NADPH Oxidase Protein Expression Research Articles

Mitochondrial reactive oxygen species impact human fibroblast responses to protracted γ-ray exposures

Purpose: Continuous exposure to ionizing radiation at a low dose rate poses significant health risks to humans on deep space missions, prompting the need for mechanistic studies to identify countermeasures against its deleterious effects. Mitochondria are a major subcellular locus of radiogenic injury, and may trigger secondary cellular responses through the production of reactive oxygen species (mtROS) with broader biological implications. Methods and Materials: To determine the contribution of mtROS to radiation-induced cellular responses, we investigated the impacts of protracted γ-ray exposures (IR; 1.1 Gy delivered at 0.16 mGy/min continuously over 5 days) on mitochondrial function, gene expression, and the protein secretome of human HCA2-hTERT fibroblasts in the presence and absence of a mitochondria-specific antioxidant mitoTEMPO (MT; 5 µM). Results: IR increased fibroblast mitochondrial oxygen consumption (JO2) and H2O2 release rates (JH2O2) under energized conditions, which corresponded to higher protein expression of NADPH Oxidase (NOX) 1, NOX4, and nuclear DNA-encoded subunits of respiratory chain Complexes I and III, but depleted mtDNA transcripts encoding subunits of the same complexes. This was associated with activation of gene programs related to DNA repair, oxidative stress, and protein ubiquination, all of which were attenuated by MT treatment along with radiation-induced increases in JO2 and JH2O2. IR also increased secreted levels of interleukin-8 and Type I collagens, while decreasing Type VI collagens and enzymes that coordinate assembly and remodeling of the extracellular matrix. MT treatment attenuated many of these effects while augmenting others, revealing complex effects of mtROS in fibroblast responses to IR. Conclusion: These results implicate mtROS production in fibroblast responses to protracted radiation exposure, and suggest potentially protective effects of mitochondrial-targeted antioxidants against radiogenic tissue injury in vivo.

Aliskiren-Loaded Nanoparticles Downregulate (Pro)renin Receptor and ACE Gene Expression in the Heart of Spontaneously Hypertensive Rats: Effect on NADPH Oxidase.

We aimed to determine effects of aliskiren, a direct renin inhibitor, loaded onto polymeric nanoparticles on the (pro)renin receptor (Atp6ap2), angiotensin II type 1 receptor (Agtr1), and angiotensin-converting enzyme (ACE) gene expression in the heart of spontaneously hypertensive rats (SHR). Twelve-week-old male SHRs were divided into an untreated group and groups treated with powdered aliskiren or aliskiren-loaded nanoparticles (25 mg/kg/day). After three weeks, the accumulation of aliskiren, distribution of polymeric nanoparticles, gene expression of Atp6ap2 and Agtr1 receptors and ACE, and protein expression of NADPH oxidase along with the conjugated diene (CD) concentration were analyzed. The accumulation of aliskiren in the heart was higher in the aliskiren-loaded nanoparticle group than in the powdered group. The fluorescent signals of nanoparticles were visible in cardiomyocytes, vessel walls, and erythrocytes. Aliskiren-loaded nanoparticles decreased the gene expression of Atp6ap2 and ACE, while not affecting Agtr1. Both forms of aliskiren decreased the protein expression of NADPH oxidase, with a more pronounced effect observed in the aliskiren-loaded nanoparticle group. CD concentration was decreased only in the aliskiren-loaded nanoparticle group. We hypothesize that aliskiren-loaded nanoparticle-mediated downregulation of Atp6ap2 and ACE may contribute to a decrease in ROS generation with beneficial effects in the heart. Moreover, polymeric nanoparticles may represent a promising tool for targeted delivery of aliskiren.

The renoprotective effects of taurine against diabetic nephropathy via the p38 MAPK and TGF-β/Smad2/3 signaling pathways.

Diabetic nephropathy (DN), a severe diabetes complication, causes kidney morphological and structural changes due to extracellular matrix accumulation. This accumulation is caused mainly by oxidative stress. Semi-essential amino acid derivative taurine has powerful antioxidant and antifibrotic effects. The aim of this study was to investigate the renoprotective effects of taurine through its possible roles in oxidative stress, extracellular matrix proteins, and the signaling pathways associated with the accumulation of extracellular matrix proteins in DN rats. 29 Wistar albino rats were randomly separated into control, taurine, diabetes, and diabetes + taurine groups. Diabetes animals were injected 45mg/kg streptozosine. Taurine is given by adding to drinking water as 1% (w/v). Urine, serum, and kidney tissue were collected from rats for biochemical and histological analysis after 12weeks. According to the studies, taurine significantly reduces the levels of malondialdehyde (MDA), total oxidant status (TOS), and protein expression of NADPH oxidase 4 (NOX4) that increase in diabetic kidney tissue. Also, decreased superoxide dismutase (SOD) activity levels significantly increased with taurine in diabetic rats. Moreover, increased mRNA and protein levels of fibronectin decreased with taurine. The matrix metalloproteinase (MMP)-2 and MMP-9 activities and their mRNA levels increased significantly, and this increase was significantly summed with taurine. There was a decrease in mRNA expression of Extracellular matrix metalloproteinase inducer (EMMPRIN). Taurine significantly increased this decrease. Diabetes increased mRNA expressions of transforming growth factor (TGF)-β and Smad2/3. Taurine significantly reduced this induction. TGF-β protein expression, p38, and Smad2/3 activations were also inhibited, but taurine was suppressed significantly. All these findings indicate that taurine may be an effective practical strategy to prevent renal diabetic injury.

Acid sphingomyelinase promotes diabetic cardiomyopathy via NADPH oxidase 4 mediated apoptosis

BackgroundIncreased acid sphingomyelinase (ASMase) activity is associated with insulin resistance and cardiac dysfunction. However, the effects of ASMase on diabetic cardiomyopathy (DCM) and the molecular mechanism(s) underlying remain to be elucidated. We here investigated whether ASMase caused DCM through NADPH oxidase 4-mediated apoptosis.Methods and resultsWe used pharmacological and genetic approaches coupled with study of murine and cell line samples to reveal the mechanisms initiated by ASMase in diabetic hearts. The protein expression and activity of ASMase were upregulated, meanwhile ceramide accumulation was increased in the myocardium of HFD mice. Inhibition of ASMase with imipramine (20 mg Kg−1 d−1) or siRNA reduced cardiomyocyte apoptosis, fibrosis, and mitigated cardiac hypertrophy and cardiac dysfunction in HFD mice. The similar effects were observed in cardiomyocytes treated with high glucose (HG, 30 mmol L−1) + palmitic acid (PA, 100 μmol L−1) or C16 ceramide (CER, 20 μmol L−1). Interestingly, the cardioprotective effect of ASMase inhibition was not accompanied by reduced ceramide accumulation, indicating a ceramide-independent manner. The mechanism may involve activated NADPH oxidase 4 (NOX4), increased ROS generation and triggered apoptosis. Suppression of NOX4 with apocynin prevented HG + PA and CER incubation induced Nppb and Myh7 pro-hypertrophic gene expression, ROS production and apoptosis in H9c2 cells. Furthermore, cardiomyocyte-specific ASMase knockout (ASMaseMyh6KO) restored HFD-induced cardiac dysfunction, remodeling, and apoptosis, whereas NOX4 protein expression was downregulated.ConclusionsThese results demonstrated that HFD-mediated activation of cardiomyocyte ASMase could increase NOX4 expression, which may stimulate oxidative stress, apoptosis, and then cause metabolic cardiomyopathy.

UV-B- triggered H2O2 production mediates isoflavones synthesis in germinated soybean.

In this study, the functions of Hydrogen peroxide (H2O2) on the synthesis of isoflavones in germinated soybean under UV-B radiation were investigated. Results showed that the activity, gene, and protein expression of NADPH oxidase were up-regulated by 1.46, 6.92, and 1.34 times with UV-B radiation, while endogenous H2O2 content was also significantly increased. UV-B radiation and exogenous H2O2 treatment significantly increased the activities, gene and protein expression of phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), and isoflavone synthase (IFS) involved in isoflavones synthesis, and there was a synergistic effect with combining treatment. However, these up-regulation effects were suppressed by the supplementary diphenylene iodonium (DPI), which is the inhibitor of NADPH oxidase. Interestingly, the inhibition effect was largely reversed by exogenous H2O2, indicating that H2O2 was indispensable in regulating the isoflavones synthesis in germinated soybeans under UV-B radiation. Overall, H2O2 is an essential signaling molecule, mediating UV-B-induced isoflavone accumulation.

Association Between NOX4 And Nrf2 Genes in Non-Small-Cell Lung Carcinoma: A Case-Control Study.

Epithelial malignancy in lung cancer, which is initiated with myofibroblast differentiation and remodeling, promotes hypoxia and intracellular ROS generation most affected by the prototypical enzyme, NADPH oxidase 4 (NOX4). In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) acts as a critical transcription factor by stimulating antioxidant proteins as redox homeostasis regulators. The aim of this study was to investigate a possible correlation between lung tissue NOX4 and Nrf2 genes (NOX4 and Nrf2) mRNA expression and bronchoalveolar lavage fluid (BALF) protein expression in non-small-cell lung carcinoma (NSCLC) patients. Samples from 25 patients with various NSCLC types and stages and 20 healthy controls were collected. NOX4 and Nrf2 mRNA were measured by qRT-PCR, and protein by western blot analysis. NOX4 mRNA and protein expression was significantly up-regulated in NSCLC patients' lung tissues and BALFs (p= 0.03 and 0.01, respectively). In addition, by adjusting for age, sex, and NSCLC types and stages, a significant and positive correlation was observed between NOX4 and Nrf2 mRNA expression (r= 0.927, p= 0.001). This was also true when not adjusted as above (r= 0.944, p< 0.001). NOX4 mRNA and protein expression is significantly up-regulated in NSCLC patients' lung tissues and BALFs, and NOX4 and Nrf2 mRNA expression is positively correlated in NSCLC tissues.

Increased Blood Pressure Causes Lymphatic Endothelial Dysfunction via Oxidative Stress in Spontaneously Hypertensive Rats.

The lymphatic system is involved in the pathogenesis of edema, inflammation, and cancer metastasis. Because lymph vessels control fluid electrolytes and volume balance, changes in lymphatic activity can be expected to alter systemic blood pressure. This study examined possible changes in lymphatic contractile properties in spontaneously hypertensive rats (SHR). Thoracic ducts isolated from 10- to 12-week-old SHR exhibited either decreased acetylcholine-induced endothelium-dependent relaxation or sodium nitroprusside-induced endothelium-independent relaxation compared with age-matched Wister-Kyoto rats. The impairment in acetylcholine responsiveness was more pronounced than sodium nitroprusside responsiveness. N-Nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor blunted acetylcholine-induced relaxation in Wister-Kyoto rats, indicating an involvement of endothelial nitric oxide production. Endothelial dysfunction in lymph vessels of SHR was attenuated by tempol (a superoxide dismutase mimetic), apocynin, or VAS-2870 (NADPH oxidase inhibitors). Consistent with these observations, nitrotyrosine levels were significantly elevated in SHR, indicative of increased oxidative stress. In addition, protein expression of NADPH oxidase 2 and phosphorylation of p47phox (Ser345) were significantly increased in SHR. Further, SB203580 (a p38 MAPK inhibitor) restored the acetylcholine-induced relaxation in SHR. It is notable that 4-week-old SHR, which exhibited normal blood pressure, did not show any decreased activity of acetylcholine- or sodium nitroprusside-induced relaxation. Additionally, antihypertensive treatment of 4-week-old SHR with hydrochlorothiazide and reserpine or hydrochlorothiazide and hydralazine for 6 weeks completely restored lymphatic endothelial dysfunction. We conclude that contractile activity of lymphatic vessels is functionally impaired with the development of increasing blood pressure, which is mediated through increased oxidative stress via the p38 MAPK/NADPH oxidase 2 pathway.

Sevoflurane anesthesia-mediated oxidative stress and cognitive impairment in hippocampal neurons of old rats can be ameliorated by expression of brain derived neurotrophic factor

Postoperative cognitive dysfunction in elderly patients has been related to neurodegenerative disorders and mortality. Sevoflurane anesthesia has been implicated in both postoperative cognitive dysfunction and neurotoxicity. Given the advantages of using inhaled anesthetics like sevoflurane, it is important to understand how their usage results in neurotoxicity and subsequently devise ways to circumvent or attenuate the anesthetic-mediated induction in neurotoxicity. We have used an aged rat model to investigate the molecular mechanisms by which sevoflurane inhalation results in neurotoxicity and whether modulation of these molecular mechanisms can inhibit or attenuate neurotoxicity and cognitive learning and memory impairment in these animals. Low- or high-dose of sevoflurane resulted in reactive oxygen species generation, increased NADPH oxidase protein expression, apoptosis and autophagy. Sevoflurane inhalation resulted in significant inhibition of brain derived neurotrophic factor (BDNF) and cognitive impairment. And the activation of PI3K/Akt/mTOR signaling pathways are attenuated in sevoflurane-mediated anesthesia. Adeno-associated virus (AAV)-mediated expression of Bdnf, but not controls EGFP, attenuated sevoflurane-induced oxidative stress and cognitive impairment in the rats. Our results highlight that AAV-mediated gene therapy might offer a potential therapeutic opportunity to treat post-operative cognitive impairment resulting from inhaled anesthetics.

Plumbagin attenuated oxygen-glucose deprivation/reoxygenation-induced injury in human SH-SY5Y cells by inhibiting NOX4-derived ROS-activated NLRP3 inflammasome.

Plumbagin (PLB), an alkaloid obtained from the roots of the plants of Plumbago genus, is an inhibitor of NADPH oxidase 4 (NOX4). This study aimed to investigate the beneficial effect of PLB against oxygen-glucose deprivation/reoxygenation (OGDR)-induced neuroinjury in human SH-SY5Y neuronal cultures. Our results showed that OGD/R stimulated NOX4 protein expression and reactive oxygen species (ROS) production in SH-SY5Y cells, whereas increased 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) production, resulting in the activation of the NLRP3 inflammasome. And PLB pretreatment reduced the ROS production by regulating the expression of NOX4 and downregulated NF-κB signaling which was induced by OGDR. Furthermore, PLB inhibited OGDR induced NLRP3 inflammasome activation but not PARP1. Overall, PLB improved OGDR induced neuroinjury by inhibiting NOX4-derived ROS-activated NLRP3 inflammasome.

Inhibition of microRNA-155 Alleviates Neurological Dysfunction Following Transient Global Ischemia and Contribution of Neuroinflammation and Oxidative Stress in the Hippocampus.

Central pro-inflammatory cytokine (PIC) signal is involved in neurological deficits after transient global ischemia induced by cardiac arrest (CA). The present study was to examine the role of microRNA- 155 (miR-155) in regulating IL-1β, IL-6 and TNF-α in the hippocampus of rats with induction of CA. We further examined the levels of products of oxidative stress 8-isoprostaglandin F2α (8-iso PGF2α, indication of oxidative stress); and 8-hydroxy-2'-deoxyguanosine (8-OHdG, indication of protein oxidation) after cerebral inhibition of miR-155. CA was induced by asphyxia and followed by cardiopulmonary resuscitation in rats. ELISA and western blot analysis were used to determine the levels of PICs and products of oxidative stress; and the protein expression of NADPH oxidase (NOXs) in the hippocampus. In addition, neurological severity score and brain edema were examined to assess neurological functions. We observed amplification of IL-1β, IL-6 and TNF-α along with 8-iso PGF2α and 8-OHdG in the hippocampus of CA rats. Cerebral administration of miR-155 inhibitor diminished upregulation of PICs in the hippocampus. This also attenuated products of oxidative stress and upregulation of NOX4. Notably, inhibition of miR-155 improved neurological severity score and brain edema and this was linked to signal pathways of PIC and oxidative stress. We showed the significant role of blocking miR-155 signal in improving the neurological function in CA rats likely via inhibition of signal pathways of neuroinflammation and oxidative stress, suggesting that miR-155 may be a target in preventing and/or alleviating development of the impaired neurological functions during CA-evoked global cerebral ischemia.

NADPH Oxidase 2-Mediated Insult in the Auditory Cortex of Zucker Diabetic Fatty Rats.

Clinical data has confirmed that auditory impairment may be a secondary symptom of type 2 diabetes mellitus (T2DM). However, mechanisms underlying pathologic changes that occur in the auditory system, especially in the central auditory system (CAS), remain poorly understood. In this study, Zucker diabetic fatty (ZDF) rats were used as a T2DM rat model to observe ultrastructural alterations in the auditory cortex and investigate possible mechanisms underlying CAS damage in T2DM. The auditory brainstem response (ABR) of ZDF rats was found to be markedly elevated in low (8 kHz) and high (32 kHz) frequencies. Protein expression of NADPH oxidase 2 (NOX2) and its matching subunits P22phox, P47phox, and P67phox was increased in the auditory cortex of ZDF rats. Expression of 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of DNA oxidative damage, was also increased in the neuronal mitochondria of the auditory cortex of ZDF rats. Additionally, decreases in the mitochondrial total antioxidant capabilities (T-AOC), adenosine triphosphate (ATP) production, and mitochondrial membrane potential (MMP) were detected in the auditory cortex of ZDF rats, suggesting mitochondrial dysfunction. Transmission electron microscopy results indicated that ultrastructural damage had occurred to neurovascular units and mitochondria in the auditory cortex of ZDF rats. Furthermore, cytochrome c (Cyt c) translocation from mitochondria to cytoplasm and caspase 3-dependent apoptosis were also detected in the auditory cortex of ZDF rats. Consequently, the study demonstrated that T2DM may cause morphological damage to the CAS and that NOX2-associated mitochondrial oxidative damage and apoptosis may be partly responsible for this insult.

Hydroxysafflor yellow A attenuates high glucose-induced human umbilical vein endothelial cell dysfunction.

High glucose (HG) induces vascular injury in diabetes. Hydroxysafflor yellow A (HSYA) has been used to ameliorate ischemic cardiovascular diseases in China for many years. In the present study, we assessed whether HSYA has a potential protective role in HG-induced human umbilical vein endothelial cell (HUVEC) injury. Cell viability was determined with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Cell apoptosis was detected by fluorescein isothiocyanate/propidium iodide staining assay. The endothelial cell permeability was measured with a permeability assay. Cell adhesion molecule (CAM) expression, vascular endothelial growth factor, and basic fibroblast growth factor levels were detected with an enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) formation was measured with a DCF-DA assay. Protein expression of NADPH oxidase 4 (NOX4) was measured by Western blotting. Our data indicated that HG increases HUVEC apoptosis, vascular permeability, monocyte adhesion, the level of CAMs, the formation of ROS, and NOX4 expression. Our data revealed that HG increases vascular injury, which is attenuated by HSYA. Because vascular inflammation has a key role in the development of diabetes mellitus, our results implied that HSYA is considered as a potential agent for diabetic vascular injury treatment.

Downregulated NOX4 underlies a novel inhibitory role of microRNA-137 in prostate cancer.

Prostate cancer is the second highest caused by cancer-related death among males. microRNAs (miRs) have been reported to participate in carcinogenesis, yet their roles in prostate cancer are rarely studied or investigated. Therefore, the present study attempted to explore the effect of miR-137 in prostate cancer via regulating NADPH oxidase 4 (NOX4). Initially, microarray analysis was performed to obtain prostate cancer-related differentially expressed genes and miRs that regulated NOX4, followed by detecting the expression of miR-137 and NOX4 and its target relationship. Moreover, PC-3 cells were transfected with small interfering RNA (siNOX4) and miR-137 mimic for exploring the effect of miR-137 on glycolysis, cell proliferation, and apoptosis in prostate cancer by evaluating lactate production, glucose uptake, adenosine triphosphate (ATP) production, viability rate, and expression of cleaved caspases 3, 8, and 9, cytochrome c, cleaved poly ADP ribose polymerase (PARP), Bax, and Bcl-2. miR-137 was vital to prostate cancer progression via regulating NOX4. Besides, miR-137 expressed poorly while NOX4 expressed highly in prostate cancer. NOX4 was the target gene of miR-137. Additionally, overexpression of miR-137 and silencing of NOX4 were observed to decrease NOX4 and Bcl-2 protein expression, but increase cleaved caspases 3, 8, and 9, cytochrome c, cleaved-PARP, and Bax protein expression. Furthermore, miR-137 overexpression and NOX4 silencing contributed to decreased lactate production, glucose uptake, ATP production, and cell proliferation, but increased apoptosis rate. Collectively, the present study showed that miR-137 repressed glycolysis in prostate cancer through knockdown of NOX4, which might be a potential theoretical target for prostate cancer treatment.

Modulation of NOX4 and MAPK Signaling Pathways by Parkia speciosa Empty Pods in H9c2 Cardiomyocytes Exposed to H2O2

Parkia speciosa, a plant that grows abundantly in Southeast Asia region, has been reported to exhibit antioxidant property. This study aimed to investigate the mechanism of antioxidant effects of empty pod extract of Parkia speciosa on hydrogen peroxide-induced oxidative stress in H9c2 cardiomyocytes. There were 4 groups of cardiomyocytes, the negative control, hydrogen peroxide, extract of Parkia speciosa+hydrogen peroxide and quercetin+hydrogen peroxide. Cardiomyocytes of groups 3 and 4 were preincubated for 1 h with 500 µg/ml of the ethyl acetate fraction of the Parkia speciosa extract or 1000 µM quercetin prior to exposure to 500 μM hydrogen peroxide for 1 h. Hydrogen peroxide significantly increased the protein expression of NADPH oxidase 4, superoxide dismutase 1 and p38 mitogen-activated protein kinase, in comparison to the negative control. NADPH oxidase 4 and superoxide dismutase 1 activities as well as reactive oxygen species level were also elevated. These elevations were significantly decreased in the pretreated groups. The Parkia speciosa extract group had comparable effects to that of quercetin. Parkia speciosa extract showed antioxidant property against hydrogen peroxide-induced oxidative stress in H9c2 cardiomyocytes by regulating mitogen-activated protein kinase and NADPH oxidase 4 signaling pathways.

NADPH Oxidase 4 Regulates Inflammation in Ischemic Heart Failure: Role of Soluble Epoxide Hydrolase.

Aims: Oxidative stress is implicated in cardiomyocyte cell death and cardiac remodeling in the failing heart. The role of NADPH oxidase 4 (NOX4) in cardiac adaptation to pressure overload is controversial, but its function in myocardial ischemic stress has not been thoroughly elucidated. This study examined the function of NOX4 in the pathogenesis of ischemic heart failure, utilizing mouse models, cell culture, and human heart samples. Results:Nox4-/- mice showed a protective phenotype in response to permanent left anterior descending coronary artery ligation with smaller infarction area, lower cardiomyocyte cross-sectional area, higher capillary density, and less cell death versus wild-type (WT) mice. Nox4-/- mice had lower activity of soluble epoxide hydrolase (sEH), a potent regulator of inflammation. Nox4-/- mice also showed a 50% reduction in the number of infiltrating CD68+ macrophages in the peri-infarct zone versus WT mice. Adenoviral overexpression of NOX4 in cardiomyoblast cells increased sEH expression and activity and CCL4 and CCL5 levels; inhibition of sEH activity in NOX4 overexpressing cells attenuated the cytokine levels. Human hearts with ischemic cardiomyopathy showed adverse cardiac remodeling, increased NOX4 and sEH protein expression and CCL4 and CCL5 levels compared with control nonfailing hearts. Innovation and Conclusion: These data from the Nox4-/- mouse model and human heart tissues show for the first time that oxidative stress from increased NOX4 expression has a functional role in ischemic heart failure. One mechanism by which NOX4 contributes to ischemic heart failure is by increasing inflammatory cytokine production via enhanced sEH activity.

EGFR mutation decreases FDG uptake in non‑small cell lung cancer via the NOX4/ROS/GLUT1 axis.

[18F]fluoro‑2‑deoxyglucose (FDG) positron emission tomography (PET)‑computed tomography(CT) is a functional imaging modality based on glucose metabolism. The association between the maximum standardized uptake value (SUVmax) from 18F‑FDG PET‑CT scanning and epidermal growth factor receptor (EGFR) mutation status has, to the best of our knowledge, not previously been fully elucidated, and the potential mechanisms by which EGFR mutations alter FDG uptake are largely unknown. A total of 157patients who were pathologically diagnosed with non‑small cell lung cancer (NSCLC) who underwent EGFR mutation testing and PET‑CT pretreatment between June 2015 and October 2017 were retrospectively analyzed. χ2 and univariate analyses were performed to identify the contributors to EGFR mutation. The receiver operating characteristic (ROC) curve was analyzed, and the area under the curve (AUC) was calculated. Glucose transporter1 (GLUT1) and NADPH oxidase4 (NOX4) expression, and reactive oxygen species (ROS) activity were detected in the A549 (wild‑type), PC‑9 (EGFR mutation‑positive, EGFR exon 19del) and NCI‑H1975 (EGFR mutation‑positive, combined with L858R and T790M substitution) cell lines. A total of 109patients who met the criteria were enrolled, and 63 of those tested as EGFR mutation‑positive. The SUVmax values were significantly lower in patients with EGFR mutations (mean, 6.52±0.38) compared with in patients with wild‑type EGFR (mean, 9.37±0.31; P<0.001). Using univariate analysis, EGFR mutation status was significantly associated with sex, smoking status, tumor histology and SUVmax of the primary tumor. In the multivariate analysis, smoking status (never‑smoking), histopathology (adenocarcinoma) and SUVmax (≤9.91) were the statistically significant predictors of EGFR mutations. ROC curve analysis identified that the SUVmax cut‑off point was 9.92, for which the AUC was 0.75 (95% confidence interval, 0.68‑0.83). Reverse transcription‑polymerase chain reaction indicated that the GLUT1 mRNA decreased in the PC‑9 and NCI‑H1975 cell lines compared with the A549 cell line (0.82±0.07 and 0.72±0.04 vs. 0.98±0.04, respectively; P<0.05) and decreased ROS activity was observed in the PC‑9 cell line. Furthermore, the expression of NOX4 mRNA decreased by 20% in PC‑9 (P<0.01) and by 14% (P<0.05) in NCI‑H1975 cells. In addition, NOX4 protein expression decreased by 13% in PC‑9 and by 16% in NCI‑H1975 cells (both P<0.05) compared with the A549 cell line. The SUVmax could be considered to effectively predict EGFR mutation status of patients with NSCLC, and the EGFR mutation status may alter FDG uptake partially via the NOX4/ROS/GLUT1 axis.

Sulforaphane protects rabbit corneas against oxidative stress injury in keratoconus through activation of the Nrf-2/HO-1 antioxidant pathway.

The aim of the present study was to examine whether activation of the nuclear factor E2-related factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) antioxidant pathway in the cornea was involved in the protective effect of sulforaphane (SF) following keratoconus (KC) injury. Following epithelial debridement, collagenase type II was applied in KC groups at room temperature for 30 min. Following this, rabbits were administered with a subconjunctival (s.c.) injection of SF or placebo (maize oil) daily for a total of 2 weeks. To investigate whether HO-1 was involved in the Nrf-2-related antioxidant pathway, rabbits were injected with zinc (II) protoporphyrin IX (ZnPP IX, s.c.) treatment in combination with SF 24 h following the application of collagenase type II. The protective effects of SF were evaluated by examining the mean keratometry (Km) and central cornea thickness (CCT), measuring reactive oxygen species (ROS) production using immunofluorescent staining, and analyzing the protein expression of NADPH oxidase (Nox) family members Nox-2 and Nox-4, and Nrf-2 and HO-1 using immunohistochemistry and western blot analysis. The mRNA levels of Nox-2, Nox-4, Nrf-2 and HO-1 were quantitatively detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. No significant difference in Km or CCT was observed among groups prior to surgery (P=0.700 and P=0.982, respectively). KC induced an apparent increase of ROS generation, and caused a significant increase in Km and a significant decrease in CCT. These changes were neutralized or reversed by SF treatment. Simultaneously, SF treatment decreased the expression of Nox-2 and Nox-4, and enhanced the expression of Nrf-2 and HO-1 in the KC corneas. The RT-qPCR results indicated that SF induced downregulation of the mRNA expression of Nox-2 and Nox-4, and upregulation of the mRNA expression of Nrf-2 and HO-1 following KC injury. The HO-1 inhibitor, ZnPP IX, counteracted the protective effects of SF on KC corneas. Therefore, the present study provided evidence that activation of the Nrf-2/HO-1 signal transduction pathway may partially promote the protective effect of the antioxidant SF in the KC cornea.

Nephroprotective effects of nebivolol in 2K1C rats through regulation of the kidney ROS-ADMA-NO pathway.

To evaluate the protective effect of nebivolol against kidney damage and elucidate the underlying mechanism in a two-kidney, one-clip (2K1C) rat model. 2K1C rats were obtained by clipping left renal artery of male Wistar rats and were considered hypertensive when systolic blood pressure (SBP) was ≥160mmHg 4 weeks after surgery. The 2K1C hypertensive rats were divided into untreated, nebivolol (10mg/kg, ig), and atenolol (80mg/kg, ig) treatment groups. The treatments lasted for 8 weeks. SBP, kidney structure and function, plasma and kidney angiotensin (Ang) II, nitric oxide (NO), asymmetric dimethylarginine (ADMA), and the oxidant status were examined. Kidney protein expression of NADPH oxidase (Nox) isoforms and its subunit p22phox, nitric oxide synthase (NOS) isoforms, protein arginine N-methyltransferase (PRMT) 1, and dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 was tested by western blotting. Nebivolol and atenolol exerted similar hypotensive effects. However, atenolol had little effect while nebivolol significantly ameliorated the functional decline and structural damage in the kidney, especially in non-clipped kidney (NCK), which was associated with the reduction of Ang II in NCK. Moreover, nebivolol inhibited the NCK production of reactive oxygen species (ROS) by decreasing Nox2, Nox4, and p22phox expression. Further, nebivolol reduced the plasma and kidney ADMA levels by increasing DDAH2 expression and decreasing PRMT1 expression. Nebivolol also increased the NCK NO level by ameliorating the expression of kidney NOS isoforms. Our results demonstrated that long-term treatment with nebivolol had renoprotective effect in 2K1C rats partly via regulation of kidney ROS-ADMA-NO pathway.

Cholinergic anti-inflammatory pathway inhibits neointimal hyperplasia by suppressing inflammation and oxidative stress

Neointimal hyperplasia as a consequence of vascular injury is aggravated by inflammatory reaction and oxidative stress. The α7 nicotinic acetylcholine receptor (α7nAChR) is a orchestrator of cholinergic anti-inflammatory pathway (CAP), which refers to a physiological neuro-immune mechanism that restricts inflammation. Here, we investigated the potential role of CAP in neointimal hyperplasia using α7nAChR knockout (KO) mice. Male α7nAChR-KO mice and their wild-type control mice (WT) were subjected to wire injury in left common carotid artery. At 4 weeks post injury, the injured aortae were isolated for examination. The neointimal hyperplasia after wire injury was significantly aggravated in α7nAChR-KO mice compared with WT mice. The α7nAChR-KO mice had increased collagen contents and vascular smooth muscle cells (VSMCs) amount. Moreover, the inflammation was significantly enhanced in the neointima of α7nAChR-KO mice relative to WT mice, evidenced by the increased expression of tumor necrosis factor-α/interleukin-1β, and macrophage infiltration. Meanwhile, the chemokines chemokine (C-C motif) ligand 2 and chemokine (CXC motif) ligand 2 expression was also augmented in the neointima of α7nAChR-KO mice compared with WT mice. Additionally, the depletion of superoxide dismutase (SOD) and reduced glutathione (GSH), and the upregulation of 3-nitrotyrosine, malondialdehyde and myeloperoxidase were more pronounced in neointima of α7nAChR-KO mice compared with WT mice. Accordingly, the protein expression of NADPH oxidase 1 (Nox1), Nox2 and Nox4, was also higher in neointima of α7nAChR-KO mice compared with WT mice. Finally, pharmacologically activation of CAP with a selective α7nAChR agonist PNU-282987, significantly reduced neointima formation, arterial inflammation and oxidative stress after vascular injury in C57BL/6 mice. In conclusion, our results demonstrate that α7nAChR-mediated CAP is a neuro-physiological mechanism that inhibits neointima formation after vascular injury via suppressing arterial inflammation and oxidative stress. Further, these results imply that targeting α7nAChR may be a promising interventional strategy for in-stent stenosis.

Ursolic acid suppresses TGF-β1-induced quiescent HSC activation and transformation by inhibiting NADPH oxidase expression and Hedgehog signaling.

Activation of quiescent hepatic stellate cells (q-HSCs) and their transformation to myofibroblasts (MFBs) is a key event in liver fibrosis. Hedgehog (Hh) signaling stimulates q-HSCs to differentiate into MFBs, and NADPH oxidase (NOX) may be involved in regulating Hh signaling. The author's preliminary study demonstrated that ursolic acid (UA) selectively induces apoptosis in activated HSCs and inhibits their proliferation in vitro via negative regulation of NOX activity and expression. However, the effect of UA on q-HSCs remains to be elucidated. The present study aimed to investigate the effect of UA on q-HSC activation and HSC transformation and to observe alterations in the NOX and Hh signaling pathways during q-HSC activation. q-HSC were isolated from adult male Sprague-Dawley rats. Following culture for 3 days, the cells were treated with or without transforming growth factor-β1 (TGF-β1; 5 µg/l); intervention groups were pretreated with UA (40 µM) or diphenyleneiodonium chloride (DPI; 10 µM) for 30 min prior to addition of TGF-β1. mRNA and protein expression of NOX and Hh signaling components and markers of q-HSC activation were examined by western blotting and reverse transcription-polymerase chain reaction. TGF-β1 induced activation of q-HSCs, with increased expression of α-smooth muscle actin (α-SMA) and type I collagen. In addition, expression of NOX subunits (gp91phox, p67phox, p22phox, and Rac1) and Hh signaling components, including sonic Hh, sterol-4-alpha-methyl oxidase, and Gli family zinc finger 2, were upregulated in activated HSCs. Pretreatment of q-HSCs with UA or DPI prior to TGF-β1 significantly downregulated expression of NOX subunits and Hh signaling components and additionally inhibited expression of α-SMA and type I collagen, thereby preventing transformation to MFBs. UA inhibited TGF-β1-induced activation of q-HSCs and their transformation by inhibiting expression of NOX subunits and the downstream Hh pathway.