一氧化氮合成酶在不同压力氧气导致的肺型氧中毒中的表达变化

目的：临床上过量使用氧气可导致肺型氧中毒的发生,目前有文献认为常压氧和高压氧导致的肺型氧中毒具有不同的发病机制。本实验拟探讨一氧化氮合成酶在不同压力氧气导致的肺型氧中毒中的表达变化。方法：60只雄性SD大鼠,随机分为6组（n=10）,分别暴露于1绝对压（atmosphere absolute,ATA）、1．5ATA、2ATA、2．5ATA、3ATA,100％氧气中56、20、10、8、6h,暴露于空气组作为对照。出舱后测定各组大鼠肺组织湿干比、支气管肺泡灌洗液蛋白含量。收集肺组织,裂解提取蛋白。行Western blot检测内皮一氧化氮合酶（eNOS）、神经型一氧化氮合酶（eNOS）的表达变化。结果：相对于正常对照组,1．0ATA组肺湿干比、支气管肺泡灌洗液蛋白量表达明显增高。随着氧分压的增高,这种改变减弱。和1．0ATA组相比,高压氧组的肺湿干比、肺泡灌洗液蛋白含量显著降低。各个氧气压力暴露组大鼠肺组织中nNOS的含量没有明显改变。而eNOS含量则在氧气压力为2ATA时明显降低（P〈0．05）,氧气压力为2．5ATA及3ATA时明显增高（P〈0．05）。结论：eNOS在肺中的表达量随着氧气压力的变化而改变。     

A novel cis-element that is responsive to oxidative stress regulates three antioxidant defense genes in rice

All organisms have defense systems against oxidative stress that include multiple genes of antioxidant defense. These genes are induced by reactive oxygen species under condition of oxidative stress. In this study, we found that a 28-bp motif is conserved on the promoter regions of three antioxidant defense genes in rice (Oryza sativa): cytosolic superoxide dismutase (sodCc1), cytosolic thioredoxin (trxh), and glutaredoxin (grx). We demonstrated that the 28-bp sequence acts as a cis-element responsive to oxidative stress by transient expression assay and designated it as CORE (coordinate regulatory element for antioxidant defense). The CORE was activated by methyl viologen treatment and induced a 3.1-fold increase in expression of the reporter gene, but it did not respond to hydrogen peroxide. The expressions of the sodCc1, trxh, and grx genes were coordinately induced by methyl viologen, suggesting that multiple genes involved in antioxidant defense are controlled by a common regulatory mechanism via CORE. Application of the mitogen-activated protein kinase kinase inhibitor caused the constitutive induction of the sodCc1, trxh, and grx genes and the activation of CORE without methyl viologen treatment. These results indicate that a mitogen-activated protein kinase cascade is involved in the gene regulation mediated by CORE.     

The acute antinociceptive effect of hyperbaric oxygen is not accompanied by an increase in markers of oxidative stress

Aims

Exposure to hyperbaric oxygen (HBO₂) causes an antinociceptive response in mice. However, breathing oxygen (O₂) at an elevated pressure can potentially cause oxygen toxicity. The aim of this study was to identify the determinants of HBO₂ antinociception and the toxicity profile of HBO₂.

Main methods

Male NIH Swiss mice were assessed for acute antinociceptive responsiveness under room air or 100% O₂ at 1.0 or 3.5 atmospheres absolute (ATA), using the acetic acid-induced abdominal constriction test. For the oxygen toxicity test, mice were exposed to 3.5 ATA oxygen for 11 min, 60 min, and 60 min daily for 2 days (120 min) or 60 min daily for 4 days (240 min), then assessed by analyzing the levels of two oxidative stress markers, MDA (malondialdehyde) and protein carbonyl in brain, spinal cord and lung.

Key findings

Only the combination of 100% O₂ and 3.5 ATA caused significant antinociception. The antinociceptive effect of 100% O₂ was pressure-dependent up to 3.5 ATA. In the oxygen toxicity test, mice exposed to HBO₂ for different time intervals had levels of brain, spinal cord and lung MDA and protein carbonyl that were comparable to that of control animals exposed to room air.

Significance

Treatment with 100% O₂ evokes a pressure-dependent antinociceptive effect. Since there was no significant increase in levels of the oxidative stress markers in the tested tissues, it is concluded that HBO₂ at 3.5 ATA produces antinociception in the absence of oxidative stress in mice.     

Transcriptional analysis of normal human fibroblast responses to microgravity stress

To understand the molecular mechanism（s） of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the spaceflown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization （SSH） to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several Gl-phase cell cycle traverse genes. Other genes showing upregulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the ＂hub＂ for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.     

Morphological and physiological changes in Saccharomyces cerevisiae by oxidative stress from hyperbaric air

Increase in air or oxygen pressure in microbial cell cultures can cause oxidative stress and consequently affect cell physiology and morphology. The behaviour of Saccharomyces cerevisiae grown under hyperbaric atmospheres of air and pure oxygen was studied. A limit of 1.0 MPa for the air pressure increase (i.e. 0.21 MPa of oxygen partial pressure) in a fed-batch culture of S. cerevisiae was established. Values of 1.5 MPa air pressure and 0.32 MPa pure oxygen pressure strongly inhibited the metabolic activity and the viability of the cells. Also, morphological changes were observed, especially cell-size distribution and the genealogical age profile. Pressure caused cell compression and an increase in number of aged cells. These effects were attributed to oxygen toxicity since similar results were obtained using air or oxygen, if oxygen partial pressure was equal to or higher than 0.32 MPa. The activity of the antioxidant enzymes, catalase and superoxide dismutase (SOD) (cytosolic and mitochondrial isoformes) indicated that the enzymes have different roles in oxidative stress cell protection, depending on other factors that affect the cell physiological state.     

利用RNA-seq技术分析淹水胁迫下转BnERF拟南芥差异表达基因

为探究淹水胁迫下BnERF调节的耐淹防御相关途径,应用RNA-seq技术,对淹水6小时后的拟南芥(Arabidopsis thaliana)野生型(WT)和转BnERF株系(E33)幼苗进行基因表达分析。结果表明,淹水3天后,E33表现出较强的耐淹性,地上部生长状况和根系发育均明显强于野生型。E33幼苗未淹水处理时相对于野生型单独上调的基因有9个,4个为膜结合蛋白,其中2个参与MAPK级联途径,其它5个参与氧化胁迫及水分调节途径;与未淹水野生型相比,无论是未淹水处理还是淹水6小时后的E33幼苗中缺氧响应、抗氧化防护及细胞、器官发育相关基因的表达量均上调。另外,淹水6小时后E33的差异基因并未完全覆盖淹水6小时后野生型的差异基因;E33幼苗中缺氧响应、氧化胁迫响应、能量的产生与转变、乙醇代谢途径中的基因以及乙烯响应因子基因的表达量都明显高于野生型。上述结果表明,BnERF直接或间接调节植物的淹水胁迫相关生理代谢途径,参与淹水胁迫的防御过程。     

Stimulation of perivascular nitric oxide synthesis by oxygen

We hypothesized that elevated partial pressures of O(2) would increase perivascular nitric oxide (*NO) synthesis. Rodents with O(2)- and.NO-specific microelectrodes implanted adjacent to the abdominal aorta were exposed to O(2) at partial pressures from 0.2 to 2.8 atmospheres absolute (ATA). Exposures to 2.0 and 2.8 ATA O(2) stimulated neuronal (type I) NO synthase (nNOS) and significantly increased steady-state.NO concentration, but the mechanism for enzyme activation differed at each partial pressure. At both pressures, elevations in.NO concentration were inhibited by the nNOS inhibitor 7-nitroindazole and the calcium channel blocker nimodipine. Enzyme activation at 2.0 ATA O(2) appeared to be due to an altered cellular redox state. Exposure to 2.8 ATA O(2), but not 2.0 ATA O(2), increased nNOS activity by enhancing nNOS association with calmodulin, and an inhibitory effect of geldanamycin indicated that the association was facilitated by heat shock protein 90. Infusion of superoxide dismutase inhibited.NO elevation at 2.8 but not 2.0 ATA O(2). Hyperoxia increased the concentration of.NO associated with hemoglobin. These findings highlight the complexity of oxidative stress responses and may help explain some of the dose responses associated with therapeutic applications of hyperbaric oxygen.     

Mechanism of okadaic acid-induced neuronal death and the effect of estrogens

Serine/threonine protein phosphatases are important mediators of general cellular function as well as neurodegenerative processes. We have previously shown inhibition of protein phosphatases to be as neurotoxic as glutamate-induced neuronal death but resistant to neuroprotection by estrogens. In this study, the mechanism by which phosphatase inhibition via okadaic acid (OA) induced neurotoxicity is explored. Neurons were exposed to OA or glutamate in the presence or absence of various protein kinases inhibitors, and/or one of four estrogens. Both OA and glutamate induced cell death via increased reactive oxygen species, protein carbonylation, lipid peroxidation, caspase-3 activity, and mitochondrial dysfunction. All estrogens attenuated glutamate-mediated responses, but not OA-induced responses. In addition, inhibition of protein kinase C and mitogen-activated protein kinase pathway was neuroprotective against glutamate but not OA toxicity. Interestingly, inhibition of mitogen-activated protein kinase pathway with PD98096 or U0126 caused a decrease in reactive oxygen species production suggesting that activation of ERK1/2 could further exacerbate the oxidative stress caused by glutamate-induced toxicity; however, these inhibitors had no effect on OA-induced toxicity. Collectively, these results indicate that both glutamate and OA neurotoxicities are mediated by persistent activation of ERK1/2 and/or protein kinase C and a resulting oxidative stress, and that protein phosphatase activity is an important and necessary aspect of estrogen-mediated neuroprotection.     

NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-alpha and -beta, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.     

Inhibition of toxin production inClostridium perfringens in vitro by hyperbaric oxygen

Since 1960, gas gangrene has been treated by the administration of pure oxygen at 3 atmospheres absolute (3 ATA).Small animals are useless for research at this pressure because of oxygen intoxication.In vitro, the growth ofClostridium perfringens (C. welchii) was not arrested by 1 1/2 hr of hyperbaric oxygen at 3 ATA, but the production of toxin is inhibited during this period. It is not inhibited at 2 ATA. If the results of the conservative treatment of patients with hyperbaric oxygen depend upon a comparable process, such treatment at less than 3 ATA would probably be ineffective.     

Dependence of Escherichia coli hyperbaric oxygen toxicity on the lipid acyl chain composition.

This study examines certain membrane-related aspects of oxygen poisoning in Escherichia coli K1060 (fabB fadE lacI) and its parent strain, K-12 Ymel. Cells were grown to exponential or stationary phase in a minimal medium and exposed to air plus 300 lb/in2 of O2 as a suspension in minimal salts. After an initial lag, both strains lost viability with apparent first-order kinetics. Hypebaric oxygen was more toxic to cells harvested during the exponential phase of growth than to cells harvested from the stationary phase of growth for both strains K-12 Ymel and K1060. Control suspensions exposed to air plus 300 lb/in2 of N2 did not lose viability during a 96-h exposure. The sensitivity of the unsaturated fatty acid auxotroph, strain K1060, to hyperbaric oxygen increased as the degree of unsaturation of the fatty acid supplement increased. Cells grown with a cyclopropane fatty acid (9,10=methylenoctadecanoate) were the most resistant; cells grown with a monounsaturated fatty acid (oleate) were intermediate; and those grown with polyunsaturated fatty acids (linoleate and linolenate) were most sensitive to hyperbaric oxygen. The parent strain, K-12 Ymel, lost viability in hyperbaric oxygen most similarly to strain K1060 supplemented with oleate. To determine the relative effect of hyperbaric oxygen on the survival of E. coli with saturated membranes, substrains of K1060 were selected for growth on 12-methyltetrade-canoate or on 9 or 10-monobromostearate. Substrains grown with a saturated fatty acid supplement were equally or more sensitive to hyperbaric oxygen than when the same substrains were grown with a cyclopropane fatty acid supplement. The lipid acyl chain composition was determined in E. coli K1060 before and after exposure to hyperbaric oxygen or hyperbaric nitrogen. The proportion of nonsaturated acyl chain lipid of either the oleate- or the 9,10-methyleneoctade-canoate-supplemented K1060 remained unchanged after hyperbaric gas exposure. In strain K1060 supplemented with linoleate and grown to stationary phase, however, the relative unsaturated acyl chain content after hyperbaric exposure decreased in both gases. This finding prompted an investigation of the role of lipid oxidation in hyperbaric oxygen toxicity. Assays of potential lipid oxidation products were performed with linoleate-grown cells. The lipid hydroperoxide and peroxide content of the lipid extract increased by 6.9 times after 48 h of air plus 300 lb/in2 of O2; malondialdehyde and fluorescent complex lipid oxidation products showed much smaller or no changes. Lipid extracts from hyperbaric oxygen-exposed cells were not toxic to viable E. coli K1060, nor did they increase the rate of loss of viability in cells simultaneously exposed to hyperbaric oxygen. Linoleic acid hydroperoxide at 1.0 mM had no effect on the viability of E. coli K-12 Ymel and only marginally decreased the viability of E. coli K1060 supplemented with linoleate. We conclude that the kinetics of oxygen toxicity in E...     

NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-α and -β, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.     

Contribution of mitochondrial DNA repair to cell resistance from oxidative stress

Numerous studies have revealed that a part of the cellular response to chronic oxidative stress involves increased antioxidant capacity. However, another defense mechanism that has received less attention is DNA repair. Because of the important homeostatic role of mitochondria and the exquisite sensitivity of mitochondrial DNA (mtDNA) to oxidative damage, we hypothesized that mtDNA repair plays an important role in the protection against oxidative stress. To test this hypothesis mtDNA damage and repair was evaluated in normal HA1 Chinese hamster fibroblasts and oxidative stress-resistant variants isolated following chronic exposure to H2O2 or 95% O2. Reactive oxygen species were generated enzymatically using xanthine oxidase and hypoxanthine. When treated with xanthine oxidase reduced levels of initial mtDNA damage and enhanced mtDNA repair were observed in the cells from the oxidative stress-resistant variants, relative to the parental cell line. This enhanced mtDNA repair correlated with an increase in mitochondrial apurinic/apyrimidinic endonuclease activity in both H2O2- and O2-resistant HA1 variants. This is the first report showing enhanced mtDNA repair in the cellular response to chronic oxidative stress. These results provide further evidence for the crucial role that mtDNA repair pathways play in protecting cells against the deleterious effects of reactive oxygen species.     

Alteration of Striatal Dopamine Levels Under Various Partial Pressure of Oxygen in Pre-convulsive and Convulsive Phases in Freely-Moving rats

The purpose of this study was to investigate the change in the striatal dopamine (DA) level in freely-moving rat exposed to different partial pressure of oxygen (from 1 to 5 ATA). Some works have suggested that DA release by the substantia nigra pars compacta (SNc) neurons in the striatum could be disturbed by hyperbaric oxygen (HBO) exposure, altering therefore the basal ganglia activity. Such changes could result in a change in glutamatergic and GABAergic control of the dopaminergic neurons into the SNc. Such alterations could provide more information about the oxygen-induced seizures observed at 5 ATA in rat. DA-sensitive electrodes were implanted into the striatum under general anesthesia. After 1 week rest, awaked rats were exposed to oxygen–nitrogen mixture at a partial pressure of oxygen of 1, 2, 3, 4 and 5 ATA. DA level was monitored continuously (every 3 min) by in vivo voltammetry before and during HBO exposure. HBO induced a decrease in DA level in relationship to the increase in partial pressure of oxygen from 1 ATA to 4 ATA (?15 % at 1 ATA, ?30 % at 2 ATA, ?40 % at 3 ATA, ?45 % at 4 ATA), without signs of oxygen toxicity. At 5 ATA, DA level strongly decreases (?75 %) before seizure which occurred after 27 min ± 7 HBO exposure. After the epileptic seizure the decrease in DA level disappeared. These changes and the biphasic effect of HBO were discussed in function of HBO action on neurochemical regulations of the nigro striatal pathway.     

Role of p38 MAPK in CYP2E1-dependent arachidonic acid toxicity

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 (CYP2E1) and in HepG2 E47 cells, which express CYP2E1. The possible role of mitogen-activated protein kinase (MAPK) members in this process was evaluated. SB203580, a p38 MAPK inhibitor, and PD98059, an ERK inhibitor, but not wortmannin a phosphatidylinositol 3-kinase (PI3K) inhibitor, prevented AA toxicity in pyrazole hepatocytes and E47 cells. SB203580 prevented the enhancement of AA toxicity by salicylate. SB203580 neither lowered the levels of CYP2E1 nor affected CYP2E1-dependent oxidative stress. The decrease in mitochondrial membrane potential produced by AA was prevented by SB203580. Treating CYP2E1-induced cells with AA activated p38 MAPK but not ERK or AKT. This activation was blocked by antioxidants. AA increased the translocation of NF-kappaB to the nucleus. Salicylate blocked this translocation, which may contribute to the enhancement of AA toxicity by salicylate. SB203580 restored AA-induced NF-kappaB translocation, which may contribute to protection against toxicity. In conclusion, AA toxicity was related to lipid peroxidation and oxidative stress, and to the activation of p38 MAPK, as a consequence of CYP2E1-dependent production of reactive oxygen species. Activation of p38 MAPK by AA coupled to AA-induced oxidative stress may synergize to cause cell toxicity by affecting mitochondrial membrane potential and by modulation of NF-kappaB activation.     

Similar but not the same: normobaric and hyperbaric pulmonary oxygen toxicity, the role of nitric oxide

Pulmonary manifestations of oxygen toxicity were studied and quantified in rats breathing >98% O(2) at 1, 1.5, 2, 2.5, and 3 ATA to test our hypothesis that different patterns of pulmonary injury would emerge, reflecting a role for central nervous system (CNS) excitation by hyperbaric oxygen. At 1.5 atmosphere absolute (ATA) and below, the well-recognized pattern of diffuse pulmonary damage developed slowly with an extensive inflammatory response and destruction of the alveolar-capillary barrier leading to edema, impaired gas exchange, respiratory failure, and death; the severity of these effects increased with time over the 56-h period of observation. At higher inspired O(2) pressures, 2-3 ATA, pulmonary injury was greatly accelerated but less inflammatory in character, and events in the brain were a prelude to a distinct lung pathology. The CNS-mediated component of this lung injury could be attenuated by selective inhibition of neuronal nitric oxide synthase (nNOS) or by unilateral transection of the vagus nerve. We propose that extrapulmonary, neurogenic events predominate in the pathogenesis of acute pulmonary oxygen toxicity in hyperbaric oxygenation, as nNOS activity drives lung injury by modulating the output of central autonomic pathways.     

Changes in inflammatory gene expression induced by hyperbaric oxygen treatment in human endothelial cells under chronic wound conditions

Hyperbaric oxygen (HBO) therapy involves the inhalation of 100% oxygen, whilst inside a chamber at greater than atmospheric pressure. It is an effective treatment for chronic diabetic wounds, although the molecular mechanisms involved remain unclear. We hypothesised that HBO could alter inflammatory gene expression in human endothelial cells via a reactive oxygen/nitrogen species-mediated pathway. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O(2) at 1 atmosphere absolute (ATA); PO(2) ~2 kPa) in the presence of lipopolysaccharide and TNF-α for 24h, then treated with HBO for 90 min (97.5% O(2) at 2.4 ATA; PO(2) ~237 kPa). 5h post-HBO, 19 genes involved in adhesion, angiogenesis, inflammation and oxidative stress were downregulated. Notably, only angiogenin gene expression, which promotes both angiogenesis and nitric oxide production (reflected by increased eNOS protein expression in this study), was upregulated. This led to a decrease in endothelial IL-8 mRNA and protein, which could help alleviate inflammatory processes during chronic wound healing. This was no longer evident 22.5h post-HBO, demonstrating the importance of daily exposures in HBO treatment protocols. These studies indicate that elevated oxygen transiently regulates inflammatory gene expression in endothelial cells, which may enhance chronic wound healing.