6 pages

Effect of long term, non cholesterol lowering dose of fluvastatin treatment on oxidative stress in brain and peripheral tissues of streptozotocin-diabetic rats

Please download to get full document.

View again

of 6
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Effect of long term, non cholesterol lowering dose of fluvastatin treatment on oxidative stress in brain and peripheral tissues of streptozotocin-diabetic rats
  Pulmonary, Gastro ı ntest ı nal and Urogen ı tal Pharmacology Effect of long term, non cholesterol lowering dose of   󿬂 uvastatin treatment onoxidative stress in brain and peripheral tissues of streptozotocin-diabetic rats Ahmet Cumao ğ lu a , Gülgun Ozansoy b , Ali Murat Irat b , Aysel Ar ı c ı o ğ lu a , Çimen Karasu c , Nuray Ar ı  b, ⁎ a Gazi University, Faculty of Medicine, Department of Medical Biochemistry, Ankara,Turkey b  Ankara University, Faculty of Pharmacy, Department of Pharmacology, Ankara,Turkey c Gazi University, Faculty of Medicine, Department of Medical Pharmacology, Ankara,Turkey a b s t r a c ta r t i c l e i n f o  Article history: Received 18 May 2010Received in revised form 23 November 2010Accepted 26 November 2010Available online 21 December 2010 Keywords: DiabetesOxidative stressStatinStreptozotocin(Rat) Oneofthemaingoalsoftreatmentofdiabetesmellitusistopreventitscomplications.Oxidativestressisuniversalindiabetes,beingultimatelyinvolvedwiththedevelopmentcomplications.Asaresultofhyperglycemia,reactiveoxygen/nitrogen species are producedinvarioustissues thatleads to tissue damage with lipidperoxidation andproteinoxidation,alongwithdisruptionincellularhomeostasisandaccumulationofdamagedmolecules.Hence,supplementation with antioxidant compounds may offer some protection against diabetic complications. Thepleiotropic effects of statins, including antioxidant and anti-in 󿬂 ammatory properties, represent an area of greatinterest in prevention and therapy of cardiovascular and neurological disorders. Using biomarkers of oxidativestress,inthisstudyweexaminedtheeffectofnoncholesterolloweringdose,longterm 󿬂 uvastatintreatmentonoxidative stress in streptozotocin-diabetic rats. Experiments were conducted in 24 Wistar adult male rats.Diabetic and non-diabetic rats were treated orally for 6 months with  󿬂 uvastatin(2 mg/kg/day, p.o) starting oneweekafterstreptozotocininjection(55 mg/kg,i.p.),(preventivestudy).Inbrain,heart,liver,pancreasandkidneyhomogenates malondialdehyde, lipid hydroperoxide, protein carbonyl content, advanced oxidation proteinproducts,3-nitrotyrosinelevelsandsuperoxidedismutase,catalaseactivitiesweremeasured.Hyperglycemiaanddyslipidemia in diabetic groups remained unchanged after  󿬂 uvastatin treatment. The drug act as antioxidant inthetissues.Hence,antioxidantpropertyof  󿬂 uvastatin,independentofcholesterolloweringeffect,mayplayarolein prevention of diabetic complications. Clinical relevance of this effect of   󿬂 uvastatin seems worthy of furtherstudies.© 2010 Elsevier B.V. All rights reserved. 1. Introduction Overthepastdecade,therehasbeensubstantialinterestinoxidativestress and its potential role in development of diabetic complications.Diabetes mellitus, in left untreated, initiate degenerative processes intissues because of excess oxidative stress. In diabetes and its complica-tions,oxidativestressresultfromanoverproductionofreactiveoxygen/nitrogen species generated by glucose autoxidation, mitochondriadysfunction,polyolpathwayandproteinglycation,andfromdecreasedantioxidant defenses (Valko et al., 2007; Negre-Salvayre et al., 2009).One of the main goals of treatment of diabetes is to prevent itscomplicationsandthereisaccumulatingevidencethatsupplementationantioxidant compounds may offer some protection against diabeticcomplications (Maritim et al., 2003; Da Ros et al., 2004; Ceriello 2006; Juranek et al., 2010; Cumaoglu et al., 2010; Sivitz and Yörek, 2010).Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)reductase inhibitors, are widely used in clinical practice for theircholesterol lowering effect and in reducing morbidity and mortalityfrom cardiovascular disease. In recent years, it becomes clear that allthe clinical bene 󿬁 ts of statins therapy can not be explained solely bytheir lipid lowering properties because a variety of experimental datarevealed that these drugs have direct antioxidant/anti-in 󿬂 ammatoryeffectsthatwereunrelatedtothelipidloweringeffects(Rikitakeetal.,2001; Stoll et al., 2004; Zhou and Liao, 2010). These bene 󿬁 cialcholesterol-independent or pleiotropic effects of statins likelycontribute to their clinical ef  󿬁 cacy in treating cardiovascular diseaseas well as other chronic conditions associated with increasedoxidative stress. Many of these pleiotropic effects are mediatedthroughinhibitionof isoprenoid synthesiswithsubsequentinhibitionofisoprenoid-mediatedactivationofsmallGTP-bindingproteins,suchas Rho family members, Rac1 and RhoA. Rac1 binds to and leads toactivationoftheNADPHoxidasesystemandsubsequentgenerationof reactive oxygen species in many cells (Zhou and Liao, 2010).Fluvastatin is one of the frequently prescribed statins worldwideand has a structure similar to alpha-tocopherol, a natural antioxidantand, its major metabolites have also a direct scavenging activity on European Journal of Pharmacology 654 (2011) 80 – 85 ⁎  Corresponding author. Department of Pharmacology, Faculty of Pharmacy, AnkaraUniversity, 06100 Tandogan, Ankara,Turkey. Tel.:+90 312 213 44 78, +90 538 2159596 (GSM); fax: +90 312 213 10 81. E-mail address: (N. Ar ı ).0014-2999/$  –  see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2010.11.035 Contents lists available at ScienceDirect European Journal of Pharmacology  journal homepage:  hydroxyl radicals and superoxide anions (Suzumura et al., 1999a,b;Nakashima et al., 2001a,b; Guan et al., 2004). The direct inhibitoryeffect on LDL oxidation has also been reported (Suzumura et al.,1999a,b; Rikitake et al., 2001; Nakashima et al., 2001b; Kaneko et al.,2003). Thus, the antioxidative effect of   󿬂 uvastatin causes by not onlyinhibitionofNADPHoxidaseactivity,butalsothescavengingactionof reactive oxygen species. Although a large number of groups haveinvestigated the antihiperlipidemic effect of   󿬂 uvastatin, its effect onoxidative stress have been examined very little in diabetic tissues.Consideringtheimportanceofoxidativestressinthepathophysiologyof diabetic complications, in this study, using markers of oxidativestress, we investigated the effects of non cholesterol lowering dose,long term  󿬂 uvastatin treatment on lipid peroxidation, proteinoxidation and antioxidant defense enzymes and low molecularweight antioxidant molecules in brain, heart, liver, pancreas andkidney tissues from streptozotocin-diabetic rats. 2. Materials and methods  2.1. Animals and treatment  Diabetes was induced in male Wistar rats weighing 250 – 300 g bysingle dose i.p. injection of 55 mg/kg of streptozotocin (n: 12).Streptozotocin freshly dissolved in 0.1 M sodium citrate buffer (pH4.5).Controlrats(n:12)wereonlyreceivedvehicle.Bloodsamplesweretaken from the tail vein, glucose levels were determined using Accu-Chek Go © glucometer (Roche Diagnostics, Germany) and the rats withblood glucose levels  N 250 mg/dl at one week after streptozotocininjection were considered diabetic. Then,  󿬂 uvastatin was administeredtothediabetic(n:6) and control (n:6)ratsbygavage ata doseof 2 mg/kg body weight daily for 6 months (preventive study). The dose of  󿬂 uvastatin choosen to not affectcholesterol levels, based onpreviouslypublished studies (Rikitake et al., 2001; Sumi et al., 2001; Mitani et al.,2003). Fluvastatin sodium (Novartis Pharma, Basel, Switzerland)dissolved in water. The animals were supplied from Ankara University,Faculty of Pharmacy, Animal Care Unit, were kept in temperaturecontrolled facilities on 12-h light/dark cycle, standart rat chow and topwater were provided  ad libidum  throught the study. The animal useprotocol was approved by Ankara University Animal Care EthicCommittee. The principles of laboratory animals care (NIH publicationNo. 85-23, revised 1985) were observed.  2.2. Blood and tissue analyses At the end of the treatment period, blood glucose levels weredetermined using glucometer, then the rats were anaesthetised withketamine (100 mg/kg), blood samples were collected from leftventricule by cardiac puncture and plasma samples were obtained.Cholesterol and triglyceride concentrations in plasma were deter-mined using analyser (Roche Diagnostic). Brain, heart, liver, pancreasand kidney were removed, washed with ice-cold physiological salinesolution, dried on  󿬁 lter paper and stored in − 70 °C. Oxidative stressparameters were quanti 󿬁 ed as follows:Malondialdehyde, an end product of unsaturated fatty acid perox-idation, can react with thiobarbituric acid to form a colored complexcalled thiobarbituric acid reactive substances. Thiobarbituric acidreactivity was assayed by the method of  Uchiyama and Mihara(1978). Butanol phase was measured with microplate reader at532 nm. The results were expressed with tetramethoxypropanestandart curve within the range from 0 to 20nmol.Lipid hydroperoxide levels were determined according to themethod of ferrous oxidation with xylenol orange (Zadeh-Nouroozetal.,1994).Homogenatesweretransferredintomicrocentrifugetubes.10mM triphenylphosphine was added to vials to remove hydroper-oxides. Methanol alone was added to the remaining vials. The lipidhydroperoxide content in the homogenates were determined as afunction of the absorbance difference of samples with and withoutelimination of lipid hydroperoxides by triphenylphosphine. FOX2reagentwasaddedinto samples.Afterincubation atroomtemperaturefor30 min,thesampleswerecentrifugedandsupernatantwascarefullyplaced into well plate. A Bio-Tek ELX800 absorbance microplate reader(Bio-TekInstrumentsInc.,USA)wasusedtomeasuretheabsorbanceata wavelength 560 nm. Concentration of lipid hydroperoxide wascalculated by using the extinction coef  󿬁 cient of blue-purple complex,1.5×10 4 M − 1 cm − 1 .Proteincarbonylcontentwasmeasuredinhomogenatesaccordingto the method of  Levine et al. (1990). Protein carbonyl groups reactwith 2,4-dinitrophenylhydrazine to generate chromophoric dinitro-phenylhydrazones. 800  μ  l volume of 2,4-dinitrophenylhydrazine wasadded to the sample tube and 800  μ  l of 2.5 M HCl to the blank tube.After 1 h incubation, 1 ml tricloroacetic acid was added for precipi-tationofproteins.Bothtubeswerecentrifugedagaintoremovedebrisand placed in the well plate. A plate reader was used to measure theabsorbance at a wavelength between 340 and 370 nm. Concentrationof protein carbonyl was calculated by using the extinction coef  󿬁 cientof 2,4-dinitrophenylhydrazine, 22×10 4 − 1 cm − 1 .3-nitrotyrosine levels were measured by a commercially availableELISA kit (HyCult Biotechnology, Uden, NL). Tissue samples werehomogenized (1:10, w/v) in ice-cold phosphate buffer (pH: 7.4)completeproteaseinhibitorcocktailusingaglasste 󿬂 onhomogenizer.AdvancedoxidationproteinproductswithcharacteristicabsorbancewerebasedonspectrophotometricdetectionaccordingtoWitko-Sarsatetal.(1996).Tissuehomogenatesdiluted1:10withphosphatebufferedsaline, and only phosphate buffered saline as blank were applied on amicrotiter plate. 10  μ  l of 1.16 M Kl and 20  μ  l of acetic acid were added,and absorbance at 340 was measured immediately. Concentration of advanced oxidation protein products were calculated by using theextinction coef  󿬁 cent of 261 mM − 1 cm − 1 .Glutathione levels in homogenates were measured by a commer-cially available assay kit (Cayman,Ann Arbor, MI, USA). This kit utilisesan enzymatic recycling method based on the reaction betweenglutathione and 5.5 ′ -dithiobis-2-nitrobenzoic acid that produces ayellow colored compound 5-thio-2-nitrobenzoic acid. The rate of 5-thio-2-nitrobenzoic acid production is directly proportional to theconcentration of glutathione in the sample. Measurement of theabsorbance of 5-thio-2-nitrobenzoic acid at 412 nm provides anaccurate estimation of glutathione in the sample. Due to the presenceof glutathione reductase, which reduces the disulphide dimer of glutathione to glutathione, in the reaction buffer, both glutathione anddisulphide dimer of glutathione are measured and the assay re 󿬂 ectstotal glutathione present in the sample.Superoxide dismutase activity was assayed using nitro bluetetrazolium by a previously reported method of  Sun et al. (1988).The samples were subjected to ethanol – chloroform (5:3) extractionbefore the enzyme activity assay. The nitro blue tetrazolium wasreduced to blue formazan by O 2 − , which has a strong absorbance at560 nm. The calculated superoxide dismutase activity was expressedas U/mg of protein in the tissue.Catalase activity in homogenates was assayed following theprocedure of  Aebi (1984). The molar extinction coef  󿬁 cient of 43.6 M/cmwasusedtodeterminecatalaseactivity.Oneunitofactivityisequalto the moles of H 2 O 2  degraded/min per mg protein. Total proteinconcentration in the homogenates were determined by the Bradford(1976) method using bovine serum albumin as a standard.  2.3. Statistical analyses The reported datas are means of measurements with their standarterror of means (S.E.M) values. ANOVA test was used for comparisonsof the groups. Student-Newman-Keul's test was used as the post hocanalyses. A values of   P  b 0.05 was set as the limit of statisticalsigni 󿬁 cance. 81  A. Cumao  ğ  lu et al. / European Journal of Pharmacology 654 (2011) 80 – 85  3. Results  3.1. Basic parameters Signi 󿬁 cant weight reduction, elevated blood glucose, triglycerideand total cholesterol levels were observed in rats following period of diabetes. Fluvastatin treatment did not affect the general parametersin both control and diabetic rats (Table 1).  3.2. Oxidative/nitrosative stress markers Oxidative/nitrosative stress markers measured in all group aregiven in Table 2.Thiobarbituric acid reactive substances and lipid hydroperoxidelevels were signi 󿬁 cantly higher and glutathione levels were signi 󿬁 -cantly lower in diabetic brain tissue, when compared with those of control group. Fluvastatin treatment prevented the alterations onthiobarbituric acid reactive substances and glutathione levels indiabeticbraintissuehomogenates, butnosigni 󿬁 cantdifferencesweredetected in the other measured parameters.When compared with control group, thiobarbituric acid reactivesubstances,lipidhydroperoxide,proteincarbonylcontent,3-nitrotyrosinelevels and catalase activity were signi 󿬁 cantly increased and glutathionelevelsweresigni 󿬁 cantlydecreasedinhearthomogenatesofdiabeticrats.Fluvastatin treatment did not affect catalase activity and glutathionelevels,butsigni 󿬁 cantlyattenuatedthiobarbituricacidreactivesubstances,lipidhydroperoxide,proteincarbonylcontent,3-nitrotyrosinelevelsaftertreatment of diabetic animals.In diabetic liver homogenates, superoxide dismutase, thiobarbi-turic acid reactive substances, protein carbonyl content, advancedoxidation protein products, 3-nitrotyrosine levels were signi 󿬁 cantlyhigher when compared with those of control group. Fluvastatintreatment was only normalized superoxide dismutase activity.In diabetic pancreas homogenates we found increased superoxidedismutase and catalase activities, thiobarbituric acid reactive sub-stances, protein carbonyl content and 3-nitrotyrosine levels. Fluvas-tatin treatment did not change superoxide dismutase activity, but  Table 1 General charactheristics of rat groups.UntreatedcontrolFluvastatintreated controlUntreateddiabeticFluvastatintreated diabeticBody weight (g) 352±20 342±12 240±25 ⁎ 250±28 ⁎ Blood glucose (mg/dl) 122±10 129±13 543±32 ⁎ 555±27 ⁎ Total cholesterol (mg/dl) 48±5 51±3 65±3 ⁎ 60±3 ⁎ Triglyceride (mg/dl) 55±4 51±3 80±6 ⁎ 81±7 ⁎ Values are mean±S.E.M,  n =6 for each group. *  p b 0.05  vs.  untreated controls .  Table 2 Effects of   󿬂 uvastatin treatment on oxidative stress and antioxidant defense system in tissues of rat groups.Untreated control Fluvastatin treated control Untreated diabetic Fluvastatin treated diabeticBra ı n TBARS 2.79±0.27 3.55±0.45 4.98±0.81 ⁎ 3.25±0.22 # LHP 5.39±0.93 4.79±0.20 8.96±1.21 ⁎ 6.17±1.60 # PCC 8.41±0.69 10.74±0.75 10.09±0.93 9.14±0.60AOPPs 1.68±0.17 1.92±0.17 1.78±0.10 1.79±0.113-NT 51.04±4.73 53.31±5.62 51.16±2.26 46.44±1.77SOD 0.14±0.02 0.17±0.01 0.16±0.006 0.15±0.01CAT 2.85±0.23 2.91±0.18 2.74±0.15 3.01±0.22GSH 8.45±0.27 7.17±0.51 6.17±0.31 ⁎ 8.44±0.27 # Heart TBARS 1.80±0.20 2.50±0.42 3.10±0.35 ⁎ 2.12±0.18 # LHP 9.32±0.33 9.04±0.58 15.71±1.23 ⁎ 9.73±0.80 # PCC 12.48±1.31 14.42±2.73 19.75±1.98 ⁎ 16.12±1.20 ⁎ AOPPs 3.78±0.27 3.73±0.31 4.41±0.33 4.20±0.373-NT 82.13±2.81 73.56±6.05 96.66±4.21 ⁎ 60.98±3.28 ⁎ , #SOD 0.075±0.01 0.077±0.011 0.089±0.005 0.089±0.005CAT 24.58±2.80 36.23±1.95 40.45±2.38* 39.93±1.36 ⁎ , #GSH 18.77±1.30 16.26±0.95 14.00±1.01 ⁎ 16.13±0.80L  ı ver TBARS 0.85±0.03 0.88±0.11 1.29±0.05 ⁎ 1.08±0.19LHP 4.39±0.37 5.31±0.96 4.89±0.16 4.61±0.4PCC 10.01±0.80 9.80±1.08 14.21±1.50 ⁎ 9.05±1.33AOPPs 2.25±0.24 2.50±0.23 3.55±0.29 ⁎ 3.23±0.373-NT 74.45±4.04 85.05±3.90 100.35±5.16 ⁎ 78.49±5.20 # SOD 0.13±0.01 0.18±0.005 0.16±0.009 ⁎ 0.15±0.003 # CAT 1093±70 1216±44 1251±60 1196±64GSH 24.90±2.06 24.19±0.98 22.08±1.97 25.01±1.86Pancreas TBARS 0.48±0.029 0.38±0.039 0.70±0.083 ⁎ 0.47±0.033 # LHP 4.71±1.66 3.61±0.72 5.80±0.62 5.80±0.62PCC 23.07±3.34 31.18±6.19 35.90±2.67 ⁎ 29.93±1.41 # AOPPs 2.12±0.21 2.62±0.69 2.48±0.23 2.53±0.513-NT 83.38±8.99 101.73±8.46* 121.21±11.95 ⁎ 74.13±4.13 # SOD 0.098±0.022 0.152±0.020 0.178±0.020 ⁎ 0.181±0.021 ⁎ CAT 312.87±24.66 306.64±13.73 436.57±28.34 ⁎ 339.62±12.11 # GSH 10.19±1.01 9.57±0.77 9.19±0.69 11.62±0.42K ı dney TBARS 1.65±0.22 1.99±0.40 2.25±0.19 ⁎ 1.71±0.19 # LHP 9.40±0.18 12.06±2.47 11.10±1.48 8.38±0.78PCC 10.25±0.60 12.45±0.79 14.72±0.43 ⁎ 11.47±1.10 # AOPPs 2.65±0.24 2.65±0.16 3.47±0.23 2.96±0.343-NT 139.24±2.13 146.31±7.08 162.17±5.94 140.44±4.96SOD 0.106±0.006 0.114±0.009 0.144±0.036 0.130±0.087CAT 917.28±64.27 952.48±92.84 792.67±42.81 888.06±24.26GSH 19.73±0.46 19.77±1.92 16.28±1.33 ⁎ 16.36±0.62Thiobarbituric Acid Reactive Substance (TBARS: nmol/mg protein); Lipid Hydroperoxide (LHP: nmol/mg protein); Protein Carbonyl Content (PCC: nmol/mg protein); AdvancedOxidationProteinProducts(AOPP:,nmol/mgprotein);3-Nitrotyrosine(3-NT:pmol/mgprotein);SuperoxideDismutaseactivity(SOD:IU/mgprotein);Catalaseactivity(CAT:IU/mgprotein); Glutathione levels (GSH: nmol/mg protein). n =6 for each group. Values are mean±S.E.M.*  p b 0.05 vs. untreated controls,  #  p b 0.05 vs. untreated diabetic.82  A. Cumao  ğ  lu et al. / European Journal of Pharmacology 654 (2011) 80 – 85  signi 󿬁 cantly prevented the alterations on thiobarbituric acid reactivesubstances, protein carbonyl content, 3-nitrotyrosine levels andcatalaseactivityindiabeticgroup.Interestingly,3-nitrotyrosinelevelswere increased signi 󿬁 cantly in pancreas homogenates from controlrats after the treatment.Finally, in diabetic kidney tissue homogenates, we found signi 󿬁 -cantly increased 3-nitrotyrosine, protein carbonyl content andthiobarbituric acid reactive substances, and signi 󿬁 cantly decreasedglutathione levels when compared with those of control group.Treatment with  󿬂 uvastatin prevented augmentations on thiobarbi-turic acid reactive substances, protein carbonyl content and 3-nitrotyrosine levels. We determined diversi 󿬁 ed antioxidant enzymeactivities in diabetic kidney tissue homogenates, but the differencesdid not reach to statistical signi 󿬁 cance. 4. Discussion Streptozotocin-diabetic rat model is a prototype of type 1 diabetesmellitus with an intense oxidative stress due to hyperglycemia(Kakkar et al., 1995, 1998). Fluvastatin dosage used in the presentstudy did not signi 󿬁 cantly affect the cholesterol levels, as expected,allowing us to investigate  in vivo  direct antioxidant effect of the drug.Thus, our results indicate that the lipid lowering effect did notcontribute to the antioxidant effect of   󿬂 uvastatin in the tissues.Chronichyperglycemiapromotesendogenfreeradicalgenerationanddeplete antioxidant defense systems. Free radicals play an importantrole as endogenous initiators and promoters of lipid and proteinoxidation that contribute to diabetes and its complications. Thus,uncontrolled diabetes leads to diabetic complications includingcardiovascular disease, nephropathy, neuropathy and retinopathy(West, 2000; Ceriello, 2006). Antioxidant effects of   󿬂 uvastatin,independent of its hypolipidemic effect, have already been demon-strated in several hypercholesterolemic rabbits (Rikitake et al., 2001;Sumi et al., 2001; Yamaguchi et al., 2002; Mitani et al., 2003), reactiveoxygenspecies-generatedratmodel(Bandohetal.,2003),myocardialinfarction rat model (Zhou et al., 2008), hyperhomosysteinemic rat(Morita et al., 2005), oxygen-induced retinophatic mice (Bartoli et al., 2009), and recently, in streptozotocin-diabetic rat model (Matsukiet al., 2010). The molecular basis of antioxidant/anti-in 󿬂 ammatoryeffects of statins relate to their ability block the production and/oractivity of reactive oxygen species. Statins have been shown to blockthe isoprenylation (geranylgeranylation) and activation of membersof the Rho family, such as RhoA and Rac1. Rac1 regulates NADPHoxidase, which is a major source of reactive oxygen species in cells.Studies have shown that statins attenuate oxidative stress throughinhibition of Rac1 (Stoll et al., 2004; Davignon, 2004). It has beenreported that treatment of 5 mg/kg/day (noncholesterol loweringdose)  󿬂 uvastatin decreases protein kinase C activator-dependentreactive oxygen species generation in rat peritoneal neutrophils andthis effect was reversed by the combined administration withmevolanate. This indicates that  󿬂 uvastatin inhibits reactive oxygenspecies generation via the inhibition of isoprenylation (geranylger-anylation) and the HMG-CoA reductase and the downstreammevalonate pathwayevenat noncholesterolloweringdoses(Bandohet al., 2003). Importantly,  󿬂 uvastatin can inhibit angiotensin II-induced vascular reactive oxygen species production and in 󿬂 amma-tionat3 mg/kg/dayinatheroscleroticmousemodel.Further,lowdose 󿬂 uvastatin actes synergistically with an angiotensin AT 1  receptorblockerontheinhibitions(Lietal.,2004).Ontheotherhand,thereareseveral reports that  󿬂 uvastatin supresses lipid peroxidation byscavenging reactive oxygen species  in vivo  and  in vitro  (Bandoh etal., 2003). Thus, the antioxidative effect of   󿬂 uvastatin is not only byinhibition of reactive oxygen species generation, but also by thescavenging action of the radicals. Fluvastatin has chemical structuresimilar to alpha-tocopherol (Nakashima et al., 2001a,b), its metabo-lites also have scavenging activity against reactive oxygen species(Suzumura et al., 1999a,b). Statins can also up-regulate antioxidantenzymesbutthestudiesarefewinnumberandnotentirelyconsistent(Davignon, 2004; Stoll et al., 2004).Fluvastatin, is synthetic, relatively hydrophilic, compared withthesemisyntheticinhibitors.Brainperfusionstudiesinratsindicatedthat small but measurable brain uptake occurres (Guillot et al.,1993). Thus, the drug has been studied little for it's central effects.For ex., at 4 mg/kg/day dosage causes signi 󿬁 cant remodeling of thebasilararteryinhypertensive rats(LedinghamandLaverty,2002),at7.5 mg/kg dosage alters psychomotor performance in rats (Baytanet al., 2006). Cerebral infarction and hemorrhage are more commonin diabetic patients, and the central complications of hyperglycemiainclude the potentiation of neuronal damage. It has been reported thatsuperoxidedismutase,butnotcatalaseactivity,wasreducedinthebrainfrom4-weekstreptozotocin-diabeticrat(Nazarogluetal.,2009).Inourprevious study, superoxide dismutase activity of brain was unchangedby the long term diabetes and the lipid peroxidation was increased(Ulusuetal.,2003).Theantioxidantenzymeactivityistissuedependentand varies from tissue to tissue and that the duration and severity of diabetes are major contributing factors for the alterations. Althoughlargely unknown, the alterations is also related to the level of reactiveoxygen species and/or location of the enzymes (Gumieniczek et al.,2002; Ulusu et al., 2003). In the present study we did not detect anysigni 󿬁 cant changes on catalase and superoxide dismutase activities indiabetic brain tissue. But thiobarbituric acid reactive substances andlipid hydroperoxide levels were signi 󿬁 cantly higher and glutathionelevels were lower in diabetic group compared to control group.Fluvastatin treatment prevented these alterations. In addition to thevascular bene 󿬁 cial pleiotropic effects of statins (improving endothelialfunction, attenuating vascular remodeling, stabilizing atheroscleroticplaques), there are increasing data to suggest that these agents haveneuroprotective effects with antioxidant and anti-in 󿬂 ammatory prop-erties. Studies are going in this area for the potential role of statins intreating stroke and neurological diseases (Reiss and Wirkowski, 2007;Zhou and Liao, 2010).Thiobarbituric acid reactive substances, lipid hydroperoxide,protein carbonyl content, 3-nitrotyrosine levels and catalase activitywere signi 󿬁 cantly higher and glutathione levels were lower in heartof diabetic rats. Fluvastatin treatment did not affect glutathionelevels, but signi 󿬁 cantly attenuated the other measured parameters.Inarecentstudy, 󿬂 uvastatin,atnoncholesterolreducingdoses(5and10 mg/kg) has been found to be effective on the protection of heart against isoproterenol-induced myocardial infarction throughmaintaining endogenous antioxidant enzyme activities (Zhou et al.,2008). Obata et al. (2009) tested the effect of 5 mg/kg/day  󿬂 uvastatintreatment in rat and they found that  󿬂 uvastatin scavenges hydroxilradical and block the LDL oxidation in heart perfusion study.Cardiac hypertrophy is mediated, in part, by myocardial oxidativestress, and Rac is an important mediator of this pathology (Zhou andLiao, 2010). Thus, Rac1 activity and reactive oxygen species gener-ationcanbeattenuatedby 󿬂 uvastatin.Inarecentstudy,10 mg/kg/dayof   󿬂 uvastatin treatment for 2 weeks signi 󿬁 cantly reduced themyocardial levels of NADPH oxidase subunit p22 phox mRNA expres-sion in streptozotocin-diabetic rat (Matsuki et al., 2010). Hence, 󿬂 uvastatin can prevent diabetic cardiomyopathy by its antioxidativeproperty.In diabetic kidney,  󿬂 uvastatin treatment prevented the alterationsonthiobarbituricacidreactivesubstances,proteincarbonylcontentand3-nitrotyrosine levels. Antioxidant enzyme activities did not changesigni 󿬁 cantly.Previously,inratkidneydamagemodel,podocytedamageand macrophage in 󿬁 ltration were found to be reduced by six week- 󿬂 uvastatin treatment at 5 mg/kg/day dosage (McKenney, 2003).Fluvastatin also prevents the oxidative DNA damage in kidney andliver of streptozotocin-diabetic mice (Imaeda et al., 2002). Peripheralartery disease and end-stage renal disease are an important diabeticcomplications and  󿬂 uvastatin seems to be effective in this tissue. In 83  A. Cumao  ğ  lu et al. / European Journal of Pharmacology 654 (2011) 80 – 85  clinical trials renoprotective effects of statins are uncertain because of relatively sparse data (Strippoli et al., 2008).Pancreatic damage is also important point in the development of diabetes and its complications. Fluvastatin treatment prevented thealterations in pancreas of diabetic group, except on superoxidedismutaseactivity.Elevationinthiobarbituricacidreactivesubstancesin the oxidative damage model of hamster pancreas was alsosuppressed by low dose  󿬂 uvastatin administration (Kaneko et al.,2003). Thus,  󿬂 uvastatin appears to be effective in preventingoxidative stress and damage in diabetic pancreas. Interestingly, wefound that 3-nitrotyrosine levels were increased in pancreas of  󿬂 uvastatin treated control rats. This unexpected augmentation seemsto be speci 󿬁 c for this tissue and the reason needs to be clari 󿬁 ed.Experimental streptozotocin-model is well-established model, how-ever, it should be taken into account that this insulinopenic model isassociated with extremly high blood glucose levels, which are notcountered in humans. Interestingly, in a recent meta-analysis it wasreported that statin therapy was associated with a small, butsigni 󿬁 cant, increased risk of diabetes. The mechanisms are notunderstood. However, the risk might be affected by the amount of drug-induced cholesterol reduction and/or high doses (Sattar et al.,2010). On the contrary, a few clinical studies have indicated that useof statins can delay the progression of diabetes. Though themechanism underlying this phenomenon is still elusive, but elevatedserumcholesterolcanimpairinsulinsecretionandincreasetherateof apoptosis in beta cells (Qian et al., 2010). In our intensive oxidativestress model chronic  󿬂 uvastatin treatment, at non cholesterolreducing dosage, did not affect the blood glucose levels.In diabetic liver homogenates,  󿬂 uvastatin treatment only normal-ized superoxide dismutase activity. In rat liver microsomes, it hasbeen shown that  󿬂 uvastatin scavenges reactive oxygen species andinhibits lipid peroxidation (Yamamoto et al., 1998).The clinical dosage of   󿬂 uvastatin for human adults is usually 20 – 30mg/day, which corresponds to a dose per g body weight of 0.33 – 0.50mg/kg, given to a patient with an average body weight of 60 kg(Kanekoetal.,2003).Thus,thedosageof  󿬂 uvastatinusedinourstudyiscloser, but not comparable(higher) to usual dosage usedclinically. Thepeculiaritiesspeci 󿬁 ctolipidmetabolisminrodentsmakethesedrugstouse high dosage, but, experiments on rodents is suitable for examiningthe extra-lipid effect of statins. For ex., Rikitake et al. (2001) reportedthat 󿬂 uvastatinatthedoseof2 mg/kg/daydidnotreduceplasmalipidsin their study, although 12.5 – 50mg/kg/day dosage has been shown toeffectively reduce plasma lipids in hyperlipidemic rabbits (Kurokawaet al., 1995). 5. Conclusions Theantioxidativepleiotropicactionof  󿬂 uvastatinmightcontributeto its bene 󿬁 cial effects, by slowing organ damage in diabetes. But, thedifferences betweenrodent and human lipoprotein metabolism makeadirecttranslationofour 󿬁 ndings,withthedoseof  󿬂 uvastatinusedinthis study, to humans impossible. Therefore, further studies arenecessary to prove whether our results can be translated to humanswithdiabetes.Ifso, additionofearly,noncholesterolloweringdoseof  󿬂 uvastatin, irrespective of the plasma cholesterol levels, in conven-tional antidiabetic regime may prevent diabetic complications.Further,  󿬂 uvastatin deserves special attention which oxidative stressplay a major role in the ethiology of a wide variety of chronic diseasesincluding central nervous system, and comparable dose is must benonlipid lowering dose.  Acknowledgements This study was supported by the Ankara University ResearchFoundation (No: 2008-0803061). We would like to thank NovartisPharmafor the genorous supply of  󿬂 uvastatin. We thankto Mr. IsmailBasar for his help during the experiments. References Aebi, H., 1984. Catalase. In: Packer, L. (Ed.), Methods in Enzymology. Academic Press,Orlando, FL, pp. 125 – 126.Bandoh, T., Sato, E.F., Mitani, H., Nakashima, A., Hoshi, K., Inoue, M., 2003. Antioxidativepotentialof   󿬂 uvastatin via the inhibition of nicotinamide adenine dinucleotidephosphate (NADPH)oxidase activity. Biol. Pharm. Bull. 26, 818 – 822.Bartoli, M., Al-Shabrawey, M., Labazi, M., Behzadian, M.A., Istanboli, M., El-Remessy, A.B.,Caldiven, R.W., Marcus, D.M., Caldwell, R.B., 2009. HMG-CoA reductase inhibitors(statin)preventsretinalneovascularizationinamodelofoxygen-inducedretinopathy.Invest. Ophthalmol. Vis. Sci. 50, 4934 – 4940.Baytan, S.H., Alkanat, M., Özeren, M., Ekinci, M., Akgün, A., 2006. Fluvastatin alterspsychomotorperformance and daily activity but not the spatial memory in rats.Tohoku J. Exp. Med. 209, 311 – 320.Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248 – 254.Ceriello, A., 2006. Controlling oxidative stress as a novel molecular approach toprotecting thevascular wall in diabetes. Curr. Opin. Lipidol. 17, 510 – 518.Cumaoglu, A., Stefek, M., Bauer, V., Ar ı , N., Ar ı c ı o ğ lu, A., Karasu, C., 2010. Glycoxidativeandnitrosative stresses in kidney of experimental diabetic rats: effects of thepyridoindoleantioxidant stobadine. Neuroendocrinol. Lett. 31, 101 – 106.DaRos,R.,Assaloni,R.,Ceriello,A.,2004. Antioxidanttherapyindiabeticcomplications:whatis new? Curr. Vasc. Pharmacol. 2, 335-4.Davignon, J., 2004. Bene 󿬁 cial cardiovascular pleiotropic effects of statins. Circulation109 (suppl 111), 39 – 43.Guan, J.Z., Murakami, H., Yamato, K., Tanabe, J., Matsui, J., Tamasawa, N., Suda, T., 2004.Effectsof   󿬂 uvastatin in type 2 diabetic patients with hyperlipidemia: reduction incholesterol oxidationproducts and VCAM-1. J. Atheroscler. Thromb. 11, 56 – 61.Guillot, F., Misslin, P., Lemaire, M., 1993. Comparison of   󿬂 uvastatin and lovastatinblood – brain barrier transfer using in vitro and in vivo methods. J. Cardiovasc.Pharmacol. 21, 339 – 346.Gumieniczek, A., Hopkala, H., Wojtowicz, Z., Nikolajuk, J., 2002. Changes in antioxidantstatus ofheart muscletissues inexperimental diabetes inrabbits. ActaBiochim. Pol.49, 529 – 535.Imaeda,A.,Kaneko,T.,Aoki,T.,Kondo,Y.,Nakamura,N.,Nagase,H.,Yoshikawa,T.,2002.Antioxidative effects of   󿬂 uvastatin and its metabolites against DNA damage instreptozotocin-treated mice. Food Chem. Toxicol. 40, 1415 – 1422. Juranek, I., Horakova, L., Rackova, L., Stefek, M., 2010. Antioxidants in treatingpathologiesinvolving oxidative damage: an update on medicinal chemistry andbiological activity of stobadine and related pyridoindoles. Curr. Med. Chem. 17,552 – 570.Kakkar, R., Karla, J., Mantha, S.V., 1995. Lipid peroxidation and activity of antiox-idantenzymes in diabetic rats. Mol. Cell. Biochem. 151, 113 – 119.Kakkar, R., Mantha, S.V., Radhi, J., Prasad, K., Kalra, J., 1998. Increased oxidative stress inrat liverand pancreas during progression of streptozotocin-induced diabetes. Clin.Sci. 94, 623 – 632.Kaneko, T., Tahara, S., Tokabayashi, F., 2003. Protective effect of   󿬂 uvastatine, an HMG-CoAreductase inhibitor on the formation of 8-oxo-21 ′ -deoxyguanosine in thenuclear DNA ofhamster pancreas. Biol. Pharm. Bull. 26, 1245 – 1248.Kurokawa, J., Hayashi, K., Toyota, Y., Shingu, T., Shiomi, M., Kajiyama, G., 1995. Highdose of  󿬂 uvastatin sodium (XU62 – 320), a new inhibitor of HMG-CoA reductase,lowers plasmacholesterol levels in homozygous Watanebe-heritable hyperlipi-demic rabbits. Biochim. Biophys. Acta 1259, 99 – 104.Ledingham, J.M., Laverty, R., 2002. Fluvastatin remodels resistance arteries ingeneticallyhypertensive rats, even in the absence of any effect on blood pressure.Clin. Exp. Pharmacol. Physiol. 29, 931 – 934.Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.G., Ahn, B.W., Shaltiel,S., Stadtman, E.R., 1990. Determination of carbonyl content in oxidatively modi 󿬁 edproteins. Methods Enzymol. 186, 464 – 478.Li, Z., Iwai, M., Wu, L., Liu, H.W., Chen, R., Jinno, T., Suzuki, J., Tsuda, M., Gao, X.Y.,Okumura, M., Cui, T.X., Huriuchi, M., 2004. Fluvastatin enhances the inhibitoryeffects of a selective AT1receptor blocker, valsartan, on atherosclerosis. Hyperten-sion 44, 758 – 763.Maritim, A.C., Sanders, R.A., Watkins, B., 2003. Diabetes, oxidative stress andantioxidants: a review. J. Biochem. Mol. Toxicol. 17, 24 – 38.Matsuki,A.,Nozawa,T.,Igarashi,N.,Sobajima,M.,Ohon,T.,Suzuki,T.,Fujii,N.,Igawa,A.,Inoue, H., 2010. Fluvastatin attenuates diabetes-induced cardiac symphatheticneuropathy in association with a decrease in oxidative stress. Circ. J. 74, 468 – 475.McKenney, M.J., 2003. Potential nontraditional applications of statins. Ann. Pharmac-other. 37, 1063 – 1071.Mitani, H., Egashira, K., Ohashi, N., Yoshikawa, M., Niwa, S., Nonomura, K., Nakashiwa,A., Kimura, M., 2003. Preservation of endothelial function by the HMG-CoAreductase inhibitor 󿬂 uvastatin through its lipid-lowering independent antioxidantproperties in atherosclerotic rabbits. Pharmacology 68, 121 – 130.Morita, H., Saito, Y., Ohashi, N., Yoshikawa, M., Katoh, M., Ashida, T., Kurihara, H.,Nakamura, T., Kurabayashi, M., Nagai, R., 2005. Fluvastatin ameliorates thehyperhomocysteinemia-induced endothelial dysfunction: the antioxidative prop-erties of   󿬂 uvastatin. Circ. J. 69, 475 – 480.Nakashima, A., Ohtawa, M., Iwasaki, K., Wada, M., Kuroda, N., Nakashima, K., 2001a.Inhibitory effects of   󿬂 uvastain and its metabolites on the formation of severalreactive oxygen species. Life Sci. 69, 1381 – 1389.84  A. Cumao  ğ  lu et al. / European Journal of Pharmacology 654 (2011) 80 – 85
Related Documents
View more...
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!