231 +/- 0.211, P = 0.000]. Ald induced up-regulated NOX4 protein expression in HSC-T6 cells in dose- and concentration-dependent manners, with the peak expression being induced by the 10-5 mol/L concentration and 24 h exposure. The Ald-treated cells expressed significantly more NOX4 protein than the untreated control cells (0.710 +/- 0.011 vs. 0.316 +/- 0.015, P = 0.000]. and the EPLE-treated cells showed significantly less Ald-induced up-regulated NOX4 expression (0.615 +/- 0.014 vs. 0.710 +/- 0.011, P = 0.000].

CONCLUSIONAldosterone antagonists inhibit the fibrosis-induced NOX4 protein expression in rat hepatic cells.

Animals ; Cell Line ; Liver Cirrhosis, Experimental ; metabolism ; Male ; Mineralocorticoid Receptor Antagonists ; pharmacology ; NADPH Oxidase 4 ; NADPH Oxidases ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo investigate the method and mechanism for exercise-related immunosuppression via the inhibitor of NADPH oxidase diphenyleneiodonium(DPI) and glutamine supplementation and on the function of neutrophils after overtraining.

METHODSFifty male Wistar rats were randomly divided into five groups: a negative control group (C), an overtraining group (E), an overtraining + DPI intervention group (D), an overtraining+ glutamine supplementation group(G) and combined glutamine + DPI intervention group(DG). After 36 - 40 h from the last training, eight rats were randomly selected from each group, and blood was sampled from the orbital vein. ELISAs were used to measure serum cytokine levels and lipid peroxidation in blood plasma. Flow cytometry was used to measure neutrophil respiratory burst and phagocytosis. The activity of NADPH oxidase was assessed by chemiluminescence and the gene expression of gp91(phox) and p47(phox) of the NADPH-oxidase subunit was checked by Western blot.

RESULTSCompared with group C, the plasma concentrations of NO increased in group G, and the NO, cytokine-induced neutrophil chemoattractant (CINC) concentrations in group DG increased significantly. The respiratory burst and phagocytosis function of neutrophils were decreased in group E, but in group DG were increased when compared with those of group E. After overtraining the expression of gp91(phox) and p47(phox) was up regulated in group E. There were no significant changes in other groups except group DG, in which the expression of gp91(phox) was down regulated. Compared with group E, the expression of gp91(phox) and p47(phox) was up regulated in group D, group G and group DG.

CONCLUSIONThe activation of NADPH oxidase is responsible for the production of superoxide anions, which may be related to the decrease in neutrophil function after over training and is the mechanism of exercise-related immunosuppression. The DPI treatment combined glutamine supplementation can reverse the decrease neutrophils function after overtraining in vitro.

Animals ; Dietary Supplements ; Glutamine ; pharmacology ; Hyperkinesis ; physiopathology ; Male ; Membrane Glycoproteins ; metabolism ; NADPH Oxidase 2 ; NADPH Oxidases ; antagonists & inhibitors ; metabolism ; Neutrophils ; metabolism ; physiology ; Onium Compounds ; pharmacology ; Oxidation-Reduction ; Rats ; Rats, Wistar ; Respiratory Burst ; physiology

OBJECTIVETo investigate the role of NADPH oxidase (Nox) in the oxidative stress injury of human dermal fibroblasts (HFbs).

METHODSAn oxidative stress injury model was established in HFbs by exposure to H(2)O(2). Normal HFbs and HFbs exposed to H(2)O(2) with and without pretreatment with NADPH oxidase inhibitor were tested for cell viability using MTT assay, and the intracellular reactive oxygen species (ROS) were determined with a DCFH-DA fluorescent probe. Western blotting was used to measure the protein expressions of membrane-bound subunit gp91phox of NADPH oxidase in the cells.

RESULTH(2)O(2) time- and concentration-dependently induced oxidative stress injury in the fibroblasts, causing a reduction of the cell viability to 40% after a 24-h exposure at 700 µmol/L (P<0.05) and an increase of ROS by 2 folds after a 2-h exposure at 700 µmol/L (P<0.05). Compared with the cells with oxidative stress injury, the cells with NADPH oxidase inhibitor pretreatment showed a 20% higher cell viability (P<0.05) and normal ROS level (P<0.05) following H(2)O(2) exposure. Western blotting demonstrated increased expression of gp91phox in the cells exposed to increasing H(2)O(2) concentrations, but gp91phox expression remained normal in cells pretreated with NADPH oxidase inhibitor.

CONCLUSIONH(2)O(2) can induce oxidative stress injury in the fibroblasts by affecting NADPH oxidase, especially its membrane-bound subunit gp91phox.

Cell Survival ; Cells, Cultured ; Fibroblasts ; cytology ; enzymology ; Humans ; Hydrogen Peroxide ; Membrane Glycoproteins ; metabolism ; NADPH Oxidase 2 ; NADPH Oxidases ; antagonists & inhibitors ; metabolism ; Oxidation-Reduction ; Oxidative Stress ; Reactive Oxygen Species ; metabolism

OBJECTIVEThe roles of cerebrovascular oxidative stress in vascular functional remodeling have been described in hindlimb-unweighting (HU) rats. However, the underlying mechanism remains to be established.

METHODSWe investigated the generation of vascular reactive oxygen species (ROS), Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and manganese superoxide dismutase (MnSOD) and glutathione peroxidase-1 (GPx-1) mRNA levels in cerebral and mesenteric smooth muscle cells (VSMCs) of HU rats.

RESULTSROS production increased in cerebral but not in mesenteric VSMCs of HU rats compared with those in control rats. Nox2 and Nox4 protein and mRNA levels were increased significantly but MnSOD/GPx-1 mRNA levels decreased in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly more in cerebral but not in mesenteric arteries of HU rats. NADPH oxidase inhibition with apocynin attenuated cerebrovascular ROS production and partially restored Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and MnSOD/GPx-1 mRNA levels in cerebral VSMCs of HU rats.

CONCLUSIONThese results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravit.

Acetophenones ; Animals ; Cerebral Arteries ; metabolism ; Glutathione Peroxidase ; metabolism ; Hindlimb Suspension ; Male ; Membrane Glycoproteins ; metabolism ; Mesenteric Arteries ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; NADPH Oxidase 2 ; NADPH Oxidase 4 ; NADPH Oxidases ; antagonists & inhibitors ; metabolism ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; Superoxide Dismutase ; metabolism

In vascular smooth muscle cells, reactive oxygen species (ROS) were known to mediate platelet-derived growth factor (PDGF)-induced cell proliferation and NADH/NADPH oxidase is the major source of ROS. NADH/NADPH oxidase is controlled by rac1 in non-phagocytic cells. In this study, we examined whether the inhibition of rac1 by adenoviral-mediated gene transfer of a dominant negative rac1 gene product (Ad.N17rac1) could reduce the proliferation of rat aortic vascular smooth muscle cells (RASMC) stimulated by PDGF via decreasing intracellular ROS. RASMC were stimulated by PDGF (80 ng/mL) with or without N-acetylcysteine 1 mM or infected with 100 mutiplicity of infection of Ad.N17rac1. Intracellular ROS levels were measured at 12 hr using carboxyl-2', 7'-dichlorodi-hydrofluorescein diacetate confocal microscopy. At 72 hr, cellular proliferation was evaluated by cell number counting and XTT assay. Compared with control, ROS levels were increased by 2-folds by PDGF. NAC and Ad.N17rac1 inhibited PDGF-induced increase of ROS by 77% and 65%, respectively. Cell number was increased by PDGF by 1.6-folds compared with control. NAC and Ad.N17rac1 inhibited PDGF-induced cellular growth by 45% and 87%, respectively. XTT assay also showed similar results. We concluded that inhibition of rac1 in RASMCs could reduce intracellular ROS levels and cellular proliferation induced by PDGF.
Adenoviridae/genetics ; Animal ; Aorta, Thoracic/cytology ; Cell Division/drug effects/physiology ; Cells, Cultured ; Gene Expression/physiology ; Gene Transfer Techniques ; Multienzyme Complexes/antagonists & inhibitors ; Muscle, Smooth, Vascular/*cytology/*metabolism ; NADH, NADPH Oxidoreductases/antagonists & inhibitors ; NADPH Oxidase/antagonists & inhibitors ; Platelet-Derived Growth Factor/*pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/*metabolism ; rac1 GTP-Binding Protein/*genetics/metabolism

BACKGROUND AND OBJECTIVES: Elevated endothelin (ET)-1 level is strongly correlated with the pathogenesis of pulmonary arterial hypertension (PAH). Expression level of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 4 is increased in the PAH patients. Ambrisentan, a selective endothelin receptor A (ERA) antagonist, is widely used in PAH therapy. The current study was undertaken to evaluate the effects of ambrisentan treatment in the monocrotaline (MCT)-induced PAH rat model. METHODS: Rats were categorized into control group (C), monocrotaline group (M) and ambrisentan group (Am). The M and Am were subcutaneously injected 60 mg/kg MCT at day 0, and in Am, ambrisentan was orally administered the day after MCT injection for 4 weeks. The right ventricle (RV) pressure was measured and pathological changes of the lung tissues were observed by Victoria blue staining. Protein expressions of ET-1, ERA, endothelial nitric oxide synthase (eNOS) and NOX4 were confirmed by western blot analysis. RESULTS: Ambrisentan treatment resulted in a recovery of the body weight and RV/left ventricle+septum at week 4. The RV pressure was lowered at weeks 2 and 4 after ambrisentan administration. Medial wall thickening of pulmonary arterioles and the number of intra-acinar arteries were also attenuated by ambrisentan at week 4. Protein expression levels of ET-1 and eNOS were recovered at weeks 2 and 4, and ERA levels recovered at week 4. CONCLUSIONS: Ambrisentan administration resulted in the recovery of ET-1, ERA and eNOS protein expression levels in the PAH model. However, the expression level of NOX4 remained unaffected after ambrisentan treatment.
Animals ; Arteries ; Arterioles ; Blotting, Western ; Body Weight ; Endothelin Receptor Antagonists ; Endothelins ; Gene Expression ; Heart Ventricles ; Humans ; Hypertension ; Hypertension, Pulmonary ; Lung ; Models, Animal ; Monocrotaline ; NADP ; NADPH Oxidase ; Nitric Oxide Synthase Type III ; Oxidoreductases ; Rats ; Receptors, Endothelin ; Victoria

The aim of the present study was to evaluate the protective effect of catalpol on vascular endothelial function in STZ-induced type 2 diabetes mellitus (T2DM) rats. 40 high-fat diet with STZ-induced diabetes rats were randomly divided into model group, catalpol low-dose, middle-dose and high-dose group (10, 50, 100 mg x kg(-1) x d(-1)), 10 normal Wistar rats were used as the normal group. The normal and model groups were given an equivalent amount of saline. All reagents were administered by oral gavage for 6 weeks. After 6 weeks, blood glucose and lipids were detected by an automatic biochemical analyzer. The endothelium-dependent vasodilation response of thoracic aortar was detected. The pathological changes of the thoracic aorta were observed by HE staining. Ser- um nitric oxide (NO), 8-iso prostaglandin F2α (8-iso-PGF2α) and superoxide dismutase (SOD) were detected by ELISA. Reactive oxygen species (ROS) level of thoracic aorta was detected by fluorescence method. The expression of Nox4 and p22phox mRNA and protein in aortic tissue were detected by RT-PCR and Western-blot respectively. After catalpol treatment, endothelial damage of thoracic aorta was attenuated significantly; ROS level of thoracic aorta and serum level of 8-iso-PGF2α were decreased significantly; serum NO and SOD levels were remarkably elevated; expression of Nox4, p22phox mRNA and protein in thoracic aorta were significantly reduced (P < 0.05). Therefore, catalpol has protective effect on endothelial of T2DM, its mechanism may be associated with the down-regulation of Nox4 and p22phox expression, inhibiting oxidative stress reaction response.
Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; pathology ; Diabetes Mellitus, Type 2 ; pathology ; Dinoprost ; analogs & derivatives ; metabolism ; Endothelium, Vascular ; drug effects ; metabolism ; pathology ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; drug effects ; Iridoid Glucosides ; pharmacology ; Male ; NADPH Oxidase 4 ; NADPH Oxidases ; antagonists & inhibitors ; genetics ; metabolism ; Nitric Oxide ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism

OBJECTIVETo study the protective effect of epalrestat against endothelial cell injuries induced by high glucose.

METHODSHuman umbilical vein endothelial cells were pretreated with epalrestat (0.1 µmol/L) for 30 min followed by exposure to high glucose for 8 h. NO concentration in the cell culture supernatant was assayed using chemiluminescence method following the exposure. Real-time PCR and Western blotting were used to detect eNOS mRNA and protein expression levels and the protein expressions of AR gene (the target gene of epalrestat) and NOX4 (the upstream gene of NO).

RESULTSCompared with mannitol treatment, an 8-h exposure to high glucose caused significantly decreased NO levels and eNOS mRNA and protein expression in the vascular endothelial cells (P<0.05). Pretreatment with epalrestat prior to high glucose exposure resulted in elevated eNOS mRNA and protein expression levels and NO up-regulation in the cell culture as compared with the glucose exposure alone group (P<0.05), causing also decreased expression of AR and NOX4 in the cells.

CONCLUSIONSHigh glucose can induce endothelial cell damage characterized by a lowered level of NO secretion. Epalrestat can protect the endothelial cells against high glucose-induced injury by inhibiting the expression of AR and NOX4.

Aldehyde Reductase ; antagonists & inhibitors ; Cells, Cultured ; Endothelium, Vascular ; drug effects ; metabolism ; Enzyme Inhibitors ; pharmacology ; Glucose ; adverse effects ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; NADPH Oxidase 4 ; NADPH Oxidases ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; RNA, Messenger ; genetics ; Rhodanine ; analogs & derivatives ; pharmacology ; Thiazolidines ; pharmacology

The incidence rates of urinary bladder cancer continue to rise yearly, and thus new therapeutic approaches and early diagnostic markers for bladder cancer are urgently needed. Thus, identifying the key mediators and molecular mechanisms responsible for the survival of bladder cancer has valuable implications for the development of therapy. In this study, the role of BLT2, a receptor for leukotriene B4 (LTB4) and 12(S)-hydroxyeicosatetraenoic acid (HETE), in the survival of bladder cancer 253J-BV cells was investigated. We found that the expression of BLT2 is highly elevated in bladder cancer cells. Also, we observed that blockade of BLT2 with an antagonist or BLT2 siRNA resulted in cell cycle arrest and apoptotic cell death, suggesting a role of BLT2 in the survival of human bladder cancer 253J-BV cells. Further experiments aimed at elucidating the mechanism by which BLT2 mediates survival revealed that enhanced level of reactive oxygen species (ROS) are generated via a BLT2-dependent up-regulation of NADPH oxidase members NOX1 and NOX4. Additionally, we observed that inhibition of ROS generation by either NOX1/4 siRNAs or treatment with an ROS-scavenging agent results in apoptotic cell death in 253J-BV bladder cancer cells. These results demonstrated that a 'BLT2-NOX1/4-ROS' cascade plays a role in the survival of this aggressive bladder cancer cells, thus pointing to BLT2 as a potential target for anti-bladder cancer therapy.
*Apoptosis ; Blotting, Western ; Cell Proliferation ; Cells, Cultured ; Gene Expression Regulation, Neoplastic/*physiology ; Humans ; Leukotriene Antagonists/pharmacology ; NADPH Oxidase/antagonists & inhibitors/genetics/metabolism ; Phosphorylation ; RNA, Messenger/genetics ; RNA, Small Interfering/genetics ; Reactive Oxygen Species/*metabolism ; Receptors, Leukotriene B4/antagonists & inhibitors/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Tetrazoles/pharmacology ; Up-Regulation ; Urinary Bladder Neoplasms/*genetics/mortality

Cardiac fibrosis occurs after pathological stimuli to the cardiovascular system. One of the most important factors that contribute to cardiac fibrosis is angiotensin II (Ang II). Accumulating studies have suggested that reactive oxygen species (ROS) plays an important role in cardiac fibrosis and sodium tanshinone IIA sulfonate (STS) possesses antioxidant action. We therefore examined whether STS depresses Ang II-induced collagen type I expression in cardiac fibroblasts. In this study, Ang II significantly enhanced collagen type I expression and collagen synthesis. Meanwhile, Ang II depressed matrix metalloproteinase-1 (MMP-1) expression and activity. These responses were attenuated by STS. Furthermore, STS depressed the intracellular generation of ROS, NADPH oxidase activity and subunit p47(phox) expression. In addition, N-acetylcysteine the ROS scavenger, depressed effects of Ang II in a manner similar to STS. In conclusion, the current studies demonstrate that anti-fibrotic effects of STS are mediated by interfering with the modulation of ROS.
Acetylcysteine/pharmacology ; Angiotensin II/*antagonists & inhibitors/pharmacology ; Animals ; Blotting, Western ; Cells, Cultured ; Collagen Type I/*metabolism ; Drugs, Chinese Herbal/*pharmacology ; Fibroblasts/*drug effects/metabolism ; Free Radical Scavengers/pharmacology ; Matrix Metalloproteinase 1/metabolism ; Myocardium/*cytology ; NADPH Oxidase/metabolism ; Oxidative Stress/drug effects ; Phenanthrenes/*pharmacology ; Rats ; Rats, Wistar ; Reactive Oxygen Species/metabolism