Inhibition of Production of Reactive Oxygen Species and Gene Expression Profile by Treatment of Ethanol Extract of Moutan Cortex Radicis in Oxidative Stressed PC12 Cells

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Inhibition of Production of Reactive Oxygen Species and Gene Expression Profile by Treatment of Ethanol Extract of Moutan Cortex Radicis in Oxidative Stressed PC12 Cells
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  It is known that reactive oxygen species (ROS) have a cen-tral role in causing many types of neuronal damage such asAlzheimer’s disease, Parkinson’s disease, ischemia, etc. 1,2) ROS comprise a group of unstable molecules: super oxideradical (O 2  ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical(OH) and hypochlorous acid (HOCl), that are generated in allcells as a by-product of cellular metabolism during conver-sion of molecular oxygen (O 2 ) to water (H 2 O). Additionally, phagocytes generate oxidative bursts of ROS to combat mi-croorganisms. Cells possess endogenous systems (super-oxide dismutase, catalase, glutathione peroxide) and diet de-rived (vitamin C and vitamin E) anti-oxidants to prevent or limit ROS induced tissue damage. H 2 O 2 is often used to in-vestigate the mechanism of ROS-induced cell death, 3) and the mechanism of H 2 O 2 -induced PC12 cell death has beenexamined. 4,5) Hydroxyl radicals form when it comes in con-tact with a range of transition metal ions. Oxidative stress oc-curs during an imbalance between ROS and anti-oxidants.Excess production of ROS may lead to cellular injurythrough nonspecific modification and disruption of proteins, phospholipids and nucleic acids. 6) Critical sites of ROS at-tack are the cell membrane and the membranes of intracellu-lar organelles. The disruptive effects of ROS involve mem- brane lipid per-oxidation and membrane protein modifica-tion, which may produce alterations in the membrane struc-ture and function including fluidity, permeability, enzyme ac-tivity, ion channels, transporter and receptor proteins. 7) Therole of ROS has been implicated in many human degenera-tive diseases of aging. Various antioxidants have been found to have some preventive and therapeutic effects on these dis-eases induced by ROS. 8) Therefore, substantial efforts have been made in recent years to identify both natural and syn-thetic antioxidants.Moutan Cortex Radicis (MCR), the root cortex of  Paeonia suffruticosa A  NDREWS , is a Chinese herbal medicine widelyused as an analgesic, antispasmodic, and anti-inflammatoryagent. The drug has also long been used in remedies for fe-male diseases. MCR is reported to inhibit oxidative DNAcleavage 9) and to have a scavenging effect on 1,1-diphenyl-2- picrylhydrazyl (DPPH) radicals and superoxide anion radi-cals generated by the xanthine–xanthine oxidase system. 10) The chemical components of MCR are known to be paeonol, paeonoside, paeonolide, paeoniflorin, benzoylpaeoniflorin,oxypaeoniflorin, benzoyl-oxy-paeoniflorin, and apiopaeono-side.PC12 cells have been widely used as an in vitro experi-mental model to study the effects of various neurotoxicagents, including 6-hydroxydopamine, 1-methyl-4-phenyl- pyridinium (MPP  ), paraquat, and manganese on dopaminer-gic cells. 11,12) PC12 cells are electrically excitable and neurosecretory (dopamine, norepinephrine, and/or acetyl-choline), and contain many membrane-bound and cytosolicmacromolecules associated with neurons. 11) The present study was designed to investigate whether ethanol extract of MCR is capable of reducing the hydrogen peroxide-induced ROS generation, the major cause of neu-ronal degenerating diseases in PC12 cells. Measurement wasmade to learn which genes are differentially expressed in re-lation to the effect of MCR using oligonucleotide microarrayassay and this was confirmed using real time RT- PCR. April 2005  Biol. Pharm. Bull. 28 (4) 661—666 (2005)661 ∗ To whom correspondence should be addressed.e-mail: hbae@khu.ac.kr© 2005 Pharmaceutical Society of Japan Inhibition of Production of Reactive Oxygen Species and Gene ExpressionProfile by Treatment of Ethanol Extract of Moutan Cortex Radicis in Oxidative Stressed PC12 Cells Samwoong R  HO , a Hwan-Suck C HUNG , b Moonkyu K  ANG , b Euna L EE , a Chongwoon C HO , b Hyunhee K  IM , a Seongkyu P ARK  , a Hong-Yeoul K  IM , a Moonchang H ONG , a Minkyu S HIN , a and Hyunsu B AE * , a,b a College of Oriental Medicine, Kyung-Hee University; #1 Hoeki-Dong, Dongdaemun-Ku, Seoul 130–701, Republic of  Korea: and b  Purimed R&D Institute, Kyung-Hee University; #1 Hoeki-Dong, Dongdaemun-Ku, Seoul 130–701, Republicof Korea. Received September 10, 2004; accepted November 11, 2004 Moutan Cortex Radicis (MCR) is one of the most widely used Oriental medicines. In this study, we assessedthe reducing effect of ethanol extract of MCR on hydrogen peroxide-induced reactive oxygen production, themain cause of cell damage or death in PC12 cells. The viability of cells treated with 1mg/ml of MCR was signifi-cantly restored from that of oxidative-stressed PC12 cells. Measurement of intracellular reactive oxygen species(ROS) generation was determined using the H 2 DCFDA assay. MCR at 1—0.01mg/ml concentration inhibitedROS production in oxidative-stressed cells. To identify candidate genes responsible for the anti-oxidative effectsof MCR on PC12 cells, an oligonucleotide microarray analysis was performed. The result of gene expression pro-files showed that 10 genes were up-regulated and 7 were down-regulated in MCR plus hydrogen peroxide treatedcells compared with hydrogen peroxide treated cells. Among them, heme oxygenase (HO) and cathechol- O -methyltransferase (COMT) are related to regulation of ROS generation and the others are known to regulate cellsurvival and progression. Subsequently, we performed real-time RT-PCR to quantify the ROS related gene.MCR treatment increased the expression of HO by 370% and COMT by 280% at the concentration of 1mg/ml.These findings suggest that MCR inhibits the production of ROS and cytotoxicity by oxidative-stressed PC12cells through over-expression of HO and COMT. Key words Moutan Cortex Radicis; reactive oxygen species; oligonucleotide microarray; cathechol- O -methyltransferase, hemeoxygenase  MATERIALS AND METHODS Materials RPMI 1640 media, penicillin–streptomycin,Superscript Choice System for cDNA Synthesis and acety-lated bovine serum albumin (BSA) were purchased from In-vitrogen Life Technologies (Rockville, U.S.A.).  N   ,  N  -Di-methyl formamide, b  -mercaptoethanol and fetal bovineserum were obtained from Sigma (St. Louis, MO, U.S.A.).2  ,7  -Dichlorodihydrofluorescein (H 2 DCFDA) was pur-chased from Molecular Probes Inc. (Eugene, U.S.A.).Paeonol (2-hydroxy-4-methoxyacetophenone) was purchased from Wako Pure Chemical Industries (Japan). Syringe filters(pore size: 0.2 m  m), 96-well tissue culture plates and 100-mmdiameter dishes were purchased from Nunc (Naperville, IL,U.S.A.). Preparation of Herbal Extract The MCR was pur-chased from Kyunghee Oriental Hospital (Seoul, SouthKorea) and authenticated by Professor Seongkyu Park, Col-lege of Oriental Medicine, Kyunghee University. Dried MCR 300g was pulverized to powder and extracted by 70%, 85%and 100% ethanol for 10min using a sonicator. All of theethanol extracts were mixed and concentrated by evaporator and then lyophilized by lyophilizer (both from EYELA Co.,Japan). The weight of the resulting dried MCR extracts was62.8g (yield 26%). The dried extract was deposited in theDepartment of Physiology, College of Oriental Medicine,Kyunghee University. Two grams of the dried extract wasdissolved in distilled water and centrifuged at 10000  g for 10min. The supernatant was passed through a 0.22 m  m filter and reached a final concentration of 100mg/ml. The solutionwas aliquoted and stored at 4°C for future use. High-Performance Liquid Chromatography (HPLC)Analysis of MCR  A dried ethanol extract of about 10mgof MCR was accurately weighed, put in a test tube, then dis-solved in 5ml of 50 % methanol (HPLC reagent, J.T. Baker Co., Ltd., U.S.A.) then filtered using a 0.45 m  m syringe filter (PVDF, Waters, U.S.A.). Paeonol as a standard materialweight was 10mg, and was dissolved according to the analy-sis condition of standard materials. The dissolved standard solution was diluted as 0.1, 0.5, 1.0, 1.5, 2.0mg/ml, respec-tively, and then a standard HPLC chromatogram was ob-tained. The relationship between the concentration and the peak-area was measured by the minimum square method ( r  2 value). The HPLC apparatus was a Waters Breeze System(717  Autosampler, 2487 dual l  absorbance detector, 1525 binary HPLC Pump, Waters Co., Milford, U.S.A.), and theassociated Waters Breeze System (Ver. 3.20) was used for data acquisition and integration. Cell Culture PC12, rat pheochromocytoma cells (KCLB#21721, South Korea) were cultured in RPMI 1640 mediawith 10% fetal bovine serum (FBS), 1% penicillin and 1%streptomycin. PC12 cells were incubated in 100ng/ml nervegrowth factor (NGF) for differentiation. Cells were main-tained in a humidified atmosphere with 5% CO 2 at 37°C. Intracellular Reactive Oxygen Species Assay The levelof intracellular ROS was determined by the change in fluo-rescence resulting from the oxidation of the fluorescent probeH 2 DCFDA. Briefly, 5  10 5 cells/well were exposed to MCR with various concentrations for 24h. After incubation, cellswere washed once with FBS free media and then incubated with 100 m  M H 2 O 2 (as inducer for ROS production) at 37°Cfor 30 min. Cells were incubated with 50 m  M of the fluores-cent probe H 2 DCFDA for 1h at 37°C. The degree of fluores-cence, corresponding to intracellular ROS, was determined using Fluoroscan Ascent FL (Type 374, Labsystems, Fin-land) (excitation 485nm; emission 538nm). Cell Viability Cell growth was measured by 3-[4,5-di-methylthiazol-2-yl-5]-[3-carboxymethoxyphenyl]-2-[4-sul-fophenyl]-2  H  tetrazolium (MTS) assay using the Cell Titer 96 ® Aqueous One Solution Cell Proliferation Assay Kit(Promega, Madison, WI, U.S.A.). Briefly, 100 m  l of the su- pernatant was added to each 96-well plate. Twenty micro-liters of MTS solution was added to each of the 96-well plateand incubated at 37°C in a humidified 5% CO 2 atmospherefor 1h. The absorbance was read at 490nm using a mi-croplate reader (Molecular Device, U.S.A.). Each sample wasduplicated. RNA Extraction Total RNA was extracted from cul-tured cells using the RNeasy mini kit (Qiagen, Valencia, CA,U.S.A.) according to the manufacturer’s instructions. The ex-tract was assayed for RNA quality and the concentration wasdetermined with a spectrophotometer (DU500, Beckman In-struments Inc., Fullerton, U.S.A.). The extract was stored at -20°C until analysis. Affymetrix GeneChip ® Array Processing cRNA label-ing, hybridization and washing were performed using stan-dard procedures as previously described. 13) Microarray Data Analysis Microarrays were scanned at 570nm, 3 m  m resolution with a gene chip scanner (Affymetrix) and analyzed as previously described. 14) For quantification of relative transcript abundance, the averagedifference value (Avg Diff) was used. The signal intensity for each gene was calculated as the average intensity differencerepresented by ((PM-MM)/(number of probe pairs)). Allchips were normalized by the mean of the total sums of AvgDiff values across all chips used in the experiment allowing acomparison between conditions. An Avg Diff level lower than 200 fluorescence units was considered below the limitof accurate quantification based on extensive quality controlexperiments performed in-house. Above 200 fluorescenceunits, based on our in-house validation dataset, any gene hav-ing an absolute ratio or change factor  2, was considered significantly differentially expressed. Real-Time RT-PCR  Using the real-time RT-PCR tech-nique, we verified our microarray results in two importantgenes, heme oxygenase (HO) and catechol- O -methyltrans-ferase (COMT). First, rare contaminating genomic DNA wasremoved using Deoxyribonuclease I (Invitrogen, U.S.A.).Then the RNA (integrity confirmed) samples were reversetranscribed using oligo (dT) primers and SuperScript IIRnase H  reverse transcriptase (Invitrogen, U.S.A.)PCR reactions were performed as follows: TemplatecDNA was incubated with 500p M of gene-specific primers(Genotech, Co., Korea), and a SYBR Green PCR Master Mixcontaining PCR buffer, AmpliTaq gold DNA polymerase,dNTP mix, MgCl 2 , ROX passive reference dye and SYBR Green I dye (Applied Biosystems, U.K.) in a GeneAmp 5700Sequence Detection System (PE Biosystems, U.K., U.S.A.).The thermal cycling program had an initial denaturation(95°C for 10min) and then 40 cycles of denaturation (95°Cfor 15s), annealing and extension (62°C for 45s). The tem- perature for annealing and extension was varied from 62 to 662Vol. 28, No. 4  67°C according to the primers. The primer sets used were:glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5  -GGCATGGACTGTGGTCATGA-3  and 5  -TTCACCAC-CATGGAGAAGGC-3  ; HO, 5  -GCCCAGCTGGAATTTC-TTTACTCT-3  and 5  -AGCCCCCAACCTCCTGATTT-3  ;COMT, 5  -AATGTGGGTGACGCGAAA-3  and 5  -GGGT-GATGGCAGCGTAGT-3  . A fluorescence signal was col-lected at the end of each cycle. After the cycles were termi-nated, the signal of each temperature from 60 to 95°C wasalso collected for the dissociation curve analysis. All reac-tions were performed in triplicate to confirm reproducibility,and included a negative control (without template) to verifythat no primer-dimers were being generated. The standard curve for each primer was constructed using serial dilutionsof cDNA. The amount of target mRNA in each sample wasnormalized with that of mean GAPDH, an endogenous con-trol. Statistical Analysis All data except microarray resultswere expressed as the mean  S.E.M. of independent experi-ments Statistical significance was compared between MCR treatment group and control by the Student’s t  -test. Resultswith  p  0.05 were considered statistically significant.RESULTS HPLC Spectrum of MCR  Paeonol is known as one of the main active compounds of MCR. We performed HPLC-fingerprinting to authenticate MCR. As shown in Fig. 1, theretention time of paeonol showed a similar retention time of MCR. The amount of paeonol contained in MCR was calcu-lated by the following formula: amount (mg) of paeonol  {quantitative amount (mg) of standard materials  AT/AS}/ n ( n  3) (cf., AT: the peak-area of MCR containing paeonol, AS: the peak-area of paeonol).When we calculated the amount of paeonol, 1g of MCR contained 12.57  0.05mg of paeonol. Effect of MCR on ROS Production in PC12 Cells Theintracellular concentration of ROS in PC12 cells, as assessed  by H 2 DCFDA oxidation, was increased by hydrogen perox-ide treatments. Prior treatment of various concentrations of MCR for 24h significantly inhibited the increase of intracel-lular ROS when cells were stimulated with hydrogen perox-ide. MCR at 0.01 and 0.1mg/ml inhibited the ROS genera-tion to 30.5% and 29.5% of the control, respectively, in PC12cells. Interestingly, ROS generation was almost completelyinhibited at 1 mg/ml of MCR (Fig. 2). Effects of MCR on Hydrogen Peroxide-Induced Toxic-ity Treatment with 100 m  M hydrogen peroxide resulted indecreasing the cell viability to approximately 53% of thePC12 cells compared with the non-treated cells (  p  0.001).Pre-treatment with 1mg/ml of MCR significantly increased cell viability to 26% from oxidative-stressed PC12 cells(79% of control,  p  0.001, see Fig. 3). This result stronglysuggests that MCR treatment restores oxidative-induced neu-ronal cell damage, so that it may be helpful for treatingneuro-degenerative diseases. Effect of MCR on Gene Expression in PC12 Cells Re-sults obtained from oligonucleotide microarrays indicated that treatment of MCR in PC12 stressed by hydrogen perox-ide changed mRNA expression. We examined over 1300genes, and 17 of them showed dynamic changes of that ex- pression to MCR addition compared to hydrogen peroxideonly treatment (see Table 2). Among them, the expressionsof 7 genes were over 2 fold decreased, whereas those of 10genes were over 2 fold increased in MCR treatment com- pared to hydrogen peroxide only treatment (see Table 2). Real-Time RT-PCR  Using the real-time quantitativePCR technique, we verified our oligonucleotide microarrayresults of the two important genes which were known to beinvolved in inhibiting the generation of ROS. April 2005663Fig.1.(A) HPLC Chromatogram of Paeonol (0.1mg/ml) and (B) MCR Extract (2mg/ml)Fig.2.Effects of MCR on the Inhibition of ROS by Various Concentra-tions of the Ethanol Extract in Hydrogen Peroxide Stimulated PC12 Cells PC12 cells were pre-incubated with various concentrations of MCR extract for 24h.Subsequently the medium was washed away and the cells were stressed with hydrogen peroxide for 30min. The production of ROS of each sample was quantified by DCF flu-orescence intensity (excitation 485nm/emission 538nm) as described previously. Val-ues are the mean  S.E.M. of three independent experiments duplicated in each run. ∗∗∗  p  0.001 compared to control.  The results presented in Figs. 4 and 5 show that the ex- pression patterns obtained with real-time RT-PCR were con-sistent with the oligonucleotide microarray results. Asshown, the expression levels of HO and COMT mRNA inMCR treatment were increased in a dose and time-dependentmanner compared to hydrogen peroxide treated cells. Theyreached maximum effects at 1mg/ml of MCR for a 24htreatment.DISCUSSIONIn this study, we showed that MCR significantly inhibited the increment of intracellular ROS and cell cytotoxicity uponhydrogen peroxide stimulation. Subsequently, the oligonu-cleotide microarray assay was performed to understand themolecular mechanism of MCR’s effect on ROS inhibition.Results of the oligonucleotide microarray showed that 17genes were differentially expressed, and among them HO and COMT were known to regulate ROS production.HO is a microsomal enzyme involved in the degradationof heme to biliverdin, iron and carbon monoxide, the first 664Vol. 28, No. 4Fig.3.Effects of MCR on the Cell Viability by Various Concentrations of MCR in the Hydrogen Peroxide Stimulated PC12 Cells Cell viability was measured by the MTS assay as depicted in Materials and Methods.Cells were treated as previously described in Fig. 1. Values are the mean  S.E.M. of three independent experiments duplicated in each run. ∗∗∗  p  0.001 compared to con-trol. Fig.4.Change of HO mRNA in PC12 Cells after MCR Treatment The amounts of HO mRNA were quantified by real-time RT-PCR after the respectiveMCR concentration (A) or the respective incubation periods at 1mg/ml of MCR (B).The expression levels of HO mRNA were normalized by dividing with GAPDH inten-sity. Values are the mean  S.E.M. of three independent experiments duplicated in eachrun. Str: 100 m  M hydrogen peroxide treatment; MCR: 100 m  M hydrogen peroxide  1mg/ml of MCR. Table1.Genes Which Significantly Differentially Expressed in Control or Hydrogen Peroxide Treated PC12 CellsGene nameFold  a ) IDP2X2-5 receptor (P2X2-5)   3.1AF020758P2X2-4 receptor (P2X2-4)   2.6AF020757c-fos   2.3X06769A2 adenosine receptor (A2AR)2.2S47609Cathechol- O -methyltransferase (COMT)3.9M93257Glucose transporter 1 (GLUT1)5.7S68135EST2233334.0AI179610Heme oxygenase (HO)3.8J02722VGF2.2M74223UI-R-C0-ja-a-07-0-UI.s12.3AI029917 Neuronatin alpha2.6U08290Macrophage migration inhibitory factor (MIF)2.0S73424 a ) Fold means ratio of hybridization intensity. The genes with negative value areabundant in non-treated PC12 cells, while positive values are abundant in hydrogen peroxide treated PC12 cells. Table2.Genes Which Significantly Differentially Expressed in the MCR Plus Hydrogen Peroxide or Hydrogen Peroxide Only Treated PC12 CellsGene nameFold  a ) IDPutative potassium channel subunit protein (RCK4)   2.1X16002P2X2-5 receptor (P2X2R-5)   2.8AF020758P2X2-4 receptor (P2X2R-4)   2.4AF020757Interferon-gamma inducing factor isoform  2.7U77777alpha precursor (IL-18 a  )P2x receptor (P2XR)   2.5U14414AP2X2 purinoceptor isoform e (P2X2R-e)   2.6AF028603Precursor interleukin 18 (IL-18)   2.2AJ222813Myelin/oligodendrocyte glycoprotein (MOG)3.9M99485EST2233333.2AI179610Glucose transporter 1 (GLUT1)4.4S68135UI-R-C0-ja-a-07-0-UI.s12.0AI029917A2 adenosine receptor (A2AR)2.2S47609Cathechol- O -methyltransferase (COMT)2.7M93257 Neuron-specific enolase (NSE)2.0X07729Heme oxygenase (HO)3.4J02722VGF2.0M74223A rat novel protein which is expressed 2.7E12625with nerve injury (E12625) a ) Fold means ratio of hybridization intensity. The genes with negative value areabundant in hydrogen peroxide treated cells, while positive values are abundant inMCR plus hydrogen peroxide treated cells.   being subsequently converted to bilirubin by cytosolic biliverdin reductase. Three isoforms of HO, HO-1, HO-2 and HO-3 have been identified which are products of distinctgenes. 15) HO-2 and HO-3 are constitutively expressed,whereas HO-1 is highly induced by a wide range of stressfulstimuli. Up-regulation of HO-1 may be among the most criti-cal cytoprotective mechanisms that are activated during timesof cellular stress, such as inflammation, ischemia, hypoxia,hyperoxia, hyperthermia, or radiation, 16) and is thought to play a key role in maintaining antioxidant and oxidant home-ostasis during times of cellular injury. 17) Prevention of ROS generation by catecholoestrogens isclosely related to the activity of the COMT. This enzyme cat-alyzes the methylation of hydroxylated sites on the aromaticring of catechol compounds which prevents their conversionto semiquinones and quinones, and therefore blocks the gen-eration of ROS. 18) Glucose transporter 1 (GLUT1) was the most up-regulated gene when we performed the oligonucleotide microarray inMCR plus hydrogen peroxide treated PC12 cells (Table 2).GLUT1 is widely distributed and is expressed in most tis-sues. 19) It is regulated under conditions which require adjust-ment of the metabolic rate, such as during cell division, 20) differentiation 21) transformation and in response to variousstresses. 22,23) Previous study demonstrated that exposure of L6 cells to ROS generating system increases the steady statelevels of GLUT1 mRNA, either by increased transcriptionrate and/or by increased stability, resulting in increased glu-cose uptake and metabolism. 24) It is assumed that oxidativestress imposed upon cells causes an adaptive response whichresults in increased energy production, presumably to recruitcellular defense mechanisms.P2X2-4 receptor and P2X2-5 receptor were down-regu-lated genes over two folds in MCR treated cells. P2X recep-tors are highly expressed throughout the nervous system,where ATP has been shown to be a neurotransmitter. SevenP2X receptor genes (P2X 1—7 ) have been identified; the func-tional receptors are thought to form as either homo- or het-erotrimers, and a range of phenotypes has been described for recombinant receptors based on their subunit composition. 25) Studies of P2X receptor distributions show that P2X2 and P2X3 receptor subtypes are selectively expressed in struc-tures associated with pain signal processing, including small-and medium-sized dorsal root ganglion neurons, peripheraland central sensory terminals, and superficial dorsalhorns. 26—28) MCR has been used for pain relief in traditionaloriental medicine. So we assumed that the P2X2 down-regu-lation might be related with the pain relief effect of MCR.At present, the precise regulatory mechanism of ROS inhi- bition by MCR is unknown. However, an important role for the inhibition of ROS in protection of neurotoxicity has beenrecognized. 28,29) In all types of neuro-degenerative diseasethere is consistent evidence of enhanced production of freeradicals and/or significant decrease of antioxidant defense.As a consequence, a large number of studies have focused onthe pathogenic significance of oxidative stress in neuronal in- jury as well as on therapeutic intervention with antioxidantand metabolic scavengers. 30,31) In conclusion, our results demonstrated that MCR de-creased ROS generation and cytotoxicity in hydrogen perox-ide stimulated PC12 cells. And we found that HO and COMT which have been known as ROS regulation were dif-ferentially expressed in this experimental condition. MCR might have a neuron protective effect in ROS mediated neu-ronal disease such as Alzheimer’s, Parkinson’s and ischemiathrough the reduction of ROS by HO and COMT. However,the precise mechanism of MCR to inhibit ROS productionand the major bioactive component remains to be elucidated.REFERENCES 1)Castellani R., Hirai K., Aliev G., Drew K. L., Nunomura A., TakedaA., Cash A. D., Obrenovich M. E., Perry G., Smith M. A.,  J. Neurosci. Res. , 70 , 357—360 (2002).2)Hartmann A., Hirsch E. C.,  Adv. Neurol. , 86 , 143—153 (2001).3)Goldshmit Y., Erlich S., Pinkas-Kramarski R.,  J. Biol. Chem. , 276 ,46379—46385 (2001).4)Yamakawa H., Ito Y., Naganawa T., Banno Y., Nakashima S.,Yoshimura S., Sawada M., Nishimura Y., Nozawa Y., Sakai N.,  Neurol. Res. , 22 , 556—564 (2000).5)Yoshimura S., Banno Y., Nakashima S., Takenaka K., Sakai H., Nishimura Y., Sakai N., Shimizu S., Eguchi Y., Tsujimoto Y., NozawaY.,  J. Biol. Chem. , 273 , 6921—6927 (1998).6)Hunt J. V., Dean R. T., Wolff S. P.,  Biochem. J. , 256 , 205—212 (1988).7)Casaril M., Corso F., Corrocher R.,  Recenti. Prog. Med. , 82 , 39—44(1991).8)Ames B. N., Shigenaga M. K., Hagen T. M.,  Proc. Natl. Acad. Sci.U.S.A. , 90 , 7915—7922 (1993).April 2005665Fig.5.Change of COMT mRNA in PC12 Cells after MCR Treatment The amounts of HO mRNA were quantified by real-time RT-PCR after the respectiveMCR concentration (A) or the respective incubation periods at 1mg/ml of MCR (B).The expression levels of COMT mRNA were normalized by dividing by GAPDH inten-sity. Values are the mean  S.E.M. of three independent experiments duplicated in eachrun. Str: 100 m  M hydrogen peroxide treatment; MCR: 100 m  M hydrogen peroxide  1mg/ml of MCR.
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