Contribution of adenoviral-mediated superoxide dismutase gene transfer to the reduction in nitric oxide-induced cytotoxicity on human islets and INS-1 insulin-secreting cells

Aims/hypothesis. Vulnerability of pancreatic islets to oxygen free radicals and nitric oxide contributes to islet transplantation obstacles. This susceptibility can be linked to the low expression levels of antioxidant enzymes in islets. Our aim was to investigate the effect of overexpressing Cu/Zn superoxide dismutase in human islets through a simple procedure on the cytotoxic effects of two nitric oxide donors: 3-morpholinosydnonimine (SIN-1) and S-Nitroso-N-acetyl-d,l-penicillamine (SNAP). Methods. Cultured human islets and INS-1 rat-derived insulin-secreting cells were transfected by an E1-deleted adenovirus carrying Cu/Zn SOD cDNA under the control of a cytomegalovirus (CMV) promoter (AdSOD). The viability of the cells was tested by the WST-1 assay (Roche, Indianapolis, Ind., USA). Results. The AdSOD procedure allowed SOD activity to increase by twofold to threefold for 2 to 8 days following transfection. Adenovirus-driven SOD overexpression was associated with a significant reduction of SIN-1-induced cytotoxicity on human islets (69.9 ± 10.5 % protection at 200 μmol/l and 40.5 ± 8.9 % protection at 400 μmol/l) and INS-1 cells (82.2 ± 8.8 % protection at 200 μmol/l and 31.1 ± 5.8 % protection at 400 μmol/l). Protection against increasing doses of SNAP was AdSOD dose-dependent. Transfected islets released significantly more insulin than control islets in glucose-theophyllin-stimulated conditions, without or following exposure to SNAP. Conclusions/interpretation. We thus established that adenoviral-induced overexpression of Cu/Zn SOD can be beneficial to human islet endocrine function and resistance to nitric oxide cytotoxicity. These data could be relevant for the development of new strategies aimed at preventing NO-induced beta-cell damage in an islet transplantation setting. [Diabetologia (2000) 43: 625–631] Received: 9 September 1999 and in revised form: 5 February 2000     

Superoxide dismutase and catalase activity in naturally derived commercial surfactants

Despite the role of reactive oxygen species in the development of respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in preterm infants, the anti‐oxidant properties of commercial surfactants have never been studied. We measured the superoxide dismutase (SOD) and catalase (CAT) activity, the scavenger activity against hydrogen peroxide (H₂O₂), and its changes after the addition of SOD and CAT in four natural surfactants, namely Infasurf, Curosurf, Survanta, and Alveofact. We found that they contain measurable amount of SOD and CAT. Curosurf and Survanta seem to have higher antioxidant effect than Infasurf and Alveofact. Moreover, the highest phospholipid concentration and recommended dose of Curosurf imply that its scavenger activity for each treatment dose in preterm infants is likely higher than that of Survanta. Finally, the supplementation with SOD and CAT induced a remarkable increase of antioxidant action in all studied surfactants. Pediatr Pulmonol. 2009; 44:1125–1131. ©2009 Wiley‐Liss, Inc.     

Oxidative stress in blood platelets from schizophrenic patients

Oxidative stress in blood platelets is observed in various diseases, including neuropsychiatric disorders. The aim of our study was to evaluate oxidative stress in blood platelets from patients with schizophrenic disorders by measuring the activity of the platelet antioxidative enzyme, superoxide dismutase (SOD), concomitant with the level of thiobarbituric acid reactive species (TBARS). In blood platelets obtained from schizophrenic patients (with paranoid schizophrenia according to DSM-IV criteria) and from healthy volunteers the level of reactive oxygen species was also measured via chemiluminescence. In resting blood platelets from schizophrenic patients the chemiluminescence was higher than in platelets from control subjects (P?<?0.05), but in thrombin-activated platelets an increase (about 53%) of chemiluminescence was observed, however this increase was lower than in thrombin-stimulated platelets from healthy subjects (101.5%). The results indicate that in platelets from schizophrenic patients generation of reactive oxygen species is enhanced. Moreover, we observed that SOD activity in blood platelets from schizophrenic patients was significantly lower than in control platelets and that a correlation exists between increased lipid peroxidation and inhibition of the activity of this antioxidative enzyme in schizophrenic platelets.     

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

AIM: To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro. METHODS: Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene (Ad-HO-1) or enhanced green fluorescent protein gene (Ad-EGFP), and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS: After seven days culture, the viability of cultured rat islets decreased significantly (92% +/- 6% vs 52% +/- 13%, P < 0.05), and glucose-stimulated insulin release also decreased significantly (6.47 +/- 0.55 mIU/L/30IEQ vs 4.57 +/- 0.40 mIU/L/30IEQ, 14.93 +/- 1.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). Transfection of rat islets with adenoviral vectors at an MOI of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets (71% +/- 15% vs 52% +/- 13%, P < 0.05). There was no significant difference in insulin release upon low glucose stimulation (2.8 mmol/L) among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group (P > 0.05), while when stimulated by high glucose (16.7 mmol/L) solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively (12.50 +/- 2.17 mIU/L/30IEQ vs 8.87 +/- 0.65 mIU/L/30IEQ; 12.50 +/- 2.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively (2.21 +/- 0.02 vs 2.08 +/- 0.05; 2.21 +/- 0.02 vs 2.11 +/- 0.03, P < 0.05). CONCLUSION: The viability and function of rat islets decrease over time in in vitro culture, and heme oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.     

Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors

In humans, errors in meiotic chromosome segregation that produce aneuploid gametes increase dramatically as women age, a phenomenon termed the “maternal age effect.” During meiosis, cohesion between sister chromatids keeps recombinant homologs physically attached and premature loss of cohesion can lead to missegregation of homologs during meiosis I. A growing body of evidence suggests that meiotic cohesion deteriorates as oocytes age and contributes to the maternal age effect. One hallmark of aging cells is an increase in oxidative damage caused by reactive oxygen species (ROS). Therefore, increased oxidative damage in older oocytes may be one of the factors that leads to premature loss of cohesion and segregation errors. To test this hypothesis, we used an RNAi strategy to induce oxidative stress in Drosophila oocytes and measured the fidelity of chromosome segregation during meiosis. Knockdown of either the cytoplasmic or mitochondrial ROS scavenger superoxide dismutase (SOD) caused a significant increase in segregation errors, and heterozygosity for an smc1 deletion enhanced this phenotype. FISH analysis indicated that SOD knockdown moderately increased the percentage of oocytes with arm cohesion defects. Consistent with premature loss of arm cohesion and destabilization of chiasmata, the frequency at which recombinant homologs missegregate during meiosis I is significantly greater in SOD knockdown oocytes than in controls. Together these results provide an in vivo demonstration that oxidative stress during meiotic prophase induces chromosome segregation errors and support the model that accelerated loss of cohesion in aging human oocytes is caused, at least in part, by oxidative damage.Chromosome segregation errors during female meiosis are the leading cause of birth defects and miscarriages in humans and their incidence increases dramatically with age (1). Over 90% of Down syndrome cases are the result of an extra copy of chromosome 21 inherited from the mother (2). Although the probability of a meiotic missegregation event is relatively low during a woman’s twenties, by the time she reaches her early forties, she has a one in three chance of conceiving an aneuploid fetus (3). Work in the last decade has begun to shed light on the molecular mechanisms that underlie this phenomenon known as the “maternal age effect.”Proper chromosome segregation during both mitosis and meiosis requires that physical linkages between sister chromatids (cohesion) be formed, maintained, and released in a regulated manner (4, 5). Sister chromatid cohesion, mediated by the evolutionarily conserved cohesin complex, is established during DNA replication. During meiosis, in addition to holding sister chromatids together, cohesion is required to maintain the physical association of recombinant homologs and is therefore essential for proper segregation during the first as well as the second meiotic division (6–8). Normally, a crossover ensures proper segregation as long as cohesion distal to a crossover stabilizes the chiasma and keeps the four-chromatid bivalent intact (Fig. 1A). Cohesion-mediated association of homologous chromosomes promotes proper orientation and microtubule attachments on the meiosis I (MI) spindle. However, if arm cohesion is lost before spindle assembly, premature separation of homologs can result in segregation errors. In addition, centromeric cohesion must be protected during anaphase I to ensure that sisters remain associated until anaphase II.Open in a separate windowFig. 1.SOD1 or SOD2 KD during midprophase I in Drosophila oocytes causes an increase in meiotic NDJ. (A) Cohesion is depicted by black bars between pink or blue sister chromatids. Accurate segregation depends on arm cohesion to hold recombinant homologs together until anaphase I and centromeric cohesin to hold sisters together until anaphase II. Premature loss of cohesion can lead to segregation errors and aneuploid eggs. (B) Percent NDJ for each SOD KD genotype and its respective control. SI Appendix, Table S1 provides raw data. (C) Percent NDJ for SOD2^GD KD and control in wild type and smc1∆/+ backgrounds. SI Appendix, Table S5 provides raw data. For both B and C, we simultaneously drove expression of a UAS-Dicer-2 transgene to maximize the efficiency of the RNAi in germline cells. *P < 0.025, using the statistical test described in ref. 72.The hypothesis that deterioration of meiotic cohesion over a woman’s lifetime contributes to the maternal age effect has gained considerable support over the last decade (9, 10). One reason this theory is attractive is that in human oocytes, cohesion is established and meiotic recombination is completed during early fetal development. Oocytes then arrest in midprophase I (dictyate) until they are recruited for ovulation starting at puberty. Therefore, to maintain the physical association of homologs and promote accurate chromosome segregation, meiotic cohesion must remain intact for decades. Several lines of investigation have uncovered age-dependent cohesion defects in human oocytes as well as those of model organisms (8, 11–18). However, the mechanism(s) leading to loss of cohesion remains largely unexplored.One hallmark of the aging cell is an increase in oxidative damage caused in large part by reactive oxygen species (ROS) (19–22). Produced primarily in the mitochondria as byproducts of metabolism, ROS can damage proteins, lipids, and DNA throughout the cell (22–25). Whereas low levels of ROS have been implicated in signaling pathways (25), failure to tightly control ROS levels can disrupt the redox status of the cell and result in oxidative stress (23). Such stress is thought to arise in aging cells due to their diminished ability to efficiently neutralize ROS (24, 26, 27) and there is strong evidence in different organisms and cell types that oxidative damage increases with age (26, 28–30). Given the extensive aging that human oocytes experience during a woman’s lifetime, accumulation of oxidative damage has been proposed as one factor that may contribute to the maternal age effect (27, 31–33), but so far only correlative evidence exists to support this theory.Testing the hypothesis that oxidative stress can induce meiotic segregation errors requires an experimental system in which ROS levels can be manipulated and the effect on chromosome segregation measured. One way to increase ROS levels within the oocyte is to lower the level of ROS scavengers, which detoxify ROS and help maintain a physiologically safe redox state. The superoxide dismutase (SOD) family of enzymes provides an important defense against oxidative stress by catalyzing the reduction of superoxide to oxygen and hydrogen peroxide. Two cellular isoforms of SOD have been extensively characterized: cytoplasmic SOD1 (Cu/ZnSOD) and mitochondrial SOD2 (MnSOD). In flies and mice, a decrease in SOD activity in the whole organism can increase oxidative damage, reduce lifespan, and lower tolerance to exogenous sources of oxidative stress (34–43). Furthermore, SOD levels have been shown to decline with age (29, 44).Here we test the hypothesis that elevated levels of ROS lead to chromosome segregation errors due to premature loss of cohesion. To mimic the age-dependent increase in oxidative damage that occurs naturally in the human oocyte, we performed conditional knockdown (KD) of ROS scavengers in the Drosophila oocyte. When we knock down either SOD1 or SOD2 during midprophase I, we observe a significant increase in chromosome nondisjunction (NDJ). In addition, meiotic segregation errors in SOD2 KD oocytes increase when the dosage of the cohesin subunit SMC1 is decreased. Direct analysis of cohesion using FISH revealed that arm cohesion defects are more prevalent in SOD KD oocytes than in controls. Consistent with premature loss of arm cohesion and destabilization of chiasmata, the relative frequency at which recombinant homologs missegregate during MI is significantly higher for SOD KD oocytes than for controls. These data provide in vivo evidence that oxidative stress can cause meiotic chromosome segregation errors due to premature loss of cohesion and destabilization of chiasmata. Our findings provide support for the hypothesis that oxidative damage in the aging oocyte contributes to the maternal age effect in humans.     

Role of 12-lipoxygenase and oxidant stress in hyperglycaemia-induced acceleration of atherosclerosis in a diabetic pig model

Aims/hypothesis: We previously showed that vascular smooth muscle cells and endothelial cells cultured under high glucose conditions produced more 12(S)-hydroxyeicosatetraenoic acid (12-HETE), the 12-lipoxygenase (12-LO) product of arachidonate metabolism, relative to normal glucose. Because the lipoxygenase (LO) pathway has been associated with oxidant stress and the pathogenesis of atherosclerosis, we now examined 12-LO activation in vivo in a pig model of diabetes-induced accelerated atherosclerosis which displays human characteristics. Methods: The animal model was developed in pigs who were fed a normal or high fat diet and given streptozotocin injections to produce normolipemic-normoglycaemic (NLNG), normolipemic-hyperglycaemic (NLHG), hyperlipemic-normoglycaemic (HLNG) and hyperlipemic-hyperglycaemic (HLHG) pigs. Tissue samples were obtained from key arterial beds to examine 12-LO expression at 20 weeks after the pigs began their diet. Results: All HG pigs maintained threefold higher serum glucose concentrations. The HL groups developed atherosclerosis but diabetic HLHG pigs showed markedly accelerated atherosclerosis (twofold) relative to non-diabetic HLNG pigs. Immunostaining showed progressive increases in 12-LO in arteries in the order NLNG, NLHG, HLNG and HLHG. Leukocyte-type 12-LO protein (immuno-blotting) as well as mRNA expression (by competitive PCR) in abdominal and coronary arteries were significantly greater in HLHG pigs than in all the other three groups. Furthermore, increased oxidant stress was observed in monocytes from NLHG and HLNG pigs, and greatly potentiated in HLHG pigs. Conclusions/interpretation: These results are consistent with the hypothesis that 12-LO activation plays a key role in accelerated atherosclerosis due to diabetes and hyperlipemia. [Diabetologia (2002) 45: 125–133] Received: 10 July 2001 and in revised form: 21 September 2001     

Amyloid formation in human IAPP transgenic mouse islets and pancreas, and human pancreas, is not associated with endoplasmic reticulum stress

Aims/hypothesis  Supraphysiological levels of the amyloidogenic peptide human islet amyloid polypeptide have been associated with beta cell endoplasmic reticulum (ER) stress. However, in human type 2 diabetes, levels of human IAPP are equivalent or decreased relative to matched controls. Thus, we sought to investigate whether ER stress is induced during amyloidogenesis at physiological levels of human IAPP. Methods  Islets from human IAPP transgenic mice that develop amyloid, and non-transgenic mice that do not, were cultured for up to 7 days in 11.1, 16.7 and 33.3 mmol/l glucose. Pancreases from human IAPP transgenic and non-transgenic mice and humans with or without type 2 diabetes were also evaluated. Amyloid formation was determined histologically. ER stress was determined in islets by quantifying mRNA levels of Bip, Atf4 and Chop (also known as Ddit3) and alternate splicing of Xbp1 mRNA, or in pancreases by immunostaining for immunoglobulin heavy chain-binding protein (BIP), C/EBP homologous protein (CHOP) and X-box binding protein 1 (XBP1). Results  Amyloid formation in human IAPP transgenic islets was associated with reduced beta cell area in a glucose- and time-dependent manner. However, amyloid formation was not associated with significant increases in expression of ER stress markers under any culture condition. Thapsigargin treatment, a positive control, did result in significant ER stress. Amyloid formation in vivo in pancreas samples from human IAPP transgenic mice or humans was not associated with upregulation of ER stress markers. Conclusions/interpretation  Our data suggest that ER stress is not an obligatory pathway mediating the toxic effects of amyloid formation at physiological levels of human IAPP.     

Comprehensive geriatric assessment of elderly highlanders in Qinghai,China, III: Oxidative stress and aging in Tibetan and Han elderly highlanders

Background: Although there are several factors which may contribute to oxidative stress at high altitude, little is known about the association between oxidative stress and aging in the community‐dwelling elderly in the Tibetan Plateau. Methods: Reactive oxygen species (ROS) and comprehensive geriatric functions were examined among 235 community‐dwelling elderly subjects aged 60 years or more (146 Hans and 89 Tibetans). As a marker of ROS, the levels of reactive oxygen metabolites (ROM) were measured using the d‐ROM test. Results: The rate of dependence of basic activities of daily living (basic ADL) among Tibetan elderly highlanders was significantly higher than that among Han elderly highlanders. The d‐ROM level was higher among the Tibetan elderly than those among the Han elderly (Tibetan 465.6 ± 97.9 Carr U, Han 415.3 ± 72.0 Carr U, P = 0.003). The ROM level was higher among women than those among men. Stepwise multiple regression analysis showed that being Tibetan, female, and oxygen saturation were independent predictors of increasing d‐ROM level (Tibetan β, 0.241; female β, 0.206; oxygen saturation β, 0.218). The high levels of ROM (d‐ROM >500 Carr U) were significantly associated with dependence of basic ADL after adjustment for age, sex and ethnicity (odds ratio = 2.51, P = 0.028). Conclusion: The findings of this study imply the possibility that ROS is higher among Tibetan elderly highlanders than that of Han, which related to the geriatric items. Further studies are needed to show the impact of oxidative stress on the aging of highlanders.     

Effects of combining simvastatin with rosiglitazone on inflammation, oxidant stress and ambulatory blood pressure in patients with the metabolic syndrome: the SIROCO study

Aim: Individually, statins and thiazolidinediones (TZDs) show positive effects on atherosclerosis progression in cellular and animal models as well as patients with diabetes; however, their combined effects have not been studied. This study examines the effects of simvastatin combined with rosiglitazone on vascular inflammation, oxidant stress, ambulatory blood pressure (BP) and other atherosclerotic factors in patients with the metabolic syndrome. Methods: This is a randomized, double blind, placebo‐controlled study in 53 subjects with the metabolic syndrome. Participants were randomized to simvastatin 40 mg/day plus placebo vs. simvastatin 40 mg/day plus rosiglitazone 4 mg/day for 6 months. The primary endpoint was the between‐group difference in high‐sensitivity C‐reactive protein (hs‐CRP) and secondary variables including urinary isoprostanes, serum malondialdehyde (MDA), ambulatory BP, adiponectin, and lipid and glycaemic profiles. Results: At study end, the group randomized to the simvastatin/rosiglitazone combination had a greater reduction in hs‐CRP of 1.33 mg/dl, (p = 0.029) and showed a trend for a greater reduction in urinary isoprostane (?39%), (p = 0.056) compared to simvastatin/placebo group. Changes in MDA levels did not differed between groups (p = 0.81). 24‐h systolic blood pressure (SBP) also showed a 4.5 mmHg reduction at 6 months (p = 0.06). Adiponectin levels increased by 3.91 µg/ml in the combination group over placebo, (p = 0.03) and blood glucose decreased in combination group vs. placebo. Conclusion: Our data show that patients with the metabolic syndrome given a statin/TZD combination manifest greater reductions in markers of vascular inflammation and oxidant stress, 24‐h ambulatory BP and increases in adiponectin as well as improved glycaemic indices.     

Reduced extracellular phagocyte oxidative activity, antioxidant level changes and increased oxidative damage in healthy human blood as a function of age

Age-related changes in the blood antioxidant status, in the prooxidative activity of peripheral phagocytes and in the markers of oxidative injury were simultaneously examined in the circulation of 45 middle-aged and elderly healthy volunteers. The results showed a decrease in the opsonin-dependent and -independent extracellular-phagocyte oxidative activity, evaluated by means of luminol chemiluminescence. An increase in the portion of the mitochondrial superoxide generation within the total oxidative phagocyte response was evaluated by means of lucigenin chemiluminescence. The erythrocyte copper/zinc superoxide dismutase increased with age, while blood catalase and glutathione peroxidase activities remained unchanged. The levels of blood SH-groups decreased with age. An age-related increase in blood concentration of thiobarbituric acid-reactive material, a marker of oxidative damage, was detected. Some data, illustrating the existence of a delicate balance between oxidants and prooxidants, were also obtained. Further studies on the interrelationship between the components determining pro/antioxidative status in an organism may prove useful for developing a complex strategy in combating ageing.     

Mitochondrial alterations, cellular response to oxidative stress and defective degradation of proteins in aging

Respiratory function decline and increase ofoxidative stress in mitochondria have beenproposed as important contributors to humanaging. A wide spectrum of alterations in agedindividuals and senescent cells are similar andare correlated to cellular response tosublethal dose of oxidative stress. Thesealterations and responses include: (1) declinein mitochondrial respiratory function; (2)increase in the rate of production of reactiveoxygen species (ROS); (3) accumulation ofmitochondrial DNA (mtDNA) mutations; (4)increase in the levels of oxidative damage toDNA, protein, and lipids; and (5) decrease inthe capacities of degradation of oxidativelydamaged proteins and other macromolecules. Responses to oxidative stress and theirsubsequent interactions in tissues result inthe deleterious effect of ROS on the cellularfunction, which culminate in aging anddegenerative diseases. In this review, wefocus on the roles that ROS play in age-relatedoxidative damage to mtDNA and proteins andoxidative stress responses at the molecular andcellular levels. The alterations of geneexpression profiles elicited by oxidativestress in aging animals are discussed. Wesuggest that the increase in mitochondrialproduction of ROS and decline in the cellularcapacity to cope with oxidative stress andsubsequent accumulation of mtDNA mutations andoxidized proteins play an important role in theaging process.     

Respiration,copper availability and SOD activity in <Emphasis Type="Italic">P. anserina</Emphasis> strains with different lifespan

P. anserina mutants with impairments in complex IV (COX) of the respiratory chain are characterized by an increase in lifespan. Examples are the nuclear grisea mutant with a moderate lifespan extension (60%) and the immortal extranuclear ex1 mutant. Here we report data demonstrating that in mutant ex1 the level of the alternative oxidase (PaAOX) is significantly higher than in mutant grisea. PaAOX levels appear to be reversely dependent on COX activity. The activity profile of superoxide dismutases in the ex1 mutant resembles the profile in senescent wild-type cultures with a high cytoplasmic copper/zinc superoxide dismutase (PaSOD1) and a low mitochondrial manganese superoxide dismutase (PaSOD2) activity. In the grisea mutant, PaSOD1 activity is only detectable in cultures grown in copper-supplemented medium. The two copper-regulated genes PaCtr3 (coding for a high affinity copper transporter) and PaSod2 are not expressed in the two mutants grown in standard medium. The repression of these genes as well as the activity of PaSOD1 is dependent on the availability of cellular copper, which appears to be high in COX-deficient strains such as mutant ex1 and in the senescent wild-type strain. In the wild-type, changes in the cellular localization of copper and in the delivery of this metal to different proteins appear to occur during senescence. Collectively, the data explain the characteristic lifespan of the investigated strains as the result of differences in energy transduction and in the machinery protecting against oxidative stress. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of mesenchymal stromal cells play a role the oxidant/antioxidant balance in a murine model of asthma

Asthma is a heterogeneous disease characterised by chronic airway inflammation. One of the most devastating consequences of this inflammatory process is the generation of reactive oxygen and nitrogen species responsible for oxidative stress. The aim of this study is to analyse the efficiency of treatment with human bone marrow-derived mesenchymal stromal cells (hMSC) in maintaining the oxidative balance in a murine model of allergic asthma by quantifying nitrotyrosine in lung tissues. After confirmation of asthma in the experimental model, samples of lung parenchyma were submitted to immunohistochemical assessment. Intravenous administration of hMSC reduced the levels of nitrotyrosine in the ASTHMA-hMSC group compared to those in the ASTHMA-SAL group. In conclusion, therapeutic administration of hMSC had a beneficial effect on oxidative stress, reducing the levels of nitrotyrosine in lung tissues in a model of allergic asthma.     

赤芍总苷对高血压大鼠血管重构基质金属蛋白酶9、金属蛋白酶组织抑制因子1和血管壁氧化应激的干预作用

目的探讨赤芍总苷(TPG)对自发性高血压大鼠(SHR)血管重构基质金属蛋白酶9(MMP-9)、金属蛋白酶组织抑制因子1(TIMP-1)和血管壁氧化应激的干预作用。方法 30只8周龄雄性SHR大鼠随机分为3组:低剂量TPG组(低剂量组,n=10)、高剂量TPG组(高剂量组,n=10)、对照组(n=10);另外以Wistar大鼠作为空白组(n=8)。低剂量组、高剂量组分别以100、200 mg/(kg·d)的TPG灌胃,对照组、空白组以2 mL生理盐水灌胃。8周后,酶联免疫吸附测定法测定血清MMP-9、TIMP-1水平;活性氧(ROS)荧光探针检测胸主动脉壁细胞氧化应激水平;免疫组织化学染色检测主动脉MMP-9、TIMP-1蛋白表达。结果与空白组比较,对照组MMP-9、TIMP-1血清水平及血管壁MMP-9、TIMP-1蛋白表达均明显升高(P0.05)。TPG治疗后,MMP-9有所下降,高剂量组与对照组比较,差异有统计学意义(P0.05);但TIMP-1在低剂量组、高剂量组、对照组间差异无统计学意义(P0.05)。对照组胸主动脉ROS水平明显高于空白组,而低剂量组、高剂量组ROS水平较对照组下降。结论高血压血管重构存在血管氧化应激和MMP-9水平升高;TPG能够抑制动脉氧化应激和MMP-9水平,改善高血压血管重构,可作为高血压治疗的辅助药物。     

Age-related changes in renal expression of oxidant and antioxidant enzymes and oxidative stress markers in male SHR and WKY rats

Oxidative stress has been hypothesized to play a role in aging and age-related disorders, such as hypertension. This study compared levels of oxidative stress and renal expression of oxidant and antioxidant enzymes in male normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) at different ages (3 and 12 months). In the renal cortex of 3-month old SHR increases in hydrogen peroxide (H₂O₂) were accompanied by augmented expression of NADPH oxidase subunit Nox4 and decreased expression of antioxidant enzymes SOD1 and SOD3. A further increase in renal H₂O₂ production and urinary TBARS was observed in 12-month old WKY and SHR as compared with 3-month old rats. Similarly, expressions of NADPH oxidase subunit p22^phox, SOD2 and SOD3 were markedly elevated with age in both strains. When compared with age-matched WKY, catalase expression was increased in 3-month old SHR, but unchanged in 12-month old SHR. Body weight increased with aging in both rat strains, but this increase was more pronounced in WKY. In conclusion, renal oxidative stress in 12-month old SHR is an exaggeration of the process already observed in the 3-month old SHR, whereas the occurrence of obesity in 12-month old normotensive rats may partially be responsible for the age-related increase in oxidative stress.     

Melatonin enhances antioxidative enzyme gene expression (CAT,GPx, SOD), prevents their UVR‐induced depletion,and protects against the formation of DNA damage (8‐hydroxy‐2'‐deoxyguanosine) in ex vivo human skin

UV radiation (UVR) induces serious structural and functional alterations in human skin leading to skin aging and carcinogenesis. Reactive oxygen species are key players in UVR‐mediated photodamage and induce the DNA‐base‐oxidized, intermediate 8‐hydroxy‐2'‐deoxyguanosine (8‐OHdG). Herein, we report the protective action of melatonin against UVR‐induced 8‐OHdG formation and depletion of antioxidative enzymes using ex vivo human full‐thickness skin exposed to UVR in a dose (0, 100, 300 mJ/cm²)‐ and time‐dependent manner (0, 24, 48 hr post‐UVR). Dynamics of depletion of antioxidative enzymes including catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD), or 8‐OHdG formation were studied by real‐time PCR and immunofluorescence/immunohistochemical staining. UVR‐treated skin revealed significant and immediate (0 hr 300 mJ/cm²) reduction of gene expression, and this effect intensified within 24 hr post‐UVR. Simultaneous increase in 8‐OHdG‐positive keratinocytes occurred already after 0 hr post‐UVR reaching 71% and 99% up‐regulation at 100 and 300 mJ/cm², respectively (P < 0.001). Preincubation with melatonin (10^?3 m ) led to 32% and 29% significant reductions in 8‐OHdG‐positive cells and the prevention of antioxidative enzyme gene and protein suppression. Thus, melatonin was shown to play a crucial role as a potent antioxidant and DNA protectant against UVR‐induced oxidative damage in human skin.