Guanosine protects C6 astroglial cells against azide-induced oxidative damage: a putative role of heme oxygenase 1.

JOURNAL OF NEUROCHEMISTRY

| 2014 | 130 | 61–74

doi: 10.1111/jnc.12694

ao em Ciências Biol ogicas: Bioquımica, Departamento de Bioquımica, Programa de P os-Graduacß~ Instituto de Ciências Basicas da Sa ude, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

Abstract Guanosine, a guanine-based purine, is an extracellular signaling molecule that is released from astrocytes and shows neuroprotective effects in several in vivo and in vitro studies. Our group recently showed that guanosine presents antioxidant properties in C6 astroglial cells. The heme oxygenase 1 signaling pathway is associated with protection against oxidative stress. Azide, an inhibitor of the respiratory chain, is frequently used in experimental models to induce oxidative and nitrosative stress. Thus, the goal of this study was to investigate the effect of guanosine on azide-induced oxidative damage in C6 astroglial cells. Azide treatment of these cells resulted in several detrimental effects, including induction of cytotoxicity and mitochondrial dysfunction, increased levels of reactive oxygen/nitrogen species, inducible nitric oxide synthase

expression and NADPH oxidase, decreased glutamate uptake and EAAC1 glutamate transporter expression, decreased glutathione (GSH) levels, and decreased activities of glutamine synthetase (GS), superoxide dismutase and catalase (CAT). The treatment also increased nuclear factor-jB activation and the release of proinflammatory cytokines tumor necrosis factor a and IL-1b. Guanosine strongly prevented these effects, protecting glial cells against azide-induced cytotoxicity and modulating glial, oxidative and inflammatory responses through the activation of the heme oxygenase 1 pathway. These observations reinforce and support the role of guanosine as an antioxidant molecule against oxidative damage. Keywords: azide, C6 astroglial cells, glioprotective molecule, guanosine, heme oxygenase 1. J. Neurochem. (2014) 130, 61–74

Guanosine, a guanine-based purine, is known to be an extracellular signaling molecule (Ciccarelli et al. 2001; Schmidt et al. 2007; Burnstock 2009). Guanosine may be released from astrocytes and shows neuroprotective effects in several in vivo and in vitro studies (Ciccarelli et al. 1999; Lara et al. 2001; Vinade et al. 2003, 2005; Schmidt et al. 2007, 2009; Chang et al. 2008). This nucleoside can effectively protect cells against hypoxia (Oleskovicz et al. 2008; Dal-Cim et al. 2013), cytotoxicity induced by the b-amyloid peptide (Pettifer et al. 2004), chronic cerebral hypoperfusion (Ganzella et al. 2012), ischemic insults (Moretto et al. 2005, 2009; Rathbone et al. 2011), and quinolinic acid-induced seizures (Schmidt et al. 2000, 2005, 2007; Vinade et al. 2003). Recent studies by our group show that guanosine possesses important antioxidant properties, possibly derived from its ability to directly scavenge

Received November 19, 2013; revised manuscript received January 31, 2014; accepted February 12, 2014. Address correspondence and reprint requests to Andre QuincozesSantos, Ph.D., Departamento de Bioquımica, Programa de PosGraduacß~ao em Ciências Biologicas: Bioquımica, Instituto de Ciências Basicas da Saude, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600 – Anexo, Bairro Santa Cecılia, 90035 –003 Porto Alegre, RS, Brazil. E-mail: [email protected] Abbreviations used: CAT, catalase; CNS, central nervous system; EAAT, excitatory amino acid transporters; GPx, glutathione peroxidase; GS, glutamine synthetase; GSH, glutathione; GUO, guanosine; HO1, heme oxygenase 1; IL-1b, interleukin 1b; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; NADPH, nicotinamide adenine dinucleotide phosphate-oxidase; NOX, NADPH oxidase; NF-jB, nuclear factor-jB; NO, nitric oxide; O2• , superoxide anion; ONOO , peroxynitrite; PI3K, phosphatidylinositide 3-kinase; PKC, protein kinase C; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; TNF-a, tumor necrosis factor a; ZnPP IX, Zinc protoporphyrin IX.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 61--74

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oxidative species and/or from the activation of pathways involved in antioxidant defenses (Quincozes-Santos et al. 2013a). Although there is increasing evidence for the protective effects of guanosine in neural cells, the mechanisms of these effects are not fully understood. In addition, guanosine may activate heme oxygenase 1 (HO1), a fundamental defense mechanism for cells exposed to oxidant challenges (Scapagnini et al. 2004; Bau et al. 2005). Heme oxygenase (HO) is the rate-limiting enzyme in the pathway through which pro-oxidant heme is degraded into the antioxidants biliverdin and bilirubin. The enzyme presents three isoforms: the inducible HO1, the constitutive HO2, and the not catalytically active HO3 (Dore 2005). HO also synthesizes carbon monoxide (CO) from the biological substrate biliverdin (Bau et al. 2005). CO can modulate activities attributed to cyclic guanosine monophosphate in the central nervous system (CNS) (Bau et al. 2005). Increases in HO1 activity are associated with protection against stressful conditions, such as oxidative stress and hypoxia (Sakata et al. 2010; Bramanti et al. 2012). HO1 counteracts nitric oxide (NO) toxicity by inhibiting inducible nitric oxide synthase (iNOS) activity (Wakabayashi et al. 2010). Nitric oxide synthase catalyzes the synthesis of NO from L-arginine. NO can interact with superoxide anions (generated by mitochondria), leading to the overproduction of the powerful oxidant species peroxynitrite (Moncada and Bolanos 2006). This compound belongs to a family of molecules known as reactive nitrogen species (RNS), whose members cause cellular damage (Coyle and Puttfarcken 1993; Calabrese et al. 2007). Oxidative stress in the brain is involved in several brain disorders, such as epilepsy, ischemia, and Parkinson’s and Alzheimer’s diseases, and it has been shown that mitochondrial metabolic dysfunction is present in these pathologies (Waldbaum and Patel 2010; Rogawski 2013; Rowley and Patel 2013; Russo et al. 2013). Mitochondria are the primary site of reactive oxygen species (ROS) production. Overproduction of ROS can affect cellular macromolecules such as lipids, proteins, and nucleic acids (Halliwell 2006, 2007). Moreover, increasing evidence suggests that a superoxidegenerating enzyme, NADPH oxidase (NOX), might play a role in ROS production in the CNS (Brennan et al. 2009; Sorce and Krause 2009). Azide is a known inhibitor of complex IV of the respiratory chain (Tulpule and Dringen 2012). Sodium azide (referred to in the text as azide) is a rapid inhibitor of cytochrome c oxidase, the enzyme of the electron transporter chain that catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen (Leary et al. 2002). Azide is also a potent inhibitor of superoxide dismutase (SOD) and catalase (CAT). Thus, azide is frequently used in experimental models because it induces increases in oxidative/ nitrosative stress, mitochondrial membrane potential (DΨm) disruption, failures in signal transduction pathways and, consequently, cellular damage (Leary et al. 2002; Bowler

et al. 2006). Furthermore, azide may inhibit HO1 activity (Sugishima et al. 2002; Ogura et al. 2009). C6 astroglial cells are widely used as an astrocyte-like cell line to study astrocytic parameters, such as glutamate uptake, glutamine synthetase (GS) activity (EC 6.3.1.2), glutathione (GSH) content, oxidative and inflammatory responses, and signaling pathways (dos Santos et al. 2006; Bramanti et al. 2008; Quincozes-Santos et al. 2009, 2013b; QuincozesSantos and Gottfried 2011; Bobermin et al. 2012; KleinkaufRocha et al. 2013). Guanosine modulates important glial functions such as glutamate uptake, and also demonstrates antioxidant and anti-inflammatory activities (Bau et al. 2005; Schmidt et al. 2007; Quincozes-Santos et al. 2013a). Thus, the aim of this study was to investigate the effects of guanosine on azide-induced oxidative damage in C6 astroglial cells. Therefore, the following astroglial roles were evaluated: (i) mitochondrial activity, (ii) ROS levels, (iii) NADPH oxidase activity and cellular superoxide levels, (iv) nitrite production and iNOS expression, (v) glutamate uptake activity, (vi) EAAC1 immunocontent, (vii) GS activity, (viii) GSH levels, (ix) antioxidant enzymatic defenses [SOD, CAT, and glutathione peroxidase (GPx)], and (x) inflammatory response. With this approach, we indicated that the glioprotective mechanism of guanosine involves the activation of the HO1 signaling pathway.

Materials and Methods Chemicals Dulbecco’s Modified Eagle’s Medium (DMEM), other materials for cell cultures and JC-1 were purchased from Gibco/Invitrogen (Carlsbad, CA, USA). Sodium azide, 2’-7’-dichorofluorescein diacetate (DCFH-DA), propidium iodide, methylthiazolyldiphenyltetrazolium bromide (MTT) Formazan, c-glutamylhydroxamate, GSH Standard Stock Solution, o-phthaldialdehyde, Zinc protoporphyrin IX (ZnPP IX), NADP/NADPH, lucigenin, superoxide anion assay kit, anti-iNOS antibody, anti-GS antibody, peroxidase-conjugated anti-rabbit immunoglobulin (IgG), and guanosine were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-EAAC1, anti-glutamate aspartate transporter (GLAST), and anti-GLT1 were obtained from Alpha Diagnostic (San Antonio, TX, USA). L[2,3-3H]-glutamate, nitrocellulose membranes, and enhanced chemiluminescence kits were purchased from Amersham (Buckinghamshire, UK). RANSOD and RANSEL were purchased from Randox (Autrim, UK). Anti-HO1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from common commercial suppliers. C6 cells C6 astroglial cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and were maintained essentially according to our previous publication (dos Santos et al. 2006). The cells were seeded in flasks and maintained in culture in DMEM with 6 mM glucose (pH 7.4), containing 5% fetal bovine serum (FBS), 2.5 mg/mL Fungizoneâ (Gibco/Invitrogen, Carlsbad, CA, USA), and 100 U/l gentamicin, at 37°C in an atmosphere of 5% CO2/95% air.


Guanosine protects C6 cells against azide toxicity

Exponentially growing cells were detached from the culture flasks using 0.05% trypsin/ethylene-diaminetetracetic acid (EDTA) and seeded 1 9 104 cells/cm2 in 96-, 24-, or 6-well plates. The cells were then maintained in DMEM (5% FBS) at 37°C in an atmosphere of 5% CO2/95% air until they reached confluence (on the 3rd day under in vitro conditions). Animals Male Wistar rats (90 days old) were obtained from our breeding colony (Department of Biochemistry, UFRGS, Porto Alegre, Brazil), maintained under controlled environment (12-h light/12-h dark cycle; 22 1°C; ad libitum access to food and water). All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Federal University of Rio Grande do Sul Animal Care and Use Committee (process number 21215). Primary astrocyte cultures These protocols were in accordance with Souza et al. 2013 and Bellaver et al. 2014. Briefly, male Wistar rats (90 days old) had their cortices and hippocampi aseptically removed from cerebral hemispheres. The tissues were then mechanically dissociated and centrifuged at 100 g for 5 min. The cells were resuspended in Hanks’ balanced salt solution (HBSS) containing DNase (0.003%) and left for decantation for 20 min (hippocampi) and 30 min (cortices). The supernatants were collected and centrifuged for 7 min (400 g). The cells from supernatant were resuspended in DMEM/F12 [10% FBS, 15 mM HEPES, 14.3 mM NaHCO3, 1% fungizone and 0.04% gentamicin], plated in 6- or 24-well plates pre-coated with poly-Llysine at a density of 3–5 9 105 cells/cm2. The cells were cultured at 37°C in a 95% air/5% CO2 incubator. The first medium exchange occurred 24 h after obtaining a culture. During the 1st week, the medium change occurred once every 2 days and, from the 2nd week on, once every 4 days. From the 3rd week on, the cells received medium supplemented with 20% FBS and around the 4th to 5th week the cells reached the confluence. Guanosine and azide treatments After the cells reached confluence, the culture medium was exchanged with serum-free DMEM and cells were pre-incubated in the absence or presence of 100 lM guanosine for 1 h. After preincubation, guanosine was maintained and 5 mM azide was added for 3 h. During all procedures, cells were maintained at 37°C in an atmosphere of 5% CO2/95% air. To study the involvement of the HO1 signaling pathway in the effects of guanosine on azide-induced oxidative damage, we coincubated ZnPP IX (10 lM), a HO1 inhibitor, with guanosine. In our conditions, this inhibitor did not change the HO1 levels (data not shown). For primary astrocytes, after confluence the cells followed the same treatment described above. After cellular treatments, the evaluations described below were performed. To measure MTT reduction, DΨm (determined using the JC-1 assay), DCFH oxidation, and NO levels, cells were incubated with the reagents specified for each methodology. For western blot analysis, cells were solubilized with the specified lysis solution. For glutamate uptake, after L-[2,3-3H]-glutamate incorporation, cells were lysed in NaOH. For GS activity, GSH and superoxide levels, and NADPH, SOD, CAT, and GPx activities, cells were lysed in a sodium phosphate buffer with KCl (140 mM).

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To measure tumor necrosis factor a (TNF-a) and interleukin 1b (IL-1b) levels, the extracellular medium was used. To determine nuclear factor-jB (NF-jB) levels, the nuclear fraction was isolated and NF-jB p65 activation was measured. MTT reduction assay MTT was added to the medium at a concentration of 50 lg/mL and cells were incubated for 30 min at 37°C in an atmosphere of 5% CO2/95% air (Bobermin et al. 2012). Subsequently, the medium was removed and the MTT crystals were dissolved in dimethylsulfoxide. Absorbance values were measured at 560 nm and 650 nm. The results are expressed as percentages relative to the control conditions. Mitochondrial membrane potential–DΨm (JC-1 assay) To determine the DΨm, cells were incubated for 30 min with JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide, 2 lg/mL) (Reers et al. 1995). Cells were then homogenized and centrifuged, washed once with HBSS, and transferred to a 96well plate. Fluorescence was measured using an excitation wavelength of 485 nm and emission wavelengths of 540 and 590 nm. The DΨm was calculated using the ratio of 590 nm (red fluorescent J-aggregates) to 540 nm (green monomers). The results are expressed as percentages relative to the control conditions. DCFH oxidation Intracellular ROS levels were detected using DCFH-DA. DCFH-DA was added to the medium at a concentration of 10 lM and cells were incubated for 30 min at 37°C. Following DCFH-DA exposure, the cells were scraped into phosphate-buffered saline with 0.2% Triton X-100. The fluorescence was measured in a plate reader (Spectra Max GEMINI XPS, Molecular Devices, USA) with excitation at 485 nm and emission at 520 nm (Quincozes-Santos et al. 2009). The results are expressed as percentages relative to the control conditions. NADPH oxidase activity NADPH oxidase activity was measured in cell lysate suspended in a sodium phosphate buffer with 140 mM KCl and protease mixture inhibitor using a modified assay (Abid et al. 2007). Briefly, this luminescence assay used lucigenin as the electron acceptor generated by the NADPH oxidase complex. NADPH oxidase assay solution with 5 lM of lucigenin was used and the concentration of NADPH (1 lM–1 mM), used as the substrate, fell well within the linear range of the assay. The data were converted to relative light units/min/mg of protein, using a standard curve generated with xanthine/xanthine oxidase. Lucigenin activity (light units/min/mg of protein) of control cells was arbitrarily set at 100%. The results are expressed as percentages relative to the control conditions. Cellular superoxide levels Cellular superoxide levels were determined using the superoxide anion assay kit from Sigma. The kit method is based on the oxidation of luminol by superoxide anions resulting in the formation of chemiluminescence light. The chemiluminescence measurement in lysed cells increases with superoxide formation. The control cells were arbitrarily set at 100%. The kit includes a superoxide anion


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producing system (xanthine/xanthine oxidase) for a positive control and the superoxide dismutase enzyme for the repression of the system, used as a negative control. The results are expressed as percentages relative to the control levels. Nitrite levels NO levels were determined by measuring the amount of nitrite (a stable oxidation product of NO), as indicated by the Griess reaction. The Griess reagent was prepared by mixing equal volumes of 1% sulfanilamide in 0.5 M HCl and 0.1% N-(1-naphthyl) ethylenediamine in deionized water. Briefly, the Griess reagent was added directly to the cell culture, which was incubated in the dark for 15 min, at 22°C (Quincozes-Santos et al. 2013b). Samples were analyzed at 550 nm on a microplate spectrophotometer. Nitrite concentrations were calculated using a standard curve prepared with concentrations of sodium nitrite ranging from 0 lM to 50 lM. The results are expressed as percentages relative to the control conditions. Western blot analysis Cells were solubilized with a lysis solution containing 4% sodium dodecyl sulfate (SDS), 2 mM EDTA, and 50 mM Tris-HCl at pH 6.8. Equal amounts of proteins from each sample were boiled in a sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 5% b-mercaptoethanol, 10% (v/v) glycerol, 0.002% (w/v) bromophenol blue) and submitted to electrophoresis in a 10% (w/v) SDSpolyacrylamide gel. The separated proteins were transferred to a nitrocellulose membrane. Equal loading of each sample was confirmed with Ponceau S staining (Sigma). The membrane was incubated with polyclonal antibodies targeting HO1 (1 : 1000), iNOS (1 : 10 000), EAAC1 (1 : 1000), GLAST (1 : 5000), GLT1 (1 : 1000), GS (1 : 10 000), and b-actin (1 : 1000). b-actin was used as a loading control. After incubating overnight with the primary antibody at 22°C, the membrane was washed and incubated with peroxidase-conjugated anti-rabbit immunoglobulin (IgG) at a dilution of 1 : 1000 for 1 h. The chemiluminescent signal was detected using enhanced chemiluminescence; the resulting films were scanned and the bands were quantified using the Scion Image software (Scion Corp., Frederick, MD, USA). Glutamate uptake The glutamate uptake was performed as described previously (dos Santos et al. 2006). Briefly, C6 cells were incubated at 37°C in HBSS containing the following components (in mM): 137 NaCl, 5.36 KCl, 1.26 CaCl2, 0.41 MgSO4, 0.49 MgCl2, 0.63 Na2HPO4, 0.44 KH2PO4, 4.17 NaHCO3, and 5.6 glucose, adjusted to pH 7.4. The assay was started by the addition of 0.1 mM L-glutamate (pH 7.4) and 0.33 lCi/mL L-[2,3-3H] glutamate. The incubation was stopped after 10 min by removing the medium and rinsing twice with ice-cold HBSS. The cells were then lysed in a solution containing 0.5 M NaOH. Incorporated radioactivity was measured in a scintillation counter. Sodium-independent uptake was determined using N-methyl-D-glucamine instead of sodium chloride. Sodium-dependent glutamate uptake, considered specific uptake, was obtained by subtracting the sodium-independent uptake from the total uptake. The results are expressed as percentages relative to the control conditions. Glutamine synthetase (GS) activity The enzymatic assay was performed as described previously (dos Santos et al. 2006). Briefly, 0.1 mL of the cell lysate, suspended in

140 mM KCl, was added to 0.1 mL of the reaction mixture containing (in mM): 10 MgCl2, 50 L-glutamate, 100 imidazole-HCl buffer (pH 7.4), 10 2-mercaptoethanol, 50 hydroxylamine-HCl, and 10 ATP, and incubated for 15 min at 37°C. The reaction was stopped by the addition of 0.4 mL of a solution containing (in mM): 370 ferric chloride, 670 HCl, and 200 Trichloroacetic acid. After centrifugation, the absorbance of the supernatant was measured at 530 nm and compared to the absorbance generated using standard quantities of c-glutamylhydroxamate treated with a ferric chloride reagent. The results are expressed in lmol/mg protein/h. Glutathione (GSH) levels GSH levels were assessed as described previously (Browne and Armstrong 1998). Cell lysate suspended in a sodium phosphate buffer with 140 mM KCl was diluted with a 100 mM sodium phosphate buffer (pH 8.0) containing 5 mM EDTA, and the protein was precipitated with 1.7% meta-phosphoric acid. The supernatant was assayed with o-phthaldialdehyde (at a concentration of 1 mg/ mL methanol) at 22°C for 15 min. Fluorescence was measured using excitation and emission wavelengths of 350 nm and 420 nm, respectively. A calibration curve was performed with standard GSH solutions at concentrations ranging from 0 lM to 500 lM. GSH concentrations were calculated as nmol/mg protein. Superoxide dismutase (SOD) activity SOD activity was determined using the RANSOD kit from Randox (Autrim, UK). The SOD activity (measured by the degree of inhibition of formazan dye formation) in lysed cells was assayed spectrophotometrically at 505 nm. The inhibition of the produced chromogen is proportional to the activity of SOD present in the sample. The results are expressed as percentages relative to the control levels. Catalase (CAT) activity CAT activity was assayed by the method described by Aebi (Aebi 1984). The decrease absorbance at 240 nm was measured in cell lysate suspended in a reaction medium containing 20 mM H2O2, 0.1% Triton X-100, 10 mM potassium phosphate buffer, pH 7.0, and 50 lg protein. One unit (U) of enzyme activity is defined as 1 lmol of H2O2 consumed per minute. The results are expressed as percentages relative to the control levels. Glutathione peroxidase (GPx) activity GPx activity was measured using the RANSEL kit from Randox (Autrim, UK). The concentration of GPx in lysed cells is assessed by measuring the decrease in absorption at 340 nm owing to the oxidation of NADPH to NADP+, which occurs during the conversion of GSH to GSSG. The results are expressed as percentages relative to the control levels. Tumor Necrosis Factor a (TNF-a) levels TNF-a levels in extracellular medium were assayed using a rat TNFa ELISA from PeproTech (Rocky Hill, NJ, USA). The results are expressed as percentages relative to the control levels. Interleukin 1b (IL-1b) levels IL-1b levels in the extracellular medium were assayed using a rat IL-1b ELISA from eBioscience (USA). The results are expressed as percentages relative to the control levels.



Nuclear factor-jB levels The levels of NF-jB p65 in the nuclear fraction, which had been isolated from lysed cells by centrifugation, were measured using an ELISA commercial kit from Invitrogen (Carlsbad, CA, USA). The results are expressed as percentages relative to the control levels. Protein assay Protein content was measured using Lowry’s method, with bovine serum albumin as a standard (Lowry et al. 1951). Statistical analyses Differences among groups were statistically analyzed using two-way analysis of variance (ANOVA), followed by Tukey’s test. All analyses were performed using the Statistical Package for Social Sciences (SPSS) software, version 15.0 (SPSS Inc., Chicago, IL, USA).

Results Effects of guanosine on mitochondrial activity The mitochondrial activity was measured by MTT reduction and DΨm. Azide exposure induced a decrease of approximately 20% in MTT reduction and DΨm (Table 1). Guanosine completely prevented these effects, restoring both parameters to control values, with no effect on the control condition. It is important to note that there was no loss in membrane integrity, as measured by propidium iodide incorporation and lactate dehydrogenase extracellular content (data not shown). Guanosine inhibited ROS accumulation induced by azide through HO1 ROS levels (measured by DCFH oxidation) increased 55% following azide exposure (Fig. 1). Guanosine prevented this effect, decreasing the ROS levels from 155 10% to 99 7% (p < 0.01). This indicates that guanosine may play an antioxidative role. Because guanosine modulates HO1 protein activity, we investigated whether the antioxidant effect of guanosine was dependent on HO1 (Bau et al. 2005).

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The HO1 inhibitor, ZnPP IX (10 lM), abolished the guanosine effect, indicating that the antioxidant activity of guanosine occurred through HO1. Guanosine had no effect on the control condition. Guanosine decreased NADPH oxidase activity and cellular superoxide levels NADPH oxidase produces superoxide anion and, consequently, is involved in ROS production. Azide increased NADPH oxidase activity by 25% (p < 0.01) and guanosine prevented this effect (Fig. 2a). In addition, the superoxide production was measured. Azide increased the cellular superoxide levels by about 45% (p < 0.01) and this effect was also prevented by guanosine (2B). Both effects were abolished in the presence of HO1 inhibitor. Guanosine had no effect on the control condition. Guanosine decreased nitrite levels and prevented azideinduced iNOS expression through HO1 The production of NO was indirectly measured by the formation of nitrite (Fig. 3a). Azide increased the nitrite levels up to 25% compared to controls (p < 0.01), whereas guanosine prevented this effect. HO1 may regulate NO production. Accordingly, the positive effect of guanosine on NO levels was blocked in the presence of the HO1 inhibitor. The excessive NO production was associated with iNOS expression and azide increased iNOS expression levels when compared to controls (Fig. 3b). Guanosine also prevented this effect, returning the iNOS immunocontent to control values (Fig. 3b). Guanosine had no effect on the control condition.

Table 1 The effects of guanosine on mitochondrial activity Treatments

MTT (% of control)

DΨm (% of control)

GUO AZD GUO + AZD

98 8 80 6* 101 8

101 8 80 7* 104 9

Cells were pre-treated for 1 h with 100 lM guanosine (GUO), followed by the addition of 5 mM azide for 3 h. MTT reduction and DΨm were measured as described in the Materials and Methods section. The data are expressed as percentages relative to the control conditions and represent the mean SEM of three independent experimental determinations, performed in triplicate, and analyzed statistically by two-way ANOVA followed by Tukey’s test. *p < 0.05 refers to statistically significant differences from the control.

Fig. 1 The effects of guanosine on reactive oxygen species (ROS) levels. Cells were pre-treated for 1 h with 100 lM guanosine (GUO), followed by the addition of 5 mM azide for 3 h. Cells were also preincubated with Zinc protoporphyrin IX (ZnPP IX) (10 lM). ROS levels (measured by DCFH oxidation) were evaluated as described in the Materials and Methods section. The data are expressed as percentages relative to the control conditions and represent the mean SEM of three independent experimental determinations, performed in triplicate, and analyzed statistically by two-way ANOVA followed by Tukey’s test. **p < 0.01 refer to statistically significant differences from the control.


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(a)

(a)

(b)

(b)

Fig. 2 Guanosine decreased NADPH oxidase activity and cellular superoxide levels. Cells were pre-treated for 1 h with 100 lM guanosine (GUO), followed by the addition of 5 mM azide for 3 h. Cells were also pre-incubated with Zinc protoporphyrin IX (ZnPP IX) (10 lM). (a) NADPH oxidase activity and (b) cellular superoxide levels were measured as described in the Materials and Methods section. The data are expressed as percentages relative to the control conditions and represent the mean SEM of three independent experimental determinations, performed in triplicate, and analyzed statistically by two-way ANOVA followed by Tukey’s test. **p < 0.01 refer to statistically significant differences from the control.

Guanosine prevented the azide-induced impairment of glutamate uptake through HO1 As the glutamate uptake system is the target of ROS/RNS activities, the effects of azide and guanosine on glutamate uptake were evaluated (Fig. 4a). Azide decreased the glutamate uptake by 20% (p < 0.01) and guanosine prevented this effect. EAAC1, the major glutamate transporter present in C6 cultured cells, decreased its expression after azide incubation (Fig. 4b). Guanosine also prevented this effect. However, in both cases, the effects were blocked by the HO1 inhibitor. We recently developed a protocol of primary astrocyte cultures from adult Wistar rats (Souza et al. 2013; Bellaver et al. 2014). Thus, in another set of experiments, we aimed to determine if the effect of guanosine here observed (on glutamate uptake) was also exerted in this primary astrocytes. As expected azide decreased glutamate uptake in cortical and hippocampal astrocytes from adult Wistar rats by 25% (p < 0.01), an effect prevented by guanosine (Table 2).

Fig. 3 Guanosine decreased nitrite production. Cells were pre-treated for 1 h with 100 lM guanosine (GUO), followed by the addition of 5 mM azide for 3 h. Cells were also pre-incubated with Zinc protoporphyrin IX (ZnPP IX) (10 lM). (a) Nitrite levels and (b) immunocontent of inducible nitric oxide synthase (iNOS) were measured as described in the Materials and Methods section. The data are expressed as percentages relative to the control conditions and represent the mean SEM of three independent experimental determinations, performed in triplicate. Western blot analysis is represented as arbitrary units. The data are analyzed statistically by two-way ANOVA followed by Tukey’s test. *p < 0.05 and **p < 0.01 refer to statistically significant differences from the control.

The expression of GLAST and GLT1 did not change with azide/guanosine treatments (not shown). The HO1 inhibitor abolished the positive effect of guanosine. Effects of guanosine on GS activity and GSH levels As shown in Fig. 5a, azide decreased GS activity from 0.50 0.04 lmol/mg protein/h to 0.40 0.03 (p < 0.01). Guanosine prevented this effect. Guanosine also increased the basal GS activity from 0.50 0.04 lmol/mg protein/h to 0.60 0.04 (p < 0.05). The immunocontent of GS did not change after treatments (Fig. 5b). Azide also decreased GSH levels (control: 11.0 0.9 nmol/mg protein vs. azide:



(a)

(b)

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Table 2 The effects of guanosine on glutamate uptake in cortical and hippocampal primary astrocytes from adult Wistar rats

Treatments

Cortical primary astrocytes (% of control)

GUO AZD GUO + AZD GUO + AZD + ZnPP IX

103 75 106 73

9 9** 9 9**

Hippocampal primary astrocytes (% of control) 100 75 102 79

8 8** 8 10*

Primary astrocyte cultures were pre-treated for 1 h with 100 lM guanosine (GUO), followed by the addition of 5 mM azide for 3 h. Cells were also pre-incubated with ZnPP IX (10 lM). Glutamate uptake was measured as described in the Materials and Methods section. The data are expressed as percentages relative to the control conditions and represent the mean SEM of three independent experimental determinations, performed in triplicate. The data are analyzed statistically by two-way ANOVA followed by Tukey’s test. *p < 0.05 and **p < 0.01 refer to statistically significant differences from the control.

to near-control levels. The HO1 inhibitor, ZnPP IX, abolished all of the effects of guanosine.

Fig. 4 Guanosine modulated glutamate uptake. Cells were pretreated for 1 h with 100 lM guanosine (GUO), followed by the addition of 5 mM azide for 3 h. Cells were also pre-incubated with Zinc protoporphyrin IX (ZnPP IX) (10 lM). (a) Glutamate uptake and (b) immunocontent of EAAC1 were measured as described in the Materials and Methods section. The data are expressed as percentages relative to the control conditions and represent the mean SEM of three independent experimental determinations, performed in triplicate. Western blot analysis is represented as arbitrary units. The data are analyzed statistically by two-way ANOVA followed by Tukey’s test. *p < 0.05 and **p < 0.01 refer to statistically significant differences from the control.

8.0 0.7 nmol/mg protein – Fig. 5c). Guanosine prevented this decrease. Effect of guanosine on superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities To determine the effects of guanosine and azide on antioxidant enzymatic cellular defenses, we studied the activities of SOD, CAT, and GPx (Table 3). As expected, C6 astroglial cells exposed to azide showed a significant decrease in SOD (18%) and CAT (21%) activities and an increase in GPx (16%) activity. Guanosine prevented the effects of azide on enzymatic defenses, restoring the values

Guanosine prevented the azide-stimulated TNF-a, IL-1b, and NF-jB levels ROS play a critical role in inflammatory response. Thus, we measured the levels of the classical proinflammatory cytokines, TNF-a (Fig. 6a), and IL-1b (Fig. 6b). Azide increased TNF-a and IL-1b levels by 40% and 35%, respectively, compared to controls (p < 0.01). Guanosine completely prevented these effects. Azide also increased the activation of NF-jB, a transcription factor involved in many biological responses, including the inflammatory response, by 25% (p < 0.01) (Fig. 6c). Guanosine decreased the levels of NF-jB p65 from 125 9% to 103 10%. We investigated whether the effects of guanosine were dependent on HO1. In the presence of the HO1 inhibitor, guanosine no longer prevented the increase of azide-induced TNF-a, IL-1b, and NF-jB levels.

Discussion Glioprotection refers to the specific protection of glial cells after CNS injury/damage. ‘Glioprotective’ molecules are compounds that modulate glial functions and consequently preserve neuronal structural and/or functional integrity. Guanosine, a glioprotective molecule, has been studied in a variety of experimental neuropathological contexts, including brain trauma (Rathbone et al. 1999), brain ischemia (Moretto et al. 2005; Rathbone et al. 2011; Connell et al. 2013; Dal-Cim et al. 2013), and seizures (Schmidt et al. 2000; Lara et al. 2001). These conditions may involve glutamatergic


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Table 3 The effects of guanosine on antioxidant enzymatic cellular defenses

(a)

Treatments GUO AZD GUO + AZD GUO + AZD + ZnPP IX

SOD (% of control) 98 82 101 79

9 7** 9 8**

CAT (% of control) 102 79 99 81

9 8** 7 8**

GPx (% of control) 102 116 96 112

10 7* 9 7*

Cells were pre-treated for 1 h with 100 lM guanosine (GUO), followed by the addition of 5 mM azide for 3 h. Cells were also pre-incubated with ZnPP IX (10 lM). SOD, CAT, and GPx activities were measured as described in the Materials and Methods section. The data are expressed as percentages relative to the control conditions and represent the mean SEM of three independent experimental determinations, performed in triplicate, and analyzed statistically by two-way ANOVA followed by Tukey’s test. *p < 0.05 and **p < 0.01 refer to statistically significant differences from the control.

(b)

(c)

Fig. 5 The effects of guanosine on glutamine synthetase (GS) activity and GSH levels. Cells were pre-treated for 1 h with 100 lM guanosine (GUO), followed by the addition of 5 mM azide for 3 h. Cells were also pre-incubated with Zinc protoporphyrin IX (ZnPP IX) (10 lM). (a) GS activity; (b) GS expression, and (c) GSH levels were measured as described in the Materials and Methods section. The data represent the mean SEM of three independent experimental determinations, performed in triplicate. Western blot analysis is represented as arbitrary units. The data are analyzed statistically by two-way ANOVA followed by Tukey’s test. *p < 0.05 and **p < 0.01 refer to statistically significant differences from the control.

excitotoxicity, oxidative stress, and inflammatory response. Here, we studied the glioprotective effects of guanosine against azide-induced oxidative damage in C6 astroglial cells, with special consideration of the following targets in the CNS: the glutamatergic system, antioxidant defenses, and anti-inflammatory response. Although guanosine modulates

several signaling pathways in the CNS, such as mitogenactivated protein kinase and phosphatidylinositide 3-kinase, the cellular and molecular mechanisms by which guanosine induces selective protection for neural cells are not fully understood. Thus, in this study, we reported a putative role of HO1 in the glioprotective effect of guanosine. Astrocytes are key cells in the CNS and serve a wide range of adaptive functions. Specifically, astrocytes regulate energy metabolism, neurotransmitter systems, ionic homeostasis, inflammatory response, and defense against oxidative stress (Anderson and Swanson 2000; Nedergaard et al. 2003; Maragakis and Rothstein 2006; Wang and Bordey 2008; Belanger et al. 2011; Parpura et al. 2012; Ransom and Ransom 2012). Glial cells also modulate the glutamatergic homeostasis within the brain, contributing to the maintenance of the extracellular concentration of glutamate below toxic levels, thus avoiding glutamatergic excitotoxicity (Trotti et al. 1998; Anderson and Swanson 2000; Danbolt 2001; Maragakis and Rothstein 2006; Coulter and Eid 2012). In this sense, our group has demonstrated that guanosine modulates glutamate uptake activity and glutamate transporter expression levels to prevent cytotoxicity (Schmidt et al. 2007; Quincozes-Santos et al. 2013a). Here, C6 astroglial cells were used as an astrocyte-like cell line to study the impairment of astrocytic functions because of azide exposure. These cells respond quickly to external stimuli that generate oxidative/nitrosative stress, such as azide. Azide has traditionally been used in vitro as an inhibitor of cytochrome c oxidase activity (Yoshikawa and Caughey 1992; Leary et al. 2002); it also induces ROS overproduction and inhibition of SOD and CAT activities. This generates oxidative stress, which can affect the function of cellular macromolecules, such as lipids, proteins, and nucleic acids



(a)

(b)

(c)

Fig. 6 Guanosine prevented the azide-stimulated tumor necrosis factor a (TNF-a), IL-1b, and Nuclear Factor-jB (NF-jB) levels. Cells were pre-treated for 1 h with 100 lM guanosine (GUO), followed by the addition of 5 mM azide for 3 h. Cells were also pre-incubated with Zinc protoporphyrin IX (ZnPP IX) (10 lM). (a) TNF-a; (b) IL-1b; and (c) NF-jB were measured as described in the Materials and Methods section. The data are expressed as percentages relative to the control conditions and represent the mean SEM of three independent experimental determinations, performed in triplicate, and analyzed statistically by two-way ANOVA followed by Tukey’s test. **p < 0.01 refer to statistically significant differences from the control.

(Halliwell 2006, 2007). In addition, Alzheimer’s disease-like symptoms have been observed in rats chronically infused with azide (Bennett et al. 1996a,b; Leary et al. 2002). It is important to emphasize that azide did not cause cell death in this context, but induced an intense cellular dysfunction. Under azide exposure, mitochondrial metabolic status (as measured by MTT) and DΨm were affected, indicating an impairment of mitochondrial function. Mitochondria and NADPH oxidase are the primary sites of ROS production in the CNS, and there is increasing evidence supporting the association between mitochondrial damage/NADPH oxidase

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and neuropathological disorders (Sorce and Krause 2009; Waldbaum and Patel 2010). Moreover, NADPH oxidase and cellular superoxide can lead to oxidative stress and neural death (Suh et al. 2008; Brennan et al. 2009; Reyes et al. 2012). Guanosine strongly prevented mitochondrial impairment and the increase in NADPH oxidase after azide exposure. Thus, in agreement with other reports, guanosine has emerged as an important antioxidant molecule that is involved in the removal of pathological ROS (Dal-Cim et al. 2011, 2012; Albrecht et al. 2013). Reinforcing this antioxidant effect, guanosine reduced azide-induced ROS/RNS production. Guanosine may possess direct radical scavenging activity and/or may modulate signaling pathways that control antioxidant defenses. Oxidative and nitrosative stress may be responsible for cytotoxicity from azide exposure. ROS/RNS up-regulate the expression of iNOS and presumably lead to an increase in NO concentrations (Huang et al. 2011). Our results indicate that guanosine also decreased iNOS expression in C6 cells. Because guanosine can modulate HO1 expression (Bau et al. 2005; Dal-Cim et al. 2012), and HO1 is a part of a signaling pathway that initiates antioxidant response, we studied whether the gliopreventive effects of guanosine could be mediated by this protein. ZnPP IX, an HO1 inhibitor, abolished the antioxidant effect of guanosine on ROS/RNS levels. HO is an essential enzyme in cellular metabolism and ZnPP IX is a potent and selective inhibitor of its activity (Vargas et al. 2008; Calkins et al. 2009, Quincozes-Santos et al. 2013a,b). In addition, the excess of NO acts as a signal to increase HO1 expression and is then able to scavenge NO and block the activity of iNOS, thus preventing further NO production. Therefore, there is a close and reciprocal interaction between the HO1 and iNOS-NO signaling systems. Here, for the first time, it has been described that guanosine possesses antioxidant activity that is mediated through HO1 signaling. Guanosine may exert neuroprotection by stimulating the glutamate uptake activity in astroglial cells (Schmidt et al. 2007). Glutamate uptake is mainly performed by astrocytic excitatory amino acid transporters. These transporters are vulnerable to the action of biological oxidants, which reduce their uptake activity (Volterra et al. 1994a,b; Trotti et al. 1998). In this study, under azide exposure, guanosine modulated glutamate uptake activity in C6 cells and in cortical and hippocampal primary astrocyte cultures from adult rats as well as EAAC1 (excitatory amino acid transporters 3) levels. EAAC1 is the main glutamate transporter present in C6 cells (Davis et al. 1998; KrizmanGenda et al. 2005; Bianchi et al. 2006) and is also sensitive to oxidative stress. The effects of guanosine on glutamate uptake were abolished in the presence of the HO1 inhibitor. These data are in accordance with the model that cellular redox status is closely associated with glutamate uptake activity. Furthermore, glutamate uptake dysfunction and


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Fig. 7 Schematic illustration of the protective effect of guanosine against azide-induced oxidative damage in C6 astroglial cells. Azide-induced mitochondrial dysfunction (1); increased reactive oxygen species (ROS)/reactive nitrogen species (RNS) levels (2); decreased glutamate uptake (3), glutamine synthetase (GS) activity (4), GSH levels (5), and superoxide dismutase (SOD) (6) and catalase (CAT) (7) activities; increased glutathione peroxidase (GPx) (8) and NADPH oxidase (9) activities and cellular superoxide levels (10); increased nuclear factor-jB (NF-jB) activation (11), tumor necrosis factor a (TNF-a) and IL-1b levels (12); and induced inducible nitric oxide synthase (iNOS) expression (13). Guanosine prevented these effects through the heme oxygenase 1 (HO1) signaling pathway. These findings reinforce the antioxidant effects of guanosine.

oxidative stress converge to provide a common final pathway for cell vulnerability. Several brain pathologies, such as seizures, brain ischemia, Parkinson’s and Alzheimer’s diseases, emerge as impaired glutamate metabolism and antioxidant defense, leading to cellular damage (Coyle and Puttfarcken 1993; Halliwell 2006; Lalo et al. 2006; Fukui et al. 2010). Once taken up by astrocytes, glutamate may be converted to glutamine by GS (Mates et al. 2002; Hertz and Zielke 2004; McKenna 2007). GS is very sensitive to oxidative/ nitrosative stress and guanosine increased its activity in both control and toxic conditions. This activation may represent an important pathway that reinforces the action of guanosine on the glutamatergic system in the brain. However, we did not observe changes in GS expression caused by guanosine. Our data are supported by other studies indicating no association between changes in GS activity and GS expression (Desjardins et al. 1999; Bidmon et al. 2008). The antioxidative effect of guanosine was also supported by modulating the homeostasis of GSH, the major nonenzymatic antioxidant in the CNS (Banerjee et al. 2008). The increase in GSH levels in glial cells confers protection against neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases (Halliwell 2006; Lee et al. 2010). As in C6 cells, glutamate and cystine share the same transport system, specifically the cystine/glutamate antiporter, system xc . Thus, the elevation of extracellular glutamate levels may contribute to the impairment of cystine transport and,

consequently, intracellular GSH biosynthesis (Lewerenz et al. 2006). Accordingly, the depletion of GSH in glial cells induces cytotoxicity and impairment of glutamate transporter activity (Schulz et al. 2000; Lee et al. 2010). In this context, the antioxidant effect of guanosine during azidemediated oxidative stress could decrease the oxidation of GSH, providing glioprotection that is associated with scavenger activity. Moreover, it is important to note that the EAAC1 glutamate transporter, which is modulated by guanosine, is responsible for the support of de novo GSH synthesis (Escartin et al. 2011). The levels of free radicals can be determined by the balance between the rates of production and clearance of free radicals by antioxidants and enzymes, such as SOD, CAT, and GPx (Gutteridge and Halliwell 2000; Droge 2002). Azide inhibited the activities of SOD and CAT, while guanosine completely prevented these effects. The decrease in SOD and CAT activities induces an overproduction of superoxide and H2O2, molecules which cause severe cellular injury. However, guanosine decreased the cellular superoxide levels. Azide also increased the GPx activity. The increase in GPx activity may compensate for the increase in endogenous H2O2 and decreased levels of GSH, the major brain antioxidant (Halliwell 2001; Droge 2002). Guanosine prevented this augmentation of GPx activity, possibly by increasing the GSH content. Our group has studied that guanosine counteracts the glutamate excitotoxicity (Schmidt et al. 2007) and recently Reyes et al. demonstrated that excitotoxic neuronal death is



mediated in part by NOX activation (Reyes et al. 2012). Here, we showed that guanosine decreased NOX activity through the HO1 signaling pathway. As expected, the antioxidant effect of guanosine is dependent on HO1. In this sense, HO1 mediates neuroprotection, down-regulates NOX and modulates several detoxification genes that encode antioxidant proteins, such as the GSH/GPx systems and thioredoxin, both of which are regulators of the intracellular redox environment (Calabrese et al. 2008; Arredondo et al. 2010; Bastianetto and Quirion 2010; Sakata et al. 2010; Rousset et al. 2013). Guanosine regulates HO1 activity (Bau et al. 2005), as well as enzymatic and non-enzymatic antioxidant defenses. Lee et al. (2010) demonstrated that the depletion of GSH in glial cells induces inflammatory responses. Thus, ROS production plays a critical role in inflammatory response, increasing TNF-a levels (Shen and Pervaiz 2006). TNF-a potentiates NO production in astrocytes (Hamby et al. 2008). In the work reported here, azide increased the levels of the main proinflammatory cytokines, TNF-a and IL-1b. Guanosine prevented this effect. Guanosine also decreased the levels of NF-jB p65. Thus, our data showed that guanosine modulates NO, iNOS, TNF-a, IL-1b, and NF-jB through HO1 signaling. The transduction factor NF-jB is considered the major inflammatory mediator in the CNS and may activate iNOS (Gloire et al. 2006; Wakabayashi et al. 2010). It is important to note that in 2007, D’Alimonte et al. (2007) showed the participation of guanosine in inflammatory response by inhibiting of TNF-a, IL-1b, and NF-jB nuclear translocation in microglial cells. Furthermore, HO1 is able to inhibit iNOS expression, and consequently NO production as well as NF-jB translocation from the cytoplasm to the nucleus (Wakabayashi et al. 2010). Guanosine is emerging as an important focus for both in vitro and in vivo studies of neurotoxicity and neuroprotection. Understanding the influence of guanosine on glial functions is critical for elucidating the cellular and molecular mechanisms of this endogenous nucleoside. Recently, we demonstrated that the gliopreventive effect of guanosine against glucose deprivation involves the activation of protein kinase C, phosphatidylinositide 3-kinase, and mitogenactivated protein kinase pathways (Quincozes-Santos et al. 2013a). The main conclusions of this study are depicted in the Fig. 7, which demonstrates that the cytotoxicity, oxidative and nitrosative stress, and inflammatory response induced by azide were prevented by guanosine through the HO1 signaling pathway. Overall, these observations reinforce the antioxidant effect of guanosine in astroglial cells. Thus, guanosine may potentially be useful as a glioprotective molecule against oxidative damage.

Acknowledgments and conflict of interest disclosure This work was supported by the Conselho Nacional de Desenvolvimento Cientıfico e Tecnol ogico (CNPq), Coordenacß~ao de

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Aperfeicßoamento de Pessoal de Nıvel Superior (CAPES), Fundacß~ao de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Financiadora de Estudos e Projetos (FINEP) – Instituto Brasileiro de Neurociências (IBN Net) 01.06.0842-00, Federal University of Rio Grande do Sul (UFRGS), and Instituto Nacional de Ciência e Tecnologia para Excitotoxicidade e Neuroprotecß~ao (INCTEN/CNPq). All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.

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ARE-Dependent Heme Oxygenase-1 Expression.

Red Yeast Rice Protects Circulating Bone Marrow-Derived Proangiogenic Cells against High-Glucose-Induced Senescence and Oxidative Stress: The Role of Heme Oxygenase-1.

Pinacidil protects osteoblastic cells against antimycin A-induced oxidative damage.

Heme oxygenase-1 protects against Alzheimer's amyloid-β(1-42)-induced toxicity via carbon monoxide production.

reperfusion injury.

Upregulation of Heme Oxygenase-1 in Response to Wild Thyme Treatment Protects against Hypertension and Oxidative Stress.

Induction of heme oxygenase‑1 expression protects articular chondrocytes against cilostazol‑induced cellular senescence.

Heme Oxygenase-1 Promoter Polymorphism Protects Liver Allograft.

Fibroblast growth factor 10 protects neuron against oxygen-glucose deprivation injury through inducing heme oxygenase-1.

α-Tocopherol protects renal cells from nicotine- or oleic acid-provoked oxidative stress via inducing heme oxygenase-1.

Remote ischemic preconditioning protects against liver ischemia-reperfusion injury via heme oxygenase-1-induced autophagy.

Guanosine inhibits LPS-induced pro-inflammatory response and oxidative stress in hippocampal astrocytes through the heme oxygenase-1 pathway.

Heme oxygenase is a heat shock protein and PEST protein in rat astroglial cells.

Heme oxygenase-1 protects endothelial cells from the toxicity of air pollutant chemicals.

Nrf2 pathway in human dopaminergic cells.

Heme oxygenase-1 and heme oxygenase-2 expression in bruises.

Edaravone protected PC12 cells against MPP(+)-cytoxicity via inhibiting oxidative stress and up-regulating heme oxygenase-1 expression.

Delayed remote preconditioning induces cardioprotection: role of heme oxygenase-1.

Milk thistle seed extract protects rat C6 astroglial cells from acute cocaine toxicity.

Guanosine protects glial cells against 6-hydroxydopamine toxicity.

Ammonia-induced oxidative damage in neurons is prevented by resveratrol and lipoic acid with participation of heme oxygenase 1.

Heme oxygenase-1-mediated autophagy protects against pulmonary endothelial cell death and development of emphysema in cadmium-treated mice.

Induction of heme oxygenase-1 by hemin protects lung against orthotopic autologous liver transplantation-induced acute lung injury in rats.

Hyperbaric oxygen preconditioning protects lung against hyperoxic acute lung injury in rats via heme oxygenase-1 induction.