NADPH Oxidase 2-Derived Reactive Oxygen Species Promote CD8+ T Cell Effector Function.

Oxidants participate in lymphocyte activation and function. We previously demonstrated that eliminating the activity of NADPH oxidase 2 (NOX2) significantly impaired the effectiveness of autoreactive CD8+ CTLs. However, the molecular mechanisms impacting CD8+ T cell function remain unknown. In the present study, we examined the role of NOX2 in both NOD mouse and human CD8+ T cell function. Genetic ablation or chemical inhibition of NOX2 in CD8+ T cells significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effector genes such as IFN-γ and granzyme B. Inhibition of NOX2 in both human and mouse CD8+ T cells prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CD8+ T cells. Furthermore, we show that NOX2-generated oxidants deactivate the tumor suppressor complex leading to activation of RheB and subsequently mTOR complex 1. These results indicate that NOX2 plays a nonredundant role in TCR-mediated CD8+ T cell effector function.

Antibiotic-induced intestinal microbiota depletion can attenuate the acute kidney injury to chronic kidney disease transition via NADPH oxidase 2 and trimethylamine-N-oxide inhibition.

Intestinal microbiota and their metabolites affect systemic inflammation and kidney disease outcomes. Here, we investigated the key metabolites associated with the acute kidney injury (AKI)-to chronic kidney disease (CKD) transition and the effect of antibiotic-induced microbiota depletion (AIMD) on this transition. In 61 patients with AKI, 59 plasma metabolites were assessed to determine the risk of AKI-to-CKD transition. An AKI-to-CKD transition murine model was established four weeks after unilateral ischemia-reperfusion injury (IRI) to determine the effects of AIMD on the gut microbiome, metabolites, and pathological responses related to CKD transition. Human proximal tubular epithelial cells were challenged with CKD transition-related metabolites, and inhibitory effects of NADPH oxidase 2 (NOX2) signals were tested. Based on clinical metabolomics, plasma trimethylamine N-oxide (TMAO) was associated with a significantly increased risk for AKI-to-CKD transition [adjusted odds ratio 4.389 (95% confidence interval 1.106-17.416)]. In vivo, AIMD inhibited a unilateral IRI-induced increase in TMAO, along with a decrease in apoptosis, inflammation, and fibrosis. The expression of NOX2 and oxidative stress decreased after AIMD. In vitro, TMAO induced fibrosis with NOX2 activation and oxidative stress. NOX2 inhibition successfully attenuated apoptosis, inflammation, and fibrosis with suppression of G2/M arrest. NOX2 inhibition (in vivo) showed improvement in pathological changes with a decrease in oxidative stress without changes in TMAO levels. Thus, TMAO is a key metabolite associated with the AKI-to-CKD transition, and NOX2 activation was identified as a key regulator of TMAO-related AKI-to-CKD transition both in vivo and in vitro.

NADPH-Oxidase 2 Promotes Autophagy in Spinal Neurons During the Development of Morphine Tolerance.

Repeated morphine administration results in analgesic tolerance. However, the underlying mechanism of morphine analgesic tolerance remains unclear. NADPH-oxidase 2 (NOX2) is the first discovered NADPH oxidase, which mainly functions to produce reactive oxygen species. Its specific role in morphine tolerance has not been fully investigated. In this work, we found that chronic morphine administration significantly increased the expression of NOX2 in spinal cord. Pretreatment of NOX2 inhibitor blocked the upregulation of NOX2 and autophagy markers, including LC3B and P62, and consequently the development of morphine tolerance. NOX2 and LC3B were both colocalized with NeuN in spinal dorsal horn in morphine-tolerant rats. Our results suggest that the increased autophagy activity in spinal neurons promoted by NOX2 activation contributes to the development of morphine tolerance. NOX2 may be considered as a new therapeutic target for morphine tolerance.

Neuroprotective properties of ethanolic extract of Citrus unshiu Markovich peel through NADPH oxidase 2 inhibition in chemotherapy-induced neuropathic pain animal model.

The present study aimed to determine the antioxidant effect of Citrus unshiu Markovich (CUM) extract in neuronal cell lines under oxidative stress and to investigate the effect of chemotherapy-induced peripheral neuropathy (CIPN) on the nociceptive response in a preclinical mice model. We tested the inhibition of H2 O2 in Neuro2A cells treated with CUM. Experimental animals were treated with oxaliplatin to induce CINP, and then administered oral CUM for 4 weeks in order to observe the effect of CUM. Animals were evaluated weekly for thermal hyperalgesia and digital motor nerve conduction velocity (NCV). Lumbar dorsal root ganglia (DRG) isolated from each animal were evaluated through immunochemical and western blot analysis for nerve damage, inflammatory response, and expression of redox signaling factors. The main mechanisms were determined to be decreased inducible nitric oxide synthase (iNOS) production due to the inhibition of NADPH oxidase 2 (NOX2). To determine the functional role of NOX2 in CINP, we administrated CUM into NOX2-deficient mice with neuropathic pain. Therefore, we suggest that CUM controls the expression levels of inflammatory factors in CINP via NOX2 inactivation. This study demonstrated that a complementary medicine such as CUM might be a potential novel therapeutic agent for the treatment of CINP.

Nox2 Inhibition Regulates Stress Response and Mitigates Skeletal Muscle Fiber Atrophy during Simulated Microgravity.

Insufficient stress response and elevated oxidative stress can contribute to skeletal muscle atrophy during mechanical unloading (e.g., spaceflight and bedrest). Perturbations in heat shock proteins (e.g., HSP70), antioxidant enzymes, and sarcolemmal neuronal nitric oxidase synthase (nNOS) have been linked to unloading-induced atrophy. We recently discovered that the sarcolemmal NADPH oxidase-2 complex (Nox2) is elevated during unloading, downstream of angiotensin II receptor 1, and concomitant with atrophy. Here, we hypothesized that peptidyl inhibition of Nox2 would attenuate disruption of HSP70, MnSOD, and sarcolemmal nNOS during unloading, and thus muscle fiber atrophy. F344 rats were divided into control (CON), hindlimb unloaded (HU), and hindlimb unloaded +7.5 mg/kg/day gp91ds-tat (HUG) groups. Unloading-induced elevation of the Nox2 subunit p67phox-positive staining was mitigated by gp91ds-tat. HSP70 protein abundance was significantly lower in HU muscles, but not HUG. MnSOD decreased with unloading; however, MnSOD was not rescued by gp91ds-tat. In contrast, Nox2 inhibition protected against unloading suppression of the antioxidant transcription factor Nrf2. nNOS bioactivity was reduced by HU, an effect abrogated by Nox2 inhibition. Unloading-induced soleus fiber atrophy was significantly attenuated by gp91ds-tat. These data establish a causal role for Nox2 in unloading-induced muscle atrophy, linked to preservation of HSP70, Nrf2, and sarcolemmal nNOS.

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage.

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE-/-) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE-/- and Nox2 (ESMIRO/ApoE-/-/Nox2-/y) were generated and compared with ESMIRO/ApoE-/-/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE-/- mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE-/-/Nox2-/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE-/-/Nox2-/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE-/- mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

Diabetes aggravates myocardial ischaemia reperfusion injury via activating Nox2-related programmed cell death in an AMPK-dependent manner.

Cardiovascular diseases such as myocardial ischaemia have a high fatality rate in patients with diabetes. This study was designed to expose the crosstalk between oxidative stress and AMPK, a vital molecule that controls biological energy metabolism, in myocardial ischaemia reperfusion injury (I/RI) in diabetic rats. Diabetes was stimulated in rats using streptozotocin injection. Rats were separated on random into control, control + I/R, Diabetes, Diabetes + I/R, Diabetes + I/R + N-acetylcysteine and Diabetes + I/R + Vas2870 groups. Myocardial infarct size was determined, and the predominant Nox family isoforms were analysed. In vitro, the H9C2 cells were administered excess glucose and exposed to hypoxia/reoxygenation to mimic diabetes and I/R. The AMPK siRNA or AICAR was used to inhibit or activate AMPK expression in H9C2 cells, respectively. Then, myocardial oxidative stress and programmed cell death were measured. Diabetes or high glucose levels were found to aggravate myocardial I/RI or hypoxia/reoxygenation in H9C2 cells, as demonstrated by an increase in myocardial infarct size or lactate dehydrogenase levels, oxidative stress generation and induction of programmed cell death. In diabetic rat hearts, cardiac Nox1, Nox2 and Nox4 were all heightened. The suppression of Nox2 expression using Vas2870 or Nox2-siRNA treatment in vivo or in vitro, respectively, protected diabetic rats from myocardial I/RI. AMPK gene knockout increased Nox2 protein expression while AMPK agonist decreased Nox2 expression. Therefore, diabetes aggravates myocardial I/RI by generating of Nox2-associated oxidative stress in an AMPK-dependent manner, which led to the induction of programmed cell death such as apoptosis, pyroptosis and ferroptosis.

Activation of NADPH/ROS pathway contributes to angiogenesis through JNK signaling in brain endothelial cells.

Recent evidences have shown that reactive oxygen species (ROS) are involved in regulating angiogenesis and preventing tissue injury. However, the precise molecular mechanisms behind ROS-induced angiogenesis are still unknown. The aim of the present study was to investigate the effects of ROS-induced angiogenesis in rat brain microvessel endothelial cells (rBMECs) and identify involving the signal pathways. For initial experiments, the rBMECs were incubated with different concentrations of hydrogen peroxide (H2O2). For the second experiments, the rBMECs were respectively treated with ROS scavenger dimethylthiourea (DMTU), NADPH oxidase (Nox) inhibitor apocynin, small interfering RNAs-mediated knock down Nox2 or Nox4, or pretreated with c-Jun N-terminal kinase (JNK) inhibitor SP600125. The cell proliferation, migration, tube formation, and the expressions of several important neuroangiogenic factors including vascular endothelial growth factor (VEGF), brain derived neurotrophic factor (BDNF), matrix metalloproteinase (MMP) -9 and phos-JNK were measured. Low level of H2O2 significantly promoted endothelial cell (EC) proliferation, migration and tube formation and upregulated levels of VEGF, BDNF, MMP-9 and phos-JNK. DMTU and apocynin significantly inhibited endothelial angiogenesis and downregulated these protein levels. As expected, knockdown of Nox2 or Nox4 expression blocked endothelial angiogenesis and downregulated the expressions of pro-neuroangiogenic factors. Furthermore, H2O2-induced endothelial angiogenesis and high expressions of pro-neuroangiogenic factors were decreased by SP600125. In conclusion, Nox-derived ROS were required for endothelial angiogenesis. Low level of ROS may activate JNK signaling pathway and upregulate pro-neuroangiogenic factors, ultimately mediating endothelial angiogenesis.

An Effective NADPH Oxidase 2 Inhibitor Provides Neuroprotection and Improves Functional Outcomes in Animal Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) has become a leading cause of death and disability all over the world. Pharmacological suppression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) can inhibit oxidative stress which is implicated in the pathology of TBI. GSK2795039 was reported to target NOX2 to inhibit [Formula: see text] and ROS production. The present study aimed to investigate the effect of GSK2795039 on NOX2 activity and neurological deficits in a TBI mouse model. TBI mouse model was established by a weight-drop to mouse skull. GSK2795039 at a dose of 100 mg/kg was administrated to mice 30 min before TBI. NOX2 expression and activity were detected by Western blot and biochemical method. Neurological damage and apoptosis were detected by behavioral test and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. GSK2795039 significantly inhibited NOX2 expression and activity in the TBI mouse model. It also attenuated TBI-induced neurological deficits, apoptosis, and neurological recovery. The results indicate that GSK2795039 can be used as a potential drug for TBI treatment.

Nox2 and Nox4 Participate in ROS-Induced Neuronal Apoptosis and Brain Injury During Ischemia-Reperfusion in Rats.

BACKGROUND: Previously studies have shown that Nox2 and Nox4, as members of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, Nox), participate in brain damage caused by ischemia-reperfusion (I/R). The aim of this study is to investigate the effects of specific chemical inhibitors of Nox2 and Nox4 on cerebral I/R-induced brain injury in rats. METHODS: At 0.5 h before MCAO surgery, the rats were pretreated with vehicle, Nox2 inhibitor (gp91ds-tat), and Nox4 inhibitor (GKT137831), respectively. After reperfusion for 24 h, the infarct sizes of brain tissues in rats in various groups are determined. The penumbra (ischemic) tissues are collected to measure ROS levels, neuronal apoptosis, and degeneration, as well as the integrity of the blood-brain barrier (BBB) in brain tissues of rats. RESULTS: gp91ds-tat and GKT137831 pretreatment significantly reduced the infarct sizes in brain tissues of rats, effectively suppressed I/R-induced increase in ROS levels, neuronal apoptosis and degeneration, and obviously alleviated BBB damage. CONCLUSION: Under cerebral I/R conditions, Nox2 inhibitor (gp91ds-tat) and Nox4 inhibitor (GKT137831) can effectively play a protective role in the brain tissues of rats.

Quercetin suppressed NADPH oxidase-derived oxidative stress via heme oxygenase-1 induction in macrophages.

NADPH oxidase-derived superoxide (O2.-) generation and oxidative stress is usually considered as an important factor to the pathogenesis of inflammatory diseases. Quercetin, widely known for their anti-oxidant and anti-inflammatory properties in vitro and in vivo, is recently identified to induce expression of antioxidant enzyme heme oxygenase-1 (HO-1). Previous studies suggest that HO-1 induction and/or subsequent HO-1 end product generation in vitro and in vivo may suppress NADPH oxidase-derived oxidative stress. In this study, we tested whether quercetin might modulate NADPH oxidase activity in macrophages via induction of HO-1. In RAW264.7 macrophages, quercetin significantly attenuated NADPH oxidase-derived O2.- generation via a HO-1-dependent mechanism. Mechanistically, the protective effects of quercetin were (1) linked to increased expression of HO-1 in the presence or absence of lipopolysaccharide (LPS), (2) similar to that observed with the NADPH oxidase inhibitor apocynin, and (3) could be abolished by the specific small-interfering RNA against HO-1 expression or HO-1 activity inhibitor tin protoporphyrin. The induction of HO-1 by quercetin was associated with the nuclear accumulation of Nrf2 and downregulation of Keap1, a negative regulator of Nrf2. In addition, this flavonoid also inhibited the overproduction of nitric oxide and inflammatory cytokines in LPS-stimulated macrophages via simultaneous induction of HO-1 expression. In agreement with the observations in macrophages, pretreatment with quercetin significantly alleviated LPS-induced inflammation in mice which was concomitant with decreased NADPH oxidase activity and increased HO-1 expression. Our results suggested that quercein could modulate NADPH oxidase-derived O2.- production in macrophages at least partly through HO-1 induction. Suppression of NADPH oxidase-dependent oxidative stress may represent a novel mechanism underlying the anti-oxidant and anti-inflammatory properties of quercetin/HO-1 pathway.

Trimetazidine prevents diabetic cardiomyopathy by inhibiting Nox2/TRPC3-induced oxidative stress.

Diabetic cardiomyopathy (DCM) is characterized by cardiac hypertrophy, fibrosis, oxidative stress and inflammation. Trimetazidine (TMZ), a potent metabolism modulator, has been shown to be cardioprotective in experimental models of ischaemia-reperfusion and type 2 diabetes-induced cardiomyopathy. The present study examined whether TMZ inhibits cardiomyopathy induced by insulin-dependent type 1 diabetes. Wistar rats were randomly divided into control group (vehicle alone), diabetes mellitus (DM; induced by streptozocin (STZ) injection) group and DM treated with TMZ (DM/TMZ) group. Cardiac function, histology, plasma biochemistry and molecular mechanism were assessed. STZ induced diabetes in rats as indicated by hyperglycemia, increased and decreased levels of advanced glycation end products (AGEs) and insulin respectively. Diabetic rats were characterized by left ventricular dysfunction, cardiachypertrophy and fibrosis and signs of inflammation and oxidative stress in the myocardium, which were accompanied by elevated levels of NADPH oxidase 2 (Nox2) and transient receptor potential channel 3 (TRPC3) in the heart. TMZ treatment ameliorated diabetes-associated structural and functional alterations by inhibiting Nox2 and TRPC3 without having any effects on glucose, insulin and AGEs levels. These results suggest that TMZ could be used as a therapy to treat cardiomyopathy associated with type 1 induced diabetes mellitus.

Putative Nox2 inhibitors worsen homocysteine-induced impaired acetylcholine-mediated relaxation.

BACKGROUND AND AIM: Increased homocysteine (Hcy) is associated with coronary artery disease (CAD). Hcy increases reactive oxygen species (ROS) via NADPH oxidases (Nox), reducing acetylcholine-mediated vasorelaxation. We aimed to determine if putative Nox2 inhibitors prevent Hcy-impaired acetylcholine-mediated vasorelaxation. METHODS AND RESULTS: New Zealand White rabbit and wild-type (C57BL/6) and Nox2-/- (NOX) mice aortic rings were mounted in organ baths. Rabbit rings were incubated with either apocynin (10 µM), gp91ds-tat (GP, 1 µM) or PhoxI2 (1 µM) and mice rings GP (1 µM) only. Some rabbit rings were incubated with 3 mM Hcy, before pre-contraction, followed by dose-response relaxation to acetylcholine (ACh; 0.01µM-10µM). In rabbit rings treated with Hcy and GP, O2‾ donor pyrogallol (1 µM) or Akt activator SC79 (1 µM) was added 5 min before ACh. Mice rings were used to compare Nox2 deletion to normal acetylcholine-mediated relaxation. In rabbits, Hcy reduced acetylcholine-mediated relaxation vs. control (p < 0.0001). Treatment + Hcy reduced relaxation compared with treatment alone (p < 0.0001). Pyrogallol and SC79 reversed the response of GP + Hcy (p = 0.0001). In mice, Nox2 deletion reduced acetylcholine-mediated vasorelaxation. Rabbit tissue analysis revealed that Hcy reduced eNOS phosphorylation at Thr495 and increased eNOS phosphorylation at Ser1177; no further alteration at Thr495 was observed with GP. In contrast, GP prevented increased phosphorylation at Ser1177. CONCLUSIONS: Apocynin, GP and PhoxI2 worsens acetylcholine-mediated vascular relaxation in rabbit aorta, which is supported by results from mouse Nox2 deletion data. These inhibitors worsen Hcy-induced vascular dysfunction, suggesting that current putative Nox2 inhibitors might not be useful in treating HHcy.

Novel endosomal NOX2 oxidase inhibitor ameliorates pandemic influenza A virus-induced lung inflammation in mice.

BACKGROUND AND OBJECTIVE: Influenza A viruses (IAV) cause respiratory tract infections that can be fatal when the virus spreads to the alveolar space (i.e. alveolitis), and this is mainly observed with highly pathogenic strains. Reactive oxygen species (ROS) production by the NOX2 NADPH oxidase in endosomes has been directly implicated in IAV pathology. Recently, we demonstrated that treatment with a novel endosome-targeted NOX2 oxidase inhibitor, cholestanol-conjugated gp91dsTAT (Cgp91ds-TAT), attenuated airway inflammation and viral replication to infection with a low pathogenic influenza A viral strain. Here, we determined whether suppression of endosome NOX2 oxidase prevents the lung inflammation following infection with a highly pathogenic IAV strain. METHODS: C57Bl/6 mice were intranasally treated with either DMSO vehicle (2%) or Cgp91ds-TAT (0.2 mg/kg/day) 1 day prior to infection with the high pathogenicity PR8 IAV strain (500 PFU/mouse). At Day 3 post-infection, mice were culled for the evaluation of airway and lung inflammation, viral titres and ROS generation. RESULTS: PR8 infection resulted in a marked degree of airway inflammation, epithelial denudation, alveolitis and inflammatory cell ROS production. Cgp91ds-TAT treatment significantly attenuated airway inflammation, including neutrophil influx, the degree of alveolitis and inflammatory cell ROS generation. Importantly, the anti-inflammatory phenotype affected by Cgp91ds-TAT significantly enhanced the clearance of lung viral mRNA following PR8 infection. CONCLUSION: Endosomal NOX2 oxidase promotes pathogenic lung inflammation to IAV infection. The localized delivery of endosomal NOX2 oxidase inhibitors is a novel therapeutic strategy against IAV, which has the potential to limit the pathogenesis caused during epidemics and pandemics.

Blocking Nox2 improves mesenchymal stem cells therapy in myocardial infarction via antagonizing oxidant and promoting survival.

An increase in reactive oxygen species (ROS) plays a key role in aging and apoptosis in mesenchymal stem cells derived from bone marrow (BMSCs). NADPH oxidase Nox2 serves as an important source of intracellular ROS formation. This study is designed to determine if blocking Nox2 enhances anti-apoptotic and anti-aging ability of BMSCs to oxidant stress, and thus improves therapeutic efficacy in myocardial infarction (MI). Nox2 inhibitor (Acetovanillone) and Nox2 siRNA were used to block Nox2 in BMSCs, and the cell viability, apoptosis, senescence and survival of BMSCs were determined by CCK-8, Edu staining, TUNEL staining, ß-galactosidase (ß-gal) assay and DAPI labeling. Here we found that both Nox2 inhibitor and Nox2 knockdown remarkably countered the decrease of viability, and the increase of aging and apoptosis of BMSCs by H2 O2 . Whereas, Nox2 overexpression exacerbated the viability reduction, senescence and apoptosis of BMSCs. The ROS accumulation in BMSCs was also suppressed by Nox2 blocking. Further study uncovered that Nox2 inhibitor caused the downregulation of p-p53, p21, p-FoxO1 and Bax, and the upregulation of anti-apoptotic protein Bcl-2. In vivo, Nox2 knockdown in grafted BMSCs led to the improvement of EF and FS in infarcted myocardium than BMSCs without Nox2 knockdown. Consistently, more retention and survival of BMSCs were found after Nox2 knockdown. Taken together, Nox2 inhibition enhances anti-aging and anti-apoptotic ability of BMSCs, and thus promotes survival and retention of BMSCs, which provides a new strategy for improving BMSCs-based therapy.

Neutrophil extracellular traps in acrolein promoted hepatic ischemia reperfusion injury: Therapeutic potential of NOX2 and p38MAPK inhibitors.

Neutrophil is a significant contributor to ischemia reperfusion (IR) induced liver tissue damage. However, the exact role of neutrophils in IR induced innate immune activation and liver damage is not quite clear. Our study sheds light on the role of chronic oxidative stress end products in worsening the IR inflammatory process by neutrophil recruitment and activation following liver surgery. We employed specific inhibitors for molecular targets-NOX2 (NADPH oxidase 2) and P38 MAPK (Mitogen activated protein kinase) signal to counteract neutrophil activation and neutrophil extracellular trap (NET) release induced liver damage in IR injury. We found that acrolein initiated neutrophil chemotaxis and induced NET release both in vitro and in vivo. Acrolein exposure caused NET induced nuclear and mitochondrial damage in HepG2 cells as well as aggravated the IR injury in rat liver. Pretreatment with F-apocynin and naringin, efficiently suppressed acrolein induced NET release in vitro. Notably, it suppressed the expression of inflammatory cytokines, P38MAPK-ERK activation, and apoptotic signals in rat liver exposed to acrolein and subjected to IR. Moreover, this combination effectively attenuated acrolein induced NET release and hepatic IR injury. In the current study we have shown that the acrolein accumulation in liver due to chronic stress, is responsible for neutrophil recruitment and its activation leading to NET induced liver damage during surgery. Our study shows that therapeutic targeting of NOX2 and P38MAPK signaling in patients with chronic hepatic disorders would improve post operative hepatic function and survival.

Gp91phox (NOX2) in Activated Microglia Exacerbates Neuronal Damage Induced by Oxygen Glucose Deprivation and Hyperglycemia in an in Vitro Model.

BACKGROUND/AIMS: Peri-operative cerebral ischemia reperfusion injury is one of the most serious peri-operative complications that can be aggravated in patients with diabetes. A previous study showed that microglia NOX2 (a NADPH oxidase enzyme) may play an important role in this process. Here, we investigated whether increased microglial derived gp91phox, also known as NOX2, reduced oxygen glucose deprivation (OGD) after induction of hyperglycemia (HG). METHODS: A rat neuronal-microglial in vitro co-culture model was used to determine the effects of gp91phox knockdown on OGD after HG using six treatment groups: A rat microglia and neuron co-culture model was established and divided into the following six groups: high glucose + scrambled siRNA transfection (HG, n = 5); HG + gp91phoxsiRNA transfection (HG-gp91siRNA, n = 5); oxygen glucose deprivation + scrambled siRNA transfection (OGD, n = 5); OGD + gp91phoxsiRNA transfection (OGD-gp91siRNA, n = 5); HG + OGD + scrambled siRNA transfection (HG-OGD, n = 5); and HG + OGD + gp91phoxsiRNA transfection (HG-OGD-gp91siRNA, n = 5). The neuronal survival rate was measured by the MTT assay, while western blotting was used to determine gp91phox expression. Microglial derived ROS and neuronal apoptosis rates were analyzed by flow cytometry. Finally, the secretion of cytokines, including IL-6, IL-8, TNF-α, and 8-iso-PGF2α was determined using an ELISA kit. RESULTS: Neuronal survival rates were significantly decreased by HG and OGD, while knockdown of gp91phox reversed these rates. ROS production and cytokine secretion were also significantly increased by HG and OGD but were significantly inhibited by knockdown of gp91phoxsiRNA. CONCLUSION: Knockdown of gp91phoxsiRNA significantly reduced oxidative stress and the inflammatory response, and alleviated neuronal damage after HG and OGD treatment in a rat neuronal-microglial co-culture model.

Regional up-regulation of NOX2 contributes to the differential vulnerability of outer hair cells to neomycin.

In hearing loss induced by aminoglycoside antibiotics, the outer hair cells (OHCs) in the basal turn are always more susceptible than OHCs in the apical turn, while the underlying mechanisms remain unknown. In this study, we reported that NAPDH oxidase 2 (NOX2) played an important role in the OHCs damage preferentially in the basal turn. Normally, NOX2 was evenly expressed in OHCs among different turns, at a relatively low level. However, after neomycin treatment, NOX2 was dominantly induced in OHCs in the basal turn. In vivo and in vitro studies demonstrated that inhibition of NOX2 significantly alleviated neomycin-induced OHCs damages, as seen from both the cleaved caspase-3 and TUNEL staining. Moreover, gp91 ds-tat delivery and DHE staining results showed that NOX2-derived ROS was responsible for neomycin ototoxicity. Taken together, our study shows that regional up-expression of NOX2 and subsequent increase of ROS in OHCs of the basal turn is an important factor contributing to the vulnerability of OHCs there, which should shed light on the prevention of hearing loss induced by aminoglycoside antibiotics.

NADPH oxidases as potential pharmacological targets against increased seizure susceptibility after systemic inflammation.

BACKGROUND: Systemic inflammation associated with sepsis can induce neuronal hyperexcitability, leading to enhanced seizure predisposition and occurrence. Brain microglia are rapidly activated in response to systemic inflammation and, in this activated state, release multiple cytokines and signaling factors that amplify the inflammatory response and increase neuronal excitability. NADPH oxidase (NOX) enzymes promote microglial activation through the generation of reactive oxygen species (ROS), such as superoxide anion. We hypothesized that NOX isoforms, particularly NOX2, are potential targets for prevention of sepsis-associated seizures. METHODS: To reduce NADPH oxidase 2-derived ROS production, mice with deficits of NOX regulatory subunit/NOX2 organizer p47phox (p47phox-/-) or NOX2 major subunit gp91phox (gp91phox-/-) were used or the NOX2-selective inhibitor diphenyleneiodonium (DPI) was used to treat wild-type (WT) mice. Systemic inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS). Seizure susceptibility was compared among mouse groups in response to intraperitoneal injection of pentylenetetrazole (PTZ). Brain tissues were assayed for proinflammatory gene and protein expression, and immunofluorescence staining was used to estimate the proportion of activated microglia. RESULTS: Increased susceptibility to PTZ-induced seizures following sepsis was significantly attenuated in gp91phox-/- and p47phox-/- mice compared with WT mice. Both gp91phox-/- and p47phox-/- mice exhibited reduced microglia activation and lower brain induction of multiple proconvulsive cytokines, including TNFα, IL-1ß, IL-6, and CCL2, compared with WT mice. Administration of DPI following LPS injection significantly attenuated the increased susceptibility to PTZ-induced seizures and reduced both microglia activation and brain proconvulsive cytokine concentrations compared with vehicle-treated controls. DPI also inhibited the upregulation of gp91phox transcripts following LPS injection. CONCLUSIONS: Our results indicate that NADPH oxidases contribute to the development of increased seizure susceptibility in mice after sepsis. Pharmacologic inhibition of NOX may be a promising therapeutic approach to reducing sepsis-associated neuroinflammation, neuronal hyperexcitability, and seizures.

Turning off NADPH oxidase-2 by impeding p67phox activation in infected mouse macrophages reduced viral entry and inflammation.

BACKGROUND: Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O2- and releasing H2O2. Herein, we investigated whether targeting membrane -embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages. METHODS: Confocal microscopy and western blots monitored levels of the viral nucleoprotein NP and p67phox, NOX2 activator subunit, Elisa assays quantified TNF-α levels in LPS or IAV-activated mouse or porcine alveolar macrophages pretreated with a fluorescent NOX inhibitor, called nanoshutter NS1. RESULTS: IAV infection in macrophages promoted p67phox translocation to the membrane, rafts clustering and activation of the NOX2 complex at early times. Disrupting rafts reduced intracellular viral NP. NS1 markedly reduced raft clustering and viral entry by binding to the C-terminal of NOX2 also characterized in vitro. NS1 decrease of TNF-α release depended on the cell type. CONCLUSION: NOX2 participated in IAV entry and raft-mediated endocytosis. NOX2 inhibition by NS1 reduced viral entry. NS1 competition with p67phox for NOX2 binding shown by in silico models and cell-free assays was in agreement with NS1 inhibiting p67phox translocation to membrane-embedded NOX2 in mouse and porcine macrophages. GENERAL SIGNIFICANCE: We introduce NS1 as a compound targeting NOX2, a critical enzyme controlling viral levels and inflammation in macrophages and discuss the therapeutic relevance of targeting the C-terminal of NADPH oxidases by probes like NS1 in viral infections.