Enhanced IRE1α Phosphorylation/Oligomerization-Triggered XBP1 Splicing Contributes to Parkin-Mediated Prevention of SH-SY5Y Cell Death under Nitrosative Stress.

Mutations in parkin, a neuroprotective protein, are the predominant cause of autosomal recessive juvenile Parkinson's disease. Neuroinflammation-derived nitrosative stress has been implicated in the etiology of the chronic neurodegeneration. However, the interactions between genetic predisposition and nitrosative stress contributing to the degeneration of dopaminergic (DA) neurons remain incompletely understood. Here, we used the SH-SY5Y neuroblastoma cells to investigate the function of parkin and its pathogenic mutants in relation to cell survival under nitric oxide (NO) exposure. The results showed that overexpression of wild-type parkin protected SH-SY5Y cells from NO-induced apoptosis in a reactive oxygen species-dependent manner. Under nitrosative stress conditions, parkin selectively upregulated the inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1) signaling axis, an unfolded protein response signal through the sensor IRE1α, which controls the splicing of XBP1 mRNA. Inhibition of XBP1 mRNA splicing either by pharmacologically inhibiting IRE1α endoribonuclease activity or by genetically knocking down XBP1 interfered with the protective activity of parkin. Furthermore, pathogenic parkin mutants with a defective protective capacity showed a lower ability to activate the IRE1α/XBP1 signaling. Finally, we demonstrated that IRE1α activity augmented by parkin was possibly mediated through interacting with IRE1α to regulate its phosphorylation/oligomerization processes, whereas mutant parkin diminished its binding to and activation of IRE1α. Thus, these results support a direct link between the protective activity of parkin and the IRE1α/XBP1 pathway in response to nitrosative stress, and mutant parkin disrupts this function.

Effect of Normobaric Hypoxia on Alterations in Redox Homeostasis, Nitrosative Stress, Inflammation, and Lysosomal Function following Acute Physical Exercise.

Hypoxia is a recognized inducer of oxidative stress during prolonged physical activity. Nevertheless, previous studies have not systematically examined the effects of normoxia and hypoxia during acute physical exercise. The study is aimed at evaluating the relationship between enzymatic and nonenzymatic antioxidant barrier, total antioxidant/oxidant status, oxidative and nitrosative damage, inflammation, and lysosomal function in different acute exercise protocols under normoxia and hypoxia. Fifteen competitive athletes were recruited for the study. They were subjected to two types of acute cycling exercise with different intensities and durations: graded exercise until exhaustion (GE) and simulated 30 km individual time trial (TT). Both exercise protocols were performed under normoxic and hypoxic (FiO2 = 16.5%) conditions. The number of subjects was determined based on our previous experiment, assuming the test power = 0.8 and α = 0.05. We demonstrated enhanced enzymatic antioxidant systems during hypoxic exercise (GE: ↑ catalase (CAT), ↑ superoxide dismutase; TT: ↑ CAT) with a concomitant decrease in plasma reduced glutathione. In athletes exercising in hypoxia, redox status was shifted in favor of oxidation reactions (GE: ↑ total oxidant status, ↓ redox ratio), leading to increased oxidation/nitration of proteins (GE: ↑ advanced oxidation protein products (AOPP), ↑ ischemia-modified albumin, ↑ 3-nitrotyrosine, ↑ S-nitrosothiols; TT: ↑ AOPP) and lipids (GE: ↑ malondialdehyde). Concentrations of nitric oxide and its metabolites (peroxynitrite) were significantly higher in the plasma of hypoxic exercisers with an associated increase in inflammatory mediators (GE: ↑ myeloperoxidase, ↑ tumor necrosis factor-alpha) and lysosomal exoglycosidase activity (GE: ↑ N-acetyl-ß-hexosaminidase, ↑ ß-glucuronidase). Our study indicates that even a single intensive exercise session disrupts the antioxidant barrier and leads to increased oxidative and nitrosative damage at the systemic level. High-intensity exercise until exhaustion (GE) alters redox homeostasis more than the less intense exercise (TT, near the anaerobic threshold) of longer duration (20.2 ± 1.9 min vs. 61.1 ± 5.4 min-normoxia; 18.0 ± 1.9 min vs. 63.7 ± 3.0 min-hypoxia), while hypoxia significantly exacerbates oxidative stress, inflammation, and lysosomal dysfunction in athletic subjects.

Harnessing oxidative stress for anti-glioma therapy.

Glioma cells use intermediate levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) for growth and invasion, and suppressing these reactive molecules thus may compromise processes that are vital for glioma survival. Increased oxidative stress has been identified in glioma cells, in particular in glioma stem-like cells. Studies have shown that these cells harbor potent antioxidant defenses, although endogenous protection against nitrosative stress remains understudied. The enhancement of oxidative or nitrosative stress offers a potential target for triggering glioma cell death, but whether oxidative and nitrosative stresses can be combined for therapeutic effects requires further research. The optimal approach of harnessing oxidative stress for anti-glioma therapy should include the induction of free radical-induced oxidative damage and the suppression of antioxidant defense mechanisms selectively in glioma cells. However, selective induction of oxidative/nitrosative stress in glioma cells remains a therapeutic challenge, and research into selective drug delivery systems is ongoing. Because of multifactorial mechanisms of glioma growth, progression, and invasion, prospective oncological therapies may include not only therapeutic oxidative/nitrosative stress but also inhibition of oncogenic kinases, antioxidant molecules, and programmed cell death mediators.

ASSESSMENT OF THE OXIDATIVE AND NITROSATIVE STRESS IN THE SERUM OF SAUDI PATIENTS WITH CUTANEOUS LEISHMANIASIS BEFORE AND AFTER TREATMENT.

Macrophages, within which Leishmania species replicate, generate large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) to kill these parasites. The present study assessed the oxidative and nitrosative stress, and specific immune enzymes in the serum of patients with cutaneous leishmaniasis (Cl) before and after treatment and in the control individuals. Serum activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), L-arginase, myeloperoxidase (MPO), and adenosine deaminase (ADA) and the levels of reduced glutathione, malondialdehyde (MDA), and nitric oxide (NO) were studied. The activities of L-arginase, MPO, and ADA and the levels of MDA and NO were significantly elevated (P < 0.001), while the activities of SOD, CAT, and GSH-Px, and the levels of reduced glutathione (GSH) were significantly (P < 0.001) reduced in untreated patients as compared with values of patients after treatment and of control individuals. The treatment, which included intramuscular injection of sodium stibogluconate and meglumine antimoniate, ameliorated these factors in comparison to the untreated group. These results suggest that oxidative and nitrosative stress may play an important role in the pathogenesis of untreated cutaneous leishmaniasis. Furthermore, the reduction in oxidative and nitrosative stress in the treated Cl patients may be due to the drug decreasing energy production by the parasite, which eventually leads to its death.

Inhibition of nitric oxide production enhances the activity of facial nerve tubulin polymerization and the ability of tau to promote microtubule assembly after neurorrhaphy.

We previously reported that inhibition of nitric oxide (NO) production promotes rat reconnected facial nerve regeneration. However, the underlying mechanism is obscure. Microtubule assembly is known to be essential to axon regeneration; nevertheless, tubulins and microtubule-associated proteins (MAPs) have been demonstrated as targets for NO and peroxynitrite. Thus, we hypothesized that NO and/or peroxynitrite may affect facial nerve regeneration via influencing on microtubule assembly. First, tubulins and tau (a MAP) were extracted from facial nerves of normal rats, treated with NO donor or peroxynitrite, and processed for microtubule assembly assay. We found that peroxynitrite, DEA NONOate, and Angeli's salt reduced the tubulin polymerization activity to a greater extent than GSNO, SIN-1, and SNAP. Additionally, SIN-1, peroxynitrite, and Angeli's salt impaired the ability of tau to promote microtubule assembly. Next, nitrosative stress biomarkers 3-nitrotyrosine (3-NT) and S-nitrosylated cysteine (SNO-Cys) were immunolabeled in facial nerves. Both biomarkers were highly upregulated in proximal and distal stumps of reconnected facial nerves at 3 days and 1 week after neurorrhaphy. Notably, the expression of 3-NT was greatly reduced at 2 weeks, whereas that of SNO-Cys was maintained. Conversely, inhibition of NO production with L-NAME prevented the upregulation of SNO-Cys. Further, we used tubulins and tau extracted from facial nerves of sham-operated, nerve suture + vehicle treatment, and nerve suture + L-NAME treatment rats to perform microtubule assembly assay. We found that L-NAME treatment enhanced polymerization activity of tubulins and ability of tau to promote microtubule assembly. It is noteworthy that α-tubulin plays a more important role than ß-tubulin since the activity of microtubule assembly using α-tubulin extracted from L-NAME-treated rats was greatly elevated, whereas that using ß-tubulin extracted from L-NAME-treated rats was not. Overall, our findings support that inhibition of NO production reduces nitrosative stress, and may thus facilitate microtubule assembly and facial nerve regeneration.

Melatonin Ameliorates LPS-Induced Testicular Nitro-oxidative Stress (iNOS/TNFα) and Inflammation (NF-kB/COX-2) via Modulation of SIRT-1.

Lipopolysaccharide (LPS) - an endotoxin that is being extensively used in laboratory to mimic microbial infection that adversely affects male fertility. This study investigated the protective effects of melatonin on LPS-induced testicular nitro-oxidative stress, inflammation, and associated damages in the testes of male golden hamsters, Mesocricetus auratus. Hamsters were administered with melatonin and LPS for 7 days. Testes of LPS treated hamsters showed degenerative changes (appearance of vacuoles, exfoliation, and depletion of germ cells in the seminiferous tubules), adverse effects on spermatogenesis (sperm count and viability), and steroidogenesis (declined serum and testicular testosterone). Furthermore, LPS treatment decreased melatonin content, melatonin receptor (MT1), and antioxidant potential (catalase and SOD), and simultaneously increased nitro-oxidative stress (CRP, nitrate, TNFα). LPS upregulated NF-kB, COX-2, and iNOS expressions to increase testicular inflammatory load that resulted in the decrease of germ cell proliferation and survival, thus culminating into germ cell apoptosis as indicated by AO-EB staining and caspase-3 expression. Administration of melatonin with LPS showed improved testicular histoarchitecture, sperm parameters, and testosterone level. Melatonin increased testicular antioxidant status (SOD, catalase) to counteract the LPS-induced testicular ROS and thus reduced testicular nitro-oxidative stress. Furthermore, melatonin treatment upregulated testicular SIRT-1 expression to inhibit LPS-induced inflammatory proteins, i.e., NF-kB/COX-2/iNOS expression. The rescue effect of melatonin was further supported by increased germ cell survival (Bcl-2), proliferation (PCNA), and declined apoptosis (caspase-3). In conclusion, our result demonstrated that melatonin rescued testes from LPS-induced testicular nitro-oxidative stress, inflammation, and associated damages by upregulation of SIRT-1.

Acute Diallyl Disulfide Administration Prevents and Reveres Lipopolysaccharide-Induced Depression-Like Behaviors in Mice via Regulating Neuroinflammation and Oxido-Nitrosative Stress.

Neuroinflammation and oxidative stress play critical roles in pathogenesis of depression. Diallyl disulfide (DADS), an active compound in garlic oil, has been shown to exhibit obvious anti-inflammatory and anti-oxidative activities. Preliminary evidence indicates that depression is associated with high levels of pro-inflammatory cytokines and oxidative markers, suggesting that inhibition of neuroinflammatory response and oxidative stress may be beneficial for depression interruption. Here, we investigated the antidepressant effect of DADS as well as it mechanisms in a depression-like model induced by lipopolysaccharide (LPS). Similarly to imipramine (10 mg/kg), a clinical antidepressant, DADS (40 or 80 mg/kg), which was administered 1 h before LPS treatment (pre-LPS) or 1.5 h and 23.5 h after LPS treatment (post-LPS), prevented and reversed LPS (100 µg/kg)-induced increase in immobility time in the tail suspension test (TST) and forced swim test (FST) in mice. Mechanistic studies revealed that DADS pre-treatment or post-treatment at the dose of 40 and 80 mg/kg prevented and reversed (i) LPS-induced increases in interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) levels in the hippocampus and prefrontal cortex, (ii) LPS-induced increases in contents of malondialdehyde (MDA), a parameter reflecting high levels of oxidative stress, and (iii) LPS-induced decreases in contents of GSH, a marker reflecting weakened anti-oxidative ability, in the hippocampus and prefrontal cortex in mice. These results indicate that DADS is comparable to imipramine in effectively ameliorating LPS-induced depression-like behaviors in mice, providing a potential value for DADS in prevention and/or therapy of depression.

Coriandrum sativum improve neuronal function via inhibition of oxidative/nitrosative stress and TNF-α in diabetic neuropathic rats.

ETHNOPHRAMACOLOGICAL RELEVANCE: Coriandrum sativum L. is traditionally acknowledged for its use in inflammatory disorders, altered blood lipid levels, respiratory and digestive problems. AIM OF THE STUDY: The present study investigates possible role of hydro-alcoholic extract of C. sativum (CHA) seeds in the attenuation of indices of diabetic peripheral neuropathy (DPN). MATERIALS AND METHODS: Phytochemical analysis was carried out by employing chromatographic, spectroscopic as well as spectrometric techniques. Diabetes was induced by a single i.p. injection of freshly prepared STZ (65 mg/kg). The indexed markers of DPN, i.e., thermal and mechanical hyperalgesia were found to be prominent on the 60th day of STZ administration. Administration of CHA (100, 200, and 400 mg/kg, p.o.) for 30 days was started on the substantiation of DPN onset. Molecular docking study was performed by targeting TNF-α. RESULTS: Phytochemical analysis revealed the presence of flavonoids, terpenoids, and phenolic acids. Oral administration of CHA considerably attenuated hyperglycemia and decreased pain threshold in diabetic rats as well as modulated oxidative-nitrosative stress. Docking study suggested good affinity of flavonoids when docked into the binding site of TNF-α. CONCLUSION: In conclusion, using STZ model, it was successfully predicted that CHA might be beneficial in diabetes-induced neuropathic pain by inhibiting oxidative/nitrosative stress and inflammatory cytokine.

Immune-Inflammatory, Metabolic, Oxidative, and Nitrosative Stress Biomarkers Predict Acute Ischemic Stroke and Short-Term Outcome.

Immune-inflammatory, metabolic, oxidative, and nitrosative stress (IMO&NS) pathways and, consequently, neurotoxicity are involved in acute ischemic stroke (IS). The simultaneous assessment of multiple IMO&NS biomarkers may be useful to predict IS and its prognosis. The aim of this study was to identify the IMO&NS biomarkers, which predict short-term IS outcome. The study included 176 IS patients and 176 healthy controls. Modified Rankin scale (mRS) was applied within 8 h after IS (baseline) and 3 months later (endpoint). Blood samples were obtained within 24 h after hospital admission. IS was associated with increased white blood cell (WBC) counts, high sensitivity C-reactive protein (hsCRP), interleukin (IL-6), lipid hydroperoxides (LOOHs), nitric oxide metabolites (NOx), homocysteine, ferritin, erythrocyte sedimentation rate (ESR), glucose, insulin, and lowered iron, 25-hydroxyvitamin D [25(OH)D], total cholesterol, and high-density lipoprotein (HDL) cholesterol. We found that 89.4% of the IS patients may be correctly classified using the cumulative effects of male sex, systolic blood pressure (SBP), glucose, NOx, LOOH, 25(OH)D, IL-6, and WBC with sensitivity of 86.2% and specificity of 93.0%. Moreover, increased baseline disability (mRS ≥ 3) was associated with increased ferritin, IL-6, hsCRP, WBC, ESR, and glucose. We found that 25.0% of the variance in the 3-month endpoint (mRS) was explained by the regression on glucose, ESR, age (all positively), and HDL-cholesterol, and 25(OH)D (both negatively). These results show that the cumulative effects of IMO&NS biomarkers are associated with IS and predict a poor outcome at 3-month follow-up.

Acute effect of passive cycle-ergometry and functional electrical stimulation on nitrosative stress and inflammatory cytokines in mechanically ventilated critically ill patients: a randomized controlled trial.

Early mobilization is beneficial for critically ill patients because it reduces muscle weakness acquired in intensive care units. The objective of this study was to assess the effect of functional electrical stimulation (FES) and passive cycle ergometry (PCE) on the nitrous stress and inflammatory cytometry in critically ill patients. This was a controlled, randomized, open clinical trial carried out in a 16-bed intensive care unit. The patients were randomized into four groups: Control group (n=10), did not undergo any therapeutic intervention during the study; PCE group (n=9), lower-limb PCE for 30 cycles/min for 20 min; FES group (n=9), electrical stimulation of quadriceps muscle for 20 min; and FES with PCE group (n=7), patients underwent PCE and FES, with their order determined randomly. The serum levels of nitric oxide, tumor necrosis factor alpha, interferon gamma, and interleukins 6 and 10 were analyzed before and after the intervention. There were no differences in clinical or demographic characteristics between the groups. The results revealed reduced nitric oxide concentrations one hour after using PCE (P<0.001) and FES (P<0.05), thereby indicating that these therapies may reduce cellular nitrosative stress when applied separately. Tumor necrosis factor alpha levels were reduced after the PCE intervention (P=0.049). PCE and FES reduced nitric oxide levels, demonstrating beneficial effects on the reduction of nitrosative stress. PCE was the only treatment that reduced the tumor necrosis factor alpha concentration.

Depletion of brain perivascular macrophages regulates acute restraint stress-induced neuroinflammation and oxidative/nitrosative stress in rat frontal cortex.

The central nervous system can respond to peripheral immune stimuli through the activation of the neurovascular unit. One of the cellular types implicated are perivascular macrophages (PVMs), hematopoietic-derived brain-resident cells located in the perivascular space. PVMs have been implicated in the immune surveillance and in the regulation of the accumulation/trafficking of macromolecules in brain-blood interfaces. Recent studies suggested that the role of PVMs could vary depending on the nature and duration of the immune challenge applied. Here, we investigate the role of PVMs in stress-induced neuroinflammation and oxidative/nitrosative consequences. The basal phagocytic activity of PVMs was exploited to selectively deplete them by ICV injection of liposomes encapsulating the pro-apoptotic drug clodronate. Acute restraint stress-induced neuroinflammation and oxidative/nitrosative stress in rat brain frontal cortex samples were assessed by western blot and RT-PCR analyses. The depletion of PVMs: (1) decreased tumor necrosis-α levels (2) prevented the Janus kinase/signal transducers and activators of transcription pathway and increased interleukin-6 receptor protein-expression in stress conditions; (3) prevented the stress-induced Toll-like receptor 4/Myeloid differentiation primary response 88 protein signaling pathway; (4) down-regulated the pro-inflammatory nuclear factor κB/cyclooxygenase-2 pathway; (5) prevented stress-induced lipid peroxidation and the concomitant increase of the endogenous antioxidant mediators nuclear factor (erythroid-derived 2)-like 2, glutathione reductase 1 and Parkinsonism-associated deglycase mRNA expression. Our results point to PVMs as regulators of stress-induced neuroinflammation and oxidative/nitrosative stress. Much more scientific effort is still needed to evaluate whether their selective manipulation is promising as a therapeutic strategy for the treatment of stress-related neuropsychopathologies.

Oxygen and pulmonary vasodilation: The role of oxidative and nitrosative stress.

Respiratory failure complicates up to 2% of live births and contributes significantly to neonatal morbidity and mortality. Under these conditions, supplemental oxygen is required to support oxygen delivery to the brain and other organs, and to prevent hypoxic pulmonary vasoconstriction. However, therapeutic oxygen is also a source of reactive oxygen species that produce oxidative stress, along with multiple intracellular systems that contribute to the production of free radicals in pulmonary endothelium and vascular smooth muscle. These free radicals cause vasoconstriction, act on multiple sites of the nitric oxide pathway to reduce cGMP-mediated vasodilation, and nitrate and inactivate essential proteins such as surfactant. In addition to oxygen, antenatal stressors such as placental insufficiency, maternal diabetes, and fetal growth restriction increase pulmonary and vascular oxidant stress and may amplify the adverse effects of oxygen. Moreover, the effects of free radical damage may extend well beyond infancy as suggested by the increased risk of childhood malignancy after neonatal exposure to hyperoxia. Antioxidant therapy is theoretically promising, but there are not yet clinical trials to support this approach. Targeting the abnormal sources of increased oxidant stress that trigger abnormal pulmonary vascular responses may be more effective in treating disease and preventing long term consequences.

Oxidative and nitrosative stress in the neurotoxicity of polybrominated diphenyl ether-153: possible mechanism and potential targeted intervention.

Polybrominated diphenyl ethers (PBDEs) have been known to exhibit neurotoxicity in rats; however, the underlying mechanism remains unknown and there is no available intervention. In this study, we aimed to investigate the role of oxidative and nitrosative stress in the neurotoxicity in the cerebral cortex and primary neurons in rats following the BDE-153 treatment. Compared to the untreated group, BDE-153 treatment significantly induced the neurotoxic effects in rats, as manifested by the increased lactate dehydrogenase (LDH) activities and cell apoptosis rates, and the decreased neurotrophic factor contents and cholinergic enzyme activities in rats' cerebral cortices and primary neurons. When compared to the untreated group, the oxidative and nitrosative stress had occurred in the cerebral cortex or primary neurons in rats following the BDE-153 treatment, as manifested by the increments in levels of reactive oxygenspecies (ROS), malondialdehyde (MDA), nitric oxide (NO), and neuronal nitric oxide synthase (nNOS) mRNA and protein expressions, along with the decline in levels of superoxide dismutase (SOD) activity, glutathione (GSH) content, and peroxiredoxin I (Prx I) and Prx II mRNA and protein expressions. In addition, the ROS scavenger N-acetyl-l-cysteine (NAC) or NO scavenger NG-Nitro-l-arginine (L-NNA) significantly rescued the LDH leakage and cell survival, reversed the neurotrophin contents and cholinergic enzymes, mainly via regaining balance between oxidation/nitrosation and antioxidation. Overall, our findings suggested that oxidative and nitrosative stresses are involved in the neurotoxicity induced by BDE-153, and that the antioxidation is a potential targeted intervention.

Acute effect of passive cycle-ergometry and functional electrical stimulation on nitrosative stress and inflammatory cytokines in mechanically ventilated critically ill patients: a randomized controlled trial

Early mobilization is beneficial for critically ill patients because it reduces muscle weakness acquired in intensive care units. The objective of this study was to assess the effect of functional electrical stimulation (FES) and passive cycle ergometry (PCE) on the nitrous stress and inflammatory cytometry in critically ill patients. This was a controlled, randomized, open clinical trial carried out in a 16-bed intensive care unit. The patients were randomized into four groups: Control group (n=10), did not undergo any therapeutic intervention during the study; PCE group (n=9), lower-limb PCE for 30 cycles/min for 20 min; FES group (n=9), electrical stimulation of quadriceps muscle for 20 min; and FES with PCE group (n=7), patients underwent PCE and FES, with their order determined randomly. The serum levels of nitric oxide, tumor necrosis factor alpha, interferon gamma, and interleukins 6 and 10 were analyzed before and after the intervention. There were no differences in clinical or demographic characteristics between the groups. The results revealed reduced nitric oxide concentrations one hour after using PCE (P<0.001) and FES (P<0.05), thereby indicating that these therapies may reduce cellular nitrosative stress when applied separately. Tumor necrosis factor alpha levels were reduced after the PCE intervention (P=0.049). PCE and FES reduced nitric oxide levels, demonstrating beneficial effects on the reduction of nitrosative stress. PCE was the only treatment that reduced the tumor necrosis factor alpha concentration.

C/EBPδ protects from radiation-induced intestinal injury and sepsis by suppression of inflammatory and nitrosative stress.

Ionizing radiation (IR)-induced intestinal damage is characterized by a loss of intestinal crypt cells, intestinal barrier disruption and translocation of intestinal microflora resulting in sepsis-mediated lethality. We have shown that mice lacking C/EBPδ display IR-induced intestinal and hematopoietic injury and lethality. The purpose of this study was to investigate whether increased IR-induced inflammatory, oxidative and nitrosative stress promote intestinal injury and sepsis-mediated lethality in Cebpd-/- mice. We found that irradiated Cebpd-/- mice show decreased villous height, crypt depth, crypt to villi ratio and expression of the proliferation marker, proliferating cell nuclear antigen, indicative of intestinal injury. Cebpd-/- mice show increased expression of the pro-inflammatory cytokines (Il-6, Tnf-α) and chemokines (Cxcl1, Mcp-1, Mif-1α) and Nos2 in the intestinal tissues compared to Cebpd+/+ mice after exposure to TBI. Cebpd-/- mice show decreased GSH/GSSG ratio, increased S-nitrosoglutathione and 3-nitrotyrosine in the intestine indicative of basal oxidative and nitrosative stress, which was exacerbated by IR. Irradiated Cebpd-deficient mice showed upregulation of Claudin-2 that correlated with increased intestinal permeability, presence of plasma endotoxin and bacterial translocation to the liver. Overall these results uncover a novel role for C/EBPδ in protection against IR-induced intestinal injury by suppressing inflammation and nitrosative stress and underlying sepsis-induced lethality.

Induction of autophagy under nitrosative stress: A complex regulatory interplay between SIRT1 and AMPK in MCF7 cells.

Induction of nitrosative stress has been observed in various cancer types and in tumor environment. However, it is still unclear how cancer cells combat the effect of nitrosative stress. The main targets of nitrosative stress in cells are cellular lipids, proteins and DNA. Autophagy or self-cleaning generates energy for cell survival under stress conditions. In the present study we investigated the role of autophagy under nitrosative stress in MCF7, a breast cancer cell line. Interestingly, we observed induction of autophagy associated with cell death when MCF7 cells were treated with NO donor compound DETA-NONOate for eight hours. While investigating the mode of cell death under nitrosative stress in MCF7 cells, it was found that it was neither apoptotic nor necrotic. Moreover, nitrosative stress did not alter mitochondrial membrane potential and cellular redox status in MCF7 cells. But we observed an increase in NAD+/NADH and a drop in NADH level in MCF7 cells following NO donor treatment. Sirtuins having NAD+ dependent deacetylase activity, play an important role in cell survival mechanisms. So we further checked the status of SIRT1 under nitrosative stress in MCF7 cells. Surprisingly, we observed an induction of SIRT1, phospho-AMPK and p53 in MCF7 cells under nitrosative stress. Interestingly, autophagy markers were down regulated in MCF7 cells upon treatment with nicotinamide, an inhibitor of SIRT1 activity and dorsomorphin, a phospho-AMPK inhibitor when treated separately under nitrosative stress. To further confirm the role of SIRT1 in the induction of autophagy associated cell death, it was knocked down using si-RNA and nitrosative stress was applied. SIRT1 knock down led to increase in MCF7 cell viability along with down regulation of autophagic markers and phospho-AMPK as well as accumulation of acetylated p53. The increase in p53 controlled DRAM1 mRNA expression in MCF7 cells under nitrosative stress further confirmed a complex interplay between p53, SIRT1 and AMPK under nitrosative stress in MCF7 cells. Altogether our work for the first time suggests a complex inter-twined partnership between AMPK, SIRT1 and p53 in regulating autophagy in response to nitrosative stress in MCF7 cells.

Sigma-1 receptor activation ameliorates LPS-induced NO production and ROS formation through the Nrf2/HO-1 signaling pathway in cultured astrocytes.

Accumulating evidence has shown that astrocytes play a critical role in neuroinflammation and protection against oxidative stress. In this study, we investigated the effects of sigma-1 receptor (Sig-1R) activation on lipopolysaccharide (LPS)-induced inflammatory reactions and oxidative/nitrosative stress in cultured astrocytes. We found that SA4503, a selective Sig-1R agonist, attenuated LPS-induced inflammatory reactions and oxidative/nitrosative stress by downregulating the expression of iNOS and tumor necrosis factor α (TNF-α) and upregulating glutathione (GSH) in cultured astrocytes. To investigate the mechanism by which SA4503 caused these effects, we then examined the expression of nuclear factor erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO-1) through western blotting. The results revealed that SA4503 treatment increased Nrf2 and HO-1 expression significantly. These results suggested that the antioxidative/nitrosative stress and anti-inflammatory effects of Sig-1R activation in astrocytes were partially mediated by Nrf2 and HO-1 activation.

The compensatory antioxidant response system with a focus on neuroprogressive disorders.

Major antioxidant responses to increased levels of inflammatory, oxidative and nitrosative stress (ONS) are detailed. In response to increasing levels of nitric oxide, S-nitrosylation of cysteine thiol groups leads to post-transcriptional modification of many cellular proteins and thereby regulates their activity and allows cellular adaptation to increased levels of ONS. S-nitrosylation inhibits the function of nuclear factor kappa-light-chain-enhancer of activated B cells, toll-like receptor-mediated signalling and the activity of several mitogen-activated protein kinases, while activating nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2 or NFE2L2); in turn, the redox-regulated activation of Nrf2 leads to increased levels and/or activity of key enzymes and transporter systems involved in the glutathione system. The Nrf2/Kelch-like ECH-associated protein-1 axis is associated with upregulation of NAD(P)H:quinone oxidoreductase 1, which in turn has anti-inflammatory effects. Increased Nrf2 transcriptional activity also leads to activation of haem oxygenase-1, which is associated with upregulation of bilirubin, biliverdin and biliverdin reductase as well as increased carbon monoxide signalling, anti-inflammatory and antioxidant activity. Associated transcriptional responses, which may be mediated by retrograde signalling owing to elevated hydrogen peroxide, include the unfolded protein response (UPR), mitohormesis and the mitochondrial UPR; the UPR also results from increasing levels of mitochondrial and cytosolic reactive oxygen species and reactive nitrogen species leading to nitrosylation, glutathionylation, oxidation and nitration of crucial cysteine and tyrosine causing protein misfolding and the development of endoplasmic reticulum stress. It is shown how these mechanisms co-operate in forming a co-ordinated rapid and prolonged compensatory antioxidant response system.

Nitrosative stress in patients with asthma-chronic obstructive pulmonary disease overlap.

BACKGROUND: Asthma-chronic obstructive pulmonary disease overlap (ACO) has frequent exacerbations and a poor quality of life and prognosis compared with those of chronic obstructive pulmonary disease alone. However, the pathogenesis of ACO has not been fully elucidated yet. OBJECTIVES: The aim of this study was to investigate nitrosative stress, which causes a redox imbalance and tissue inflammation in the airways of patients with ACO, and to evaluate the relationship between nitrosative stress and the clinical course in study subjects. METHODS: Thirty healthy subjects and 56 asthmatic patients participated in this study. The asthmatic patients were divided into 33 asthmatic patients and 23 patients with ACO. The study subjects had been followed prospectively for 2 years to evaluate the clinical course. Nitrosative stress was evaluated based on the production of 3-nitrotyrosine (3-NT) in sputum cells. RESULTS: Production of 3-NT was significantly enhanced in patients with ACO compared with that in asthmatic patients. Amounts of reactive persulfides and polysulfides, newly identified powerful antioxidants, were significantly decreased in the ACO group. Baseline levels of 3-NT were significantly correlated with the frequency of exacerbations and decrease in FEV1 adjusted by age, smoking history, and blood eosinophil count. The 3-NT-positive cells were also significantly correlated with amounts of proinflammatory chemokines and cytokines. CONCLUSIONS: These findings suggested that greater nitrosative stress occurred in the airways of patients with ACO, and the degree of nitrosative stress was correlated with an impairment in the clinical course. Nitrosative stress might be related to the pathogenesis of ACO.

High-throughput screening identifies compounds that protect RPE cells from physiological stressors present in AMD.

Dysfunction and eventual loss of retinal pigment epithelial (RPE) cells is a hallmark of atrophic age-related macular degeneration (AMD), and linked to oxidative and nitrosative damage. Herein, we use a high-throughput screen (HTS) to identify compounds that protect human RPE cells from oxidative stress-induced cell death and elucidate the possible mechanism of action. HTS was used to identify compounds that protect RPE cells from oxidative damage. We tested the identified compound(s) in models of RPE stress, including tert-butyl hydroperoxide (TBHP) exposure, ultraviolet-B (UV-B)-mediated light damage and nitrosative stress to the basement membrane using ARPE-19 cells, primary human RPE cells and induced-pluripotent stem cell (iPSC)-derived RPE cells from patients with AMD. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect gene expression of oxidative stress- and apoptosis-related genes and mitochondrial function was measured using a Seahorse XF96 analyzer to elucidate possible mechanisms of action. Five thousand and sixty-five compounds were screened, and of these, 12 compounds were active based on their ability to improve cell viability after exposure to TBHP. After chemical structure review, we identified ciclopirox olamine as a potent inhibitor of oxidative damage to RPE cells. Ciclopirox olamine increased cell viability in ARPE-19 cells treated with TBHP, UV-B light or on nitrite-modified extracellular matrix (ECM) by 1.68-fold, 1.54-fold and 4.3-fold, respectively (p < 0.01). Treatment with TBHP altered expression of genes related to oxidative stress and apoptosis, which was reversed by pretreatment with ciclopirox olamine. Ciclopirox olamine improved mitochondrial function in TBHP-exposed ARPE-19 cells and iPSC-derived RPE cells. Ciclopirox olamine protected primary human RPE cells and iPSC-derived RPE cells from the oxidative stress or damaged basement membrane. HTS of bioactive Food and Drug Administration (FDA)-approved libraries and follow-up studies can be used to identify small molecules (including ciclopirox olamine) that protect RPE cells exposed to various stressors associated with disease progression of AMD. This strategy can be used to identify potential compounds for treatment and prevention of AMD.