Nox Activity Research Articles

Abstract 2098: A Novel Mechanism Of Focal Adhesion Kinase Activation By Integrins And Its Role In Outside-in Signaling In Platelets

Ligand binding to integrin αIIbβ3 mediates platelet adhesion/aggregation and transmits outside-in signaling, leading to platelet spreading, granule secretion, and significant expansion of platelet thrombi resulting in thrombosis. Focal Adhesion Kinase (FAK) has been recognized in many cells to play an important role during integrin outside-in signaling. Whereas FAK activation downstream of integrins is characterized by autophosphorylation of FAK at Y 397 followed by Y 576/7 phosphorylation, it is not clear how FAK phosphorylation and activation is regulated and how FAK play roles in outside-in signaling in platelets. Here we show that reactive oxygen species (ROS) plays a key role in integrin-dependent stimulation of FAK phosphorylation and activation and ROS-stimulated FAK activation is downstream of the Gα13-integrin interaction, NOX2 activation, and the Src signaling pathway. Furthermore, FAK, following ROS-dependent activation, is important for the integrin outside-in signaling-mediated activation of Syk and immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway. We further show that FAK is important in amplification of platelet activation and aggregation as well as in vivo thrombosis. Thus, we have identified a novel mechanism of FAK activation and a key role of FAK in integrin outside-in signaling and integrin-dependent ITAM pathway activation, leading to amplification of platelet activation, aggregation, and thrombosis.

Revealing the roles of Zr on enhanced H2-SCR performances on Pt/TiO2 catalyst

The development of Hydrogen Internal Combustion Engine (H2-ICEs) to replace conventional fuel engines is expected to be an effective way to reduce CO2 emissions, but some NOx emitted from the H2-ICEs still should be eliminated by the selective catalytic reduction of NOx with H2 (H2-SCR). Large amount of platinum oxide existed on Pt/TiO2 leads to its low H2-SCR activity. In contrast, the Zr-modified Pt/TiO2 catalysts exhibit a higher NOx conversion and N2 selectivity in a wider temperature window (100–180 °C) than those of Pt/TiO2. The appropriately weakened strong interaction between Pt and TiO2 by Zr induces more Pt0 species, which are active sites for the activation of NOx and H2 in H2-SCR reaction. More oxygen vacancies caused by the formation of Zr-Ov-Ti3+ bands involve in the activation of NOx and the adsorption of nitrates (NOx−) species. Furthermore, in situ Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTs) demonstrates that Pt/ZrxTi1-x catalysts possess more adsorbed NOx− species and a faster reduction rate for them. Monodentate and bridged nitrates are considered to be more reactive with H2 than bidentate nitrates at relatively low temperatures. This work provides directions for the development of high-performance H2-SCR catalysts and the modulation of the conversion temperature.

Do oral and gut microbiota communicate through redox pathways? A novel asset of the nitrate-nitrite-NO pathway.

Nitrate may act as a regulator of •NO bioavailability via sequential reduction along the nitrate-nitrite-NO pathway with widespread health benefits, including a eubiotic effect on the oral and gut microbiota. Here, we discuss the molecular mechanisms of microbiota-host communication through redox pathways, via the production of •NO and oxidants by the family of NADPH oxidases, namely hydrogen peroxide (via Duox2), superoxide radical (via Nox1 and Nox2) and peroxynitrite, which leads to downstream activation of stress responses (Nrf2 and NFkB pathways) in the host mucosa. The activation of Nox2 by microbial metabolites is also discussed. Finally, we propose a new perspective in which both oral and gut microbiota communicate through redox pathways, with nitrate as the pivot linking both ecosystems.

NADPH oxidase exerts a B cell-intrinsic contribution to lupus risk by modulating endosomal TLR signals.

Genome-wide association studies in systemic lupus erythematosus (SLE) have linked loss-of-function mutations in phagocytic NADPH oxidase complex (NOX2) genes, including NCF1 and NCF2, to disease pathogenesis. The prevailing model holds that reduced NOX2 activity promotes SLE via defective efferocytosis, the immunologically silent clearance of apoptotic cells. Here, we describe a parallel B cell-intrinsic mechanism contributing to breaks in tolerance. In keeping with an important role for B cell Toll-like receptor (TLR) pathways in lupus pathogenesis, NOX2-deficient B cells exhibit enhanced signaling downstream of endosomal TLRs, increased humoral responses to nucleic acid-containing antigens, and the propensity toward humoral autoimmunity. Mechanistically, TLR-dependent NOX2 activation promotes LC3-mediated maturation of TLR-containing endosomes, resulting in signal termination. CRISPR-mediated disruption of NCF1 confirmed a direct role for NOX2 in regulating endosomal TLR signaling in primary human B cells. Together, these data highlight a new B cell-specific mechanism contributing to autoimmune risk in NCF1 and NCF2 variant carriers.

Female rats present higher oxidative damage and inflammation during goitrogenesis.

Thyroid disorders affect more women than men, but the underlying mechanisms contributing to this disparity remain incompletely understood. Thyrotropin (TSH), the primary regulator of thyroid oxidative hormonogenesis, has been implicated as a risk factor for proliferative thyroid diseases and a predictor of malignancy. In this study, we aimed to evaluate the impact of sustained elevated TSH levels on thyroid redox homeostasis, inflammatory markers, and DNA damage response in both male and female rats. Rats were treated with methimazole for 7 or 21 days, and hormonal measurements were conducted. H2O2 levels were evaluated in thyroid membrane fractions, while enzymatic activities were assessed in total thyroid homogenates. Sex-specific differences emerged, with females displaying higher reactive oxygen species levels - increased transiently NOX and sustained DUOX activities. Lipid peroxidation marker 4-hydroxynonenal (4-HNE) was elevated in females at both time points, contrasting with males just at 21 days. Sexual dimorphism was observed in DNA damage response, with females showing higher γH2AX levels at 21 days. Elevated IL-1β, TNF-α, CD11b mRNA, and phospho-NF-κB levels at 7 days indicated a distinct inflammatory profile in females. Notably, both sexes exhibited upregulated antioxidant enzymes. Our data suggest that females are more susceptible to oxidative damage and inflammation in our goiter model, which may be associated with higher ROS production and a less-efficient antioxidant defense system. These findings provide insights into the sex-specific mechanisms underlying thyroid dysfunction and highlight the importance of considering sex disparities in thyroid disorder research.

Knockdown of ANGPTL2 promotes left ventricular systolic dysfunction by upregulation of NOX4 in mice.

Background: Angiopoietin-like 2 (ANGPTL2) is a pro-inflammatory and pro-oxidant circulating protein that predicts and promotes chronic inflammatory diseases such as atherosclerosis in humans. Transgenic murine models demonstrated the deleterious role of ANGPTL2 in vascular diseases, while deletion of ANGPTL2 was protective. The nature of its role in cardiac tissues is, however, less clear. Indeed, in adult mice knocked down (KD) for ANGPTL2, we recently reported a mild left ventricular (LV) dysfunction originating from a congenital aortic valve stenosis, demonstrating that ANGPTL2 is essential to cardiac development and function. Hypothesis: Because we originally demonstrated that the KD of ANGPTL2 protected vascular endothelial function via an upregulation of arterial NOX4, promoting the beneficial production of dilatory H2O2, we tested the hypothesis that increased cardiac NOX4 could negatively affect cardiac redox and remodeling and contribute to LV dysfunction observed in adult Angptl2-KD mice. Methods and results: Cardiac expression and activity of NOX4 were higher in KD mice, promoting higher levels of cardiac H2O2 when compared to wild-type (WT) mice. Immunofluorescence showed that ANGPTL2 and NOX4 were co-expressed in cardiac cells from WT mice and both proteins co-immunoprecipitated in HEK293 cells, suggesting that ANGPTL2 and NOX4 physically interact. Pressure overload induced by transverse aortic constriction surgery (TAC) promoted LV systolic dysfunction in WT mice but did not further exacerbate the dysfunction in KD mice. Importantly, the severity of LV systolic dysfunction in KD mice (TAC and control SHAM) correlated with cardiac Nox4 expression. Injection of an adeno-associated virus (AAV9) delivering shRNA targeting cardiac Nox4 expression fully reversed LV systolic dysfunction in KD-SHAM mice, demonstrating the causal role of NOX4 in cardiac dysfunction in KD mice. Targeting cardiac Nox4 expression in KD mice also induced an antioxidant response characterized by increased expression of NRF2/KEAP1 and catalase. Conclusion: Together, these data reveal that the absence of ANGPTL2 induces an upregulation of cardiac NOX4 that contributes to oxidative stress and LV dysfunction. By interacting and repressing cardiac NOX4, ANGPTL2 could play a new beneficial role in the maintenance of cardiac redox homeostasis and function.

Highly active and stable VOx/TiO2 nanosheets for low-temperature NH3-SCR of NO: Structure-directing role of support

Low-temperature NH3-SCR over conventional catalysts based on V2O5/TiO2 is significantly limited by the narrow range of operating temperatures and poor activity at low temperatures. Herein, a novel catalyst consisting of defective TiO2 nanosheets supported vanadia (V/TNS) is successfully developed for the low-temperature NH3-SCR of NO. The V/TNS catalyst demonstrated excellent SCR performance, achieving over 95 % conversion with high N2 selectivity (>90 %) over a wide temperature range of 140–380 °C. Importantly, V/TNS was highly resistant to H2O and SO2 poisoning during 30 h of continuous operation, maintaining an NO conversion efficiency that exceeded 92 % at 180 °C. In contrast, a high-surface-area anatase TiO2 particle-supported catalyst (V/TiO2(A)) suffered from catalytic deactivation. Detailed characterization revealed that the TNS support, with its abundant surface defects and high surface area, improved vanadia dispersion and facilitated the formation of surface V4+ species and oxygen vacancies. These factors synergistically enhanced the adsorption and activation of NH3 and NOx, boosting catalytic performance. Moreover, the presence of SO2 had a notable inhibitory effect on the adsorption and activation of NH3 and NOx and their reactions on V/TiO2(A), while these detrimental effects were considerably mitigated on V/TNS.

Nrf2-mediated redox balance alleviates LPS-induced vascular endothelial cell inflammation by inhibiting endothelial cell ferroptosis

Ferroptosis plays an important role in inflammation and oxidative stress. Whether ferroptosis is involved in the inflammation of vascular endothelial cells and its regulation mechanism remains unclear. We estimated the correlation between serum iron ion levels and the inflammation index of 33 patients with arteriosclerosis. In vitro, HUVECs with or without ferrostatin-1 were exposed to Lipopolysaccharide. Corresponding cell models to verify the target signaling pathway. The results showed that serum iron ion levels had a significant positive correlation with N ratio, N/L, LDL level, and LDL/HDL (P < 0.05), and a negative correlation with L ratio (P < 0.05) in the arteriosclerosis patients. In vitro, ferroptosis is involved in HUVECs inflammation. Ferrostatin-1 can rescue LPS-induced HUVECs inflammation by decreasing HMGB1/IL-6/TNF-α expression. Nrf2 high expression could protect HUVECs against ferroptosis by activating the GPX4/GSH system, inhibiting ferritinophagy, and alleviating inflammation in HUVECs by inhibiting HMGB1/IL-6/TNF-α expression. It also found that Nrf2 is a key adaptive regulatory factor in the oxidative damage of HUVECs induced by NOX4 activation. These findings indicated that ferroptosis contributed to the pathogenesis of vascular endothelial cell damage by mediating endothelial cell inflammation. Nrf2-mediated redox balance in vascular inflammation may be a therapeutic strategy in vascular diseases.

Role of Oxygen and Its Radicals in Peripheral Nerve Regeneration: From Hypoxia to Physoxia to Hyperoxia.

Oxygen is compulsory for mitochondrial function and energy supply, but it has numerous more nuanced roles. The different roles of oxygen in peripheral nerve regeneration range from energy supply, inflammation, phagocytosis, and oxidative cell destruction in the context of reperfusion injury to crucial redox signaling cascades that are necessary for effective axonal outgrowth. A fine balance between reactive oxygen species production and antioxidant activity draws the line between physiological and pathological nerve regeneration. There is compelling evidence that redox signaling mediated by the Nox family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases plays an important role in peripheral nerve regeneration. Further research is needed to better characterize the role of Nox in physiological and pathological circumstances, but the available data suggest that the modulation of Nox activity fosters great therapeutic potential. One of the promising approaches to enhance nerve regeneration by modulating the redox environment is hyperbaric oxygen therapy. In this review, we highlight the influence of various oxygenation states, i.e., hypoxia, physoxia, and hyperoxia, on peripheral nerve repair and regeneration. We summarize the currently available data and knowledge on the effectiveness of using hyperbaric oxygen therapy to treat nerve injuries and discuss future directions.

Structural basis of human NOX5 activation

Structural basis of human NOX5 activation

PRDX6-iPLA2 aggravates neuroinflammation after ischemic stroke via regulating astrocytes-induced M1 microglia

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.

Inhibition of NOX2 or NLRP3 inflammasome prevents cardiac remote ischemic preconditioning.

Introduction: Short episodes of ischemia-reperfusion (IR) in the heart (classical ischemic preconditioning, IPC) or in a limb (remote ischemic preconditioning, RIPC) before a prolonged ischemic episode, reduce the size of the infarct. It is unknown whether IPC and RIPC share common mechanisms of protection. Animals KO for NOX2, a superoxide-producing enzyme, or KO for NLRP3, a protein component of inflammasome, are not protected by IPC. The aim of this study was to investigate if NOX2 or NLRP3 inflammasome are involved in the protection induced by RIPC. Methods: We preconditioned rats using 4 × 5min periods of IR in the limb with or without a NOX2 inhibitor (apocynin) or an NLRP3 inhibitor (Bay117082). In isolated hearts, we measured the infarct size after 30min of ischemia and 60min of reperfusion. In hearts from preconditioned rats we measured the activity of NOX2; the mRNA of Nrf2, gamma-glutamylcysteine ligase, glutathione dehydrogenase, thioredoxin reductase and sulfiredoxin by RT-qPCR; the content of glutathione; the activation of the NLRP3 inflammasome and the content of IL-1β and IL-10 in cardiac tissue. In exosomes isolated from plasma, we quantified NOX2 activity. Results: The infarct size after IR decreased from 40% in controls to 9% of the heart volume after RIPC. This protective effect was lost in the presence of both inhibitors. RIPC increased NOX2 activity in the heart and exosomes, as indicated by the increased association of p47phox to the membrane and by the increased oxidation rate of NADPH. RIPC also increased the mRNA of Nrf2 and antioxidant enzymes. Also, RIPC increased the content of glutathione and the GSH/GSSG ratio. The inflammasome proteins NLRP3, procaspase-1, and caspase-1 were all increased in the hearts of RIPC rats. At the end of RIPC protocol, IL-1β increased in plasma but decreased in cardiac tissue. At the same time, IL-10 did not change in cardiac tissue but increased by 70% during the next 50min of perfusion. Conclusion: RIPC activates NOX2 which upregulates the heart's antioxidant defenses and activates the NLRP3 inflammasome which stimulates a cardiac anti-inflammatory response. These changes may underlie the decrease in the infarct size induced by RIPC.

Co3O4 as an efficient passive NOx adsorber for emission control during cold-start of diesel engines

The Co3O4 nanoparticles, dominated by a catalytically active (110) lattice plane, were synthesized as a low-temperature NOx adsorbent to control the cold start emissions from vehicles. These nanoparticles boast a substantial quantity of active chemisorbed oxygen and lattice oxygen, which exhibited a NOx uptake capacity commensurate with Pd/SSZ-13 at 100 °C. The primary NOx release temperature falls within a temperature range of 200-350 °C, making it perfectly suitable for diesel engines. The characterization results demonstrate that chemisorbed oxygen facilitate nitro/nitrites intermediates formation, contributing to the NOx storage at 100 °C, while the nitrites begin to decompose within the 150-200 ºC range. Fortunately, lattice oxygen likely becomes involved in the activation of nitrites into more stable nitrate within this particular temperature range. The concurrent processes of nitrites decomposition and its conversion to nitrates results in a minimal NOx release between the temperatures of 150–200 °C. The nitrate formed via lattice oxygen mainly induces the NOx to be released as NO2 within a temperature range of 200–350 °C, which is advantageous in enhancing the NOx activity of downstream NH3-SCR catalysts, by boosting the fast SCR reaction pathway. Thanks to its low cost, considerable NOx absorption capacity, and optimal release temperature, Co3O4 demonstrates potential as an effective material for PNA applications.

Procyanidin B2 Protects TR-iBRB2 Cells Against Hyperglyc emia Stress by Attenuating Oxidative Stress and Inflammasome Activation via Regulation of Redoxosomes/NF-kB Signaling.

Microvascular dysfunction is a hallmark of diabetic retinopathy (DR), which may lead to visual impairment and blindness. Procyanidin B2 (PB2) is a subclass of flavonoids and is widely known due to its anti-oxidant and antiinflammatory effects. However, little is known about the effect of PB2 on hyperglycemia stress-induced retinal microvascular dysfunction. The purpose of this study was to investigate the effect of PB2 against hyperglycemia stress in rat retinal capillary endothelial cells (TR-iBRB2) as well as the underpinning mechanism. Cell viability was determined using MTT assay. ROS, NOX activity analysis, Western blot analysis, and immunofluorescence analysis were applied in the study. The results showed that PB2 pre-treatment significantly reduced high glucose- induced cytotoxicity in TR-iBRB2 cells by suppressing oxidative stress and inflammasome activation. Mechanistical study revealed that redoxosomes were formed and activated in TR-iBRB2 cells upon hyperglycemia stress, resulting in activation of NF- κB and thus induction of oxidative stress and inflammasomes activation. However, PB2 pre-treatment dose-dependently attenuated the above events, indicating the protective effect of PB2 against hyperglycemia stress was achieved by regulating redoxosomes/ NF-kB signaling. Our findings may contribute to the potential clinical use of PB2 in treating DR and suggest redoxosomes/NF-kB signaling may be a potential therapeutic target of this disease.

Low temperature NH3 selective catalytic reduction of NOx over CeOx-biochar catalyst prepared by air oxidation

A highly efficient CeOx-BC0.6 (BC: biochar. 0.6: mass ratio of Ce/(BC + Ce)) catalyst was prepared by air oxidation for low temperature NH3 selective catalytic reduction of NOx. The catalytic performance, characterization and reaction mechanism of the CeOx-BC0.6 were investigated. The results showed that NOx conversion and N2 selectivity of CeOx-BC0.6 catalyst obtained by calcining in air were above 90 % at 150–300 °C and 100,000 h−1. The surface oxygen groups, acid sites and oxygen vacancies were generated by air oxidation treatment, which promoted the adsorption and activation of reactants. NH3 and NOx were adsorbed on the catalyst surface in the form of NH4+, NH3+, nitrite and nitrate adspecies. The redox cycles of ketonic carbonyl/phenol (CO/C–OH) pairs and Ce4+/Ce3+ pairs were responsible for the activation of NH3 and NOx. NH2NO and NH4NO2 were the main active intermediates. The reaction of NH3 selective catalytic reduction of NOx over CeOx-BC0.6 mainly followed Eley-Rideal mechanism. These results will promote the understanding of mechanism of NH3 selective catalytic reduction of NOx for biochar-based catalyst prepared by air oxidation.

Insights into enhancement of NH3-SCR activity and N2 selectivity of LDHs-derived NiMnAlOx catalysts: Combination of experiments and DFT calculations

The LDHs-derived NiMnAlOx catalysts was proposed for NH3-SCR denitration technology, which improved the overall catalytic performance. At 100–250 oC, the preferred Ni1Mn0.5Al0.5Ox had NOx conversion of exceeded 95%, and N2 selectivity of more than 89%, which was higher than the control catalysts. Additionally, Ni1Mn0.5Al0.5Ox also performed better resistance to SO2 with less sulfate species deposited on the surface. Transient reaction was conducted to illustrate that Ni1Mn0.5Al0.5Ox reduced the N2O formation by suppressing the occurrence of non-selective catalytic pathway. Physicochemical characterization analyses confirmed that Ni1Mn0.5Al0.5Ox catalyst had more active species, abundant surface oxygen, moderate acidic sites and redox properties, which promoted the adsorption and activation of NOx and NH3, and further accelerated the SCR reaction. Finally, DFT calculations provided insights into the mechanism and influence of the degradation of the Jahn-Teller effect and the reversal of the magnetic moment at the Mn site on the surface adsorption process.

Sirtuin1 Mediates the Protective Effects of Echinacoside against Sepsis-Induced Acute Lung Injury via Regulating the NOX4-Nrf2 Axis.

Currently, the treatment for sepsis-induced acute lung injury mainly involves mechanical ventilation with limited use of drugs, highlighting the urgent need for new therapeutic options. As a pivotal aspect of acute lung injury, the pathologic activation and apoptosis of endothelial cells related to oxidative stress play a crucial role in disease progression, with NOX4 and Nrf2 being important targets in regulating ROS production and clearance. Echinacoside, extracted from the traditional Chinese herbal plant Cistanche deserticola, possesses diverse biological activities. However, its role in sepsis-induced acute lung injury remains unexplored. Moreover, although some studies have demonstrated the regulation of NOX4 expression by SIRT1, the specific mechanisms are yet to be elucidated. Therefore, this study aimed to investigate the effects of echinacoside on sepsis-induced acute lung injury and oxidative stress in mice and to explore the intricate regulatory mechanism of SIRT1 on NOX4. We found that echinacoside inhibited sepsis-induced acute lung injury and oxidative stress while preserving endothelial function. In vitro experiments demonstrated that echinacoside activated SIRT1 and promoted its expression. The activated SIRT1 was competitively bound to p22 phox, inhibiting the activation of NOX4 and facilitating the ubiquitination and degradation of NOX4. Additionally, SIRT1 deacetylated Nrf2, promoting the downstream expression of antioxidant enzymes, thus enhancing the NOX4-Nrf2 axis and mitigating oxidative stress-induced endothelial cell pathologic activation and mitochondrial pathway apoptosis. The SIRT1-mediated anti-inflammatory and antioxidant effects of echinacoside were validated in vivo. Consequently, the SIRT1-regulated NOX4-Nrf2 axis may represent a crucial target for echinacoside in the treatment of sepsis-induced acute lung injury.

P300/CBP Regulates HIF-1-Dependent Sympathetic Activation and Hypertension by Intermittent Hypoxia.

Obstructive sleep apnea (OSA), a widespread breathing disorder, leads to intermittent hypoxia (IH). Patients with OSA and IH-treated rodents exhibit heightened sympathetic nerve activity and hypertension. Previous studies reported transcriptional activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) by HIF-1 (hypoxia-inducible factor-1) contribute to autonomic dysfunction in IH-treated rodents. Lysine acetylation, regulated by KATs (lysine acetyltransferases) and KDACs (lysine deacetylases), activates gene transcription and plays an important role in several physiological and pathological processes. This study tested the hypothesis that acetylation of HIF-1α by p300/CBP (CREB-binding protein) (KAT) activates Nox transcription, leading to sympathetic activation and hypertension. Experiments were performed on pheochromocytoma-12 cells and rats treated with IH. IH increased KAT activity, p300/CBP protein, HIF-1α lysine acetylation, HIF-1 transcription, and HIF-1 binding to the Nox4 gene promoter in pheochromocytoma-12 cells, and these responses were blocked by CTK7A, a selective p300/CBP inhibitor. Plasma norepinephrine (index of sympathetic activation) and blood pressures were elevated in IH-treated rats. These responses were associated with elevated p300/CBP protein, HIF-1α stabilization, transcriptional activation of Nox2 and Nox4 genes, and reactive oxygen species, and all these responses were absent in CTK7A-treated IH rats. These findings suggest lysine acetylation of HIF-1α by p300/CBP is an important contributor to sympathetic excitation and hypertension by IH.

Targeting NOX2 with Bivalent Small-Molecule p47phox-p22phox Inhibitors.

Nicotinamide adenine dinucleotide phosphate oxidase isoform 2 (NOX2) is an enzymatic complex whose function is the regulated generation of reactive oxygen species (ROS). NOX2 activity is central to redox signaling events and antibacterial response, but excessive ROS production by NOX2 leads to oxidative stress and inflammation in a range of diseases. The protein-protein interaction between the NOX2 subunits p47phox and p22phox is essential for NOX2 activation, thus p47phox is a potential drug target. Previously, we identified 2-aminoquinoline as a fragment hit toward p47phoxSH3A-B and converted it to a bivalent small-molecule p47phox-p22phox inhibitor (Ki = 20 μM). Here, we systematically optimized the bivalent compounds by exploring linker types and positioning as well as substituents on the 2-aminoquinoline part and characterized the bivalent binding mode with biophysical methods. We identified several compounds with submicromolar binding affinities and cellular activity and thereby demonstrated that p47phox can be targeted by potent small molecules.

Can endoplasmic reticulum stress observed in the PTZ-kindling model seizures be prevented with TUDCA and 4-PBA?

Epilepsy is a chronic neurological disease with recurrent seizures. Increasing evidence suggests that endoplasmic reticulum (ER) stress may play a role in the pathogenesis of epilepsy. We aimed to investigate the effects of Tauroursodeoxycholic acid (TUDCA) and 4-phenyl-butyric acid (4-PBA), which are known to suppress ER stress, on developed seizures in terms of markers of ER stress, oxidative stress, and apoptosis. The pentylenetetrazole (PTZ) kindling model was induced in Wistar albino rats (n = 48) by administering 35 mg/kg PTZ intraperitoneally (I.P.) every other day for 1 month. TUDCA and 4-PBA were administered via I.P. at a dose of 500 mg/kg dose. ER stress, apoptosis, and oxidative stress were determined in the hippocampus tissues of animals in all groups. Immunohistochemistry, qRT-PCR, ELISA, and Western Blot analyzes were performed to determine the efficacy of treatments. Expressions of ATF4, ATF6, p-JNK1/2, Cleaved-Kaspase3, and Caspase12 significantly increased in PTZ-kindled seizures compared to the control group. Increased NOX2 and MDA activity in the seizures were measured. In addition, stereology analyzes showed an increased neuronal loss in the PTZ-kindled group. qRT-PCR examination showed relative mRNA levels of CHOP. Accordingly, TUDCA and 4-PBA treatment suppressed the expressions of ATF4, ATF6, Cleaved-Caspase3, Kaspase12, NOX2, MDA, and CHOP in TUDCA + PTZ and 4-PBA + PTZ groups. ER stress-induced oxidative stress and apoptosis by reducing neuronal loss and degeneration were also preserved in these groups. Our data show molecularly that TUDCA and 4-PBA treatment can suppress the ER stress process in epileptic seizures.