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Acute myeloid leukaemia (AML) is a highly heterogeneous malignancy, with a poor 5-year overall survival rate of approximately 30%. Consequently, the search for novel therapeutic strategies is ongoing, and the identification of new vulnerabilities could accelerate progress. Oxidative stress and metabolic rewiring are established hallmarks of cancer, and recent evidence suggests that NADPH oxidases may regulate metabolism, potentially linking these two processes. Increasing research highlights the importance of NOX2 in AML, particularly its role in metabolic regulation. In this study, we investigated the effects of simultaneously inhibiting NOX2 and glycolysis in AML cells. Dual inhibition of NOX2 and glycolysis—by targeting hexokinase or lactate dehydrogenase (LDH)—significantly reduced cell proliferation, markedly impaired clonogenic potential, and induced extensive cell death in a broad panel of AML cell lines. Importantly, these findings were further validated in primary bone marrow samples derived from AML patients, where combined inhibition triggered similar potent anti-leukemic effects. Furthermore, the combined inhibition of NOX2 and LDH enhanced the efficacy of cytarabine (AraC), suggesting this approach could boost the effectiveness of conventional therapies. In an in vivo AML model, targeting NOX2 and LDH in myeloid progenitor cells delayed the onset of leukaemia and extended survival. In conclusion, our findings propose a novel therapeutic strategy for AML through the dual targeting of NOX2 and glycolysis. [ABSTRACT FROM AUTHOR]

Background: Extracellular matrix (ECM) stiffness is closely related to the progress of diabetic cardiomyopathy (DCM) and the response of treatment of DCM to anti-diabetic drugs. Dapagliflozin (Dapa) has been proven to have cardio-protective efficacy for diabetes and listed as the first-line drug to treat heart failure. But the regulatory relationship between ECM stiffness and treatment efficacy of Dapa remains elusive. Materials and methods: This work investigated the effect of ECM stiffness on DCM progression and Dapa efficacy using both in vivo DCM rat model and in vitro myocardial cell model with high glucose injury. First, through DCM rat models with various levels of myocardial injury and administration with Dapa treatment for four weeks, the levels of myocardial injury, myocardial oxidative stress, expressions of AT1R (a mechanical signal protein) and the stiffness of myocardial tissues were obtained. Then for mimicking the stiffness of myocardial tissues at early and late stages of DCM, we constructed cell models through culturing H9c2 myocardial cells on the polyacrylamide gels with two stiffness and exposed to a high glucose level and without/with Dapa intervention. The cell viability, reactive oxygen species (ROS) levels and expressions of mechanical signal sensitive proteins were obtained. Results: The DCM progression is accompanied by the increased myocardial tissue stiffness, which can synergistically exacerbate myocardial cell injury with high glucose. Dapa can improve the ECM stiffness-induced DCM progression and its efficacy on DCM is more pronounced on the soft ECM, which is related to the regulation pathway of AT1R-FAK-NOX2. Besides, Dapa can inhibit the expression of the ECM-induced integrin β1, but without significant impact on piezo 1. Conclusions: Our study found the regulation and effect of biomechanics in the DCM progression and on the Dapa efficacy on DCM, providing the new insights for the DCM treatment. Additionally, our work showed the better clinical prognosis of DCM under early Dapa intervention. [ABSTRACT FROM AUTHOR]

The crosstalk between astrocytes and microglia plays a pivotal role in neuroinflammation following ischemic stroke, and phenotypic distribution of these cells can change with the progression of ischemic stroke. Peroxiredoxin (PRDX) 6 phospholipase A2 (iPLA2) activity is involved in the generation of reactive oxygen species(ROS), with ROS driving the activation of microglia and astrocytes; however, its exact function remains unexplored. MJ33, PRDX6D140A mutation was used to block PRDX6-iPLA2 activity in vitro and vivo after ischemic stroke. PRDX6T177A mutation was used to block the phosphorylation of PRDX6 in CTX-TNA2 cell lines. NAC, GSK2795039, Mdivi-1, U0126, and SB202190 were used to block the activity of ROS, NOX2, mitochondrial fission, ERK, and P38, respectively, in CTX-TNA2 cells. In ischemic stroke, PRDX6 is mainly expressed in astrocytes and PRDX6-iPLA2 is involved in the activation of astrocytes and microglia. In co-culture system, Asp140 mutation in PRDX6 of CTX-TNA2 inhibited the polarization of microglia, reduced the production of ROS, suppressed NOX2 activation, and inhibited the Drp1-dependent mitochondrial fission following OGD/R. These effects were further strengthened by the inhibition of ROS production. In subsequent experiments, U0126 and SB202190 inhibited the phosphorylation of PRDX6 at Thr177 and reduced PRDX6-iPLA2 activity. These results suggest that PRDX6-iPLA2 plays an important role in the astrocyte-induced generation of ROS and activation of microglia, which are regulated by the activation of Nox2 and Drp1-dependent mitochondrial fission pathways. Additionally, PRDX6-iPLA2 activity is regulated by MAPKs via the phosphorylation of PRDX6 at Thr177 in astrocytes. [ABSTRACT FROM AUTHOR]

Background: According to WHO 2020, CAD is the second leading cause of death in Indonesia with death cases reaching 259,297 or 15.33% of total deaths. Unfortunately, most of the patients of CAD in Indonesia did not match the golden period or decline to be treated with Percutaneous Coronary Intervention (PCI). Based on the recent study, there were increases in MMP-9, NOX2, and TGF-β1 in STEMI patients which contribute to cardiac remodeling. Moreover, there is controversy regarding the benefit of late PCI (12-48 hours after onset of STEMI) in stable patients. Lately, colchicine is widely used in cardiovascular disease. This study was conducted to explore the effect of colchicine to reduce MMP- 9, NOX2, and TGF-β1 levels after myocardial infarction in stable patients. Method: In this clinical trial study, we assessed 129 STEMI patients, about 102 patients who met inclusion criteria were randomized into four groups. Around 25 patients received late PCI (12–48 h after the onset of chest pain), optimal medical treatment (OMT) for STEMI, and colchicine; 24 patients received late PCI and OMT; 22 patients didn't get the revascularization (No Revas), OMT, and colchicine; and 31 patients received No Revas and OMT only. The laboratory test for MMP-9, NOX2, and TGF-β1 were tested in Day-1 and Day-5. The data were analyzed using Mann-Whitney. Results: A total of 102 patients with mean age of 56 ± 9.9, were assigned into four groups. The data analysis showed significant results within No Revas + OMT + Colchicine group versus No Revas + OMT + Placebo in MMP-9 (Day-1: p = 0.001; Day-5: p = 0.022), NOX2 (Day-1: p = 0.02; Day-5: p = 0.026), and TGF-β1 (Day-1: p = 0.00; Day-5: p = 0.00) with the less three markers in OMT + Colchicine group than OMT + Placebo group. There were no significant differences within the late PCI + OMT + colchicine group and PCI + OMT + Placebo group. Conclusions: Colchicine could significantly reduce MMP-9, NOX2, and TGF-β1 levels in stable STEMI patients. So that, colchicine could be a potential agent in STEMI patients and prevent cardiac remodeling events. [ABSTRACT FROM AUTHOR]

Background: Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infections syndrome (PANDAS) identifies patients with acute onset of obsessive-compulsive and tic disorders. The objective of this study was to evaluate serum NOX2 levels, as well as 8-iso-prostaglandin F2α (8-iso-PGF2α) and lipopolysaccharide (LPS) of PANDAS patients.Methods: In this study we wanted to compare serum levels of soluble NOX2-dp (sNOX-2-dp), iso-PGF2α and LPS in 60 consecutive subjects, including 30 children affected by PANDAS and 30 controls (CT) matched for age and gender. Serum zonulin was used as intestinal permeability assay.Results: Compared with CT, PANDAS children had increased serum levels of sNOX-2-dp, 8-iso-PGF2α and LPS. Bivariate analysis showed that serum sNOX2-dp was significantly correlated with LPS (Rs = 0.359; p = 0.005), zonulin (Rs = 0.444; p < 0.001) and 8-iso-PGF2α (Rs = 0.704; p < 0.001). Serum LPS significantly correlated with zonulin (Rs = 0.610; p < 0.001), and 8-iso-PGF2α (Rs = 0.591; p = 0.001). Finally, a multiple linear regression analysis showed that serum 8-iso-PGF2α and zonulin were the only independent variables associated with sNOX2-dp (R2 = 68%).Conclusion: This study shows that children affected by PANDAS have high circulating levels of sNOX2-dp, isoprostanes and of LPS that could be involved in the process of neuroinflammation. [ABSTRACT FROM AUTHOR]

Background: Microglia function is essential to maintain the brain homeostasis. Evidence shows that aged microglia are primed and show exaggerated response to acute inflammatory challenge. Systemic inflammation signals to the brain inducing changes that impact cognitive function. However, the mechanisms involved in age-related cognitive decline associated to episodic systemic inflammation are not completely understood. The aim of this study was to identify neuropathological features associated to age-related cognitive decline in a mouse model of episodic systemic inflammation.Methods: Young and aged Swiss mice were injected with low doses of LPS once a week for 6 weeks to induce episodic systemic inflammation. Sickness behavior, inflammatory markers, and neuroinflammation were assessed in different phases of systemic inflammation in young and aged mice. Behavior was evaluated long term after episodic systemic inflammation by open field, forced swimming, object recognition, and water maze tests.Results: Episodic systemic inflammation induced systemic inflammation and sickness behavior mainly in aged mice. Systemic inflammation induced depressive-like behavior in both young and aged mice. Memory and learning were significantly affected in aged mice that presented lower exploratory activity and deficits in episodic and spatial memories, compared to aged controls and to young after episodic systemic inflammation. Systemic inflammation induced acute microglia activation in young mice that returned to base levels long term after episodic systemic inflammation. Aged mice presented dystrophic microglia in the hippocampus and entorhinal cortex at basal level and did not change morphology in the acute response to SI. Regardless of their dystrophic microglia, aged mice produced higher levels of pro-inflammatory (IL-1β and IL-6) as well as pro-resolution (IL-10 and IL-4) cytokines in the brain. Also, higher levels of Nox2 expression, oxidized proteins and lower antioxidant defenses were found in the aged brains compared to the young after episodic systemic inflammation.Conclusions: Our data show that aged mice have increased susceptibility to episodic systemic inflammation. Aged mice that showed cognitive impairments also presented higher oxidative stress and abnormal production of cytokines in their brains. These results indicate that a neuroinflammation and oxidative stress are pathophysiological mechanisms of age-related cognitive impairments. [ABSTRACT FROM AUTHOR]

Background: Macrophages stand at the forefront of both innate and adapted immunity through their capacities to recognize, engulf, and eliminate foreign particles, and to stimulate adapted immune cells. They are also involved in controlling pro- and anti-inflammatory pathways. Macrophage activity against Mycobacterium tuberculosis (M. tuberculosis) has been shown to involve Toll-like receptor (TLR) activation and ROS production. Previous studies have shown that lipopolysaccharide (LPS), through TLR4, could activate macrophages, improve their bactericidal ROS production, and facilitate anti-infective immune responses. We sought to better understand the role of the TLR4-NOX2 axis in macrophage activation during M. tuberculosis infection.Methods: THP-1 macrophages and PMA primed THP-1 macrophages [THP-1(A)] were treated with LPS and infected by M. tuberculosis. Cells were analyzed by flow cytometry for TLR4 expression, ROS production, phagocytosis, and killing of M. tuberculosis. Western blotting was used to analyze NOX2 expression. Inhibitors of the TLR4-NOX2 pathway were used to assess this pathway's role in these processes, and their role in LPS activation of macrophages.Results: We found that THP1-derived macrophages or PMA primed THP-1 macrophages exhibit higher surface TLR4 levels and increased NOX2 expression levels following LPS treatment. M. tuberculosis infection reduced these levels, but LPS was able to limit the negative effects of M.tb. Additionally, LPS increases THP-1(A) cells' bactericidal activities including phagocytosis, ROS production, and destruction of M. tuberculosis. Significantly, all of these activities are impaired when TLR4 or NOX2 are inhibited.Conclusion: These studies demonstrate the importance of the TLR4-NOX2 axis in M. tuberculosis elimination by macrophages and may lead to novel therapies for tuberculosis and other bacterial infections. [ABSTRACT FROM AUTHOR]

Background: Spinal cord injury (SCI) among people over age 40 has been steadily increasing since the 1980s and is associated with worsened outcome than injuries in young people. Age-related increases in reactive oxygen species (ROS) are suggested to lead to chronic inflammation. The NADPH oxidase 2 (NOX2) enzyme is expressed by microglia and is a primary source of ROS. This study aimed to determine the effect of age on inflammation, oxidative damage, NOX2 gene expression, and functional performance with and without SCI in young adult (3 months) and middle-aged (12 months) male rats.Methods: Young adult and middle-aged rats were assessed in two groups-naïve and moderate contusion SCI. Functional recovery was determined by weekly assessment with the Basso, Beattie, and Breshnahan general motor score (analyzed two-way ANOVA) and footprint analysis (analyzed by Chi-square analysis). Tissue was analyzed for markers of oxidative damage (8-OHdG, Oxyblot, and 3-NT), microglial-related inflammation (Iba1), NOX2 component (p47PHOX, p22PHOX, and gp91PHOX), and inflammatory (CD86, CD206, TNFα, and NFκB) gene expression (all analyzed by unpaired Student's t test).Results: In both naïve and injured aged rats, compared to young rats, tissue analysis revealed significant increases in 8-OHdG and Iba1, as well as inflammatory and NOX2 component gene expression. Further, injured aged rats showed greater lesion volume rostral and caudal to the injury epicenter. Finally, injured aged rats showed significantly reduced Basso-Beattie-Bresnahan (BBB) scores and stride length after SCI.Conclusions: These results show that middle-aged rats demonstrate increased microglial activation, oxidative stress, and inflammatory gene expression, which may be related to elevated NOX2 expression, and contribute to worsened functional outcome following injury. These findings are essential to elucidating the mechanisms of age-related differences in response to SCI and developing age-appropriate therapeutics. [ABSTRACT FROM AUTHOR]

Background: Chronic microglia-mediated inflammation and oxidative stress are well-characterized underlying factors in neurodegenerative disease, whereby reactive inflammatory microglia enhance ROS production and impact neuronal integrity. Recently, it has been shown that during chronic inflammation, neuronal integrity is compromised through targeted disruption of the axon initial segment (AIS), the axonal domain critical for action potential initiation. AIS disruption was associated with contact by reactive inflammatory microglia which wrap around the AIS, increasing association with disease progression. While it is clear that chronic microglial inflammation and enhanced ROS production impact neuronal integrity, little is known about how acute microglial inflammation influences AIS stability. Here, we demonstrate that acute neuroinflammation induces AIS structural plasticity in a ROS-mediated and calpain-dependent manner.Methods: C57BL/6J and NOX2-/- mice were given a single injection of lipopolysaccharide (LPS; 5 mg/kg) or vehicle (0.9% saline, 10 mL/kg) and analyzed at 6 h-2 weeks post-injection. Anti-inflammatory Didox (250 mg/kg) or vehicle (0.9% saline, 10 mL/kg) was administered beginning 24 h post-LPS injection and continued for 5 days; animals were analyzed 1 week post-injection. Microglial inflammation was assessed using immunohistochemistry (IHC) and RT-qPCR, and AIS integrity was quantitatively analyzed using ankyrinG immunolabeling. Data were statistically compared by one-way or two-way ANOVA where mean differences were significant as assessed using Tukey's post hoc analysis.Results: LPS-induced neuroinflammation, characterized by enhanced microglial inflammation and increased expression of ROS-producing enzymes, altered AIS protein clustering. Importantly, inflammation-induced AIS changes were reversed following resolution of microglial inflammation. Modulation of the inflammatory response using anti-inflammatory Didox, even after significant AIS disruption occurred, increased the rate of AIS recovery. qPCR and IHC analysis revealed that expression of microglial NOX2, a ROS-producing enzyme, was significantly increased correlating with AIS disruption. Furthermore, ablation of NOX2 prevented inflammation-induced AIS plasticity, suggesting that ROS drive AIS structural plasticity.Conclusions: In the presence of acute microglial inflammation, the AIS undergoes an adaptive change that is capable of spontaneous recovery. Moreover, recovery can be therapeutically accelerated. Together, these findings underscore the dynamic capabilities of this domain in the presence of a pathological insult and provide evidence that the AIS is a viable therapeutic target. [ABSTRACT FROM AUTHOR]

Background: NADPH oxidase (NOX2) is an enzyme system that generates reactive oxygen species (ROS) in microglia and macrophages. Excessive ROS production is linked with neuroinflammation and chronic neurodegeneration following traumatic brain injury (TBI). Redox signaling regulates macrophage/microglial phenotypic responses (pro-inflammatory versus anti-inflammatory), and NOX2 inhibition following moderate-to-severe TBI markedly reduces pro-inflammatory activation of macrophages/microglia resulting in concomitant increases in anti-inflammatory responses. Here, we report the signaling pathways that regulate NOX2-dependent macrophage/microglial phenotype switching in the TBI brain.Methods: Bone marrow-derived macrophages (BMDMs) prepared from wildtype (C57Bl/6) and NOX2 deficient (NOX2-/-) mice were treated with lipopolysaccharide (LPS; 10 ng/ml), interleukin-4 (IL-4; 10 ng/ml), or combined LPS/IL-4 to investigate signal transduction pathways associated with macrophage activation using western immunoblotting and qPCR analyses. Signaling pathways and activation markers were evaluated in ipsilateral cortical tissue obtained from adult male wildtype and NOX2-/- mice that received moderate-level controlled cortical impact (CCI). A neutralizing anti-IL-10 approach was used to determine the effects of IL-10 on NOX2-dependent transitions from pro- to anti-inflammatory activation states.Results: Using an LPS/IL-4-stimulated BMDM model that mimics the mixed pro- and anti-inflammatory responses observed in the injured cortex, we show that NOX2-/- significantly reduces STAT1 signaling and markers of pro-inflammatory activation. In addition, NOX2-/- BMDMs significantly increase anti-inflammatory marker expression; IL-10-mediated STAT3 signaling, but not STAT6 signaling, appears to be critical in regulating this anti-inflammatory response. Following moderate-level CCI, IL-10 is significantly increased in microglia/macrophages in the injured cortex of NOX2-/- mice. These changes are associated with increased STAT3 activation, but not STAT6 activation, and a robust anti-inflammatory response. Neutralization of IL-10 in NOX2-/- BMDMs or CCI mice blocks STAT3 activation and the anti-inflammatory response, thereby demonstrating a critical role for IL-10 in regulating NOX2-dependent transitions between pro- and anti-inflammatory activation states.Conclusions: These studies indicate that following TBI NOX2 inhibition promotes a robust anti-inflammatory response in macrophages/microglia that is mediated by the IL-10/STAT3 signaling pathway. Thus, therapeutic interventions that inhibit macrophage/microglial NOX2 activity may improve TBI outcomes by not only limiting pro-inflammatory neurotoxic responses, but also enhancing IL-10-mediated anti-inflammatory responses that are neuroprotective. [ABSTRACT FROM AUTHOR]

Background: Nonalcoholic steatohepatitis (NASH) is characterized by fat accumulation in the hepatocyte, inflammation, liver cell injury, and varying degrees of fibrosis, and can lead to oxidative stress in liver. Here, we investigated whether Salidroside, a natural phenolic antioxidant product, can protect rat from liver injury during NASH. Methods: NASH model was established by feeding the male SD rats with high-fat and high-cholesterol diet for 14 weeks. Four groups of male SD rats including, normal diet control group, NASH model group, and Salidroside treatment group with150mg/kg and 300 mg/kg respectively, were studied. Salidroside was given by oral administration to NASH in rats from 9 weeks to 14 weeks. At the end of 14 weeks, liver and serum were harvested, and the liver injury, oxidative stress and histological features were evaluated. Results: NASH rats exhibited significant increases in the following parameters as compared to normal diet control rats: fat droplets with foci of inflammatory cell infiltration in the liver. ALT, AST in serum and TG, TC in hepatocyte elevated. Oxidative responsive genes including CYP2E1 and Nox2 increased. Additionally, NASH model decreased antioxidant enzymes SOD, GSH, GPX, and CAT in the liver due to their rapid depletion after battling against oxidative stress. Compared to NASH model group, treatment rats with Salidroside effectively reduced lipid accumulation, inhibited liver injury in a does-dependent manner. Salidroside treatment restored antioxidant enzyme levels, inhibited expression of CYP2E1 and Nox2 mRNA in liver, which prevented the initial step of generating free radicals from NASH. Conclusion: The data presented here show that oral administration of Salidroside prevented liver injury in the NASH model, likely through exerting antioxidant actions to suppress oxidative stress and the free radical-generating CYP2E1 enzyme, Nox2 in liver. [ABSTRACT FROM AUTHOR]

Background: Reactive oxygen species (ROS) protect the host against a large number of pathogenic microorganisms. ROS have different effects on parasites of the genus Leishmania: some parasites are susceptible to their action, while others seem to be resistant. The role of ROS in L. amazonensis infection in vivo has not been addressed to date. Methods: In this study, C57BL/6 wild-type mice (WT) and mice genetically deficient in ROS production by phagocytes (gp91phox-/-) were infected with metacyclic promastigotes of L. amazonensis to address the effect of ROS in parasite control. Inflammatory cytokines, parasite loads and myeloperoxidase (MPO) activity were evaluated. In parallel, in vitro infection of peritoneal macrophages was assessed to determine parasite killing, cytokine, NO and ROS production. Results: In vitro results show induction of ROS production by infected peritoneal macrophages, but no effect in parasite killing. Also, ROS do not seem to be important to parasite killing in vivo, but they control lesion sizes at early stages of infection. IFN-γ, TNF-α and IL-10 production did not differ among mouse strains. Myeloperoxidase assay showed augmented neutrophils influx 6 h and 72 h post - infection in gp91phox-/- mice, indicating a larger inflammatory response in gp91phox-/- even at early time points. At later time points, neutrophil numbers in lesions correlated with lesion size: larger lesions in gp91phox-/- at earlier times of infection corresponded to larger neutrophil infiltrates, while larger lesions in WT mice at the later points of infection also displayed larger numbers of neutrophils. Conclusion: ROS do not seem to be important in L. amazonensis killing, but they regulate the inflammatory response probably by controlling neutrophils numbers in lesions. [ABSTRACT FROM AUTHOR]

Background: The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in intracerebral hemorrhage (ICH)-induced inflammatory injury, and the purinergic 2X7 receptor (P2X7R) is upstream of NLRP3 activation. This study aimed to investigate how P2X7R functions in ICH-induced inflammatory injury and how the receptor interacts with the NLRP3 inflammasome. Methods: Rats were treated with P2X7R small interfering RNA (siRNA) 24 h before undergoing collagenase-induced ICH. A selective P2X7R inhibitor (blue brilliant G, BBG) or a peroxynitrite (ONOO(-)) decomposition catalyst (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) [FeTPPS]) was injected 30 min after ICH. Brain water content, hemorrhagic lesion volume, and neurological deficits were evaluated, and western blot, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were carried out. Results: Striatal P2X7R and NLRP3 inflammasomes were activated after ICH. Gene silencing of P2X7R suppressed NLRP3 inflammasome activation and interleukin (IL)-1β/IL-18 release and significantly ameliorated brain edema and neurological deficits. Additionally, enhanced NADPH oxidase 2 (NOX2, gp91(phox)) and inducible nitric oxide synthase (iNOS), as well as their cytotoxic product (ONOO(-)) were markedly attenuated by BBG treatment following ICH. This was accompanied by downregulations of the inflammasome components, IL-1β/IL-18 and myeloperoxidase (MPO, a neutrophil marker). Most importantly, inflammasome activation and IL-1β/IL-18 release were significantly inhibited by ONOO(-) decomposition with FeTPPS. Conclusions: Our findings implicate that P2X7R exacerbated inflammatory progression and brain damage in ICH rats possibly via NLRP3 inflammasome-dependent IL-1β/IL-18 release and neutrophil infiltration. ONOO(-), a potential downstream signaling molecule of P2X7R, may play a critical role in triggering NLRP3 inflammasome activation. [ABSTRACT FROM AUTHOR]

Background: Spinal cord injury (SCI) results in the activation of the NADPH oxidase (NOX) enzyme, inducing production of reactive oxygen species (ROS). We hypothesized that the NOX2 isoform plays an integral role in post-SCI inflammation and functional deficits.Methods: Moderate spinal cord contusion injury was performed in adult male mice, and flow cytometry, western blot, and immunohistochemistry were used to assess NOX2 activity and expression, inflammation, and M1/M2 microglia/macrophage polarization from 1 to 28 days after injury. The NOX2-specific inhibitor, gp91ds-tat, was injected into the intrathecal space immediately after impact. The Basso Mouse Scale (BMS) was used to assess locomotor function at 24 h post-injury and weekly thereafter.Results: Our findings show that gp91ds-tat treatment significantly improved functional recovery through 28 days post-injury and reduced inflammatory cell concentrations in the injured spinal cord at 24 h and 7 days post-injury. In addition, a number of oxidative stress markers were reduced in expression at 24 h after gp91ds-tat treatment, which was accompanied by a reduction in M1 polarization marker expression.Conclusion: Based on our findings, we now conclude that inhibition of NOX2 significantly improves outcome after SCI, most likely via acute reductions in oxidative stress and inflammation. NOX2 inhibition may therefore have true potential as a therapy after SCI. [ABSTRACT FROM AUTHOR]

Background: Although matrix metalloproteinase (MMP)-7 expression is correlated with increased metastatic potential in human colon cancer cells, the underlying molecular mechanism of invasive phenotype remains unknown. In the current study, we investigated the regulatory effects of membrane NADPH oxidase (NOX) and AMP activated protein kinase (AMPK) on MMP-7 expression and invasive phenotype change in colon cancer cells. Methods: Production of superoxide anion was measured by lucigenin chemiluminescence assay using whole cells and protein extracts (NADPH oxidase activity), and intracellular reactive oxygen species (ROS) by fluorescence microscopy using 2',7'-dichlorofluorescein diacetate (DCF-DA). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to measure mRNA and protein levels, respectively. siRNA transfection was used to assess involvement of genes in cancer invasion, which were identified by Matrigel transwell invasion assay. Luciferase reporter assay was performed to identify transcription factors linked to gene expression. Results: Under basal conditions, less invasive human colon cancer cells (HT29 and Caco-2) showed low MMP-7 expression but high NOX1 expression and AMPK phosphorylation. Treatment of HT29 and Caco-2 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced an invasive phenotype response along with corresponding increases in ROS production and NOX2 and MMP-7 expression as well as reduced AMPK phosphorylation, which resemble basal conditions of highly invasive human colon cancer cells (SW620 and HCT116). In addition, inverse regulation between AMPK phosphorylation and NOX2 and MMP-7 expression was observed in HT29 cells treated with different concentrations of exogenous hydrogen peroxide. TPA-induced invasive phenotype in HT29 cells was abolished by treatment with Vit. E, DPI, apocynin, and NOX2 siRNA but not NOX1 siRNA, indicating NOX2-derived ROS production induced an invasive phenotype. TPA-induced induction of MMP-7 expression was suppressed by AP-1, NF-κB, and MAPK (ERK, p38, and JNK) inhibitors, whereas TPA-induced expression of NOX2 and its regulators, p47phox and p67phox, was blocked by p38 and NF-κB inhibitors. Conclusions: Molecular switch from NOX1 to NOX2 in colon cancer cells induces ROS production and subsequently enhances MMP-7 expression by deactivating AMPK, which otherwise inhibits stimulus-induced autoregulation of ROS and NOX2 gene expression. [ABSTRACT FROM AUTHOR]

Background In the last decade, reactive oxygen species (ROS) production has been shown to occur upon T-cell receptor (TCR) stimulation and to affect TCR-mediated signalling. However, the exact reactive species that are produced, how ROS are generated and their requirement for T-cell activation, proliferation or cytokine production remain unclear, especially in the case of primary human T cells. Moreover, several groups have questioned that ROS are produced upon TCR stimulation. Results To shed some light onto this issue, we specifically measured superoxide production upon TCR ligation in primary human and mouse T lymphocytes. We showed that superoxide is indeed produced and released into the extracellular space. Antioxidants, such as superoxide dismutase and ascorbate, abolished superoxide production, but surprisingly did not affect activation, proliferation and cytokine secretion in TCR-stimulated primary human T cells. It has been suggested that T cells produce ROS via the NADPH oxidase 2 (NOX2). Therefore, we investigated whether T-cell activation is affected in NOX2-deficient mice (gp91phox -/-). We found that T cells from these mice completely lack inducible superoxide production but display normal upregulation of activation markers and proliferation. Conclusions Collectively, our data indicate that primary T cells produce extracellular superoxide upon TCR triggering, potentially via NOX2 at the plasma membrane. However, superoxide is not required for T-cell activation, proliferation and cytokine production. [ABSTRACT FROM AUTHOR]

Background Human brain microvascular endothelial cells (hBMVECs) are integral part of the blood brain barrier. Post-translational modifications of adherens junction proteins regulate the permeability of human brain microvascular endothelial cells. Pro-inflammatory signals can induce tyrosine phosphorylation of adherens junction proteins. The primary objective of this work is to provide a molecular model; how the HIV-1 Tat protein can compromise the BBB integrity and eventually lead to neurological consequences. We exposed hBMVECs to recombinant HIV-1 clade C Tat protein to study the effect of HIV-1 Tat C on permeability of hBMVECs. Trans-endothelial electrical resistance and fluorescent dye migration assay have been used to check the permeability of hBMVECs. DCFDA staining has been used for intracellular reactive oxygen species (ROS) detection. Western blotting has been used to study the expression levels and co-immunoprecipitation has been used to study the interactions among adherens junction proteins. Results HIV-1 Tat C protein induced NOX2 and NOX4 expression level and increased intracellular ROS level. Redox-sensitive kinase; PYK2 activation led to increased tyrosine phosphorylation of VE-cadherin and β-catenin, leading to disruption of junctional assembly. The dissociation of tyrosine phosphatases VE-PTP and SHP2 from cadherin complex resulted into increased tyrosine phosphorylation of VE-cadherin and β-catenin in HIV-1 Tat C treated hBMVECs. Conclusion Unrestricted phosphorylation of junctional proteins in hBMVECs, in response to HIV-1 Tat C protein; leads to the disruption of junctional complexes and increased endothelial permeability. [ABSTRACT FROM AUTHOR]

Background: Although the etiology of PD remains unclear, increasing evidence has shown that oxidative stress plays an important role in its pathogenesis and that of other neurodegenerative disorders. NOX2, a cytochrome subunit of NOX, transports electrons across the plasma membrane to generate ROS, leading to physiological and pathological processes. Heme oxygenase-1 (HO-1) can be rapidly induced by oxidative stress and other noxious stimuli in the brain or other tissues. As taxanthin (ATX), a carotenoid with antioxidant properties, is 100-1000 times more effective than vitamin E. The present study investigated the neuro protective effects of ATX on MPP+-induced oxidative stress in PC12 cells. Results: MPP+ significantly decreased MTT levels in a concentration-dependent manner. Hemin, SnPPIX and ATX didn't exhibit any cyto toxic effects on PC12 cells. Pretreatment with ATX (5, 10, 20 ?M), caused intracellular ROS production in the MPP+ group to decrease by 13.06%, 22.13%, and 27.86%, respectively. MPP+ increased NOX2,NRF2 and HO-1 protein expression compared with control (p < 0.05). Co-treatment with hemin or ATX suppressed NOX2 expression (p < 0.01), and greatly increased NRF2 and HO-1 expression (p < 0.01). MPP+ treatment up-regulated both NOX2 (p < 0.01) and HO-1 (p < 0.01) mRNA levels. Co-treatment with hemin or ATX significantly increased HO-1 mRNA levels (p < 0.01), and decreased NOX2 mRNA levels (p < 0.01). MPP+ increased NOX2 and HO-1 expression with considerable fluorescence extending out from the peri uclear region toward the periphery;this was attenuated by DPI. Co-treatment with hemin or ATX significantly up-regulated HO-1 expression and decreased NOX2 expression with considerable fluorescence intensity (stronger than the control and MPP+ groups). Conclusions: ATX suppresses MPP+ -induced oxidative stress in PC12 cells via the HO-1/NOX2 axis. ATX should be strongly considered as a potential neuroprotectant and adjuvant therapy for patients with Parkinson's disease. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is characterized by progressive memory loss and cognitive impairment. The aggregation of amyloid β (Aβ) and hyperphosphorylated tau protein are two major pathological features of AD. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, NOX) has been indicated in Aβ pathology; however, whether and how it affects tau pathology are not yet clear. Methods: The role of NOX2 in cognitive function, amyloid plaque formation, and tau hyperphosphorylation were examined in APP/PS1 transgenic mice mated with p47phox-deficient mice (with deletion of the gene of neutrophil cytosolic factor 1, Ncf1) and/or in p47phox-deficient mice receiving intracerebroventricular (ICV) injection of streptozotocin (STZ). The cognitive and non-cognitive functions in these mice were assessed by Morris water maze, Rotarod test, open field, and elevated plus maze. Aβ levels, amyloid plaques, p47phox expression, and astrocyte activation were evaluated using immunofluorescence staining, ELISA, and/or Western blotting. Cultured primary neuronal cells were treated with okadaic acid or conditioned media (CM) from high glucose-stimulated primary astrocytes. The alteration in tau pathology was determined using Western blotting and immunofluorescence staining. Results: Deletion of the gene coding for p47phox, the organizer subunit of NOX2, significantly attenuated cognitive impairment and tau pathology in these mice. p47phox deficiency decreased the activation of astrocytes but had no effect on Aβ levels and amyloid plaque formation in the brains of aged APP/PS1 mice, which displayed markedly increased expression of p47phox in neurons and astrocytes. Cell culture studies found that neuronal p47phox deletion attenuated okadaic acid-induced tau hyperphosphorylation at specific sites in primary cultures of neurons. CM from high glucose-treated WT astrocytes increased tau hyperphosphorylation in primary neurons, whereas this effect was absent from p47phox-deficient astrocytes. Conclusions: These results suggest that p47phox is associated with cognitive function and tau pathology in AD. p47phox expressed in neurons contributes to tau hyperphosphorylation directly, while p47phox in astrocytes affect tau hyperphosphorylation by activating astrocytes indirectly. Our results provide new insights into the role of NOX2 in AD and indicate that targeted inhibition of p47phox may be a new strategy for the treatment of AD. [ABSTRACT FROM AUTHOR]

Background: Macrophages stand at the forefront of both innate and adapted immunity through their capacities to recognize, engulf, and eliminate foreign particles, and to stimulate adapted immune cells. They are also involved in controlling pro- and anti-inflammatory pathways. Macrophage activity against Mycobacterium tuberculosis (M. tuberculosis) has been shown to involve Toll-like receptor (TLR) activation and ROS production. Previous studies have shown that lipopolysaccharide (LPS), through TLR4, could activate macrophages, improve their bactericidal ROS production, and facilitate anti-infective immune responses. We sought to better understand the role of the TLR4-NOX2 axis in macrophage activation during M. tuberculosis infection., Methods: THP-1 macrophages and PMA primed THP-1 macrophages [THP-1(A)] were treated with LPS and infected by M. tuberculosis. Cells were analyzed by flow cytometry for TLR4 expression, ROS production, phagocytosis, and killing of M. tuberculosis. Western blotting was used to analyze NOX2 expression. Inhibitors of the TLR4-NOX2 pathway were used to assess this pathway's role in these processes, and their role in LPS activation of macrophages., Results: We found that THP1-derived macrophages or PMA primed THP-1 macrophages exhibit higher surface TLR4 levels and increased NOX2 expression levels following LPS treatment. M. tuberculosis infection reduced these levels, but LPS was able to limit the negative effects of M.tb. Additionally, LPS increases THP-1(A) cells' bactericidal activities including phagocytosis, ROS production, and destruction of M. tuberculosis. Significantly, all of these activities are impaired when TLR4 or NOX2 are inhibited., Conclusion: These studies demonstrate the importance of the TLR4-NOX2 axis in M. tuberculosis elimination by macrophages and may lead to novel therapies for tuberculosis and other bacterial infections.

Background: Nonalcoholic steatohepatitis (NASH) is characterized by fat accumulation in the hepatocyte, inflammation, liver cell injury, and varying degrees of fibrosis, and can lead to oxidative stress in liver. Here, we investigated whether Salidroside, a natural phenolic antioxidant product, can protect rat from liver injury during NASH., Methods: NASH model was established by feeding the male SD rats with high-fat and high-cholesterol diet for 14 weeks. Four groups of male SD rats including, normal diet control group, NASH model group, and Salidroside treatment group with150mg/kg and 300 mg/kg respectively, were studied. Salidroside was given by oral administration to NASH in rats from 9 weeks to 14 weeks. At the end of 14 weeks, liver and serum were harvested, and the liver injury, oxidative stress and histological features were evaluated., Results: NASH rats exhibited significant increases in the following parameters as compared to normal diet control rats: fat droplets with foci of inflammatory cell infiltration in the liver. ALT, AST in serum and TG, TC in hepatocyte elevated. Oxidative responsive genes including CYP2E1 and Nox2 increased. Additionally, NASH model decreased antioxidant enzymes SOD, GSH, GPX, and CAT in the liver due to their rapid depletion after battling against oxidative stress. Compared to NASH model group, treatment rats with Salidroside effectively reduced lipid accumulation, inhibited liver injury in a does-dependent manner. Salidroside treatment restored antioxidant enzyme levels, inhibited expression of CYP2E1 and Nox2 mRNA in liver, which prevented the initial step of generating free radicals from NASH., Conclusion: The data presented here show that oral administration of Salidroside prevented liver injury in the NASH model, likely through exerting antioxidant actions to suppress oxidative stress and the free radical-generating CYP2E1 enzyme, Nox2 in liver.

Background: Reactive oxygen species (ROS) protect the host against a large number of pathogenic microorganisms. ROS have different effects on parasites of the genus Leishmania: some parasites are susceptible to their action, while others seem to be resistant. The role of ROS in L. amazonensis infection in vivo has not been addressed to date., Methods: In this study, C57BL/6 wild-type mice (WT) and mice genetically deficient in ROS production by phagocytes (gp91(phox-/-)) were infected with metacyclic promastigotes of L. amazonensis to address the effect of ROS in parasite control. Inflammatory cytokines, parasite loads and myeloperoxidase (MPO) activity were evaluated. In parallel, in vitro infection of peritoneal macrophages was assessed to determine parasite killing, cytokine, NO and ROS production., Results: In vitro results show induction of ROS production by infected peritoneal macrophages, but no effect in parasite killing. Also, ROS do not seem to be important to parasite killing in vivo, but they control lesion sizes at early stages of infection. IFN-γ, TNF-α and IL-10 production did not differ among mouse strains. Myeloperoxidase assay showed augmented neutrophils influx 6 h and 72 h post - infection in gp91(phox-/-) mice, indicating a larger inflammatory response in gp91(phox-/-) even at early time points. At later time points, neutrophil numbers in lesions correlated with lesion size: larger lesions in gp91(phox-/-) at earlier times of infection corresponded to larger neutrophil infiltrates, while larger lesions in WT mice at the later points of infection also displayed larger numbers of neutrophils., Conclusion: ROS do not seem to be important in L. amazonensis killing, but they regulate the inflammatory response probably by controlling neutrophils numbers in lesions.