Peroxidase Là Gì

Journal of Neuroscience 18 February 2004, 24 (7) 1531-1540; DOI: https://doi.org/10.1523/gamesbaidoithuong.com.3989-03.2004
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Abstract

Oxidative mechanisms of injury are important in many neurological disorders, including hypoxic-ischemic brain damage. Cerebral palsy after preterm birth is hypothesized lớn be caused by hypoxic-ischemic injury of developing oligodendrocytes (OLs). Here we examined the developmental sensitivity of OLs khổng lồ exogenous hydrogen peroxide (H2O2) with stage-specific rat oligodendrocyte cultures. We found that H2O2 itself or that generated by glucose oxidase was more toxic to lớn developing than to lớn mature OLs. Mature OLs were able khổng lồ degrade H2O2 faster than developing OLs, suggesting that higher antioxidant enzyme activity might be the basis for their resistance. Catalase expression & activity were relatively constant during oligodendrocyte maturation, whereas glutathione peroxidase (GPx) was upregulated with a twofold lớn threefold increase in its expression and activity. Thus, it appeared that the developmental change in resistance to H2O2 was caused by modulation of GPx but not by catalase expression. To test the relative sầu roles of catalase and GPx in the setting of oxidative sầu áp lực, we measured enzyme activity in cells exposed to lớn H2O2 & found that H2O2 induced a decrease in catalase activity in developing but not in mature OLs. Inhibition of GPx by mercaptosuccinate led to lớn an increase in the vulnerability of mature OLs lớn H2O2 as well as a reduction in catalase activity. Finally, H2O2-dependent inactivation of catalase in developing OLs was prevented by the GPx mimic ebselen. These data provide evidence for a key role for GPx-catalase cooperativity in the resistance of mature OLs lớn H2O2-induced cell death.Bạn vẫn xem: Enzyme peroxidase là gì

Introduction

Ischemia/reperfusion injury to lớn the developing brain is a major cause of neurological disorders in the perinatal period. Neurological deficits in infants surviving hypoxic-ischemic insult include mental retardation, seizures, và cerebral palsy. The neuropathology of perinatal brain injury is complex, and involves gray và white matter khổng lồ varying degrees, depending on the gestational age và the developmental stage of cerebral vascularity (Kinney and Armstrong, 1997). Periventricular leukomalacia (PVL) involves injury khổng lồ the cerebral white matter resulting in a chronic disturbance of myelination; it is the predominant underlying pathology of cerebral palsy in premature infants (Volpe, 1997, 2001). During the maturational period of peak vulnerability lớn PVL, the White matter is predominantly populated with premyelinating oligodendrocytes (OLs) (Baông xã et al., 2002).

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In human PVL, there is now emerging evidence that oxidative stress plays a key role in the pathogenesis of the white-matter injury (Haynes et al., 2003). Previous work has shown that developing OLs are more vulnerable than mature OLs lớn endogenous oxidative sầu bít tất tay caused by the depletion of intracellular glutathione (GSH). (Oka et al., 1993; Yonezawa et al., 1996; Back et al., 1998, 2001). However, the sensitivity of OLs at specific stages of development to lớn exogenous sources of oxidative sầu căng thẳng remains unknown.

Peroxides, including hydroren peroxide (H2O2), are one of the main reactive sầu oxyren species (ROS) leading to oxidative sầu găng tay (Halliwell and Gutteridge, 1999). H2O2 is continuously generated by several enzymes (including superoxide dismutase, glucose oxidase, & monoamine oxidase) và must be degraded to lớn prevent oxidative sầu damage. The cytotoxic effect of H2O2 is thought lớn be caused by hydroxyl radicals generated from iron-catalyzed reactions, causing subsequent damage khổng lồ DNA, proteins, and membrane lipids (Halliwell, 1992). Recently, both glutathione peroxidase và catalase, the two main enzymes involved in H2O2 detoxification, have been shown lớn be implicated in the disposal of exogenous H2O2 by astrocytes (Dringene and Hamprecht, 1997). Both have sầu been found in the brain (De Marchena et al., 1974; Gaunt and de Duve sầu, 1976; Brannan et al., 1981) and in astrocytes và oligodendrocytes (Dringen & Hamprecht, 1997; Hirrlinger et al., 2002). Catalase is known khổng lồ be of special importance when the clearance of H2O2 in high concentrations is required. More recently, a higher capađô thị for the degradation of H2O2 has been demonstrated in myelin basic protein (MBP) expressing oligodendrocytes than in astrocytes, microglia, or neurons (Hirrlinger et al., 2002). However, the developmental variation in the vulnerability of OLs khổng lồ H2O2 has not been investigated.

In the present work, we investigated the sensitivity of OLs to lớn H2O2 at specific developmental stages of maturation. Developing OLs appeared lớn be more vulnerable lớn H2O2 than were mature OLs. In pursuing the basis for this difference in vulnerability, we found that GPx expression was upregulated in mature OLs. Although catalase expression was unchanged through development, this enzyme in developing OLs was inactivated by exposure lớn H2O2. The vulnerability of catalase lớn H2O2 appeared khổng lồ depkết thúc on the level of GPx activity. In mature OLs, the upregulation of glutathione peroxidase maintains the catalase activity at a high level in the presence of H2O2.

Materials and Methods

Materials. DMEM, HBSS, Earle"s balanced salternative text solution (EBSS), fetal bovine serum, penicillin, và streptomycin were purchased from Invitrogene (Rockville, MD). Unless otherwise specified, all other chemicals were from Sigma (St. Louis, MO).

Oligodendrocyte cultures. Primary rat OLs were prepared from the cerebral hemispheres of Sprague Dawley rats at postnatal day 1-2 using a shaking method (McCarthy and de Vellis, 1980; Oka et al., 1993) with modifications, as described previously (Li et al., 2003). Briefly, forebrains free of meninges were chopped inlớn 1 mm3 blocks & placed into HBSS containing 0.01% trypsin và 10 μg/ml DNase. After digestion for 15 min at 37°C, the tissue was collected by centrifugation và triturated in the plating medium (DMEM 20% serum) containing DMEM, 20% fetal bovine serum, 40 IU/ml penicillin, and 40 μg/ml streptomycin, & passed through a 70 μm sieve. Cells were plated onkhổng lồ polylysine-coated 75 cmét vuông flasks at a density of 1 pup brain per flask. Cultures were fed with fresh DMEM 20S medium every other day for 10-11 d at 37°C in a humid atmosphere of 5% CO2 & 95% air.

To isolate OLs, the mixed glial cultures were shaken for 1 hr at 200 rpm at 37°C to remove sầu adherent microglia/macrophages, & the cultures were washed with the same medium and subjected khổng lồ shaking at 200 rpm overnight (18-22 hr) lớn separate OLs from the astrocyte layer. The suspension was plated onkhổng lồ uncoated Petri dishes and incubated for 1 hr at 37°C to lớn further remove sầu residual microglia và astrocytes that adhere khổng lồ the dishes. The OLs were then collected by passing through a 15 μm sieve, followed by centrifugation. Isolated OLs were plated onto poly-d-ornithine (50 μg/ml)-coated 96 well culture plates (at mật độ trùng lặp từ khóa of 3.3 × 103 cells per well for cell-survival assay), 24 well plates with glass coverslips (1.74 × 104 cells per well for imaging), and 60 milimet plates (2.75 × 105 cells per plate for enzyme assays). Purified OLs were cultured for 7-8 d in a serum-miễn phí basal defined medium (BDM): DMEM, 0.1% bovine serum albumin, 50 μg/ml apo-transferrin, 50 μg/ml insulin, 30 nm sodium selenite, 10 nm d-biotin, 10 nm hydrocortisone, 200 μm l-cystine, 10 ng/ml platelet-derived growth factor, & 10 ng/ml basic fibroblast growth factor. At 7-8 d, the cultures were composed primarily of progenitors and pre-OLs (A2B5+, O4+, O1-, MBP-negative). After 7 d, culture medium was changed to serum-không lấy phí BDM containing 10 ng/ml ciliary neurotrophic factor and 15 nm 3,3′,5-triiodo-l-thyronine for 14 additional days until cells were differentiated into lớn mature OLs (MBP+). The purity of OL cultures was consistently >95% OLs with 2O2 diluted from a 9.8 m stochồng solution. Unless otherwise specified, the cells were incubated for 24 hr before being assayed for cell survival. Alternatively, H2O2 was generated in OL cultures by the addition of glucose oxidase (đôi mươi mU/ml) to lớn the medium (BDM; 25 milimet glucose) (Kyên and Kyên ổn, 1991). The cells were incubated for 24 hr followed by Alamar blue assay (Southern Biotechnology, Birmingđắm đuối, AL) for cell viability.

Cell-survival assay. Cell survival was determined after treatment for 24 hr using Alamar xanh, a tetrazolium dye that is reduced by living cells lớn a fluorescent sản phẩm. This assay is similar in principle lớn the 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell viability assay and has been previously validated as an accurate measure of survival of OLs (Back et al., 1999). For the H2O2 challenge, the results from the Alamar blue assay were matched by a calcein-based assay (Molecular Probes, Euren, OR) (data not shown). All results of cell death assays were confirmed by visual inspection using phase-contrast microscopy. Briefly, culture medium in the 96 well plate was aspirated, and cells were incubated with 200 μl of assay solution prepared by diluting 100× stoông chồng solution of Alamar blue into lớn EBSS for 2 hr at 37°C. The fluorescence of the assay solution, reflecting cell viability, was measured with a fluorescence plate reader (FluoroCount, Packard Instrument, Meridan, CT) using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. All survival assays were performed at least in triplicate.

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H2O2 colorimetric assay. A method described previously was used to characterize the kinetics of H2O2 depletion from the culture medium. The OLs were plated in polyornithine-coated 24 well plates. H2O2 depletion was monitored by a colorimetric assay that involved the oxidation of o-dianisidine dihydrochloride (o-DD) in the presence of horseradish peroxidase (Yakes and Van Houten, 1997). At the start of the experiment, H2O2 was added to each well containing 500 μl EBSS lớn yield 400 μm. In this medium, the hydroren peroxide concentration remained constant over the period studied in wells lacking any cells. As the experiment progressed, 100 μl aliquots of the treatment medium were removed at specific time points. These aliquots were then incubated with 2.5 U/ml of horseradish peroxidase & 250 μm o-DD for 1 hr at 37°C. The absorbance was then measured at 470 nm against a blank containing EBSS without the addition of H2O2. The amount of H2O2 remaining in the medium was determined from a standard curve generated by using known concentrations of H2O2. The initial rate of H2O2 clearance was obtained manually from the plotted results.

Immunocytochemistry, Tdt-mediated dUTPhường niông chồng over labeling, & immunofluorescence microscopy. Cells were washed in cold PBS & fixed with 4% paraformaldehyde for 10 min at room temperature, washed three times with PBS, & blocked with PBS containing 5% goat serum for 1 hr at room temperature. The coverslips were incubated for 1 hr with mouse monoclonal antibodies O4 và O1 (1:100 dilution; gifts of Dr. Steven E. Pfeiffer, University of Connecticut Health Center, Farmington, CT), MBPhường (1:500 dilution; Boehringer Mannheyên, Indianapolis, IN). After three khổng lồ four washes at 15 min each, the appropriate secondary antitoàn thân conjugated with Alexa red (1:1000 dilution; Molecular Probes) was added lớn the coverslips and incubated for 1 hr. After extensive washes, cells were again exposed briefly khổng lồ 4% paraformaldehyde in PBS for 5 min at room temperature. After several rinses in PBS, the coverslips were then incubated for 2 hr with PBS containing 5% goat serum and 0.1% Triton X-100 containing rabbit polyclonal anti-catalase (1:500 dilution; Abcam, Cambridge, UK) antibodies. After three washes, the appropriate secondary antitoàn thân conjugated with Oregon green (1:500 dilution; Molecular Probes) was incubated with cells for 1 hr. Nuclei were finally stained by adding Hoechst 33258 at a final concentration of 2 μg/ml for 1 min. After three more washes, the coverslips were mounted onlớn glass slides with FluoroMount (Southern Biocông nghệ, Birmingsi mê, AL) và kept in the dark at 4°C.

Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick over labeling (TUNEL) staining was performed using the In Situ Cell Death detection kit from Robít (Indianapolis, IN) và following the protocol for cell culture.

Cell images were captured with a fluorescence microscope (Eclipse E800; Nikon, Tokyo, Japan) equipped with a Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI).

Protein extracts and Western blot. OLs were rinsed with cold PBS và scraped into lysis buffer containing the following: 150 mm NaCl, 50 mm Tris, 0.5% Triton X-100, và 1× protease inhibitor mixture (Roche). For Western blotting, samples were boiled for 5 min in the presence of β-mercaptoethanol và SDS; 10 μg of protein from the cell lysates were separated by electrophoresis using a 12% polyacrylamide gel. After electroblotting khổng lồ a polyvinylidene difluoride membrane & blocking of nonspecific binding sites, membranes were exposed either lớn anti-catalase (1:500 dilution; Abcam, Cambridge, UK) or to lớn anti-GPx-1 (1:100 dilution; Novus Biological, Littleton, CO) antibodies followed by appropriate secondary antibodies conjugated with horseradish peroxidase và detection by chemiluminescence (PerkinElmer, Wellesley, MA).

Catalase, GPx, and glutathione reductase enzyme activity assays; glutathione chemical assay. Briefly, cells were harvested và in a lysate buffer containing 0.1% Triton X-100, then centrifuged lớn remove the supernatant for assay. Catalase activity measurement was based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced was measured spectrophotometrically at 540 nm. One unit of catalase was defined as the formation of 1 nmol of formaldehyde per minute per milligram of protein. GPx activity was measured indirectly by a coupled reaction with glutathione reductase. Oxidized glutathione produced on reduction of hydroperoxide by GPx was recycled to lớn its reduced form by the oxidation of NADPH to NADP+, which was accompanied by a decrease in absorbance at 340 nm. Under conditions of the assay, the rate of decrease in the absorbance at 340 nm was directly proportional to lớn the GPx activity in the sample. One unit of GPx was defined as the amount of enzyme causing the oxidation of 1 nmol of NADPH per minute and per milligram of protein. Catalase và GPx activities were measured using kits from Cayman Chemical (Ann Harbor, MI). Glutathione reductase activity was assayed in the presence of 1 milimet oxidized GSH (GSSG) by measuring the rate of NADPH oxidation, which is accompanied by a rapid decrease in absorbance at 340 nm. The assay temperature is 25°C. Results were expressed similarly to lớn those of the GPx assay.

Total intracellular reduced GSH was measured by a fluorometric method as described previously (Wang và Joseph, 1999).