Phosphate Oxidase Activity Research Articles

Oxidative stress in diabetes mellitus and its complications: From pathophysiology to therapeutic strategies.

Oxidative stress due to aberrant metabolism is considered as a crucial contributor to diabetes and its complications. Hyperglycemia and hyperlipemia boost excessive reactive oxygen species generation by elevated mitochondrial respiration, increased nicotinamide adenine dinucleotide phosphate oxidase activity, and enhanced pro-oxidative processes, including protein kinase C pathways, hexosamine, polyol, and advanced glycation endproducts, which exacerbate oxidative stress. Oxidative stress plays a significant role in the onset of diabetes and its associated complications by impairing insulin production, increasing insulin resistance, maintaining hyperglycemic memory, and inducing systemic inflammation. A more profound comprehension of the molecular processes that link oxidative stress to diabetes is crucial to new preventive and therapeutic strategies. Therefore, this review discusses the mechanisms underlying how oxidative stress contributes to diabetes mellitus and its complications. We also summarize the current approaches for prevention and treatment by targeting the oxidative stress pathways in diabetes.

Ursolic Acid from Perilla frutescens (L.) Britt. Regulates Zymosan-Induced Inflammatory Signaling in Raw 264.7

Background: Inflammation is considered one of the hallmarks of inflammatory diseases. Ursolic acid (UA) may exert therapeutic effects; however, the anti-inflammatory effects of UA require further study. Objectives: This study evaluated the effects of UA from Perilla frutescens (L.) Britt. leaves on pro-inflammatory activity induced by zymosan. Methods: Ursolic acid was extracted from P.frutescens (L.) Britt. leaves using ethanol, and thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were used for UA analysis. The effect of UA on zymosan-induced cytokine production was evaluated by enzyme-linked immunosorbent assay (ELISA). Western blotting detected phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, p38, and p47phox in Raw 264.7 cells. Reactive oxygen species (ROS) were measured using a specific immunofluorescent dye. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities were assayed using a luminometer. Results: Zymosan-induced tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12 p40 in macrophages were significantly inhibited by UA (30 μg/mL, P < 0.001). In addition, UA (30 μg/mL, P < 0.001) significantly inhibited zymosan-induced phosphorylation of ERK1/2, p38, p47phox, ROS, and NADPH oxidase in the cells. Importantly, blockade of Dectin-1 using laminarin, a pure β-glucan, markedly abrogated the UA-mediated inhibition of zymosan-induced production of inflammatory cytokines, ERK1/2, p38, ROS, and p47phox phosphorylation in macrophages. Conclusions: Collectively, these data indicate that UA regulates zymosan-induced inflammatory responses and suggest novel approaches for managing excessive inflammatory responses.

The role of reactive oxygen species in regulation of the plasma membrane H+-ATPase activity in Masson pine (Pinus massoniana Lamb.) roots responding to acid stress.

To understand the role of reactive oxygen species (ROS) in regulation of the plasma membrane (PM) H+-ATPase in acid-stressed Masson pine roots, different acidity (pH6.6 as the control, pH5.6 and pH4.6) of simulated acid rain (SAR) added with and without external chemicals (H2O2, enzyme inhibitors and ROS scavenger) was prepared. After 30days of SAR exposure, the plant morphological phenotype attributes, levels of cellular ROS and lipid peroxidation, enzymatic activities of antioxidants, PM nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and PM H+-ATPase activity in pine seedlings were measured. Compared with the control, the growth of pine seedlings exposed to SAR in the presence or absence of H2O2 was well-maintained, but the application of Na3VO4, 1,3-dimethyl-2-thiourea, N, N-dimethylthiourea (DMTU) and diphenyleneiodonium chloride (DPI) caused a substantial growth inhibition. In addition, SAR exposure, SAR with H2O2 treatment, and SAR with Na3VO4 treatment increased the cellular H2O2 content, O2- content and malondialdehyde (MDA) content, while the use of DMTU and DPI lead to relatively low levels. Similarly, the enzymatic activities of antioxidants, PM NADPH oxidase and PM H+-ATPase in acid stressed pine seedlings elevated with the increasing acidity. A significant stimulation of these enzymatic activities obtained from SAR with H2O2 treatment was observed, whereas which decreased obviously with the addition of Na3VO4, DMTU and DPI (P<0.05). Moreover, a positive correlation was found between plant morphological attributes and the PM H+-ATPase activity (P<0.05). Besides, the PM H+-ATPase activity positively correlated with the cellular ROS contents and the enzymatic activities of antioxidants and PM NADPH oxidase (P<0.05). Therefore, the PM H+-ATPase is instrumental in the growth of pine seedlings resisting to acid stress by enhancing its activity. The process involves the signaling transduction of cellular ROS and coordination with PM NADPH oxidase.

Ca2+-mediated reactive oxygen species signaling regulates cell repair after mechanical wounding in the red alga Griffithsia monilis.

Mechanical damage to a cell can be fatal, and the cell must reseal its membrane and restore homeostasis to survive. Plant cell repair involves additional steps such as rebuilding vacuoles, rearranging chloroplasts, and remodeling the cell wall. When we pierced a Griffithsia monilis cell with a glass needle, a large amount of intracellular contents was released, but the cell membrane resealed in less than a second. The turgor of the vacuole was quickly restored, and the punctured cell returned to its original shape within an hour. Organelles such as chloroplasts and nuclei migrated to the wound site for 12 h and then dispersed throughout the cell after the wound was covered by a new cell wall. Using fluorescent probes, high levels of reactive oxygen species (ROS) and calcium were detected at the wound site from 3 h after wounding, which disappeared when cell repair was complete. Wounding in a solution containing ROS scavengers inhibited cellular repair, and inhibiting nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity or blocking calcium influx reversibly inhibited cell repair. Oryzalin reversibly inhibited both chloroplast movement and ROS production during cell repair. Our results show that cell repair in G. monilis is regulated by calcium-mediated ROS signaling and that microtubules serve as mechanical effectors.

Wedelolactone from Eclipta prostrata (L) L. suppresses inflammation and improves insulin resistance

Objective: To evaluate the effects of wedelolactone, a major flavonoid from Vietnamese Eclipta prostrata (L) L., on inflammation and insulin resistance. Methods: Wedelolactone was extracted from the leaves of Vietnamese Eclipta prostrata (L.) L. with methanol by Soxhlet. The effects of wedelolactone on lipopolysaccharide (LPS)-induced cytokine production, reactive oxygen species (ROS) generation, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities in Raw 264.7 cells were measured by enzyme-linked immunosorbent assay (ELISA), specific immunofluorescent dyes and luminometric analysis, respectively. In addition, its effects on glucose uptake and the protein expression of insulin receptor substrate 1 (IRS1) and glucose transporter 4 (GLUT4) were examined in 3T3-L1 cells by immunofluorescent dyes and Western blot. Results: Wedelolactone at 30 μg/mL significantly inhibited LPS-induced production of tumor necrosis factor-α, interleukin (IL)-6, and IL-8 (P&lt;0.01) with no noticeable effects on IL-10 level. It also reduced ROS generation and NADPH oxidase activities in LPS-stimulated Raw 264.7 cells (P&lt;0.01). Furthermore, wedelolactone showed anti-insulin resistance activity, as evidenced by improved glucose uptake and the upregulated expression of IRS1 and GLUT4 in 3T3-L1 cells (P&lt;0.01). Conclusions: Wedelolactone exhibits anti-inflammation and anti-insulin resistance effects, which may be used for the treatment of diabetes and inflammation-associated diseases.

Asprosin contributes to vascular remodeling in hypertensive rats via superoxide signaling.

Proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to vascular remodeling. Asprosin, a newly discovered protein hormone, is involved in metabolic diseases. Little is known about the roles of asprosin in cardiovascular diseases. This study focused on the role and mechanism of asprosin on VSMC proliferation and migration, and vascular remodeling in a rat model of hypertension. VSMCs were obtained from the aortic media of 8-week-old male Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Asprosin was upregulated in the VSMCs of SHR. For in vitro studies, asprosin promoted VSMC proliferation and migration of WKY and SHR, and increased Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity, NOX1/2/4 protein expressions and superoxide production. Knockdown of asprosin inhibited the proliferation, migration, NOX activity, NOX1/2 expressions and superoxide production in the VSMCs of SHR. The roles of asprosin in promoting VSMC proliferation and migration were not affected by hydrogen peroxide scavenger, but attenuated by superoxide scavenger, selective NOX1 or NOX2 inhibitor. Toll-like receptor 4 (TLR4) was upregulated in SHR, TLR4 knockdown inhibited asprosin overexpression-induced proliferation, migration and oxidative stress in VSMCs of WKY and SHR. Asprosin was upregulated in arteries of SHR, and knockdown of asprosin in vivo not only attenuated oxidative stress and vascular remodeling in aorta and mesentery artery, but also caused a subsequent persistent antihypertensive effect in SHR. Asprosin promotes VSMC proliferation and migration via NOX-mediated superoxide production. Inhibition of endogenous asprosin expression attenuates VSMC proliferation and migration, and vascular remodeling of SHR.

Effect of Anagliptin on Vascular Injury in the Femoral Artery of Type 2 Diabetic Rats.

Patients with diabetes mellitus (DM) often experience complications such as peripheral arterial disease (PAD), which is thought to be caused by vascular damage resulting from increased oxidative stress. Dipeptidyl peptidase-4 inhibitors have been reported to reduce oxidative stress, although the exact mechanism remains unclear. This study aimed to investigate the impact of long-term (6 weeks) anagliptin treatment at a dose of 200 mg/kg/d against oxidative stress in the femoral artery of Otsuka Long-Evans Tokushima Fatty (OLETF) rats using a well-established animal model for type 2 DM. Serum toxic advanced glycation end-products concentrations and blood glucose levels after glucose loading were significantly elevated in OLETF rats compared to Long-Evans Tokushima Otsuka (LETO) rats but were significantly suppressed by anagliptin administration. Plasma glucagon-like peptide-1 concentrations after glucose loading were significantly increased in anagliptin-treated rats. Superoxide production and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in femoral arteries were significantly increased in OLETF rats compared to LETO rats but were significantly decreased by anagliptin administration. The expressions of NADPH oxidase components (p22phox in the intima region and p22phox and gp91phox in the media region) in the femoral artery were significantly increased in OLETF rats compared to LETO rats but were significantly suppressed by anagliptin administration. Furthermore, the femoral artery showed increased wall thickness in OLETF rats compared to LETO rats, but anagliptin administration reduced the thickening. This study suggests that long-term anagliptin administration can reduce oxidative stress in femoral arteries and improve vascular injury.

Multi-omics analysis of lung tissue metabolome and proteome reveals the therapeutic effect of Shegan Mahuang Decoction against asthma in rats

Ethnopharmacological relevanceShegan Mahuang Decoction (SMD) is a classic traditional Chinese medicine (TCM) formula for asthma treatment, but the anti-asthma mechanism of SMD is still not fully studied. Aims of the studyIn this study, we established an ovalbumin (OVA)-induced asthma rat model and treated it with SMD to observe its anti-asthma effect and explore the related mechanism. Materials and methodsWe evaluated the anti-inflammatory effect of SMD via testing the levels of immunoglobulin E (IgE), C-reactive protein (CRP), interleukin-4 (IL-4), interleukin-6 (IL-6) in serum and performing the hematoxylin-eosin (H&E) staining of lung tissue slices. We analyzed the variations of metabolites and proteins in the lung tissue of different groups using liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics and TMT-based proteomics approaches. The metabolic biomarkers and differentially expressed proteins (DEPs) were picked, and the related signal transduction pathways were also investigated. In addition, the key proteins on the signaling pathway were validated through western blotting (WB) experiment to reveal the anti-asthma mechanism of SMD. ResultsThe results showed that the SMD could significantly reduce the serum levels of IgE, CRP, IL-4, and IL-6 and attenuate the OVA-induced pathological changes in lung tissue. A total of 34 metabolic biomarkers and 84 DEPs were screened from rat lung tissue, which were mainly associated with lipid metabolism, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, the excessive production of reactive oxygen species (ROS), and lysosome pathway. Besides, SMD could inhibit the myeloid differentiation factor 88 (MyD88)/inhibitor of kappa B kinase (IKK)/nuclear factor-kappa B (NF-κB) signaling pathway to exhibit anti-inflammatory activities. ConclusionsSMD exhibited a therapeutic effect on asthma, which possibly be exerted by inhibiting the MyD88/IKK/NF-κB signaling pathway.

Prime Editing Efficiently and Precisely Corrects Causative Mutation in Chronic Granulomatous Disease, Restoring Myeloid Function: Toward Development of a Prime Edited Autologous Hematopoietic Stem Cell Therapy

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by a lack of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in phagocytic myeloid cells such as neutrophils. NADPH oxidase produces reactive oxygen species (ROS) that directly kill bacteria and fungi to control infection. CGD patients lack this ability to control infection, which results in chronic and severe infections, inflammation, autoimmunity, poor quality of life, and reduced lifespan. Current standard care for CGD is antimicrobial prophylaxis and aggressive management of infection. For patients with a suitable human leukocyte antigen (HLA) matched donor, an allogeneic hematopoietic stem cell (HSC) transplant (allo-HSCT) offers a curative approach. However, many patients do not have access to HLA matched donors and allo-HSCT also carries a risk of GvHD and graft rejection. P47phox-deficient CGD, the most common autosomal recessive CGD, is caused by mutations in the NCF1 gene, which encodes the p47phox protein, a subunit of the NADPH oxidase complex. Approximately 80% of p47phox-deficient CGD patients carry the same homozygous mutation, a 2 nucleotide GT deletion (delGT) in exon 2 of NCF1 which is correctable using Prime Editing. Transplantation of Prime Edited patient autologous CD34 + cells, in which the delGT mutation has been corrected with Prime Editing, may provide a curative treatment for p47phox-deficient CGD patients. To evaluate whether correction of the delGT NCF1 mutation in CGD patient CD34 + cells restores p47phox protein expression and NADPH oxidase activity in myeloid progeny, Prime Editors were designed to correct this mutation precisely and efficiently. The Prime Editors were delivered to CD34 + cells from two p47phox-deficient CGD patients homozygous for delGT NCF1. In both studies, ≧75% of Prime Edited patient CD34 + cells carried at least 1 corrected allele as determined by ddPCR and DNA sequencing analyses. Myeloid progeny differentiated from Prime Edited patient CD34 + cells had restored p47phox protein expression and NADPH oxidase activity to ~80% of normal healthy donors as determined by multiple ROS detection assays. To evaluate reproducibility, Prime Editors were delivered to CD34 + cells from 6 different healthy donors. On average, greater than 90% of CD34 + cells were Prime Edited across 6 donors in independent experiments. To confirm that Prime Edited CD34 + cells retain stem cell multipotency, engraftment, and hematopoietic reconstitution properties, Prime Edited and mock treated CD34 + cells from three different donors were transplanted into immunodeficient mice. Sixteen weeks after CD34 + cell transplantation, &amp;gt;87% of long-term hematopoietic stem cells (LT-HSC) that repopulated the bone marrow had at least 1 allele corrected. There were no significant differences between ‘electroporation only’ mock treated and Prime Editor electroporated human CD34 + cells with respect to long-term CD34 + cell engraftment, human CD45 + blood cell chimerism, hematopoietic multilineage blood cell reconstitution and biodistribution, or LT-HSC cell potency. Across these studies, &amp;gt;95% human CD45 + cell chimerism was achieved with Prime Edited CD34 + cells. Despite high on-target correction of the delGT mutation in NCF1, off-target editing, unintended editing, and chromosomal rearrangements (larger deletions or translocation) events were not detected using a robust suite of genome-wide assays. In summary, Prime Editing corrects a mutation that causes the most common autosomal recessive from of CGD; correction of this delGT mutation at NCF1 restores NADPH oxidase activity and function in myeloid progeny of Prime Edited CGD CD34 + cells; and Prime Edited long-term HSCs retain multipotency and functionality in vivo. These results support the development of a Prime Edited CGD patient autologous CD34 + cell therapy as a potential curative approach for p47phox-deficient CGD patients.

Malic Acid Improves Behavioral, Biochemical, and Molecular Disturbances in the Hypothalamus of Stressed Rats.

Stress can lead to emotional and mental symptoms such as anxiety, sadness, panic attacks, and depression. Malic acid was chosen due to malic acid has the ability to improve antioxidant activity and improves liver damage. This study evaluates malic acid anti-depressant activity in the hypothalamus of stressed rats. Thirty-six male albino rats were divided into 2 equal groups; Normal and chronic mild stress (CMS) rats. Normal rats were divided into 3 equal groups; control, malic acid, and venlafaxine drug groups: normal rats were administered orally with 1 mL of saline solution, 250 mg/kg of malic acid, and 20 mg/kg of venlafaxine drug, respectively. CMS rats were divided into 3 equal groups; CMS, CMS + malic acid, and CMS + venlafaxine drug: CMS rats were administered orally with 1 mL of saline solution, 250 mg/kg of malic acid, and 20 mg/kg of venlafaxine drug, respectively. All the above-mentioned treatments were administered once a day by oral gavage for 6 weeks. The obtained results revealed that the animal behavioral tests such as forced swimming test, tail suspension test, sucrose preference test, and open-field test (center square entries test, center square duration test, and distance travelled test), norepinephrine, dopamine, serotonin, γ-aminobutyric acid, nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity, oxidative index, conjugated dienes, catalase, glutathione peroxidase, superoxide dismutase, malondialdehyde, interleukin-6, tumor necrosis factor-α, interleukin-10, interleukin-1β, sodium/potassium-ATPase activity, and histamine-N-methyl transferase (Hnmt) and tyrosine hydroxylase (TH) enzymes in the hypothalamus of stressed rats, were returned to approaching the normal state in the stressed group after treating with malic acid for 6 weeks. Malic acid ameliorated stressed-related symptoms and it inhibited superoxide anion and neuro-inflammation in the hypothalamus of stressed rats.

Maintenance of cellular vitamin B6 levels and mitochondrial oxidative function depend on pyridoxal 5′-phosphate homeostasis protein

Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B6-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B6 metabolism are not well understood. To address these questions, we used PLPHP-deficient patient skin fibroblasts and HEK293cells and YBL036C (PLPHP ortholog)-deficient yeast. We showed that independent of extracellular B6 vitamer type (pyridoxine, pyridoxamine, or pyridoxal), intracellular pyridoxal 5'-phosphate (PLP) was lower in PLPHP-deficient fibroblasts and HEK293cells than controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5'-phosphate oxidase activity. Experiments utilizing 13C4-pyridoxine confirmed lower pyridox(am)ine 5'-phosphate oxidase activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP-deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C-deficient yeast, PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP-deficient HEK293cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B6 metabolism, and mitochondrial oxidative metabolism.

Isoamericanin A improves lipopolysaccharide-induced memory impairment in mice through suppression of the nicotinamide adenine dinucleotide phosphateoxidase-dependent nuclear factor kappa B signaling pathway.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Isoamericanin A (ISOA) is a natural lignan possessing great potential for AD treatment. This study investigated the efficacy of ISOA on memory impairments in the mice intrahippocampal injected with lipopolysaccharide (LPS) and the underlying mechanism. Y-maze and Morris Water Maze data suggested that ISOA (5 and 10 mg/kg) ameliorated short- and long-term memory impairments, and attenuated neuronal loss and lactate dehydrogenase activity. ISOA exerted anti-inflammatory effect demonstrating by the reduction of ionized calcium-binding adapter molecule 1 positive cells and suppression of marker protein and pro-inflammation cytokines expressions induced by LPS. ISOA suppressed the nuclear factor kappa B (NF-κB) signaling pathway by inhibiting IκBα phosphorylation and NF-κB p65 phosphorylation and nuclear translocation. ISOA inhibited superoxide and intracellular reactive oxygen species accumulation by reducing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, demonstrating by suppressing NADP+ and NADPH contents, gp91phox expression, and p47phox expression and membrane translocation. These effects were enhanced in combination with NADPH oxidase inhibitor apocynin. The neuroprotective effect of ISOA was further proved in the in vitro models. Overall, our data revealed a novel pharmacological activity of ISOA: ameliorating memory impairment in AD via inhibiting neuroinflammation.

Induced Inflammatory and Oxidative Markers in Cerebral Microvasculature by Mentally Depressive Stress.

Cerebrovascular disease (CVD) is recognized as the leading cause of permanent disability worldwide. Depressive disorders are associated with increased incidence of CVD. The goal of this study was to establish a chronic restraint stress (CRS) model for mice and examine the effect of stress on cerebrovascular inflammation and oxidative stress responses. A total of forty 6-week-old male C57BL/6J mice were randomly divided into the CRS and control groups. In the CRS group (n = 20), mice were placed in a well-ventilated Plexiglas tube for 6 hours per day for 28 consecutive days. On day 29, open field tests (OFT) and sucrose preference tests (SPT) were performed to assess depressive-like behaviors for the two groups (n = 10/group). Macrophage infiltration into the brain tissue upon stress was analyzed by measuring expression of macrophage marker (CD68) with immunofluorescence in both the CRS and control groups (n = 10/group). Cerebral microvasculature was isolated from the CRS and controls (n = 10/group). mRNA and protein expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1), and macrophage chemoattractant protein-1 (MCP-1) in the brain vessels were measured by real-time PCR and Western blot (n = 10/group). Reactive oxygen species (ROS), hydrogen peroxide (H2O2), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activities were quantified by ELISA to study the oxidative profile of the brain vessels (n = 10/group). Additionally, mRNA and protein expressions of NOX subunits (gp91phox, p47phox, p67phox, and p22phox) in the cerebrovascular endothelium were analyzed by real-time PCR and Western blot (n = 10/group). CRS decreased the total distances (p < 0.05) and the time spent in the center zone in OFT (p < 0.001) and sucrose preference test ratio in SPT (p < 0.01). Positive ratio of CD68+ was increased with CRS in the entire region of the brain (p < 0.001), reflecting increased macrophage infiltration. CRS increased the expression of inflammatory factors and oxidative stress in the cerebral microvasculature, including TNF-α (p < 0.001), IL-1β (p < 0.05), IL-6 (p < 0.05), VCAM-1 (p < 0.01), MCP-1 (p < 0.01), ROS (p < 0.001), and H2O2 (p < 0.001). NADPH oxidase (NOX) was activated by CRS (p < 0.01), and mRNA and protein expressions of NOX subunits (gp91phox, p47phox, p67phox, and p22phox) in brain microvasculature were found to be increased. To our knowledge, this is the first study to demonstrate that CRS induces depressive stress and causes inflammatory and oxidative stress responses in the brain microvasculature.

Combination Therapy of Niacin and Apocynin Attenuates Lung Injury During Sepsis in Rats

IntroductionOxidative stress contributes to tissue injury through reactive oxygen species–dependent signaling pathways during sepsis. We studied therapeutic benefits of the combination therapy of niacin, which increased reduced glutathione levels, and apocynin, which suppressed reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activity, in septic rats. Materials and methodsPolymicrobial sepsis was induced through cecal ligation and puncture (CLP) with antibiotics in male Sprague–Dawley rats (n = 189). The rats were randomly divided into sham, CLP, CLP + niacin, CLP + apocynin, and CLP + niacin + apocynin groups. Six hours after CLP, vehicle, niacin (360 mg/kg through the orogastric tube), and/or apocynin (20 mg/kg through intraperitoneal injection) were administered. The occurrence of mortality for 72 h after CLP was observed. Next, a separate set of animals was euthanized at 24 h post-CLP for lung tissue analyses. ResultsCombination therapy with niacin and apocynin significantly improved survival in rats with sepsis (75.0% versus 28.8%, P = 0.006) but monotherapy with niacin or apocynin did not. Monotherapy with niacin and apocynin appeared to increase NADPH levels and decrease Nox levels and activity, respectively, but failed to show statistical significances. However, combination therapy significantly decreased Nox levels and activity, increased NADPH and glutathione levels, decreased intranuclear nuclear factor-κB (NF-κB) p65 levels, reduced inflammatory cytokine expression and malondialdehyde levels, and attenuated histological lung injuries. ConclusionsCombination therapy with niacin and apocynin synergistically attenuated lung injuries and improved survival in rats with sepsis through niacin-induced glutathione redox cycle activation and apocynin-induced Nox suppression.

Perfluorooctane sulfonate-induced oxidative stress contributes to pancreatic β-cell apoptosis by inhibiting cyclic adenosine monophosphate pathway: Prevention by pentoxifylline

Endocrine-disrupting chemical perfluorooctane sulfonate (PFOS) acute exposure stimulates insulin secretion from pancreatic β-cells. However, chronic exposure to PFOS on pancreatic β-cells, its role in insulin secretion, and the underlying mechanisms have not been studied. We used rat insulinoma INS-1 and human 1.1b4 islet cells to investigate the chronic effects of PFOS on glucose-stimulated insulin secretion and toxicity implicated in the downregulation of β-cell functionality. Chronic exposure of INS-1 cells or human pancreatic 1.1b4 β-cells to PFOS stimulated the small G-protein RAC1-guanosine triphosphate-dependent nicotinamide adenine dinucleotide phosphate oxidase (NOX2/gp91phox) subunit expression and activation. Upregulated NOX2/gp91phox activation led to elevated reactive oxygen species (ROS) production with a decrease in the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway in both cell types. Inhibition of cAMP/PKA signaling induces β-cell mitochondrial dysfunction and endoplasmic stress via the loss of PDX1-SERCA2B and glucose-stimulated insulin release. Inhibiting RAC1-NOX2/gp91phox activation or elevating cAMP by pentoxifylline, a Food and Drug Administration-approved phosphodiesterase inhibitor, significantly reduced PFOS-induced ROS production and restored insulin secretory function of pancreatic β-cells. Enhanced secretory function in pentoxifylline-treated cells was associated with increased stability of PDX1-SERCA2B protein levels. Intriguingly, inhibition of cAMP/PKA signaling impaired pentoxifylline-induced insulin secretion caused by the activation of ROS production and mitochondrial dysfunction. Overall, our findings show that PFOS has a new and first-ever direct chronic effect on pancreatic β-cell failure through increased RAC1-NOX2/gp91phox activation and pentoxifylline-induced cAMP/PKA signaling, which inhibits PFOS-mediated mitochondrial dysfunction.

Lipoxin A4 attenuates MSU-crystal-induced NLRP3 inflammasome activation through suppressing Nrf2 thereby increasing TXNRD2.

Gout is a common inflammatory disease. The activation of NLRP3 inflammasome induced by monosodium urate (MSU) crystals has a critical role in gout, and its prevention is beneficial for patients. Lipoxin A4 (LXA4) is an endogenous lipoxygenase-derived eicosanoid mediator with powerful anti-inflammatory properties. However, whether LXA4 can suppress NLRP3 inflammasome activation induced by MSU crystals remains unclear. This study aimed to investigate the protective effect of LXA4 on MSU-crystal-induced NLRP3 inflammasome activation and its underlying molecular mechanisms. We found that LXA4 inhibited MSU-crystal-induced NLRP3 inflammasome activation, interleukin (IL)-1β maturation, and pyroptosis. More specifically, LXA4 suppressed the assembly of the NLRP3 inflammasome, including oligomerization and speck formation of ASC, and ASC-NLRP3 interaction. Furthermore, LXA4 suppressed oxidative stress, the upstream events for NLRP3 inflammasome activation, as evidenced by the fact that LXA4 eliminated total reactive oxygen species (ROS) generation and alleviated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and mitochondrial dysfunction. However, LXA4 also depressed the Nrf2 activation, a critical molecule in the antioxidant pathway, and then exerted an inhibitory impact on Klf9 expression and promotional impact on TXNRD2 expression, two molecules located downstream of Nrf2 in sequence. Knockdown of TXNRD2 reversed the LXA4-induced depression of ROS and NLRP3 inflammasome. Moreover, LXA4 alleviated joint inflammation and decreased the production of cleaved caspase-1 and matured IL-1β in gouty arthritis rats. Taken together, our findings demonstrate that LXA4 can attenuate MSU-crystal-induced NLRP3 inflammasome activation, probably through suppressing Nrf2 activation to increase TXNRD2 expression. The present study highlights the potential of LXA4 as an attractive new gout treatment candidate.

The Inhibitory Role of Hydrogen Sulfide in UII-Induced Cardiovascular Effects and the Underlying Signaling Pathways.

Urotensin II (UII) could increase blood pressure and heart rate via increased central reactive oxygen species (ROS) levels. We reported previously that hydrogen sulfide (H2S) exerts an antihypertensive effect by suppressing ROS production. The aim of the current study is to further examine the effects of endogenous and exogenous H2S on UII-induced cardiovascular effects by using an integrated physiology approach. We also use cell culture and molecular biological techniques to explore the inhibitory role of H2S on UII-induced cardiovascular effects. In this study, we found that cystathionine-β-synthase (CBS), the main H2S synthesizing enzyme in CNS, was expressed in neuronal cells of the rostral ventrolateral medulla (RVLM) area. Cellular distribution of CBS and urotensin II receptor (UT) in SH-SY5Y cells that are confirmed as glutamatergic were identified by immunofluorescent and Western blots assay. In Sprague-Dawley rats, administration of UII into the RVLM resulted in an increase in mean arterial pressure (MAP), heart rate (HR), ROS production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and phosphorylation of p47phox, extracellular signal-regulated protein kinase (ERK)1/2 and p38MAPK, but not stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK). These effects of UII were attenuated by application into the RVLM of endogenous (L-cysteine, SAM) or exogenous (NaHS) H2S. These results were confirmed in SH-SY5Y cells. UII-induced cardiovascular effects were also significantly abolished by pretreatment with microinjection of Tempol, Apocynin, SB203580, or PD98059 into the RVLM. Preincubated SH-SY5Y cells with Apocynin before administration of UII followed by Western blots assay showed that ROS is in the upstream of p38MAPK/ERK1/2. Gao activation assay in SH-SY5Y cells suggested that H2S may exert an inhibitory role on UII-induced cardiovascular effects by inhibiting the activity of Gαo. These results suggest that both endogenous and exogenous H2S attenuate UII-induced cardiovascular effects via Gαo-ROS-p38MAPK/ERK1/2 pathway.

Severe G6PD deficiency leads to recurrent infections and defects in ROS production: Case report and literature review.

Purpose: Severe glucose-6-phosphate dehydrogenase (G6PD) deficiency can lead to reduced nicotinamide adenine dinucleotide phosphate oxidase activity in phagocytes, resulting in immunodeficiency, with a limited number of reported cases. Here, we aimed to report a child with severe G6PD deficiency in China and investigate the mechanism of his recurrent infections. Methods: The clinical manifestations and immunological phenotypes of this patient were retrospectively collected. Gene mutation was detected by whole-exome sequencing and confirmed by Sanger sequencing. Dihydrorhodamine (DHR) analysis was performed to measure the respiratory burst of neutrophils. Messenger ribonucleic acid and protein levels were detected in the patient under lipopolysaccharide stimulation by real-time quantitative reverse transcription polymerase chain reaction and Western blot. A review of the literature was performed. Results: A male child with G6PD deficiency presented with recurrent respiratory infections, Epstein‒Barr virus infection and tonsillitis from 8 months of age. Gene testing revealed that the proband had one hemizygous mutation in the G6PD gene (c.496C>T, p. R166C), inherited from his mother. This mutation might affect hydrophobic binding, and the G6PD enzyme activity of the patient was 0. The stimulation indexes of the neutrophils in the patient and mother were 22 and 37, respectively. Compared with healthy controls, decreased reactive oxygen species (ROS) production was observed in the patient. Activation of nuclear factor kappa-B (NF-κB) signaling was found to be influenced, and the synthesis of tumor necrosis factor alpha (TNF-α) was downregulated in the patient-derived cells. In neutrophils of his mother, 74.71% of the X chromosome carrying the mutated gene was inactivated. By performing a systematic literature review, an additional 15 patients with severe G6PD deficiency and recurrent infections were identified. Four other G6PD gene mutations have been reported, including c.1157T>A, c.180_182del, c.514C>T, and c.953_976del. Conclusion: Severe G6PD deficiency, not only class I but also class II, can contribute to a chronic granulomatous disease-like phenotype. Decreased reactive oxygen species synthesis led to decreased activation of the NF-κB pathway in G6PD-deficient patients. Children with severe G6PD deficiency should be aware of immunodeficiency disease, and the DHR assay is recommended to evaluate neutrophil function for early identification.

Biochemical regulatory processes in the control of oxidants and antioxidants production in the brain of rats with iron and copper chronic overloads.

Iron [Fe(II)] and copper [Cu(II)] overloads in rat brain are associated with oxidative stress and damage. The purpose of this research is to study whether brain antioxidant enzymes are involved in the control of intracellular redox homeostasis in the brain of rats male Sprague-Dawley rats (80-90g) that received drinking water supplemented with either 1.0g/L of ferrous chloride (n = 24) or 0.5g/L cupric sulfate (n = 24) for 42days. Nicotinamide adenine dinucleotide phosphate(NADPH) oxidase, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione transferase (GT) activities in brain were determined by spectrophotometric methods and NO production by the content of nitrite concentration in the organ. Chronic treatment with Fe(II) and Cu(II) led to a significant decrease of nitrite content and SOD activity in brain. Activity of NADPH oxidase increased with Cu(II) treatment. Concerning Fe(II), catalase and GT activities increased in brain after 28 and 4days of treatment, respectively. In the case of Cu(II), catalase activity decreased whereas GT activity increased after 2 and 14days, respectively. The regulation of redox homeostasis in brain involves changes of the activity of these enzymes to control the steady state of oxidant species related to redox signaling pathways upon Cu and Fe overload. NO may serve to detoxify cells from superoxide anion and hydrogen peroxide with the concomitant formation of peroxynitrite. However, the latest is a powerful oxidant which leads to oxidative modifications of biomolecules. These results suggest a common pathway to oxidative stress and damage in brain for Cu(II) and Fe(II).

NADPH Oxidases in Aortic Aneurysms.

Abdominal aortic aneurysms (AAAs) are a progressive dilation of the infrarenal aorta and are characterized by inflammatory cell infiltration, smooth muscle cell migration and proliferation, and degradation of the extracellular matrix. Oxidative stress and the production of reactive oxygen species (ROS) have been shown to play roles in inflammatory cell infiltration, and smooth muscle cell migration and apoptosis in AAAs. In this review, we discuss the principles of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase/NOX) signaling and activation. We also discuss the effects of some of the major mediators of NOX signaling in AAAs. Separately, we also discuss the influence of genetic or pharmacologic inhibitors of NADPH oxidases on experimental pre-clinical AAAs. Experimental evidence suggests that NADPH oxidases may be a promising future therapeutic target for developing pharmacologic treatment strategies for halting AAA progression or rupture prevention in the management of clinical AAAs.