ABSTRACT
OBJECTIVE@#To investigate the effects of 1,25-dihydroxyvitamin D3 (1,25-VitD3) supplementation on cerebral injury after ischemia/reperfusion (I/R) in mice with middle cerebral artery occlusion (MCAO).@*METHODS@#Male C57BL6 mice were randomly divided into Sham group, Vehicle group and 1,25-VitD3 group, with 10 mice in each group. Vehicle group and 1,25-VitD3 group were given MCAO for 1 hour, and then killed after reperfusion for 24 hours. Mice in 1,25-VitD3 group were treated with 1,25-VitD3 at the dose of 100 ng/(kg·d) by injected intraperitoneally for 5 days before MCAO operation. Cerebral ischemic penumbra areas of each group were collected for TTC staining, RT-PCR, TTC staining and immunohistochemistry assay. The function defect of mice was evaluated by using neurological function score.@*RESULTS@#Compared with the sham group, the volume of cerebral infarction in Vehicle group was increased significantly, and the expressions of IL-6, IL-1beta and Gp91phox in brain tissues were increased significantly (P＜0.05); compared with Vehicle group, supplementation of 1,25-VitD3 reduced the volume of cerebral infarction by about 50% in I/R mice (P＜0.05), and the expressions of IL-6, IL-1beta and Gp91phox in brain tissues of 1,25-VitD3 group were decreased significantly (P＜0.05). The expression of Foxp3, a T-regulatory cell marker, was significantly increased in the brain of mice (P＜0.05), while the expression of Rorc, a transcription factor, was significantly decreased (P＜0.05), suggesting that Th17/gamma Delta T-cell response was reduced and the number of neutrophils in the brain injury site of mice was significantly reduced (P＜0.05).@*CONCLUSION@#Vitamin D could alleviate the development of cerebral infarction after arterial occlusion (MCAO) reperfusion, and its mechanism may be through regulating the inflammatory response in mouse brain I/R.
Subject(s)
Animals , Male , Mice , Brain , Cytokines , Metabolism , Infarction, Middle Cerebral Artery , Drug Therapy , Inflammation , Mice, Inbred C57BL , NADPH Oxidase 2 , Metabolism , Protective Agents , Pharmacology , Random Allocation , Rats, Sprague-Dawley , Reperfusion Injury , Drug Therapy , T-Lymphocytes , Th17 Cells , Vitamin D , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of NADPH oxidase (Nox) in the oxidative stress injury of human dermal fibroblasts (HFbs).</p><p><b>METHODS</b>An 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.</p><p><b>RESULT</b>H(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.</p><p><b>CONCLUSION</b>H(2)O(2) can induce oxidative stress injury in the fibroblasts by affecting NADPH oxidase, especially its membrane-bound subunit gp91phox.</p>
Subject(s)
Humans , Cell Survival , Cells, Cultured , Fibroblasts , Cell Biology , Hydrogen Peroxide , Membrane Glycoproteins , Metabolism , NADPH Oxidase 2 , NADPH Oxidases , Metabolism , Oxidation-Reduction , Oxidative Stress , Reactive Oxygen Species , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the molecular mechanism by which salidroside protects PC12 cells from HO-induced apoptosis.</p><p><b>METHODS</b>PC12 cells cultured in DMEM supplemented with 10% horse serum and 5% fetal bovine serum were pretreated with different doses of salidroside for 2 h and then stimulated with HOfor different lengths of time. The expression levels of PARP and caspase 3 and the phosphorylation of p38, ERK and JNK were determined with Western blotting. The cell nuclear morphology was observed after DAPI staining. The production of ROS was detected using a ROS detection kit, and the levels of gp91and p47in the membrane and cytoplasm were detected by membrane-cytoplasm separation experiment; the binding between gp91and p47was assayed by coimmunoprecipitation experiment.</p><p><b>RESULTS</b>Salidroside dose-dependently suppressed cell apoptosis, lowered phosphorylation levels of p38, ERK and JNK, inhibited the production of ROS, reduced the binding between gp91and p47, and inhibited the activity of NOX2 in PC12 cells exposed to HO.</p><p><b>CONCLUSION</b>Salidroside protects PC12 cells from HO-induced apoptosis at least partly by suppressing NOX2-ROS-MAPKs signaling pathway.</p>
Subject(s)
Animals , Rats , Apoptosis , Caspase 3 , Metabolism , Glucosides , Pharmacology , Hydrogen Peroxide , MAP Kinase Signaling System , Membrane Glycoproteins , Metabolism , NADPH Oxidase 2 , NADPH Oxidases , Metabolism , Neuroprotective Agents , Pharmacology , PC12 Cells , Phenols , Pharmacology , Phosphorylation , Reactive Oxygen Species , MetabolismABSTRACT
<p><b>OBJECTIVE</b>Both of gp91(phox) (an isoform of nicotinamide adenine dinucleotide phosphate-reduced oxidases) and Src (a non-receptor protein tyrosine kinase) play a prominent role in mediating hypoxia/reoxygenation injury of neurons. The present study was designed to investigate the neuroprotective effect of equol, a predominant active metabolite of daidzein, against hypoxia/reoxygenation injury in rat pheochromocytoma cell line (PC12) and explore the underlying mechanisms.</p><p><b>METHODS</b>PC12 cells exposed to hypoxia/reoxygenation injury were examined for reactive oxygen species (ROS) using dihydroethidium and 2', 7'-dichlorofluorescein diacetate and analyzed for changes in lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) content. The expression levels of gp91(phox) and phosphorylated Src-Tyr416 (p-Src) were measured using Western blotting.</p><p><b>RESULTS</b>Equol dose-dependently restored the cell viability and decreased LDH activity and MDA content in culture medium of PC12 cells exposed to hypoxia/reoxygenation. Pretreatment of the cells with 10(-5) and 10(-6) mol/L equol inhibited hypoxia/reoxygenation-induced increase of ROS. PC12 cells treated with equol prior to hypoxia/reoxygenation injury showed significant enhancement of the protein levels of gp91(phox) and p-Src.</p><p><b>CONCLUSION</b>Equol confers neuroprotection against hypoxia/reoxygenation injury in PC12 cells by inhibiting the generation of ROS very likely as a result of down-regulation of gp91(phox) and inhibition of Src phosphorylation.</p>
Subject(s)
Animals , Rats , Cell Hypoxia , Cell Survival , Down-Regulation , Equol , Pharmacology , L-Lactate Dehydrogenase , Metabolism , Malondialdehyde , Metabolism , Membrane Glycoproteins , Metabolism , NADPH Oxidase 2 , NADPH Oxidases , Metabolism , Neurons , Metabolism , Neuroprotective Agents , Pharmacology , PC12 Cells , Phosphorylation , Reactive Oxygen Species , Metabolism , src-Family Kinases , MetabolismABSTRACT
The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 μmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling.
Subject(s)
Humans , 8,11,14-Eicosatrienoic Acid , Metabolism , Pharmacology , Adaptor Proteins, Signal Transducing , Genetics , Metabolism , Apoptosis , Aryl Hydrocarbon Hydroxylases , Genetics , Metabolism , Atherosclerosis , Genetics , Metabolism , Pathology , Catalase , Genetics , Metabolism , Cytochrome P-450 CYP2C8 , Genetics , Metabolism , Gene Expression Regulation , Heme Oxygenase-1 , Genetics , Metabolism , Human Umbilical Vein Endothelial Cells , Cell Biology , Metabolism , Membrane Glycoproteins , Genetics , Metabolism , Models, Biological , NADPH Oxidase 2 , NADPH Oxidases , Genetics , Metabolism , NF-E2-Related Factor 2 , Genetics , Metabolism , Nitric Oxide Synthase Type III , Genetics , Metabolism , RNA, Small Interfering , Genetics , Metabolism , Reactive Oxygen Species , Metabolism , Signal Transduction , Tumor Necrosis Factor-alpha , Metabolism , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To observe the effect of sesamin (Ses) on pulmonary vascular remodeling in rats with monocrotaline ( MCT)-induced pulmonary hypertension (PH).</p><p><b>METHOD</b>Totally 48 male Sprague-Dawley (SD) rats were fed adaptively for one week and then divided into the normal control group, the MCT group, the MCT +Ses (50 mg x kg(-1)) group and the MCT + Ses (100 mg x kg(-1)) group, with 12 rats in each group. The PH rat model was induced through the subcutaneous injection with MCT(60 mg x kg(-1)). After the administration for four weeks, efforts were made to measure the right ventricular systolic pressure( RVSP) and mean pulmonary artery pressure (mPAP) through right jugular vein catheterization, and isolate right ventricle( RV) and left ventricle( LV) +septum (S) and measure their length to calculate RV/ ( LV + S) and ratio of RV to tibial length. Pathologic changes in arterioles were observed by HE staining. Masson's trichrome stain was used to demonstrate changes in collagen deposition of arterioles. The alpha-smooth muscle actin (alpha-SMA) expression in pulmonary arteries was measured by immunohistochemisty. The total antioxidative capacity (T-AOC) and malondialdehyde (MDA) content in pulmonary arteries were determined by the colorimetric method. The protein expressions of collagen I, NOX2 and NOX4 were analyzed by Real-time PCR and Western blot.</p><p><b>RESULT</b>After the administration for 4 weeks, Ses could attenuate RVSP and mPAP induced by MCT, RV/ (LV + S) and ratio of RV to Tibial length, alpha-SMA and collagen I expressions and remodeling of pulmonary vessels and right ventricle. Meanwhile, Ses could obviously inhibit the expressions of NOX2, NOX4 and MDA content and increase T-AOC.</p><p><b>CONCLUSION</b>Sesamin could ameliorate pulmonary vascular remodeling induced by monocrotaline in PH rats. Its mechanism may be related to expressions of NOX2 and NOX4 expression and reduction in oxidative stress injury.</p>
Subject(s)
Animals , Humans , Male , Rats , Dioxoles , Disease Models, Animal , Drugs, Chinese Herbal , Hypertension, Pulmonary , Drug Therapy , Genetics , Lignans , Lung , Metabolism , Membrane Glycoproteins , Genetics , Metabolism , Monocrotaline , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases , Genetics , Metabolism , Pulmonary Artery , Metabolism , Rats, Sprague-Dawley , Vascular RemodelingABSTRACT
<p><b>OBJECTIVE</b>The 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.</p><p><b>METHODS</b>We 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.</p><p><b>RESULTS</b>ROS 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.</p><p><b>CONCLUSION</b>These results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravit.</p>
Subject(s)
Animals , Male , Acetophenones , Cerebral Arteries , Metabolism , Glutathione Peroxidase , Metabolism , Hindlimb Suspension , Membrane Glycoproteins , Metabolism , Mesenteric Arteries , Metabolism , Myocytes, Smooth Muscle , Metabolism , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases , Metabolism , Rats, Sprague-Dawley , Reactive Oxygen Species , Superoxide Dismutase , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To explore the relationship between the expression of endothelial nitric oxide synthase (eNOS) and NADPH oxidase (NOX) in the lungs of mice treated by chronic hypoxic exposure.</p><p><b>METHODS</b>Thirty male wild-type (WT) C57Bl/6 mice and thirty male eNOS-knockout (KO) C57BL/6 mice were randomly divided into normoxic groups (exposed to normoxia for 7 days or 21 days), hypoxic groups (exposed to 10% oxygen for 7 days or 21 days), and treatment groups (exposed to 10% oxygen and orally administrated 10 mmol/L 4-hydroxy TEMPO in drinking water for 7 days or 21 days) (n=6 in each group). The remodeling of the small pulmonary arteries was evaluated by the percentage of media wall thickness (MT%). The weight ratio of right ventricle to left ventricle plus septum (RV/[LV+S]) was calculated to evaluate the hypertrophy of right ventricle. Real-time PCR was used to measure the mRNA expression of NOX2, NOX4, and eNOS in mouse lungs. ELISA was used to determine the concentration of reactive oxygen species (ROS) in mouse lungs.</p><p><b>RESULTS</b>In WT mice and KO mice, the hypoxic groups had significantly increased pulmonary vascular remodeling and RV/[LV+S] compared with the normoxic and treatment groups (P<0.05), but there were no significant differences between the normoxic and treatment groups (P>0.05). In WT mice, the hypoxic and treatment groups had significantly lower ROS concentrations than the normoxic group (P<0.05), but there were no significant differences between the hypoxic and treatment groups (P>0.05). In WT mice, the mRNA expression of eNOS, NOX2, and NOX4 was significantly higher in the hypoxic group than in the normoxic group (P<0.05), and 4-hydroxy TEMPO reversed their over-expression. In the normoxic group, the KO mice had significantly higher NOX2 and NOX4 mRNA expression than the WT mice (P<0.05); in KO mice, the hypoxic group showed no significant changes in NOX4 mRNA expression (P>0.05), but had significantly reduced NOX2 mRNA expression (P<0.05), as compared with the normoxic group; the treatment group had reduced expression of NOX2 mRNA expression and increased NOX4 mRNA expression (P<0.05), as compared with the hypoxic group.</p><p><b>CONCLUSIONS</b>eNOS plays a key role in the regulation of expression of NOX2 and NOX4 in the lungs exposed to hypoxia. It suggests that NOX and eNOS may physically interact with one another in pulmonary vascular remodeling induced by chronic hypoxia.</p>
Subject(s)
Animals , Male , Mice , Chronic Disease , Hypoxia , Lung , Membrane Glycoproteins , Genetics , Physiology , Mice, Inbred C57BL , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases , Genetics , Physiology , Nitric Oxide Synthase Type III , Genetics , Physiology , RNA, MessengerABSTRACT
<p><b>OBJECTIVE</b>To investigate the mechanism of β-amyloid protein (Aβ) in regulating the expression of the receptor for advanced glycation end products (RAGE).</p><p><b>METHODS</b>Aβ1-40 was injected into the bilateral hippocampus of rats, and 3 weeks later, the levels of reactive oxygen species (ROS) production were detected by flow cytometry. The expressions of RAGE, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (gp9l(phox) and p47(phox)), nuclear factor-κB (NF-κB), and inhibitor of κB (IκB) were measured by Western blotting.</p><p><b>RESULTS</b>Injection of Aβ1-40 caused a significant increase in the expressions of RAGE, gp9l(phox), p47(phox), phospho-p47(phox), phospho-IκBα, NF-κB and phospho-NF-κB in rat hippocampus but decreased the level of IκBα. Aβ1-40 injection also resulted in a significantly increased content of ROS in the hippocampus of the rats.</p><p><b>CONCLUSION</b>Aβ up-regulates the expression of RAGE in rat hippocampus via NADPH/ ROS/NF-κB signaling pathway.</p>
Subject(s)
Animals , Male , Rats , Amyloid beta-Peptides , Hippocampus , Metabolism , Membrane Glycoproteins , Metabolism , NADP , Metabolism , NADPH Oxidase 2 , NADPH Oxidases , Metabolism , NF-kappa B , Metabolism , Oxidative Stress , Peptide Fragments , Rats, Sprague-Dawley , Reactive Oxygen Species , Metabolism , Receptor for Advanced Glycation End Products , Receptors, Immunologic , Metabolism , Signal Transduction , Up-RegulationABSTRACT
<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Fifty 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.</p><p><b>RESULTS</b>Compared 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.</p><p><b>CONCLUSION</b>The 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.</p>
Subject(s)
Animals , Male , Rats , Dietary Supplements , Glutamine , Pharmacology , Hyperkinesis , Membrane Glycoproteins , Metabolism , NADPH Oxidase 2 , NADPH Oxidases , Metabolism , Neutrophils , Metabolism , Physiology , Onium Compounds , Pharmacology , Oxidation-Reduction , Rats, Wistar , Respiratory Burst , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To explore the role of NADPH oxidase inhibitor apocynin on ischemia/reperfusion (I/R)-induced myocardial injury.</p><p><b>METHODS</b>Male SD rat hearts were divided into the normal control group; sham group; I/R group (1 h ischemia followed by 3 h reperfusion); I/R + apocynin group (50 mg/kg, administrated at 30 min before reperfusion) and I/R + vehicle group (same volume vehicle administrated at 30 min before reperfusion). At the end of reperfusion, myocardial infarct size, apoptosis, plasma CK activity, myocardial NOX activity, myocardial caspase-3 expression and activity, myocardial mRNA and protein expressions of vascular peroxidase 1 (VPO1) and NOX2 were measured.</p><p><b>RESULTS</b>Infarct size, ratio of cardiomyocyte apoptosis, mRNA and protein expression of VOP1 and NOX2, serum CK, myocardial NOX and caspase-3 activities in the I/R group were all significantly increased compared to those in the sham group (P < 0.01). Above parameters were similar between I/R + vehicle group and I/R group (all P > 0.05). Infarct size, ratio of cardiomyocyte apoptosis, myocardial mRNA and protein expression of VOP1 and NOX2, serum CK, myocardial NOX and caspase-3 activities were significantly lower in I/R + apocynin group compared to those in I/R group (all P < 0.01).</p><p><b>CONCLUSIONS</b>NOX/VPO pathway plays an important role in mediating I/R-induced myocardial oxidative injury. NOX inhibition could reduce I/R-induced myocardial oxidative injury by attenuating myocardial apoptosis in this model.</p>