P22phox mRNA Research Articles

Effects of Chinese herbal medicine Jinmaitong-medicated serum on inducible nitric oxide synthase and NADPH oxidase p22-phox subunit of rat Schwann cells cultured in high-glucose medium

To investigate the effects of medicated serum prepared by administration of Jinmaitong (JMT), a compound Chinese herbal medicine, on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase p22-phox subunit and inducible nitric oxide synthase (iNOS) of rat Schwann cells cultured in high-glucose medium. Wistar rats were divided into normal control group (distilled water), JMT (JMT at dose of 1.31 g/(kg·d)) group and vitamin C (vitamin C at dose of 0.08 g/(kg·d)) group to prepare medicated serum. Bilateral sciatic nerves of new born Wistar rats were used to separate Schwann cells. Schwann cells cultured in high-glucose medium were divided into high glucose group (cultured with 50 mmol/L glucose medium), JMT group (cultured with JMT-medicated serum) and vitamin C (VC) group (cultured with VC-medicated serum). Schwann cells cultured in DMEM were used as the normal control. After 48 h culturing, the expression of iNOS was detected by immunofluorescence method and p22-phox mRNA was tested by real-time fluorescent quantitative polymerase chain reaction. The expression levels of iNOS and p22-phox mRNA in the high glucose group were higher than those in the normal control group (P<0.01). Compared with the high glucose group, expressions of iNOS protein and p22-phox mRNA in JMT group were significantly decreased (P<0.01) and JMT-medicated serum had better effect than VC (P<0.01). JMT-medicated serum can down-regulate the expressions of iNOS protein and NADPH oxidase p22-phox subunit mRNA of Schwann cells cultured in high-glucose medium.

Rutaecarpine prevents hypoxia–reoxygenation-induced myocardial cell apoptosis via inhibition of NADPH oxidases

It is proposed that myocardial cell apoptosis causes ventricular remodeling and heart failure. The aim of the present study was to determine the effects of rutaecarpine (Rut) on hypoxia-reoxygenation (H-R)-induced apoptosis in myocardial cell line H9c2, as well as the underlying mechanisms. Cultured H9c2 cells were exposed to hypoxia for 24 h, followed by 12 h reoxygenation. Rut (in concentrations of 0.1, 1, and 10 µmol/L) was added 1 h prior to H-R. Cell viability and lactate dehydrogenase were measured to evaluate the cell injuries. Apoptosis was evaluated by Hoechst 33258 staining and flow cytometry. NADPH oxidase activity was measured by assay kit; intracellular reactive oxygen species (ROS) generation was detected by 2',7'-dichlorofluorescein diacetate; and Nox2, Nox4, and p47(phox) mRNA and protein expression were analyzed by real-time PCR and Western blotting, respectively. The results showed that H-R significantly decreased cell viability and increased the lactate dehydrogenase release, as well as the apoptotic rate, concomitantly with enhanced NADPH oxidase activity. H-R also upregulated mRNA and protein expressions of Nox2, Nox4, and p47(phox) and increased ROS production. Treatment with Rut markedly reversed these effects introduced by H-R. These results suggest that the protective effects of Rut against H-R-induced myocardial cell injury and apoptosis might, at least partly, be due to the inhibition of the NADPH oxidase - ROS pathway.

Inhibitory Effects of T/L-type Calcium Channel Blockers on Tubulointerstitial Fibrosis in Obstructed Kidneys in Rats

To examine the effect of L- and T/L-type calcium channel blockers on interstitial fibrosis in chronic unilateral ureteral obstruction (UUO). Tubulointerstitial fibrosis is a common outcome of several progressive renal diseases. Calcium channel blockers are widely used for the treatment of hypertension with renal diseases; however, the direct effect of calcium channel blockers on renal diseases independent of lowering blood pressure has not been fully elucidated. Sprague-Dawley rats were divided into 3 treatment groups: (1) vehicle control; (2) nifedipine, an L-type calcium channel blockers; and (3) efonidipine, a T/L-type calcium channel blockers. Treatment was initiated 1 day before and continued until 6 days after creation of the UUO. Tubulointerstitial fibrosis in the obstructed kidney was significantly increased compared with that in the contralateral unobstructed kidney. Furthermore, the increased fibrosis was accompanied by increased fibrogenic signaling expressed by transforming growth factor β1 and connective tissue growth factor mRNA levels, increased oxidative stress expressed by p22phox, p47phox and gp91phox mRNA level. Moreover, treatment with a nonhypotensive dose of efonidipine but not nifedipine in the obstructed kidney significantly suppressed the fibrogenic signaling and the oxidative stress, resulting in reduced tubulointerstitial fibrosis. The plasma aldosterone level in efonidipine-treated animals was increased compared with vehicle-treated animals, although not significantly. The increased plasma aldosterone level did not increase sgk-1 mRNA level in efonidipine but not in nifedipine treated animals. Treatment with efonidipine improved tubulointerstitial fibrosis more effectively than treatment with nifedipine in UUO. The antifibrogenic effect by efonidipine was obtained through suppression of fibrogenic signaling.

Glycated albumin, a precursor of advanced glycation end‐products, up‐regulates NADPH oxidase and enhances oxidative stress in human endothelial cells: molecular correlate of diabetic vasculopathy

Hyperglycaemia induces non-enzymatic glycation reactions in proteins which generate Amadori products and advanced glycation end-products; the latter are thought to participate in the vascular complications of diabetic patients. However, the exact mechanisms concerning the effects of glycated proteins on vascular tissue remain to be determined. Therefore, the effects of glycated human serum albumin on human umbilical vein endothelial cells were studied. Reactive oxygen species production was measured by the cytochrome C reduction method and by 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (c-DCF-DA) fluorescence after treating human umbilical vein endothelial cells with glycated human serum albumin (6-200 µg/mL). The expression of Nox4 and p22phox mRNAs were analysed by reverse transcription-quantitative polymerase chain reactions and the levels of their proteins were measured by immunofluorescence. Low concentrations of glycated human serum albumin enhanced reactive oxygen species production in human umbilical vein endothelial cells after 4 h of treatment at both extracellular and intracellular sites. This enhanced production was sustained, although to a lesser extent, after 6 and 12 h of treatment. The gene expression study revealed that Nox4 and p22phox mRNA levels were elevated after 4 h of treatment with glycated human serum albumin. This mRNA elevation and enhanced reactive oxygen species production correlated with an increased expression of the Nox4 protein. The results revealed that a circulating and abundant modified glycated human serum albumin protein in diabetic patients induced a sustained reactive oxygen species production in human endothelial cells. This effect may have been due to an up-regulation of Nox4, the main subunit of NADPH oxidase in the endothelium.

Oxidative stress enhances granulocytic differentiation in HL 60 cells, an acute promyelocytic leukemia cell line

This paper studied the effects of physiologically available oxidants on HL 60 differentiation induced by all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). Hydrogen peroxide (15 μM) and taurine chloramine (200 μM) induced HL 60 differentiation, which was detected by CD11b expression and superoxide production. Cd11b and p67phox mRNA expression was also augmented by these oxidants. In contrast, reducing chemicals, such as dithiothreitol, 2,3-dimercapto-1-propanol and N-acetylcysteine inhibited CD11b expression. Notably, DMSO inhibited methionine sulfoxide reductase activity, induced heme oxygenase-1 (ho-1) mRNA and enhanced oxidant-induced cell death, which indicated that DMSO intensified oxidative stress. After the addition of oxidants, ho-1 expression preceded the cd11b expression. Vicinal dithiol-reactive phenylarsine oxide (50 nM) also increased CD11b expression induced by DMSO or ATRA. These observations suggested that oxidative stress enhanced granulocytic differentiation of HL 60 cells and that leukaemic cell differentiation was affected by cellular redox status.

Loss of ACE2 Accelerates Pressure Overload-Induced Myocardial Oxidative Stress and Ventricular Dysfunction

Introduction: Pressure overload activates the cardiac renin-angiotensin system (RAS) in association with myocardial oxidative stress and left ventricular dysfunction. The key peptidase action of angiotensin converting enzyme 2 (ACE2) is degradation of angiotensin (Ang) II to Ang (1–7), functioning effectively as a negative regulator of the RAS. Hypothesis: We hypothesized that ACE2 exerts its protective roles against myocardial oxidative stress and ventricular dysfunction by modulation of NADPH oxidase activity. Methods: Male wildtype (WT) mice, ACE2 knockout (KO) and ACE2/p47phox double KO mice of 10 weeks of age underwent aortic banding model of pressure-overload for 3 weeks. We characterized the functional, structural and molecular changes in the heart in response to pressure overload by echocardiography, TaqMan real-time PCR, Western blot analysis and assessed NADPH oxidase activity. Results: Compared with WT mice, loss of ACE2 resulted in a marked increase in pressure overload-induced myocardial oxidative stress in banded ACE2 KO mice, associated with decreased cardiac function, increased expression of hypertrophy markers (ANF and BNP) and elevated NADPH oxidase activities including p47phox, and Nox2 and Nox4 mRNA expression. These changes were linked with activation of PKCα protein and phosphorylation of the extracellular signal-regulated protein kinase (ERK1/2), Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling in ACE2 KO mice. Loss of p47 subunit prevented the increase in NADPH oxidase activity and attenuated the adverse remodeling in the ACE2 KO mice. Conclusions: Loss of ACE2 accelerates pressure-overload induced myocardial oxidative stress, ventricular dysfunction by activation of the NADPH oxidase subunits with the p47 subunit playing a key role. ACE2 suppresses the myocardial NADPH oxidase system possible by reducing Ang II levels. Introduction: Pressure overload activates the cardiac renin-angiotensin system (RAS) in association with myocardial oxidative stress and left ventricular dysfunction. The key peptidase action of angiotensin converting enzyme 2 (ACE2) is degradation of angiotensin (Ang) II to Ang (1–7), functioning effectively as a negative regulator of the RAS. Hypothesis: We hypothesized that ACE2 exerts its protective roles against myocardial oxidative stress and ventricular dysfunction by modulation of NADPH oxidase activity. Methods: Male wildtype (WT) mice, ACE2 knockout (KO) and ACE2/p47phox double KO mice of 10 weeks of age underwent aortic banding model of pressure-overload for 3 weeks. We characterized the functional, structural and molecular changes in the heart in response to pressure overload by echocardiography, TaqMan real-time PCR, Western blot analysis and assessed NADPH oxidase activity. Results: Compared with WT mice, loss of ACE2 resulted in a marked increase in pressure overload-induced myocardial oxidative stress in banded ACE2 KO mice, associated with decreased cardiac function, increased expression of hypertrophy markers (ANF and BNP) and elevated NADPH oxidase activities including p47phox, and Nox2 and Nox4 mRNA expression. These changes were linked with activation of PKCα protein and phosphorylation of the extracellular signal-regulated protein kinase (ERK1/2), Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling in ACE2 KO mice. Loss of p47 subunit prevented the increase in NADPH oxidase activity and attenuated the adverse remodeling in the ACE2 KO mice. Conclusions: Loss of ACE2 accelerates pressure-overload induced myocardial oxidative stress, ventricular dysfunction by activation of the NADPH oxidase subunits with the p47 subunit playing a key role. ACE2 suppresses the myocardial NADPH oxidase system possible by reducing Ang II levels.

Activation of Mineralocorticoid Receptors by Exogenous Glucocorticoids and the Development of Cardiovascular Inflammatory Responses in Adrenalectomized Rats

Activation of the mineralocorticoid receptor (MR) in the context of a high salt intake produces cardiovascular inflammation plus cardiac fibrosis and failure. Inactivation of vascular 11beta-hydroxysteroid dehydrogenase type 2 activity in intact animals by carbenoxolone (CBX) produces a similar pathology, presumably reflecting coronary vascular MR activation by endogenous glucocorticoids. To test this hypothesis, we have used adrenalectomized rats, without endogenous corticosteroids, and examined the consequences of corticosterone (CORT) replacement on a series of cardiovascular disease parameters. Uninephrectomized adrenalectomized Sprague Dawley rats given 1% NaCl/0.3% KCl to drink were treated for 8 d as follows: control; 20 mg deoxycorticosterone (DOC); 2 mg/d CORT; 2.5 mg/d CBX; CORT plus CBX (CORT/CBX); and CORT/CBX plus 100 mg/kg.d eplerenone. Markers of cardiac oxidative stress (p22(phox) and NOX4 mRNA) were up-regulated in the DOC and CORT/CBX groups; in contrast, inflammatory cell infiltration was increased and endothelial nitric oxide synthase down-regulated by CORT as well as by DOC and CORT/CBX. In the kidney, connective tissue growth factor mRNA levels were increased by DOC and CORT/CBX; in contrast, DOC had no effect on mRNA levels for channel inducing factor or endothelin 3, which were elevated only by CORT/CBX. All changes noted were reversed by eplerenone. Rats given 10-fold lower CORT (0.2 mg/d) with or without CBX showed no change in any parameter. These results suggest that there exist distinct but overlapping ligand-specific MR-mediated tissue responses to a classic mineralocorticoid (DOC) and to the glucocorticoid CORT, in the presence and absence of CBX to block vascular 11beta-hydroxysteroid dehydrogenase type 2.

Resveratrol prevents endothelial dysfunction and aortic superoxide production after trauma hemorrhage through estrogen receptor-dependent hemeoxygenase-1 pathway

To determine whether resveratrol provides vasculoprotection in trauma-hemorrhaged animals and whether the effects are mediated via estrogen receptor-dependent hemeoxygenase-1. Prospective, multiexperimental, randomized, controlled studies. University research laboratory. Male Sprague-Dawley rats weighing 300-350 g. Male Sprague-Dawley rats underwent trauma hemorrhage (mean arterial pressure 40 mm Hg for 90 min, then resuscitation). Resveratrol (30 mg/kg) with or without an estrogen receptor antagonist (ICI 182,780), a hemeoxygenase enzyme inhibitor (chromium-mesoporphyrin), or vehicle was injected during resuscitation. At 24 hrs after trauma hemorrhage with resuscitation or sham operation, the animals were euthanized for further evaluation. Acetylcholine-induced endothelium-dependent relaxation decreased, whereas nicotinamide adenine dinucleotide-stimulated superoxide radical production in the aorta and aortic p22phox, p47phox, gp91phox, NOX1, and NOX4 mRNA concentrations increased in trauma-hemorrhaged rats vs. sham rats. All altered parameters were normalized in resveratrol-treated trauma-hemorrhaged rats. Furthermore, there was a significant increase in hemeoxygenase-1 after trauma hemorrhage, and resveratrol treatment further increased hemeoxygenase-1 expression in trauma-hemorrhaged rats. However, administration of ICI 182,780 or chromium-mesoporphyrin abolished the resveratrol-induced prevention of shock-induced oxidative stress and endothelial damage. In the resveratrol-treated rats subjected to trauma hemorrhage, there were significant improvements in plasma aspartate aminotransferase and alanine aminotransferase levels, and mortality rate, and there was lesser damage in histology. Resveratrol treatment prevented the overproduction of superoxide radical/NADPH oxidase expression and restored the trauma-hemorrhage-impaired endothelium-dependent relaxation via estrogen receptor-dependent stimulation of hemeoxygenase-1 expression.

Reduced 22Na+ Uptake and Dysregulation of NHERF2/Akt/NOS Pathway in Rat Renal Proximal Tubule Cells Stably Overexpressing p22phox

We showed that superoxide (O2−) reduced renal proximal tubule (PT) reabsorption (Hypertension. 2009 Oct.), but, the molecular mechanisms are unknown. We tested the hypothesis that O2− impairs Na+‐H+ exchanger 3 (NHE3) activity. A stably p22phox transfected rat PT cell line (s‐p22phox), compared to empty vector transfected wild type cells (Wt), had increased p22phox mRNA and protein by 4.1‐ and 1.59‐fold, and O2− by 2.2‐ fold but reduced 22Na+uptake by 36% (all P&lt;0.05). Immunoblot assay of s‐p22phox cells showed no change in NHE3 but increased expression of inhibitory Na+–H+ exchanger regulatory factor 2 (NHERF2), neuronal and inducible nitric oxide synthase (nNOS &amp; iNOS) by 183%, 293% and 372%, respectively (all P&lt;0.05). Since activated Akt regulates NOS and NHERF2, we compared the Akt in both cell types. Total Akt (t‐Akt) protein abundance was similar but the phosphorylated Akt at ser473 (p‐Aktser473) was markedly increased leading to a 207% higher p‐Akt/t‐Akt ratio in s‐p22phox cells. Knockdown of NHEFR2 by specific siRNA in s‐p22phox cells increased significantly (25±4.9%; P&lt;0.05) 22Na+uptake. In conclusion, overexpression of p22phox in PT cells inhibits NHE3 transport activity and upregulates NHERF2, nNOS and iNOS, and phosphorylated Akt. This dysregulation of NHERF2/Akt/NOS signaling pathways by O2− impairs PT function. This provides a novel insight into the molecular mechanisms underlying the effects of oxidative stress on ion and fluid homeostasis in the kidney.

Polyphenol-containing azuki bean ( Vigna angularis) seed coats attenuate vascular oxidative stress and inflammation in spontaneously hypertensive rats

We investigated the effects of azuki bean ( Vigna angularis) seed coats (ABSC), which contain polyphenols, on the vascular oxidative stress and inflammation associated with hypertension. Spontaneously hypertensive rats (SHR) and control normotensive Wistar-Kyoto (WKY) rats were divided into 2 groups each. One group was fed 0% ABSC; the other, a 1.0% ABSC-containing diet. Tail systolic blood pressure (SBP) was examined throughout ABSC treatment. At 8 weeks, vascular superoxide (O 2 −) production was measured by lucigenin-enhanced chemiluminescence. mRNA expressions of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, macrophage chemoattractant protein-1 (MCP-1) and its receptor C-C chemokine receptor 2 (CCR2) in the aorta were analyzed by reverse transcriptase-polymerase chain reaction. Protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were determined by western blotting. Polyphenol-containing ABSC suppressed the elevation of SBP throughout the treatment period. The NADPH-stimulated O 2 − level decreased significantly in the aorta of ABSC-treated SHR compared with the level of untreated SHR. The p47phox and Nox4 mRNA expression increased significantly in untreated SHR compared with that in WKY rats. Conversely, the level of p47phox mRNA was significantly lower in ABSC-treated SHR than in untreated SHR. The protein abundance of both iNOS and COX-2 was significantly decreased in the aorta of the ABSC-treated SHR compared with this abundance in untreated SHR. The MCP-1 and CCR2 mRNA expressions increased in untreated SHR, and these levels were significantly lower in ABSC-treated SHR. In conclusion, our results suggested that polyphenol-containing ABSC could attenuate vascular oxidative stress and inflammation during the progression of hypertension, and this may lead to an improvement in hypertension.

Nicotinamide-adenine dinucleotide phosphate oxidase p22phox expression in induced sputum cells for patients with obstructive sleep apnea hypopnea syndrome

the aim of the study was to observe the change of NADPH oxidase p22phox expression in the induced sputum cells for patients with obstructive sleep apnea hypopnea syndrome (OSAHS), and its relationship with the severity of OSAHS. thirty OSAHS patients and 23 healthy controls were recruited in the study. Before sleep and in the next morning sputum induction was performed twice for each subject, cell numbers and differentials in induced sputum were counted. NADPH oxidase p22phox mRNA expression was measured in induced sputum cells by RT-PCR and the protein was measured by immunocytochemistry. the OSAHS group showed a significant higher percentage of neutrophils and a lower percentage of macrophages in the induced sputum compared to the control group (P < 0.05). Macrophages and neutrophils represented the main cell types expressing NADPH oxidase p22phox in induced sputum cells by immunocytochemistry. In the control group, level of NADPH oxidase p22phox mRNA and percentages of NADPH oxidase p22phox positive neutrophils and macrophages in sputum samples collected both in the morning and before sleep was significantly lower than in OSAHS group (P < 0.05). In OSAHS group, higher levels of NADPH oxidase p22phox mRNA and percentages of NADPH oxidase p22phox positive neutrophils and macrophages were found in the morning than at pre-sleep. A negative correlation was found between levels of NADPH oxidase p22phox mRNA and percentage of NADPH oxidase p22phox positive neutrophils and macrophages in the morning sputum were negatively associated with LspO(2), but positively associated with AHI. these findings suggest that there are changes of NADPH oxidase p22phox expression in induced sputum cells of OSAHS patients, and these changes are related to the severity of OSAHS.

Apocynin attenuate spatial learning deficits and oxidative responses to intermittent hypoxia

Rationale: The long-term intermittent hypoxia (LTIH) that characterizes sleep-disordered breathing impairs spatial learning and increases oxidative stress in rodents. We hypothesized that LTIH activated brain NADPH oxidase, which served as a critical source of superoxide in the oxidation injury, and that apocynin might attenuate LTIH-induced spatial learning deficits by reducing LTIH-induced NADPH oxidase expression. Objective: To investigate the effects of apocynin on spatial learning and oxidative responses to LTIH in rats. Methods: Forty healthy male Sprague–Dawley (SD) rats were randomly divided into four groups of 10 each: a LTIH group, an apocynin-treated LTIH group, a sham LTIH group and an apocynin-treated sham group. Spatial learning in each group was assessed with the Morris water maze test. RT-PCR and Western blot were used to examine mRNA and protein expression of NADPH oxidase subunit p47phox and p22phox in the hippocampus region. The level of MDA and SOD were detected by colorimetric method. The terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling (TUNEL) method was used to display the apoptotic cells of the hippocampus tissue. Results: Apocynin treatment prevented LTIH-induced decreases in spatial learning during the Morris water maze as well as LTIH-induced decrease in SOD levels. In untreated animals, LTIH exposure was related to increase of MDA levels in comparison to sham LTIH animals, and apocynin-treated animal exposure to LTIH showed reduction in MDA levels. Increases in hippocampus NADPH oxidase subunit p47phox mRNA and protein expression were observed in LTIH-exposed animals; there was no statistical difference of p47phox mRNA and protein expression between LTIH group and apocynin treatment group. Treatment with apocynin significantly ameliorated cell apoptosis in LTIH-exposed animals. Conclusion: These results indicate that apocynin attenuates LTIH-induced spatial learning deficits and mitigates LTIH-induced oxidative stress through multiple beneficial effects on oxidant pathways. NADPH oxidase up-expression is closely associated with oxidative processes in LTIH rats, and inhibition of NADPH oxidase activity may hopefully serve as a useful strategy for cognitive function impairment from chronic intermittent hypoxia.

Effect of Tongluo recipe on oxidative stress in kidneys of diabetic rats

Objective:To study the effect of two types of Tongluo recipe(ultrafine type and fine type) on the level of oxidative stress in kidneys of diabetic Sprague-Dawley rats.Methods: Experimental diabetes was induced by intraperitoneal injection of streptozotocin(60 mg/kg) in male Sprague-Dawley rats.One week after streptozotocin injection rats with blood glucose higher than 16.7 mmol/L were taken as diabetics.The study included the following 4 groups: normal control(NC),diabetes mellitus control(DMC),diabetics treated with ultrafine Tongluo recipe(DM+UT,1 g·kg-1·d-1),and diabetics treated with fine Tongluo recipe(DM+FT,1 g·kg-1·d-1).Four weeks after treatment,24 h urinary protein(24 h UP) and kidney weight/body weight(KW/BW) were detected as renal function indices;malondialdehyde(MDA) and antioxidant enzymes was examined with colorimetry;and NADPH oxidase subunits p47phox and p22phox mRNA were studied with real-time PCR.Results: Compared with the NC group,DMC group had significantly higher MDA,24 h UP,KW/BW,and glutathione peroxidase(GSH-Px) activity,and had significantly lower activity of total superoxide dismutase(TSOD).No significant difference was found in catalase(CAT) activity between the NC group and DMC group.Compared with the 2 Tongluo Recipe groups,the DMC group had significantly lower 24 h UP,KW/BW and MDA,and significantly higher activities of TSOD and GSH-Px,and no significant difference was found in their CAT activities.The effect of ultrafine type on 24 h UP,KW/BW,MDA,TSOD and GSH-Px was significantly greater than the fine type did.The expression of p47phox mRNA of NADPH oxidase in renal cortex group was significantly lower than that in the DMC group,and the latter was significantly higher than the 2 Tongluo of the NC recipe groups,and there was no significant difference between the 2 Tongluo recipe groups.There was no significant difference in p22phox mRNA expression between all the 4 groups.Conclusion: Tongluo recipe may improve the activities of antioxidant enzymes(including TSOD and GSH-Px),decrease p47phox expression,and therefore inhibit the oxidative stress in renal cortex of diabetic rats,reducing the early kidney injury.

Inhibitory effect of reinioside C on monocyte–endothelial cell adhesion induced by oxidized low-density lipoprotein via inhibiting NADPH oxidase/ROS/NF-κB pathway

Monocyte adhesion to activated vascular endothelial cells is the critical event in the initiation of atherosclerosis. Adhesion molecules are inflammatory markers, which are upregulated by oxidized low-density lipoprotein (ox-LDL) and play a pivotal role in atherogenesis. In present study, the effect of reinioside C, a major compound of Polygala fallax Hemsl., on adhesion of monocytes to endothelial cells induced by ox-LDL was investigated. The results showed that incubation of endothelial cells with ox-LDL (100 microg/mL) for 24 h markedly increased the expression of ICAM-1 and P-selectin and enhanced the adhesion of monocytes to endothelial cells. Pretreatment with reinioside C (1, 3, or 10 microM) dose-dependently decreased ox-LDL-induced upregulation of expression of ICAM-1 and P-selectin and the enhanced adhesion of monocytes to endothelial cells. To determine the role of NADPH oxidase/reactive oxygen species (ROS)/nuclear factor-kappaB (NF-kappaB) pathway, endothelial cells were treated with ox-LDL (100 microg/mL) for 2 h, and NADPH oxidase subunit (Nox 2 and p22phox) mRNA expression, intracellular ROS level, and NF-kappaB activity were measured. The results showed that reinioside C attenuated ox-LDL-induced NADPH oxidase subunit (Nox 2 and p22phox) mRNA expression, generation of ROS, and activation of NF-kappaB in endothelial cells in a dose-dependent manner; the two latter effects were inhibited by pyrollidine dithiocarbamate, the inhibitor of NF-kappaB. These findings suggest that reinioside C attenuates ox-LDL-induced expression of adhesion molecules (P-selectin and ICAM-1) and the adhesion of monocytes to endothelial cells by inhibiting NADPH oxidase/ROS/NF-kappaB pathway.

Low-density lipoprotein apheresis for haemodialysis patients with peripheral arterial disease reduces reactive oxygen species production via suppression of NADPH oxidase gene expression in leucocytes

Peripheral arterial disease (PAD) is a major complication of haemodialysis (HD), especially in patients with diabetes mellitus. Although previous reports have indicated that low-density lipoprotein apheresis (LDL-A) improves arteriosclerosis in PAD patients, the mechanism by which LDL-A affects PAD is still unclear. In this study, we tested the hypothesis that LDL-A attenuates reactive oxygen species (ROS) production in HD patients with PAD. Twenty HD patients with PAD were investigated in this study. Clinical effects were evaluated by thermography and angiography. Oxidative stress in serum was evaluated by thiobarbituric acid reactive substances (TBARS) and expression of p22phox mRNA. Ischaemic symptoms due to PAD were gradually improved in 13 patients (65%) after LDL-A. One session of LDL-A removed approximately 75% of LDL from serum. Some patients exhibited dramatic improvement of severe symptoms of PAD such as skin ulcers after serial performance of LDL-A. The levels of LDL cholesterol, malondialdehyde-modified LDL, high-sensitivity C-reactive protein, vascular endothelial growth factor, international normalized ratio of pro-thrombin time and bradykinin were decreased after a single session of LDL-A, although there were no additional changes after 10 sessions of LDL-A. The levels of fibrinogen and p22phox mRNA were decreased by a single session of LDL-A, and these decreases continued over the entire period of treatment. TBARS was decreased after a course of LDL-A. LDL-A improved ischaemic symptoms in HD patients with PAD by reducing ROS production in leucocytes. We conclude that LDL-A is an effective therapy for patients with HD complicated by PAD.

Role of NAD(P)H oxidase in transforming growth factor‐β1‐induced monocyte chemoattractant protein‐1 and interleukin‐6 expression in rat renal tubular epithelial cells

This study investigated the role of NAD(P)H oxidase in transforming growth factor-beta1 (TGF-beta1)-induced reactive oxygen species (ROS) generation, monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) expression in rat renal tubular epithelial NRK-52E cells. The cells were treated with 10 ng/mL TGF-beta1, either in the presence or absence of the NAD(P)H oxidase inhibitor, diphenyleneiodonium (DPI), or short hairpin RNA (shRNA) suppressing p67phox expression. Expression of NAD(P)H oxidase subunits, MCP-1, and IL-6 at the mRNA levels was detected by reverse transcription polymerase chain reaction, while expression of NAD(P)H oxidase subunit p67phox protein was analyzed by western blot and MCP-1 by enzyme-linked immunosorbent assay. The cellular ROS generation was visualized using 2',7'-dichlorodihydrofluorescein diacetate by confocal microscopy. Compared to control, TGF-beta1 upregulated NAD(P)H oxidase subunit p67phox mRNA by 3.59-fold (P < 0.01), but had no effect on p22phox, gp91phox and p47phox NAD(P)H subunits. TGF-beta1 was also able to significantly increase intracellular ROS (P < 0.05), MCP-1 (P < 0.01) and IL-6 (P < 0.05) expression in NRK-52E cells. Further studies showed that generation of ROS and upregulation of MCP-1 and IL-6 by TGF-beta1 were significantly blocked by addition of DPI or shRNA-p67phox (P < 0.01), suggesting that these effects were NAD(P)H oxidase-dependent. TGF-beta1 differentially regulates the expression of NAD(P)H oxidase subunits and mediates MCP-1 and IL-6 expression in rat renal tubular cells via the NAD(P)H oxidase/p67phox-dependent mechanism.

The study on inhabiting endothelial cell aging by targeted silencing of &lt;I&gt;p22phox&lt;/I&gt;

The aim of the study was to determine the importance and possible mechanism of NAD (P)H oxidase subunits P (superscript 22phox) involved in human umbilical endothelial cell lines ECV-304 aging by special short interference RNA (siRNA). Three siRNAs targeting p22phox were designed and synthesized in vitro, which were used to transfect ECV-304 cultured in vitro for selecting the most powerful and most suitable transfection concentration and time. The cell line ECV-304 was divided into three groups: control group, angiotensin II (Ang II) group, siRNA group, and Ang II + siRNA group. Cell aging was identified by beta-gal stain. Reactive oxygen species (ROS) and NO level in cells and medium were measured. RT-PCR and Western blot were used to analyze mRNA and protein expression of NAD(P)H oxidase subunit p22phox. Among the 3 siRNAs, siRNA-1 was the most powerful on gene silence with 50 nmol/L transfection concentration at 24 h and 36 h. The number of positive cells stained by beta-gal were increased in ECV-304 stimulated with Ang II, and p22phox mRNA and protein expression were increased in aging ECV-304 stimulated with Ang II, which had lower NO and higher ROS. Compared with Ang II group, ROS level was decreased and NO level was increased in Ang+siRNA group with decreased aging level. The result of the present study suggested that siRNA could induce NAD(P)H oxidase subunit p22phox gene silence, Ang II could induce ECV-304 aging cultured in vitro, and the possible pathway of endothelial cell aging is that Ang II upregulates p22phox expression, and then enhances the cell ROS level.

Enhanced susceptibility of cyclin kinase inhibitor p21 knockout mice to high fat diet induced atherosclerosis

Cyclin kinase inhibitor p21 is one of the most potent inhibitors of aortic smooth muscle cell proliferation, a key mediator of atherosclerosis. This study tests if p2l deficiency will result in severe atherosclerosis in a mouse model. p21-/- and strain matched wild type mice were fed with high fat diet for 21 weeks. Analysis for biochemical parameters (cholesterol, triglycerides) in serum and mRNA expression of CD36, HO-1, TGF-β, IFN-γ, TNF-α, PPAR-γ and NADPH oxidase components (p22phox, NOX-1 and Rac-1) was performed in aortic tissues by Real Time PCR. p21-/- mice gained significantly (p < 0.01) more weight than wild type mice, triglycerides (p < 0.05) and cholesterol levels (p < 0.01) were more pronounced in the sera of p21-/- compared to wild type mice fed with high fat diet. High fat diet resulted in significantly decreased TGF-β (p < 0.02), HO-l (p < 0.02) and increased CD36 (p < 0.03) mRNA expression in aortic tissues of p21-/- mice compared to animal fed with regular diet. IFN-γ mRNA expression (235 ± 11 folds) increased significantly in high fat diet fed p21-/- mice and a multifold modulation of PPAR-γ(136 ± 7), p22phox, NOX-1 and Rac-1 (15–35-folds) mRNA in aortic tissues from p21-/- mice compared to the wild type mice. Severity of atherosclerotic lesions was significantly higher in p21-/- compared to wild type mice. The results demonstrate that the deficiency of p21 leads to altered expression of pro-atherogenic genes, and severe atherosclerosis in mice fed with high fat diet. This opens the possibility of p21 protein as a therapeutic tool to control progression of atherosclerosis.

Effects of cyclic strain on endothelial cell apoptosis and tubulogenesis are dependent on ROS production via NAD(P)H subunit p22phox

Objective Vascular endothelial cells (ECs) are constantly exposed to blood flow associated forces such as cyclic strain due to blood pressure, which affects ECs survival and angiogenesis by producing ROS via NAD(P)H oxidase. NAD(P)H oxidase subunit p22phox is reported to be related to the development of atherosclerosis and increased levels of p22phox mRNA are correlated to ECs proliferation. However, the importance and signaling mechanism of p22phox on ECs survival and angiogenesis under cyclic strain are unclear. Methods 5%–20% cyclic strain were applied by the Flexercell system to simulate in vivo environment of human ECs; the effect of p22phox on mechanical ECs survival mechanism and tubulogenesis was determined by western blot and 3-D tissue culture by knocking down p22phox expression via shRNA plasmid. Results Knockdown of p22phox induced expression of cleaved caspase-3 and decreased cell viability ratio (CVR). 5% strain increased and 20% strain decreased CVR of shp22phox cells. There were complex biphasic effects of cyclic strain on ECs survival signaling. 5% strain continuously increased Akt phosphorylation; 20% strain increased after 10min stimulation and decreased Akt phosphorylation lately. 5% strain increased and 20% strain decreased eNOS phosphorylation. Knockdown of p22phox decreased Akt and eNOS phosphorylation with or without cyclic strain. ROS production was increasingly stimulated progressively by strain via the p22phox pathway. 5% strain increased and 20% strain decreased total NO production and vascular tubulogenesis via p22phox pathway. Conclusion ROS production is pivotal to responses to physiological or pathological strain. Physiological strain increases but pathological strain decreases ECs survival and tubulogenesis, and these effects occur via the NAD(P)H subunit p22phox pathway.

Regulation of Dimethylarginine Dimethylaminohydrolase (DDAH) in Rat Preglomerular Vascular Smooth Muscle Cells (PGVSMCs) by Reactive Oxygen Species (ROS)

DDAH degrades asymmetric dimethylarginine (ADMA) which is an endogenous NOS inhibitor and risk factor for cardiovascular disorders. We tested the hypothesis that ROS regulate the transcription, translation or activity of DDAH. We created a stable rat PGVSMC line overexprissing p22phox (S‐p22phox) using a p22phox‐pcDNA4/HisMax construct (a generous gift from Kathy Griendling). Quantitative real time PCR and Western blotting confirmed a marked increase in p22phox mRNA and protein of 4.7±0.6 and 2.3±0.5 fold, (p&lt;0.01), compared to wild type cells (WT). S‐p22phox cells had elevated superoxide and hydrogen peroxid generation (2.3±0.09 fold and 1.8±0.1 fold p&lt;0.01). The mRNAs for DDAH ‐1 and ‐2 were increased by 2.8 ± 0.2 and 1.8 ± 0.1 fold in S‐p22phox cells p&lt; 0.01). DDAH‐2 promoter activity, determined by transient transfection and luciferase assay, was 4.1 fold increased in S‐p22phox cells (p &lt; 0.01). Interestingly, both DDAH‐1 and ‐2 proteins were significantly lower in S‐p22phox cells (0.9±0.2 and 0.9±0.1 fold; p&lt;0.01), yet β‐actin was not different. DDAH activity measured by a colorimetric assay (Kidney Int. 72:886–9, 2007) was reduced by 62±2% in S‐p22phox(p&lt;0.01). The results reveal for the first time an important effect but complex of p22phox – dependent ROS generation on DDAH. Thus, ROS upregulate the expression of DDAH‐1 and ‐2 genes via activating a promoter, at least for DDAH‐2, yet downregulate both DDAH‐1 and ‐2 proteins and their enzyme activity. This is a specific effect since ROS do not change the expression of cell structure proteins. These findings in PGVSMCs provide a novel regulatory pathway whereby ROS may modulate renal hemodynamics, and thereby BP and salt homeostasis.