Nox4 Protein Research Articles

Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells.

BackgroundStudies have shown that both NOX4 and RhoA play essential roles in fibrosis and that they regulate each other. In lung fibrosis, NOX4/ROS is located upstream of the RhoA/ROCK1 signaling pathway, and the two molecules are oppositely located in renal fibrosis. Currently, no reports have indicated whether the above mechanisms or other regulatory mechanisms exist in liver fibrosis.ObjectivesTo investigate the effects of the NOX4/ROS and RhoA/ROCK1 signaling pathways on hepatic stellate cell (HSC)-T6 cells, the interaction mechanisms of the two pathways, and the impact of UA on the two pathways to elucidate the role of UA in the reduction of hepatic fibrosis and potential mechanisms of HSC-T6 cell proliferation, migration, and activation.MethodsStable cell lines were constructed using the lentiviral transduction technique. Cell proliferation, apoptosis, migration, and invasion were examined using the MTS, TdT-mediated dUTP nick-end labeling, cell scratch, and Transwell invasion assays, respectively. The DCFH-DA method was used to investigate the ROS levels in each group. RT-qPCR and western blotting techniques were utilized to assess the mRNA and protein expression in each group. CoIP and the Biacore protein interaction analysis systems were used to evaluate protein interactions.ResultsThe NOX4/ROS and RhoA/ROCK1 signaling pathways promoted the proliferation, migration, and activation of HSCs. UA inhibited cell proliferation, migration, and activation by inhibiting the activation of the two signaling pathways, but the mechanism of apoptosis was independent of these two pathways. The NOX4/ROS pathway was upstream of and positively regulated the RhoA/ROCK1 pathway in HSCs. No direct interaction between the NOX4 and RhoA proteins was detected.ConclusionThe NOX4/ROS and RhoA/ROCK1 signaling pathways are two critical signaling pathways in a series of behavioral processes in HSCs, and NOX4/ROS regulates RhoA/ROCK1 through an indirect pathway to control the activation of HSCs. Additionally, NOX4/ROS and RhoA/ROCK1 constitute a new target for UA antifibrosis treatment.

Thyroid hormone protects cardiomyocytes from H2O2-induced oxidative stress via the PI3K-AKT signaling pathway

Oxidative stress plays an important role in the progression of cardiac diseases, including acute myocardial infarction, ischemia/reperfusion (I/R) injury and heart failure. Growing evidence indicates that thyroid hormone has protective properties against cardiovascular diseases. However, little is known about its effect on oxidative stress in cardiomyocytes or the underlying mechanisms. This study showed that T3 pretreatment in vivo significantly reduced cardiac dysfunction by increasing the left ventricular ejection function and ameliorating the pathological changes induced by I/R-induced injury. In an in vitro experiment, T3 inhibited apoptosis in H2O2-treated cardiomyocytes, as evidenced by the decreased expression of Bax, cleaved caspase 3 and 9, and increased expression of Bcl-2. In addition, oxidative stress observed in hearts of mice with I/R injury was significantly alleviated by T3 pretreatment, intracellular ROS and mitochondrial ROS overproduction were effectively inhibited, and similar results were also detected in H2O2-treated cardiomyocytes in vitro. T3 significantly increased antioxidant protein (Nrf2 and HO-1) expression levels, and inhibited NOX2 and NOX4 protein expression levels in H2O2-treated cardiomyocytes. Moreover, T3 preserved mitochondrial functions upon H2O2-induced oxidative stress by increasing mitochondrial membrane potential and promoting the expression of mitochondrial biogenesis genes. Notably, the PI3K/AKT signaling was significantly activated by T3 pretreatment in H2O2-induced cardiomyocytes. Together, these findings revealed that T3 could be served as potential therapeutic target for protection against cardiac oxidative stress injury through its antioxidant and anti-apoptosis effects, which are mediated by the activation of the PI3K/AKT signaling pathway.

Cyclophilin A Protects Cardiomyocytes against Hypoxia/Reoxygenation-Induced Apoptosis via the AKT/Nox2 Pathway.

Hypoxia/reoxygenation (H/R) accelerates the process of cardiomyocyte apoptosis during ischemia-reperfusion. Excessive reactive oxygen species (ROS) are a critical driver of oxidative stress injury. Cyclophilin A (CyPA) is a major ROS-induced factor in atherosclerosis. There is a positive feedback mechanism between CyPA and ROS, which enables the oxidative stress response to continue and expand. However, it is unclear whether this positive feedback mechanism exists in cardiomyocytes. Through western blotting and flow cytometric assays and TUNEL assay, we found that CyPA inhibited the apoptosis of H9c2 cardiomyocytes under H/R conditions. By dihydroethidium (DHE) staining and electron spin resonance (ESR) assays, we demonstrated that CyPA reduced ROS production and suppressed O2− production dependent on reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. By western blotting, we showed that CyPA inhibited the expression of NADPH oxidase 2 (Nox2) protein by the AKT pathway. Through confocal microscopy assay, we found that CyPA reduced the expression of Nox2 membrane-bound subunits. The current study shows that a positive feedback mechanism does not exist in H9c2 cardiomyoblasts. CyPA protects H9c2 cardiomyoblasts against H/R-induced apoptosis via the AKT/Nox2 pathway. This could be a potential target for ischemia-reperfusion injury therapy.

Guttation capsules containing hydrogen peroxide: an evolutionarily conserved NADPH oxidase gains a role in wars between related fungi.

SummaryWhen resources are limited, the hypocrealean fungus Trichoderma guizhouense can overgrow another hypocrealean fungus Fusarium oxysporum, cause sporadic cell death and arrest growth. A transcriptomic analysis of this interaction shows that T. guizhouense undergoes a succession of metabolic stresses while F. oxysporum responded relatively neutrally but used the constitutive expression of several toxin‐encoding genes as a protective strategy. Because of these toxins, T. guizhouense cannot approach it is potential host on the substrate surface and attacks F. oxysporum from above. The success of T. guizhouense is secured by the excessive production of hydrogen peroxide (H2O2), which is stored in microscopic bag‐like guttation droplets hanging on the contacting hyphae. The deletion of NADPH oxidase nox1 and its regulator, nor1 in T. guizhouense led to a substantial decrease in H2O2 formation with concomitant loss of antagonistic activity. We envision the role of NOX proteins in the antagonism of T. guizhouense as an example of metabolic exaptation evolved in this fungus because the primary function of these ancient proteins was probably not linked to interfungal relationships. In support of this, F. oxysporum showed almost no transcriptional response to T. guizhouense Δnox1 strain indicating the role of NOX/H2O2 in signalling and fungal communication.

Reduction of Oxidative Stress as a Potential Mechanism Underlying Euphorbia bicolor Latex Extract‐Induced Analgesia in a Rat Model of Orofacial Pain

One target of pain management is the transient receptor potential V1 ion channel (TRPV1), a cation channel that is a pain generator located on nociceptors. Recent studies have reported that reactive oxygen species (ROS; oxidative stress) may cause pain through activation of TRPV1. ROS are key mediators in the development of peripheral and central sensitization in various pain etiologies, including neuropathic, inflammatory and opioid‐induced pain. Furthermore, TRPV1 can be activated and potentiated by NADPH oxidase (NOX), which plays a key role in the production of ROS. ROS can activate TRPV1 by inducing its conformational change or by increasing the plasma membrane permeability to calcium ions. In support, inhibition of ROS is effective in reducing pain. Our previous studies reported that Euphorbia bicolor (Euphorbiaceae) latex extract possesses long‐lasting peripheral analgesia, in part through TRPV1, in the inflamed hindpaw of both male and female rats. The present study hypothesized that E. bicolor latex extract‐evoked analgesia occurs via downregulation of oxidative stress biomarkers in a rat model of orofacial pain. Orofacial pain behavior was tested using the von Frey method to assess mechanical allodynia in the rat vibrissal pad. Baseline sensitivity was recorded in male and female rats followed by injections of complete Freund's adjuvant (CFA) into the left vibrissal pad to induce inflammatory pain. Mechanical allodynia was then confirmed 24 hours following CFA injections. Rats received one injection of either E. bicolor latex extract (300 μg/mg in 0.9% saline and <5% methanol) or vehicle (0.9% saline and <5% methanol) into the inflamed vibrissal pad. Mechanical sensitivity was then reassesed at 1 hour, 6 hours, 24 hours, and/or 72 hours. Data were recorded as mechanical threshold required to elicit a withdrawal response. All rats were then rapidly decapitated immediately following the last time point and trunk blood and trigeminal ganglia (TG) were collected. A separate group of rats received the same treatments and were used to collect blood and TGs at the 1‐ and 6‐hour time points. ROS in the TGs were quantified by the fluorescent dye, 2′,7′‐dichlorodihydrofluorescein diacetate method. Oxidative stress markers were measured in trunk blood by quantification of advanced oxidation protein product (AOPP) in plasma samples. ROS‐induced NOX4 protein expression was quantified by western blot analysis. Here we report that E. bicolor latex extract significantly reduced orofacial mechanical sensitivity in both male and female rats at 24 hours and 72 hours, respectively and in concurrence with our previous reports in hindpaw‐inflamed rats. Both ROS and NOX4 were significantly reduced in the trigeminal ganglia of extract‐treated as compared to vehicle‐treated rats at the same time points. In addition, plasma AOPP was also significantly reduced with the onset of analgesia. Overall, the latex extract reduced the oxidative stress biomarkers in both plasma and TGs of rats and downregulated NOX4 protein expression corresponding with the onset of analgesia. These data indicate that the extract contains phytochemicals that may serve as novel therapeutics for treating pain with an oxidative stress component.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NADPH Oxidase Isoforms Are Involved in Glucocorticoid-Induced Preosteoblast Apoptosis.

Oxidative stress induced by long-term glucocorticoid (GC) use weakens the repair capacity of bone tissue. Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase (NOX) is a superoxide-generating enzyme that plays an important role in regulating bone metabolism. To clarify the role of nonphagocytic NOX isoforms in osteoblast reactive oxygen species (ROS) generation and apoptosis, dexamethasone was used to establish a high-dose GC environment in vitro. A dose-dependent increase in intracellular ROS generation was demonstrated, which was accompanied by increased osteoblastic MC3T3-E1 cell apoptosis. Addition of the ROS inhibitor NAC (N-acetyl-L-cysteine) or NOX inhibitor DPI (diphenyleneiodonium) reversed this effect, indicating that NOX-derived ROS can induce osteoblast apoptosis under high-dose dexamethasone stimulation. NOX1, NOX2, and NOX4 are NOX homologs recently identified in bone tissue. To clarify the NOX isoforms that play a role in osteoblast ROS generation, Nox1, Nox2, and Nox4 mRNA expression and NOX2 and NOX4 protein expression were analyzed. Nox1 and Nox4 mRNA expression was elevated in a dose-dependent manner after culture in 100 nM, 250 nM, 500 nM, or 1000 nM dexamethasone, and the increased expression of NOX1 mRNA was more significant compared with NOX4 mRNA. Small interfering RNAs (siRNAs) were used to confirm the role of NOX1 and NOX4 in ROS generation. To clarify the signaling pathway in ROS-induced osteoblast apoptosis, mitogen-activated protein kinase (MAPK) signaling molecules were analyzed. Phosphorylated ASK1 and p38 levels were significantly higher in the 1000 nM dexamethasone group, which NAC or DPI markedly attenuated. However, the total mRNA and protein levels of ASK1 and p38 between the dexamethasone group and control were not significantly different. This is related to ROS regulating the posttranslational modification of ASK1 and p38 in MC3T3-E1 cell apoptosis. Altogether, NOX1- and NOX4-derived ROS plays a pivotal role in high-dose dexamethasone-induced preosteoblast apoptosis by increasing phosphorylated ASK1 and p38 and may be an important mechanism in steroid-induced avascular necrosis of the femoral head (SANFH).

Effects of the (Pro)renin Receptor on Cardiac Remodeling and Function in a Rat Alcoholic Cardiomyopathy Model via the PRR-ERK1/2-NOX4 Pathway.

Alcoholic cardiomyopathy (ACM) caused by alcohol consumption manifests mainly as by maladaptive myocardial function, which eventually leads to heart failure and causes serious public health problems. The (pro)renin receptor (PRR) is an important member of the local tissue renin-angiotensin system and plays a vital role in many cardiovascular diseases. However, the mechanism responsible for the effects of PRR on ACM remains unclear. The purpose of this study was to determine the role of PRR in myocardial fibrosis and the deterioration of cardiac function in alcoholic cardiomyopathy. Wistar rats were fed a liquid diet containing 9% v/v alcohol to establish an alcoholic cardiomyopathy model. Eight weeks later, rats were injected with 1 × 109v.g./100 μl of recombinant adenovirus containing EGFP (scramble-shRNA), PRR, and PRR-shRNA via the tail vein. Cardiac function was assessed by echocardiography. Cardiac histopathology was measured by Masson's trichrome staining, immunohistochemical staining, and dihydroethidium staining. In addition, cardiac fibroblasts (CFs) were cultured to evaluate the effects of alcohol stimulation on the production of the extracellular matrix and their underlying mechanisms. Our results indicated that overexpression of PRR in rats with alcoholic cardiomyopathy exacerbates myocardial oxidative stress and myocardial fibrosis. Silencing of PRR expression with short hairpin RNA (shRNA) technology reversed the myocardial damage mediated by PRR. Additionally, PRR activated phosphorylation of ERK1/2 and increased NOX4-derived reactive oxygen species and collagen expression in CFs with alcohol stimulation. Administration of the ERK kinase inhibitor (PD98059) significantly reduced NOX4 protein expression and collagen production, which indicated that PRR increases collagen production primarily through the PRR-ERK1/2-NOX4 pathway in CFs. In conclusion, our study demonstrated that PRR induces myocardial fibrosis and deteriorates cardiac function through ROS from the PRR-ERK1/2-NOX4 pathway during ACM development.

The association of NOX4 mediated nuclear colocalization and overall survival in renal cell carcinoma.

639 Background: NOX4 protein are involved in cell differentiation, migration and apoptosis. We recently demonstrated that NOX4 in renal cell carcinoma (RCC) is an energetic sensor and couples energy metabolism to drug-resistance. In this study we demonstrate that enhanced nuclear localization of NOX4 is correlated with poor survival. Methods: We identified 350 RCC patients with (n = 85) and without (n = 265) venous tumor thrombus (VTT) who underwent radical nephrectomy with or without thrombectomy at our institution between 2013-2016. Fifty patients with RCC and VTT were matched 1:1 to patients with RCC and no VTT. Tissue Microarray (TMA) consisting of primary renal tumor, adjacent renal tumor and VTT were created. These matched TMA (n = 174) were stained with NOX4 antibodies; scanned using the Aperio scanning system and analyzed with the ImageScope software. The RCC nucleus was evaluated for roundness, elongation and size. NOX4 nuclear staining was categorized based on intensity as either strong positive, moderate, weak or no localization. Differential protein expression between the primary tumor, adjacent normal parenchyma and VTT was assessed using the Chi-squared test or t-test. Progression-free survival (PFS) and overall-survival (OS), stratified by NOX4 staining intensity, was estimated using the Kaplan-Meier method and compared with the log-rank test. Cox regression model was used to investigate effect of NOX4 staining on survival in multivariate analyses. Results: NOX4 nuclear colocalization was higher in patients who had progression and death from RCC. On univariate analysis, the intensity of NOX4 protein expression in the nucleus was significantly associated with PFS (HR 1.67, 95%CI (1.08-2.57; p = 0.02) and OS (HR 2.31, 95%CI (1.26-4.24; p = 0.007). On multivariate analyses the association between NOX4 protein expression and OS remains statistical significance (HR 2.09, 95%CI (1.05-4.18, p = 0.037). Conclusions: We provide evidence that in high grade/high stage cancer, NOX4 is colocalized within the nucleus of RCC. The prognostic role of NOX4 was determined at the protein level. We provide rationale for further investigation of NOX4, which may serve as a checkpoint in RCC.

Canagliflozin, a sodium-glucose cotransporter2 inhibitor, normalizes renal susceptibility to type1 cardiorenal syndrome through reduction of renal oxidative stress in diabetic rats.

Aims/IntroductionType 2 diabetes mellitus is a risk factor of acute kidney injury after myocardial infarction (MI), a form of cardiorenal syndrome. Recent clinical trials have shown that a sodium–glucose cotransporter 2 (SGLT2) inhibitor improved both cardiac and renal outcomes in patients with type 2 diabetes mellitus, but effects of an SGLT2 inhibitor on cardiorenal syndrome remain unclear.Materials and MethodsType 2 diabetes mellitus (Otsuka Long‐Evans Tokushima Fatty rats [OLETF]) and control (Long‐Evans Tokushima Otsuka rats [LETO]) were treated with canagliflozin, an SGLT2 inhibitor, for 2 weeks. Renal tissues were analyzed at 12 h after MI with or without preoperative fasting.ResultsCanagliflozin reduced blood glucose levels in OLETF, and blood β‐hydroxybutyrate levels were increased by canagliflozin only with fasting. MI increased neutrophil gelatinase‐associated lipocalin and kidney injury molecule‐1 protein levels in the kidney by 3.2‐ and 1.6‐fold, respectively, in OLETF, but not in LETO. The renal messenger ribonucleic acid level of Toll‐like receptor 4 was higher in OLETF than in LETO after MI, whereas messenger ribonucleic acid levels of cytokines/chemokines were not significantly different. Levels of lipid peroxides, nicotinamide adenine dinucleotide phosphate oxidase (NOX)2 and NOX4 proteins after MI were significantly higher in OLETF than in LETO. Canagliflozin with pre‐MI fasting suppressed MI‐induced renal expression of neutrophil gelatinase‐associated lipocalin and kidney injury molecule‐1 in OLETF, together with reductions in lipid peroxides and NOX proteins in the kidney. Blood β‐hydroxybutyrate levels before MI were inversely correlated with neutrophil gelatinase‐associated lipocalin protein levels in OLETF. Pre‐incubation with β‐hydroxybutyrate attenuated angiotensin II‐induced upregulation of NOX4 in NRK‐52E cells.ConclusionsThe findings suggest that SGLT2 inhibitor treatment with a fasting period protects kidneys from MI‐induced cardiorenal syndrome, possibly by β‐hydroxybutyrate‐mediated reduction of NOXs and oxidative stress, in type 2 diabetic rats.

Palmitic acid-induced autophagy increases reactive oxygen species via the Ca2+/PKCα/NOX4 pathway and impairs endothelial function in human umbilical vein endothelial cells.

It is well known that the lipotoxic mechanism of palmitic acid (PA), a main constituent of triglyceride, is dependent on reactive oxygen species (ROS). Recently, it has also been reported that PA is an autophagy inducer. However, the causal association and underlying mechanism of induced autophagy and ROS in PA toxicity remain unclear. The present study demonstrates for the first time that PA-induced autophagy enhances ROS generation via activating the calcium ion/protein kinase Cα/nicotinamide adenine dinucleotide phosphate oxidase 4 (Ca2+/PKCα/NOX4) pathway in human umbilical vein endothelial cells (HUVECs). It was revealed that PA treatment resulted in a significant increase in ROS generation and autophagic activity, leading to endothelial dysfunction as indicated by downregulated nitric oxide synthesis, decreased capillary-like structure formation and damaged cell repair capability. Furthermore, PA effectively activated the Ca2+/PKCα/NOX4 pathway, which is indicative of upregulated cytosolic Ca2+ levels, activated PKCα and increased NOX4 protein expression. 3-Methyladenine was then used to inhibit autophagy, which significantly reduced PA-induced ROS generation and blocked the Ca2+/PKCα/NOX4 pathway. The endothelial dysfunction caused by PA was ameliorated by downregulating ROS generation using a NOX4 inhibitor. In conclusion, PA-induced autophagy contributes to endothelial dysfunction by increasing oxidative stress via the Ca2+/PKCα/NOX4 pathway in HUVECs.

Research on the mechanism of high glucose affecting the apoptosis of schwann cells by Nox4 NADPH oxidase

To investigate the mechanism of high glucose affecting the apoptosis of schwann cells through Nox4 NADPH oxidase. The schwann cells of newborn Wistar rats were cultured in vitro. The cultured cells were divided into four groups: control group, high-glucose group, NOX4 siRNA group and control siRNA group (n=10). The WST-1 method was used to detect the cell vitality, and the DCFH-DA method was used to detect the contents of intracellular reactive oxygen free radicals (ROS). Nox4 and Caspase3 mRNA expressions were detected by real-time fluorescence quantitative RT-PCR. Nox4 and Caspase3 protein expressions were determined by Western blot. High glucose culture up-regulated Nox4 mRNA and protein expressions of schwann cells, decreased activity of schwann cells, increased intracellular ROS content, and promoted apoptosis by increasing Caspase3 mRNA and protein expressions. NOX4 siRNA blocked the accumulation of ROS in the high glucose cultured schwann cells, and reduced the damage of glucose on cell viability, by inhibiting NOX4 gene expression. NOX4 siRNA also reduced cell apoptosis by down-regulating Caspase3 mRNA and protein expressions. Nox4 was involved in the hyperglycemic-induced apoptosis of schwann cells through ROS. The regulation of Nox4 expression or function might be a new way to treat diabetic peripheral neuropathy.

Mechanism of inhibitory effect of catalpol on TNF-α induced HAECs cell damage

Catalpol is an iridoid glycoside extracted from the root of Rehmannia glutinosa. It has been reported to have antioxidant stress effects. Adenosine 5' monophosphate-activated protein kinase( AMPK) plays an important role in inhibiting oxidative stress. This study was designed to investigate the protective effects of catalpol on TNF-α-exposed human aorta epithelial cells( HAECs) via inhibit oxidative stress,and the relationship between catalpol and AMPK was detected by RNA interference technique. Levels of superoxide dismutase( SOD),malonaldehyde( MDA),glutathione( GSH) and lactate dehydrogenase( LDH) were measured with a colorimetric assay kit. The level of ROS was measured with FACS calibur. Western blot was employed to detect the protein expression of AMPK,phosphorylated-AMPK and NOX4. Finally,RNA interference technique was used to investigate the role of AMPK in catalpol-induced protective effects. TNF-α treatment decreased the expression of phosphorylated-AMPK protein level,however,catalpol could reverse the decreased phosphorylated-AMPK level. Catalpol could inhibit NOX4 protein expression and decrease ROS overproduction. After using AMPK siRNA that effects of catalpol on ROS overproduction and NOX4 protein expression inhibition were attenuated. The above results suggest that catalpol inhibits oxidative stress in TNF-α-exposed HAECs by activating AMPK.

Prenatal hypoxia affected endothelium-dependent vasodilation in mesenteric arteries of aged offspring via increased oxidative stress

Prenatal hypoxia can affect vascular functions in young offspring. However, there is limited knowledge regarding whether and how prenatal hypoxia influences vascular functions in aged offspring. This study compared the effects of prenatal hypoxia on the mesenteric arteries (MA) between a young adult and aged offspring and investigated the underlying mechanisms. Pregnant rats were randomly divided into the control and prenatal hypoxia groups. The vascular functions and molecular levels were assessed in 5-month-old (5 M) or 20-month-old (20 M) offspring. Prenatal hypoxia decreased acetylcholine-mediated vascular relaxations in 20-M but not 5-M offspring. Sodium nitroprusside-mediated relaxation curves were not altered by prenatal hypoxia in 5- and 20-M offspring. Prenatal hypoxia enhanced the contractile responses caused by phenylephrine, phorbol 12,13-dibutyrate, and 5-hydroxytryptamine only in 5-M offspring. The endothelial NO synthase (eNOS) activities were decreased along with downregulated eNOS mRNA expression and phosphorylated eNOS/total eNOS protein expression in 20-M offspring with prenatal hypoxia. The NADPH oxidase (NOX) inhibitor apocynin and superoxide dismutase (SOD) mimetic tempol restored the acetylcholine-mediated weaker relaxations in 20-M offspring with prenatal hypoxia. Enzyme-linked immunosorbent and dihydroethidium assay showed that prenatal hypoxia enhanced oxidative stress in 20-M offspring. Transmission electron microscopy showed that prenatal hypoxia damaged mitochondrial structures in the MA endothelial cells of 20-M offspring. Increased NOX2 protein expression and decreased SOD3 expression were found in 20-M offspring. The results demonstrated that endothelial dysfunction induced by intrauterine hypoxia occurred with aging via enhanced oxidative stress and decreased nitric oxide activities in aged offspring.

Sevoflurane Effect on Cognitive Function and the Expression of Oxidative Stress Response Proteins in Elderly Patients undergoing Radical Surgery for Lung Cancer

To investigate the effects of sustained inhalation of sevoflurane on cognitive function and the expression of oxidative stress response proteins such as NADPH oxidase subunits NOX2 and NOX4 in elderly patients undergoing radical surgery for lung cancer. An experimental study. Department of Anesthesiology, Suzhou Kowloon Hospital, Shanghai Jiao Tong University School of Medicine, China, from February 2016 to October 2017. Elderly patients who underwent radical surgery for lung cancer were divided into the sevoflurane group and the propofol group, with 52 cases in each group. Sustained inhalation of sevoflurane and propofol was administered to maintain anesthesia in the respective groups. Cognitive function and lung function parameters were compared between the two groups. Serum S100 &beta; levels and expression of NOX2 and NOX4 proteins in peripheral blood mononuclear cells of the two groups were determined. At 24 hours after surgery, the lung function indices of the sevoflurane group such as FEV1, FVC and VC were higher than those of the propofol group (p<0.001, p=0.008 and p=0.002, respectively). At the end of the surgery and at 24 hours after surgery, the MMSE scores of the sevoflurane group were higher than the propofol group (all p<0.001). S100 levels were lower than the propofol group (p=0.003 and p<0.001, respectively). Levels of NADPH oxidase subunits NOX2 and NOX4 proteins in peripheral blood mononuclear cells of the sevoflurane group were lower than the propofol group (p=0.033, p<0.001, p<0.001and p<0.001, respectively). Compared with intravenous anesthesia with propofol, general anesthesia with sevoflurane inhalation has little effect on the short-term cognitive function in elderly patients undergoing radical surgery for lung cancer, and can effectively improve lung function. The mechanism may be related to the reduction of the expression of NOX2 and NOX4 proteins.

Protein-Protein Interaction Assay to Analyze NOX4/p22phox Heterodimerization.

The stabilization and activation of NOX4 through its binding with p22phox are well documented; however little is known of the precise manner by which these two proteins interact. In recent years, the field of proteomics has undergone tremendous development with the introduction of many novel methods for the identification and characterization of protein-protein interactions (PPIs). To enhance our understanding of structural determinants leading to the association between NOX4 and p22phox, we developed a binary luciferase reporter assay (NanoBiT®) to quantitatively assess NOX4-p22phox heterodimerization. The complementation reporter quantitatively determines the accurate, reduced, or failed complex assembly, which can be confirmed and further interrogated by analyzing NOX4 catalytic activity (H2O2 release), protein expression, and dimer localization. This association-based PPI technique represents both a much-needed expansion of the NOX4 lead discovery tool box and a versatile method to probe the architecture of NOX and DUOX complexes in the future.

Intersecting Stories of the Phagocyte NADPH Oxidase and Chronic Granulomatous Disease.

Neutrophils serve as the circulating cells that respond early and figure prominently in human host defense to infection and in inflammation in other settings. Optimal oxidant-dependent antimicrobial activity by neutrophils relies on the ability of stimulated phagocytes to utilize a multicomponent NADPH oxidase to generate oxidants. The frequent, severe, and often fatal infections experienced by individuals with chronic granulomatous disease (CGD), an inherited disorder in which one of the NADPH oxidase components is absent or dysfunctional, underscore the link between a functional phagocyte NADPH oxidase and robust host protection against microbial infection.The history of the discovery and characterization of the normal neutrophil NADPH oxidase and the saga of recognizing CGD and its underlying causes together illustrate how the observations of astute clinicians and imaginative basic scientists synergize to forge new understanding of both basic cell biology and pathogenesis of human disease.In this chapter, we review the events in the stepwise evolution of our understanding of the phagocyte NADPH oxidase, both in the context of normal human neutrophil function and in the setting of CGD. The phagocyte oxidase complex employs a heterodimeric transmembrane protein composed of gp91phox and p22phox to relay electrons from NADPH to molecular oxygen, while other cofactors contribute to localization and regulation of the activity of the assembled oxidase. The b-type cytochrome gp91phox, also known as NOX2, serves as the catalytic component of this multicomponent enzyme complex. Although many of the features of the composition and regulation of the phagocyte oxidase may apply as well to NOX2 expressed in non-phagocytes and to other members of the NOX protein family, exceptions exist and pose special challenges to investigators exploring the biology of NADPH oxidases.

Spectroscopy of NOX Protein Family Members.

All members of the NOX protein family contain a unique b-type cytochrome that mediates the electron transport that characterizes the activity of the multicomponent oxidase complexes. Referred to as cytochrome b558, because of its signature spectral absorbance at 558nm in reduced-minus-oxidized difference spectroscopy, or cytochrome b(-245), because of its very low midpoint potential of -245mV at pH7.0, the protein possesses two stacked inequivalent hemes ligated by pairs of histidine residues in membrane helices h3 and h5. In a flavin-dependent fashion, cytochrome b558 shuttles electrons from cytoplasmic NADPH across membranes to molecular oxygen and thereby generates superoxide anion. By performing reduced-minus-oxidized difference spectroscopy and using the millimolar extinction coefficient, E 559-540nm=21.6cm-1mM-1, one can calculate the amount of cytochrome b558 in intact cells or partially purified membrane preparations. Measurements in samples where cytochrome b558 is relatively high and the presence of unrelated heme-containing proteins low, as in neutrophils, are straightforward. However, low levels of cytochrome b558 expression combined with an abundance of mitochondria and other sources of heme proteins make spectral detection of cytochrome b558 in non-phagocytic cells extremely challenging.

The anti-inflammatory and anti-oxidative effects of conbercept in treatment of macular edema secondary to retinal vein occlusion

To investigate the effects of conbercept on inflammatory and oxidative response in macular edema secondary to retinal vein occlusion (RVO-ME). Retinal microvasculature were detected by optical coherence tomographic angiography (OCTA). The inflammation related factors including prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), prostaglandin F2a (PGF2a), intercellular cell adhesion molecule-1 (ICAM-1) and macrophage inflammatory protein-1 (MIP-1) were determined in human and mice with RVO-ME. OCTA images showed that capillary non-perfusion, enlargement of the foveal avascular zone, telangiectatic vessels and some forms of intraretinal edema in RVO-ME and all these were alleviated by conbercept treatment. PGE1, PGE2, PGF2a, ICAM-1 and MIP-1 in aqueous fluid extracted from RVO-ME patients was significantly increased compared with non-RVO subjects, intravitreal injection of conbercept partly reduced ICAM-1 and MIP-1 levels but not PGE1, PGE2 and PGF2a. The glutathione level was reduced in aqueous fluid extracted from RVO-ME patients but was restored after conbercept treatment. The inflammation, angiogenesis and ROS generation was increased in RVO-ME mice, conbercept partly inhibited these effects. Mechanistically, conbercept inhibited vascular endothelial growth factor (VEGF), ICAM-1, MIP-1, NOX-1 and NOX-4 protein expressions, but not PGE1, PGE2 and PGF2a expressions. Conbercept alleviates RVO-ME through inhibiting inflammation, angiogenesis and oxidative responses. These findings further reveals the molecular mechanism of conbercept for treatment of RVO-ME.

Estradiol ameliorates antioxidant axis SIRT1-FoxO3a-MnSOD/catalase in the heart of fructose-fed ovariectomized rats

Harmful effects of fructose-rich diet (FRD) were predominantly observed in males, suggesting protective effects of estrogens. Little is known about AMPK/sirtuin-1 (SIRT1)/forkhead box O3 (FoxO3a)/manganese superoxide dismutase (MnSOD)/catalase signaling in the heart in state of metabolic syndrome and oxidative stress induced by fructose over-consumption. We investigated the effect of 10% FRD on expression of AMPK-SIRT1-FoxO3a-MnSOD/catalase axis in myocardium and potentially beneficial effect of 17β-estradiol replacement. The expression of NADPH oxidase 4 (Nox4) and miRNA-155, unfavorable regulators of this axis, were also investigated. FRD significantly increased AMPK and decreased FoxO3a activity, decreased SIRT1, MnSOD and Nox4 protein expression while E2 reverted these changes, except for Nox4, and increased catalase protein level. E2 diminished Nox4 and MnSOD mRNA level in FRD ovariectomized rats. These results suggest independent response of AMPK and SIRT to FRD treatment. The proposed signaling in the heart should be further investigated in the prooxidative and antioxidative milieu.

5-Hydroxytryptamine (5-HT)-exacerbated DSS-induced colitis is associated with elevated NADPH oxidase expression in the colon.

This study investigated the impact of 5-hydroxytryptamine (5-HT) on the expression of NOXs in dextran sulfate sodium (DSS)-induced colitis in mice. C57BL/6J (B6) mice at 6 to 8 weeks of age were treated with 5-HT, DSS, or DSS + 5-HT. After 6-day treatment, the severity of colitis, infiltration of leukocytes, and messenger RNA (mRNA) and/or protein levels of Nox1, Nox2, Nox4, and Duox2 were analyzed in the colon by real-time quantitative polymerase chain reaction (qPCR), immunohistochemistry (IHC), and Western blot analysis. The direct effect of 5-HT on NOX gene and protein expression in HT-29 colon cancer cells and in U-937 macrophage cells were determined by qPCR and Western blot analysis. Mice treated with 5-HT alone did not develop colitis, while those treated with 1.0% DSS or DSS + 5-HT had mild and severe colitis, respectively. All treated mice had more myeloperoxidase-positive cells in the colon compared with untreated control mice. Mice treated with 5-HT or DSS alone had increased Nox2 and Nox4 mRNA and protein levels in the colon determined by qPCR, IHC, and Western blot analysis. These two Nox expressions were even higher in mice treated with DSS + 5-HT, while the expression levels of epithelium-localized Nox1 and Duox2 tended to decrease. Additionally, mice treated with 5-HT alone had elevated Nox1 and Duox2 expression as shown by qPCR and IHC. However, these gene expressions were diminished in DSS + 5-HT-treated mice likely due to erosion of epithelium. Furthermore, 5-HT induced NOX1 and DUOX2 gene and protein expression in HT-29 colon cancer epithelial cells, whereas induced NOX2 and NOX4 gene and protein expression in U-937 cells. As 5-HT induced NOX1 and DUOX2 gene and protein expression in colon epithelial and HT-29 cells, NOX2 and NOX4 in the infiltrating leukocyte in mouse colon and in U-937 cells, the exacerbate colitis induced by combined 5-HT and DSS treatment might be relevant to increased NOX expression in mice colons.