Increased Superoxide Production Research Articles

Attenuation of stroke damage by angiotensin II type 2 receptor stimulation via peroxisome proliferator-activated receptor-gamma activation.

The brain renin-angiotensin system plays a crucial role in ischemic stroke. It is known that stimulation of the angiotensin II type 2 (AT2) receptor protects against ischemic brain injury. We recently demonstrated that AT2 receptor stimulation by compound 21 (C21), a direct AT2 receptor agonist, inhibited vascular intimal proliferation with activation of peroxisome proliferator-activated receptor-gamma (PPAR-γ). However, whether direct AT2 receptor stimulation protects against ischemic brain injury via PPAR-γ activation is still unknown. 8-week-old male C57BL/6 J mice were subjected to middle cerebral artery (MCA) occlusion. 2 weeks before MCA occlusion, they were administered C21 with or without GW9662, a PPAR-γ antagonist. Neurologic deficit, ischemic size, superoxide anion, superoxide dismutase (SOD) activity, expression of NADPH subunits and blood brain barrier (BBB) stabilization were assessed 24 h after MCA occlusion. Cerebral blood flow (CBF) was measured in the core and periphery of the MCA territory before, immediately after, 1 h and 24 h after MCA occlusion. Treatment with C21 markedly decreased the neurologic deficit and ischemic size with an increase in CBF, SOD activity and BBB stabilization genes compared with the non-treated group. Co-administration of GW9662 partially attenuated this protective effect of C21 on neurologic deficit and ischemic size via an increase in superoxide anion production and a decrease of SOD activity and BBB stabilization genes, while GW9662 treatment alone had no significant effect on neurologic deficit and ischemic size. These results suggest that direct AT2 receptor stimulation has a preventive effect on stroke-induced brain injury partly due to activation of PPAR-γ.

Effect of mate tea (Ilex paraguariensis) on the expression of the leukocyte NADPH oxidase subunit p47phox and on circulating inflammatory cytokines in healthy men: a pilot study

Increased superoxide production by phagocytic NADPH oxidase has been associated with inflammatory conditions. Growing evidences suggest that dietary polyphenols may modulate the expression of NADPH oxidase subunits. Herein, we examined whether soluble mate tea (SMT) consumption – a polyphenol-rich beverage – affects the expression of the leukocyte NADPH oxidase protein p47phox and/or circulating biomarkers of inflammation and antioxidant biomarkers in humans. In a two-phase study, nine men were requested to drink water (control) for 8 d and then follow a second 8-d period drinking SMT. Blood samples were analysed for p47phox protein in CD16+/CD14− cells, interleukin (IL)-1β (IL-1β), tumour necrosis factor-alpha (TNF-α), IL-6, total phenols, and reduced and oxidised glutathione (GSH and GSSG, respectively) after each study phase. After SMT intake, CD16+/CD14− cells' p47phox protein and serum TNF-α and IL-6 levels were significantly attenuated (P < .05) while plasma phenolic compounds and blood GSH:GSSG ratio were significantly enhanced (P < .05). Consumption of SMT favourably affected leukocytes' p47phox expression and inflammatory cytokine and antioxidants levels in peripheral blood, which may help decrease oxidative stress and low-grade inflammation.

Melatonin Promotes Apoptosis of Colorectal Cancer Cells via Superoxide-mediated ER Stress by Inhibiting Cellular Prion Protein Expression

Melatonin, an endogenously secreted indoleamine hormone that is produced in the pineal gland, is known to possess antitumor effect via various mechanisms including induction of apoptosis and pro-oxidant effects in various cancer cells, including colorectal cancer (CRC). In our study, we hypothesized that melatonin enhances the anticancer effects via suppression of PrPC and PINK1 levels, thereby increasing superoxide production. To investigate the antitumor effects of melatonin in CRC cells, assessing its effects on mitochondrial dysfunction, production of superoxide, induction of endoplasmic reticulum stress, and cellular apoptosis were assessed. Melatonin was found to decrease the expression of PrPC and PINK1, and increase superoxide accumulation in the mitochondria. In addition, PrPC-knockdown potentiated the effects of melatonin resulting further in significantly reduced expression of PINK1 and increased superoxide production in CRC. si-PRNP-transfected CRC cells treated with melatonin increased the production of intracellular superoxide and induced endoplasmic reticulum stress associated protein, and apoptosis. Melatonin induces mitochondria-mediated cellular apoptosis in CRC cancer cells via a PrPC-dependent pathway. PrPC knockdown combined with melatonin amplifies the effects of melatonin, suggesting a novel therapeutic strategy in targeting CRC cells.

Immuno-Spin Trapping-Based Detection of Oxidative Modifications in Cardiomyocytes and Coronary Endothelium in the Progression of Heart Failure in Tgαq*44 Mice.

Recent studies suggest both beneficial and detrimental role of increased reactive oxygen species and oxidative stress in heart failure (HF). However, it is not clear at which stage oxidative stress and oxidative modifications occur in the endothelium in relation to cardiomyocytes in non-ischemic HF. Furthermore, most methods used to date to study oxidative stress are either non-specific or require tissue homogenization. In this study, we used immuno-spin trapping (IST) technique with fluorescent microscopy-based detection of DMPO nitrone adducts to localize and quantify oxidative modifications of the hearts from Tgαq*44 mice; a murine model of HF driven by cardiomyocyte-specific overexpression of Gαq* protein. Tgαq*44 mice and age-matched FVB controls at early, transition, and late stages of HF progression were injected with DMPO in vivo and analyzed ex vivo for DMPO nitrone adducts signals. Progressive oxidative modifications in cardiomyocytes, as evidenced by the elevation of DMPO nitrone adducts, were detected in hearts from 10- to 16-month-old, but not in 8-month-old Tgαq*44 mice, as compared with age-matched FVB mice. The DMPO nitrone adducts were detected in left and right ventricle, septum, and papillary muscle. Surprisingly, significant elevation of DMPO nitrone adducts was also present in the coronary endothelium both in large arteries and in microcirculation simultaneously, as in cardiomyocytes, starting from 10-month-old Tgαq*44 mice. On the other hand, superoxide production in heart homogenates was elevated already in 6-month-old Tgαq*44 mice and progressively increased to high levels in 14-month-old Tgαq*44 mice, while the enzymatic activity of catalase, glutathione reductase, and glutathione peroxidase was all elevated as early as in 4-month-old Tgαq*44 mice and stayed at a similar level in 14-month-old Tgαq*44. In summary, this study demonstrates that IST represents a unique method that allows to quantify oxidative modifications in cardiomyocytes and coronary endothelium in the heart. In Tgαq*44 mice with slowly developing HF, driven by cardiomyocyte-specific overexpression of Gαq* protein, an increase in superoxide production, despite compensatory activation of antioxidative mechanisms, results in the development of oxidative modifications not only in cardiomyocytes but also in coronary endothelium, at the transition phase of HF, before the end-stage disease.

P-385 - Overproduction of reactive oxygen species is the primary pathological event related to neuronal cell death in iPSC derived neurons from patients with familial Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disease characterised initially loss of dopaminergic neurons in the substantia nigra (SN) and later widespread nondopaminergic neuronal loss including in the cortex. Genetic mutations can cause familial PD, but the sequence of molecular events that trigger neuronal vulnerability is poorly understood. Of the familial forms of PD, point mutations of gene encoding alpha-synuclein (SNCA), the major component of the predominant pathological hallmark called Lewy bodies/neurites, result in dominant PD. To characterize the cellular pathophysiology, we differentiated patient-specific induced pluripotent stem cells (iPSCs) from PD patient fibroblasts carrying alpha-synuclein point mutation, A53T, into functional cortical and midbrain neurons. We demonstrated that endogenous levels of SNCA-A53T reduce cell survival and the source of vulnerability is the excess reactive oxygen species (ROS) evidenced by increased superoxide production and lipid peroxidation levels compared to control. By utilizing the power of patient specific iPSC derived neurons, this study provides an insight into the primary pathological events leading to neuronal cell death and may furthermore be an important direct tool for therapeutic development.

A novel mechanism of NO synthase uncoupling involving isolevuglandin adduction

We have previously shown that hypertension leads to modification of self‐proteins in antigen presenting cells by isolevuglandin (isoLG) lipid oxidation products. These modified proteins seem to be immunogenic. IsoLG‐modified peptides are presented in major histocompatibility complex (MHC) class 1 and drive CD8+ T cell proliferation. To understand alterations in the peptidome presented by MHC1 in hypertension, we created transgenic mice expressing a truncated form of MHC1 Kb lacking the transmembrane domain, driven by the CD11c promoter. The lack of a transmembrane domain causes MHC1 to be shed into the media when DCs are cultured. We treated these mice with either 2 weeks sham or angiotensin (ang) II infusion, isolated splenic DCs and placed these in culture for 72 hours. The shed soluble MHC1 Kb was harvested and associated peptides were analyzed by mass spectroscopy. One peptide identified in DCs from hypertensive mice was an identical match for a portion of the neuronal nitric oxide synthase (nNOS) containing an isoLG adduct at lysine 225. Because this lysine is positioned between the heme and arginine binding sites, we hypothesized that its modification would disrupt normal NO production and instead lead to superoxide production, i.e. NOS uncoupling. In subsequent studies, we found that nNOS expression is increased 5‐fold in DCs of ang II‐treated compared to sham‐treated mice (n = 6). Importantly, ang II infusion increased superoxide production of DCs from 37±3 to 111±5 pmol/mg protein (mean ± SE, p = 0.0001), and this was completely blocked by treatment of the cells with the selective nNOS inhibitor L‐vinyl NIO, suggesting that nNOS is a source of superoxide in DCs in hypertension. Next we expressed wild type nNOS and a mutant nNOS in which lysine 225 was mutated to an alanine (nNOS K225A) in human epithelial kidney (HEK) cells. Untransfected HEK cells failed to produce NO, as measured by electron spin resonance and the spin trap Fe[DETC]2. HEK cells transfected with either the WT or K225A nNOS produced 152 ± 5 vs 156 ± 6 arbitrary units of NO/mg protein (p = NS). However, exposure of the transfected HEK cells to tertbutyl hydroperoxide, which promotes isoLG formation, reduced NO production by cells expressing the wild‐type enzyme to 86 ± 6 units/mg protein, while having no effect on cells transfected with the mutant K225A (p = 0.003, n = 5 – 7), further implicating lysine 225 as a site of oxidative modification in nNOS. In summary, hypertension increases nNOS expression in DCs and this enzyme is altered by isoLG adduction at lysine 225. This leads to an increase in superoxide and reduced NO production in these immune cells. We conclude that isoLG adduction represents a novel mechanism of NOS uncoupling and a source of reactive oxygen species in dendritic cells.Support or Funding InformationAmerican Heart Association grants POST290900 and SFRN204200, and National Institutes of Health grants K01HL130497, R01HL125865, R01HL039006 and P01HL129941This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ketone salts inhibit production of superoxide anions during normobaric and hyperbaric hyperoxia in rat solitary complex neurons

The threat of central nervous system‐O2 toxicity (CNS‐OT; i.e., seizures) is what limits the use of hyperbaric oxygen (HBO2) in diving, submarine and hyperbaric medicine. We previously reported that ketone ester given orally to Sprague‐Dawley (SD) rats 30 min prior to HBO2 exposure increases the levels of ketone bodies in the blood without inducing pathological metabolic acidosis, and delays the onset of CNS‐OT by nearly 600% (D'Agostino et al., 2013 AJP 304: R829–R836). We hypothesize that therapeutic ketosis induces neuroprotection against CNS‐OT in part by decreasing production of reactive oxygen species during hyperoxia. Tissue slices of the caudal medulla oblongata (300–400mm thick) were harvested from SD rats (P12–P28; n=35 rats). The fluorogenic dye, dihydroethidium (DHE, 2.5 mM) was added to nutrient medium (36–37 °C) containing a cocktail of nitric oxide synthase inhibitors (1 mM N‐w‐nitro‐L‐arginine methyl ester &amp; 100 mM 7‐nitroindazole) and superoxide dismutase inhibitors (10mM diethyldithiocarbamate) to optimize O2‐induced changes in DHE fluorescence (Ciarlone and Dean, 2016 AJP 311: C1014–C1026). DHE fluorescence was detected in neurons of the caudal solitary complex (cSC) using a Nikon TE‐2000 inverted microscope or a custom‐built hyperbaric microscope and Texas Red filter (ex/em 525 nm/590 nm) once every 5 min and expressed in arbitrary units of change in fluorescence intensity unit/minute (DFIU/min). Nutrient media were equilibrated with gases as follows (in ATA): control O2: 0.4 O2/0.05 CO2/bal N2 (60 min); normobaric hyperoxia, NBO2: 0.95 O2/0.05 CO2 (60 min); and HBO2: 4.95 O2/0.05 CO2 (60 min). Ketone salts (KS) added to media during hyperoxia (60 min) included 2–2.5 &amp; 5 mM, 50:50 mix of b–hydroxybutyrate + acetoacetate. Data were analyzed by 1‐way ANOVA and appropriate comparisons testing (P&lt;0.05) using Nikon NIS‐Elements and Graphpad Prism. DHE fluorescence increased during 1 hr of NBO2 (139%) and HBO2 (240%). Neurons in the cSC had variable sensitivity to hyperoxia exhibiting low to high rates of increased superoxide production. During the 2nd hr of NBO2, DHE fluorescence was unchanged from that in the 1st hr. Conversely, DHE fluorescence decreased during the 2nd hr of HBO2 suggesting cellular dysfunction in 4.95 ATA O2. Thus, KS were tested during hr 2 in NBO2 in the same slices, whereas KS were tested during hr 1 of HBO2 in a separate set of brain slices. One hour of ketosis significantly decreased DHE fluorescence on average during NBO2 by 1/3 (2mM) and 1/2 (5mM), and during HBO2 by 1/3 (5mM); however, not all neurons as defined by their O2‐sensitivity for superoxide production were equally responsive to ketosis. Our findings support the hypothesis that therapeutic ketosis induces neuroprotection during hyperoxia in part by decreasing O2‐induced production of superoxide anions.Support or Funding InformationResearch supported by ONR Undersea MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Roles of elevated 20‑HETE in the breakdown of blood brain barrier and the severity of brain edema in experimental traumatic brain injury.

Breakdown of the blood brain barrier (BBB) is a secondary injury following traumatic brain injury (TBI) and can lead to the development of brain edema. However, the factors that contribute to the disruption of the BBB and increase the severity of brain edema in TBI remain to be elucidated. 20‑hydroxyeicosatetraenoic acid (20‑HETE) is a metabolite of arachidonic acid. The inhibition of 20‑HETEsynthesis by HET0016 has been suggested as a strategy to decrease brain edema. The present study aimed to investigate whether the elevated production of 20‑HETE in cerebral tissue may contribute to BBB breakdown and increase the severity of brain edema in rats with TBI. BBB permeability was quantified using dynamic contrast‑enhanced magnetic resonance imaging and brain edema was measured according to brain water content. Superoxide production in injured tissue was also assessed. Liquid chromatography‑mass spectrometry was used to evaluate 20‑HETE production in injured tissue. Western blot analysis was used to assess the expression of occludin, zonula occludens (ZO)‑1, matrix metalloproteinase (MMP)‑9, and proteins of the c‑Jun N‑terminal kinase (JNK) pathway. A total of 3, 24 and 72h following the induction of TBI, 20‑HETE levels, BBB permeability and brain edema were identified to be increased, accompanied by an increase in superoxide production. Conversely, superoxide dismutase levels, in addition to the total antioxidative capability were decreased. In addition, the expression of MMP‑9 and proteins of the JNK pathway was upregulated, whereas the expression of occludin and ZO‑1 was observed to be suppressed. These results suggested that 20‑HETE may aggravate BBB disruption following TBI, via enhancing the expression of MMP‑9 and tight junction proteins. Furthermore, oxidative stress and the JNK signaling pathway may be involved in BBB dysregulation. In conclusion, the results of the present demonstrated that the production of 20‑HETE was increased in cerebral tissue following traumatic injury, thus suggesting that it may contribute to the compromise of BBB integrity and the development of brain edema.

The Antioxidant Effect of Rosmarinic Acid by Different Delivery Routes in the Animal Model of Noise-Induced Hearing Loss.

Trans-tympanic Rosmarinic Acid (RA), as compared with the systemic administration, protects against noise-induced auditory hair cell and hearing losses in rats in vivo. ROS production, lipoperoxidative damage, and an imbalance of antioxidant defences play a significant role in noise-induced hearing loss. Several molecules with antioxidant properties have been tested to restore redox homeostasis; however, drug delivery system represents a challenge for their effectiveness. In our model, acute and intense noise exposure induces hearing loss, hair cell death, and oxidative stress, with an increase in superoxide production and over-expression of lipid peroxidation in cochlear structures. RA was administrated in male Wistar rats by trans-tympanic (20 μl) and systemic (10 mg/kg) modality. In systemic administration, RA was injected 1 hour before noise exposure and once daily for the following 3 days. ABRs were measured before and at days 1, 3, 7, and 30 after noise exposure. Rhodamine-phalloidin staining, dihydroethidium and 8-isoprostane immunostainings were performed to assess and quantify outer hair cells loss, superoxide production, and lipid peroxidation in the different experimental groups. Systemic RA administration significantly decreased noise-induced hearing loss and the improvement of auditory function was paralleled by a significant reduction in cochlear oxidative stress. The trans-tympanic modality of drug administration showed a similar degree of protection both at the functional and morphological levels. The effectiveness of RA given via trans-tympanic injection could be interesting for the future application of this minimally-invasive procedure in the treatment of ROS-induced hearing loss.

Uric acid disrupts hypochlorous acid production and the bactericidal activity of HL-60 cells

Uric acid is the end product of purine metabolism in humans and is an alternative physiological substrate for myeloperoxidase. Oxidation of uric acid by this enzyme generates uric acid free radical and urate hydroperoxide, a strong oxidant and potentially bactericide agent. In this study, we investigated whether the oxidation of uric acid and production of urate hydroperoxide would affect the killing activity of HL-60 cells differentiated into neutrophil-like cells (dHL-60) against a highly virulent strain (PA14) of the opportunistic pathogen Pseudomonas aeruginosa. While bacterial cell counts decrease due to dHL-60 killing, incubation with uric acid inhibits this activity, also decreasing the release of the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α). In a myeloperoxidase/Cl-/H2O2 cell-free system, uric acid inhibited the production of HOCl and bacterial killing. Fluorescence microscopy showed that uric acid also decreased the levels of HOCl produced by dHL-60 cells, while significantly increased superoxide production. Uric acid did not alter the overall oxidative status of dHL-60 cells as measured by the ratio of reduced (GSH) and oxidized (GSSG) glutathione. Our data show that uric acid impairs the killing activity of dHL-60 cells likely by competing with chloride by myeloperoxidase catalysis, decreasing HOCl production. Despite diminishing HOCl, uric acid probably stimulates the formation of other oxidants, maintaining the overall oxidative status of the cells. Altogether, our results demonstrated that HOCl is, indeed, the main relevant oxidant against bacteria and deviation of myeloperoxidase activity to produce other oxidants hampers dHL-60 killing activity.

Extracellular Vesicles Released from Mycobacterium tuberculosis-Infected Neutrophils Promote Macrophage Autophagy and Decrease Intracellular Mycobacterial Survival.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb). In the lungs, macrophages and neutrophils are the first immune cells that have contact with the infecting mycobacteria. Neutrophils are phagocytic cells that kill microorganisms through several mechanisms, which include the lytic enzymes and antimicrobial peptides that are found in their lysosomes, and the production of reactive oxygen species. Neutrophils also release extracellular vesicles (EVs) (100–1,000 nm in diameter) to the extracellular milieu; these EVs consist of a lipid bilayer surrounding a hydrophilic core and participate in intercellular communication. We previously demonstrated that human neutrophils infected in vitro with Mtb H37Rv release EVs (EV-TB), but the effect of these EVs on other cells relevant for the control of Mtb infection, such as macrophages, has not been completely analyzed. In this study, we characterized the EVs produced by non-stimulated human neutrophils (EV-NS), and the EVs produced by neutrophils stimulated with an activator (PMA), a peptide derived from bacterial proteins (fMLF) or Mtb, and observed that the four EVs differed in their size. Ligands for toll-like receptor (TLR) 2/6 were detected in EV-TB, and these EVs favored a modest increase in the expression of the co-stimulatory molecules CD80, a higher expression of CD86, and the production of higher amounts of TNF-α and IL-6, and of lower amounts of TGF-β, in autologous human macrophages, compared with the other EVs. EV-TB reduced the amount of intracellular Mtb in macrophages, and increased superoxide anion production in these cells. TLR2/6 ligation and superoxide anion production are known inducers of autophagy; accordingly, we found that EV-TB induced higher expression of the autophagy-related marker LC3-II in macrophages, and the co-localization of LC3-II with Mtb inside infected macrophages. The intracellular mycobacterial load increased when autophagy was inhibited with wortmannin in these cells. In conclusion, our results demonstrate that neutrophils produce different EVs in response to diverse activators, and that EV-TB activate macrophages and promote the clearance of intracellular Mtb through early superoxide anion production and autophagy induction, which is a novel role for neutrophil-derived EVs in the immune response to Mtb.

Abstract TP423: Hesperidin in Citrus Fruits Juice Improves Brain Ischemia-Induced Cognitive Impairment

Introduction: We recently reported that drinking Citrus iyo juice (CI) inhibited more effectively vascular remodeling in inflammation-induced vascular injury mouse model than Citrus unshiu juice (CU) (PLoS One. 2015). These results led us to explore the possibility that citrus fruits juice drinking could attenuate cognitive decline in transient cerebral ischemia model, focusing on the effects of flavanone, hesperidin, which is more abundantly contained in CI compared with CU and has antioxidant activity. Methods: Eight-week-old male C57BL/6 mice were administrated 10 % CI or CU in drinking water or 100 mg/kg/day hesperidin orally by gavage. Two weeks after administration, brain ischemia was induced by bilateral common carotid artery occlusion (BCCAO) for 18 minutes. The Y maze task was performed 2 weeks after BCCAO operation. After cognitive task, cerebral blood flow (CBF) was measured by a laser speckle flowmetry. Cerebral superoxide anion production 24 hours after BCCAO was measured by dihydroethidium (DHE) staining. mRNA levels were measured by quantitative real-time RT-PCR. Results: Administration of CI, CU or hesperidin did not influence systolic blood pressure, bodyweight and brain weight. Cognitive function was impaired with the increase in superoxide anion production after BCCAO. The cognitive impairment was more effectively attenuated by the administration of CI with the significant increase in CBF than sham group. Interestingly, we also observed that the treatment with hesperidin significantly prevented cognitive decline after BCCAO. Superoxide anion production determined by DHE was attenuated by CI or hesperidin, not by CU. The increases in mRNA levels of tumor necrosis factor (TNF)-α in the brain of the brain 24 hours after BCCAO was prevented by hesperidin. Two weeks after BCCAO, expression of mRNA of NADPH oxidase subunits in the brain tended to decrease in treatment with hesperidin. AT1, AT2 and Mas receptor mRNA expression in the brain did not differ among three groups. Conclusion: These results suggest that the intake of hesperidin in citrus fruits juice should prevent cognitive decline after brain ischemia at least in part due to reduction of oxidative stress, inflammatory cytokine and an increase in CBF.

P047 Superoxide production and enhanced NOX2 induction through JAK/STAT signalling mediate IL-6-induced inflammatory responses of colon epithelial cells

IL-6 plays an important role in the pathogenesis of inflammatory bowel disease (IBD), which is supported by close correlation between IL-6 production and severity of the disease in IBD patients. IL-6 enhances expression of adhesion molecules that are required for the recruitment process of neutrophils and monocytes to lesion sites. The regulation of those molecule expressions by NF-κB, a redox-sensitive transcription factor, implies that IL-6-induced adhesion molecule expression may also be associated with reactive oxygen species (ROS) producing activity of IL-6. Present study focused on whether NADPH oxidase is involved in IL-6-induced ROS production and changes in the protein expressions. The IL-6-induced adhesion of monocytes (U937 cell line) to colon epithelial cells (HT-29 cell line) was examined by co-culture of HT-29 cells with U937 cells that were already labelled with BCECF-AM (10 μg/ml). After 3 h treatment with IL-6, BCECF fluorescence from adhered cells was measured. To identify signalling pathway, siRNA transfection, RT-PCR and Western blot analyses were performed. ROS was measured by lucigenin chemiluminescence assay. IL-6 significantly increased U937 monocytic cell adhesion to HT-29 colonic epithelial cells, which was accompanied by upregulation of adhesion molecules (ICAM-1 and VCAM-1) and repression of junction proteins (E-cadherin and claudin). Concurrently, IL-6 significantly increased superoxide production in a time-dependent manner, which matched significant induction of NOX2 and its regulatory subunits. The IL-6-induced ROS production and protein expression changes were attenuated by pretreatment with NADPH oxidase inhibitors (VAS-2840, and DPI) and STAT3 inhibitor (stattic), but not by inhibitors against other enzymes, such as cytosolic COX-2 (celecoxib), mitochondria (antimycin A), xanthine oxidase (allopurinol), and iNOS (NAME). Similarly, tofacitinib, a JAK inhibitor, and sttattic blocked IL-6-induced superoxide production and NOX2 induction. Taken together, our results suggest that IL-6-activated JAK/STAT signalling is linked to superoxide production and NOX2 induction, resulting in IL-6-induced inflammatory responses of colon epithelial cells.

Could Oxidative Stress Regulate the Expression of MicroRNA-146a and MicroRNA-34a in Human Osteoarthritic Chondrocyte Cultures?

Oxidative stress and the overproduction of reactive oxygen species (ROS) play an important role in the pathogenesis of osteoarthritis (OA). Accumulating evidence has demonstrated the involvement of microRNAs (miRNAs) dysregulation in disease development and progression. In this study, we evaluated the effect of oxidative stress on miR-146a and miR-34a expression levels in human OA chondrocytes cultures stimulated by H2O2. Mitochondrial ROS production and cell apoptosis were detected by flow cytometry. The antioxidant enzymes SOD-2, CAT, GPx, the transcriptional factor NRF2 and the selected miRNAs were analyzed by qRT-PCR. The H2O2-induced oxidative stress was confirmed by a significant increase in superoxide anion production and of the apoptotic ratio. Furthermore, H2O2 significantly up-regulated the expression levels of SOD-2, CAT, GPx and NRF2, and modulated miR-146a and miR-34a gene expression. The same analyses were carried out after pre-treatment with taurine, a known antioxidant substance, which, in our experience, counteracted the H2O2-induced effect. In conclusion, the induction of oxidative stress affected cell apoptosis and the expression of the enzymes involved in the oxidant/antioxidant balance. Moreover, we demonstrated for the first time the modification of miR-146a and miR-34a in OA chondrocytes subjected to H2O2 stimulus and we confirmed the antioxidant effect of taurine.

Bisphenol A mediated effects on innate immunity in freshwater teleost spotted snakehead Channa punctatus murrel

The present study aimed to investigate the direct effect of endocrine disruptor, bisphenol A (BPA), on phagocytosis, superoxide production and nitric oxide release by splenic phagocytes of the teleost Channa punctatus. BPA is a xenoestrogen and has been identified as potent exotoxic agent in aquatic ecosystems. Our observations indicated differential immunomodulatory effects on teleost phagocyte activities. Fish splenic phagocytes were incubated in RPMI-1640 culture medium for 16 h with either BPA or E2 at varying concentrations ranging from 10−9 M to 10−6 M. Incubation with both BPA and E2 showed a significant (P < 0.05) increase in superoxide production. Conversely, phagocytosis was depressed at all concentrations, while NO release was significantly less (P < 0.05) at 10−8 M to 10−7 M concentrations of BPA and 10−9 M to 10−6 M concentrations of E2. The present study thus indicated that BPA can modulate innate immune responses in teleost C. punctatus by altering fish immune cell activities, thereby affecting general fitness of the entire population.

Uncoupling Protein 2 Inhibits Myointimal Hyperplasia in Preclinical Animal Models of Vascular Injury.

BackgroundIntracoronary stent restenosis, characterized by excessive smooth muscle cell (SMC) proliferation and myointimal hyperplasia, remains a clinical challenge. Mitochondrial membrane potential has been linked to the proliferative rate of SMCs. This study aimed to screen a critical gene regulating mitochondrial potential and to confirm its effects on myointimal formation in preclinical animal models.Methods and ResultsWe performed transcriptome screening for genes differentially expressed in ligated versus unligated mouse carotid arteries. We observed that uncoupling protein 2 gene (Ucp2) mRNA, encoding UCP2, was transiently upregulated during the first 3 days after ligation and then significantly downregulated from day 7 through day 21, during which time neointima formed remarkably. The UCP2 protein level also declined after day 7 of ligation. In ligated carotid arteries, Ucp2 −/− mice, compared with wild‐type littermates, exhibited accelerated myointimal formation, which was associated with increased superoxide production and can be attenuated by treatment with antioxidant 4‐hydroxy‐2,2,6,6‐tetramethyl‐piperidinoxyl (TEMPOL). Knockdown of UCP2 enhanced human aortic SMC migration and proliferation that can also be attenuated by TEMPOL, whereas UCP2 overexpression inhibited SMC migration and proliferation, along with decreased activity of nuclear factor‐κB. Moreover, nuclear factor‐κB inhibitor attenuated UCP2 knockdown‐enhanced SMC proliferation. Adenovirus‐mediated overexpression of UCP2 inhibited myointimal formation in balloon‐injured carotid arteries of rats and rabbits and in‐stent stenosis of porcine coronary arteries. Moreover, UCP2 overexpression also suppressed neointimal hyperplasia in cultured human saphenous vein ex vivo.ConclusionsUCP2 inhibits myointimal hyperplasia after vascular injury, probably through suppressing nuclear factor‐κB–dependent SMC proliferation and migration, rendering UCP2 a potential therapeutic target against restenosis.

Transient postnatal overfeeding causes liver stress-induced premature senescence in adult mice

Unbalanced nutrition early in life is increasingly recognized as an important factor in the development of chronic, non-communicable diseases at adulthood, including metabolic diseases. We aimed to determine whether transient postnatal overfeeding (OF) leads to liver stress-induced premature senescence (SIPS) of hepatocytes in association with liver structure and hepatic function alterations. Litters sizes of male C57BL/6 mice were adjusted to 9 pups (normal feeding, NF) or reduced to 3 pups during the lactation period to induce transient postnatal OF. Compared to the NF group, seven-month-old adult mice transiently overfed during the postnatal period were overweight and developed glucose intolerance and insulin resistance. Their livers showed microsteatosis and fibrosis, while hepatic insulin signaling and glucose transporter protein expressions were altered. Increased hepatic oxidative stress (OS) was observed, with increased superoxide anion production, glucose-6-phosphate dehydrogenase protein expression, oxidative DNA damage and decreased levels of antioxidant defense markers, such as superoxide dismutase and catalase proteins. Hepatocyte senescence was characterized by increased p21WAF, p53, Acp53, p16INK4a and decreased pRb/Rb and Sirtuin-1 (SIRT-1) protein expression levels. Transient postnatal OF induces liver OS at adulthood, associated with hepatocyte SIPS and alterations in liver structure and hepatic functions, which could be mediated by a SIRT-1 deficiency.

A novel antimicrobial peptide isolated from centipede Scolopendra subspinipes mutilans stimulates neutrophil activity through formyl peptide receptor 2

In this study, we identified scolopendrasin X, a novel antimicrobial peptide (AMP), from centipede Scolopendra subspinipes mutilans. Scolopendrasin X strongly stimulated mouse neutrophils, resulting in intracellular calcium increase, chemotactic migration through pertussis toxin-sensitive G-protein and phospholipase C pathway, and increased superoxide anion production in neutrophils. Target receptor for scolopendrasin X, formyl peptide receptor (FPR)2 mediated scolopendrasin X-induced neutrophil activation. Moreover, scolopendrasin X significantly blocked inflammatory cytokine production induced by lipopolysaccharide in mouse neutrophils. Taken together, our results suggest that the novel AMP scolopendrasin X can be used as a material to regulate neutrophil activity through FPR2.

Tributyltin chloride disrupts aortic vascular reactivity and increases reactive oxygen species production in female rats.

Organotin compounds, such as tributyltin (TBT), are environment contaminants that induce bioaccumulation and have potential toxic effects on marine species and mammals. TBT have been banned by the International Maritime Organization in 2003. However, the assessment of butyltin and metal contents in marine sediments has demonstrated high residual levels of TBT in some cases exceeding 7000ngSng-1. The acceptable daily intake (ADI) level for TBT established by the World Health Organization is 0.5μg/kg bw/day is based on genotoxicity, reproduction, teratogenicity, immunotoxicity, and mainly neurotoxicity. However, their effect on the cardiovascular system is not well understood. In this study, female rats were exposed to 0.5μg/kg/day of TBT for 15days with the goal of understanding the effect of TBT on vascular function. Female Wistar rats were treated daily by gavage and divided into control (n=10) and TBT (n=10) groups. The aortic rings were incubated with phenylephrine in both the presence and absence of endothelium. The phenylephrine concentration-response curves were generated by exposing endothelium-intact samples to NG-nitro-L-arginine methyl ester (L-NAME), apocynin, superoxide dismutase (SOD), catalase, tiron, and allopurinol. Acetylcholine (ACh) and sodium nitroprusside (SNP) were used to evaluate the relaxation response. Exposure to TBT reduced serum 17β-estradiol E2 levels and increased vascular reactivity. After incubation with L-NAME, the vascular reactivity to phenylephrine was significantly higher. Apocynin, SOD, catalase, and tiron decreased the vascular reactivity to phenylephrine to a significantly greater extent in TBT-treated rats than in the control rat. The relaxation induced by ACh and SNP was significantly reduced in TBT rats. Exposure to TBT induced aortic wall atrophy and increased superoxide anion production and collagen deposition. These results provide evidence that exposing rats to the current ADI for TBT (0.5μg/kg) for 15days induced vascular dysfunction due to oxidative stress and morphological damage and should be considered an important cardiovascular risk factor.

Vascular activation of K+ channels and Na+-K+ ATPase activity of estrogen-deficient female rats

The goal of the present study was to evaluate vascular potassium channels and Na+-K+-ATPase activity in estrogen deficient female rats. Female rats that underwent ovariectomy were assigned to receive daily treatment with placebo (OVX) or estrogen replacement (OVX+E2, 1mg/kg, once a week, i.m.). Aortic rings were used to examine the involvement of K+ channels and Na+-K+-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100μM) and K+ channels blockers: tetraethylammonium (TEA, 5mM), 4-aminopyridine (4-AP, 5mM), iberiotoxin (IbTX, 30nM), apamin (0.5mM), charybdotoxin (ChTX, 0.1mM) and iberiotoxin plus apamin. When aortic rings were pre-contracted with KCl (60mM) or pre-incubated with TEA (5mM), 4-aminopyridine (4-AP, 5mM) and iberiotoxin (IbTX, 30nM) plus apamin (0.5μM), the ACh-induced relaxation was less effective in the ovariectomized group. Additionally, 4-AP and IbTX decreased the relaxation by sodium nitroprusside in all groups but this reduction was greater in the ovariectomized group. Estrogen deficiency also increased aortic functional Na+-K+ ATPase activity evaluated by K+-induced relaxation. L-NAME or endothelium removal were not able to block the increase in aortic functional Na+-K+ ATPase activity, however, TEA (5mM) restored this increase to the control level. We also found that estrogen deficiency increased superoxide anion production and reduced nitric oxide release in aortic ring from ovariectomized animals. In summary, our results emphasize that the process underlying ACh-induced relaxation is preserved in ovariectomized animals due to the activation of K+ channels and increased Na+-K+ ATPase activity.