Mangiferin Ameliorates 6-Hydroxydopamine- Induced
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Pharmacological Reports Copyright © 2012 2012, 64, 848856 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences Mangiferinameliorates6-hydroxydopamine- inducedcytotoxicityandoxidativestressin ketaminemodelofschizophrenia VietlaS.Rao1,AnaC.Carvalho1,MariaTeresaS.Trevisan2, GeanneM.Andrade1,HelioV.NobreJúnior1,ManoelO.Moraes1, HemersonI.MagalhãesH.Iury1,TalitaC.Morais1,FlaviaA.Santos1 1 DepartmentofPhysiologyandPharmacology,FederalUniversityofCeará,CelNunesdeMelo-1127, CaixaPostal-3157,60430-270Fortaleza,CE,Brazil 2 DepartmentofOrganicandInorganicChemistry,FederalUniversityofCeara,Fortaleza,CE,Brazil Correspondence: VietlaS.Rao,e-mail:[email protected];[email protected] Abstract: Background: Accumulating evidence indicates that mangiferin (MGF), a natural xanthone, by virtue of its antioxidant and anti- inflammatory properties is neuroprotective. Here we sought to verify the cytoprotective role of MGF on cultured rat primary mesen- cephalic cells exposed to 6-hydroxydopamine (6-OHDA) in vitro, and the MGFs anti-inflammatory potential in mouse model of ketamine-inducedschizophrenia invivo. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay was performed to measure cell viability in mesen- cephalic cell cultures exposed to neurotoxin (6-OHDA, 40 µM). Schizophrenia was induced in mice by ketamine (50 mg/kg, ip, twice a day, for 7 days). The treatment effects of MGF (50 mg/kg, po, for 7 days) were verified on locomotor behavioral changes in open-field test, and on the oxidant stress-related increase in lipid-peroxidation (malondialdehyde) and interleukin-6 (IL-6) levels in brain tissues. Results: MGF (10–100 µM) produced no per se effect on cell viability as measured by MTT assay, but significantly prevented the 6-OHDA-induced cell death in a concentration-dependent manner. Acridine orange/ethidium bromide (AO/EtBr) staining con- firmed the absence of 6-OHDA-induced morphological changes characteristic of apoptosis/necrosis. In open-field test, ketamine- induced impaired locomotor activity and behavioral changes such as grooming and stereotyped but not rearing were effectively ame- liorated by MGF pretreatment. Also, ketamine-associated increase in brain tissue levels of IL-6 and MDAwere significantly lowered inMGF-pretreatedmice. Conclusion: Mangiferin has a neurocytoprotective role related, at least in part, to an antioxidant and anti-inflammatory mechanism, whichcouldbeexploredformoreeffectivetherapiesofschizophreniaandotherneurodegenerativediseases. Keywords: mangiferin,cytoprotection,mesencephaliccellculture,6-OHDA,ketamine,oxidativestress Abbreviations: AO/EtBr – acridine orange/ethidium bromide, Introduction DMSO – dimethyl sulfoxide, FBS – fetal bovine serum, IL-6 – interleukin-6, MDA – malondialdehyde, MEM – minimum essen- tial medium, MGF – mangiferin, MTT – 3-(4,5-dimethylthiazol- The brain is susceptible to oxidative damage due to its 2-yl)-2,5-diphenyltetrazolium bromide, MW – molecular weight, high lipid content and oxygen consumption. Neurode- 6-OHDA – 6-hydroxydopamine, PBS – phosphate buffer solu- tion, PI – propidium iodide, SOD – superoxide dismutase, generative diseases like Alzheimer, Parkinson, bipolar TBARS – thiobarbituric acid reactive substances disorder, epilepsy and schizophrenia are problems of 848 Pharmacological Reports, 2012, 64, 848856 Mangiferinpreventscytotoxicityandoxidativestress Vietla S. Rao et al. vast clinical importance. Free iron can react with eral traditional medicinal plants [16] has been shown H2O2 via the Fenton reaction, a primary cause of lipid to produce multiple pharmacological effects that in- peroxidation, and may be of particular importance for clude antioxidant and anti-inflammatory [2, 4, 32], these disorders [1]. Schizophrenia is a severe brain immunomodulatory [20, 26], and neuroprotective [9, disorder characterized by hallucinations, disordered 33] activities. There have been reports that MGF de- thinking and deficiencies in cognition and it has been creases glutamate-induced neurotoxicity and memory suggested that environmental factors combined with loss induced by scopolamine [23, 27, 39]. We previ- a genetic predisposition result in the manifestation of ously demonstrated that MGF affords gastroprotec- this disease [25]. Despite its high incidence in general tion against absolute ethanol or indomethacin-induced population, lack of adequate understanding on the pa- gastric ulceration through an antioxidant mechanism thophysiology of schizophrenia makes the treatment [10]. Impaired antioxidant defenses are suggested to options difficult. Extensive evidence indicates that the participate in the pathophysiology of schizophrenia glutamatergic N-methyl-D-aspartate (NMDA) neuro- and other neurodegenerative conditions. Altered su- transmission is highly disrupted resulting in severe peroxide dismutase (SOD) and increased lipid peroxi- cognitive dysfunction and this pathway seems to be dation, measured by the thiobarbituric acid reactive highly deregulated in experimental models of schizo- substances (TBARS/MDA), are increased in schizo- phrenia that simulate the symptoms of schizophrenia phrenic patients [17]. Besides, MGF has the iron com- [6, 11, 28, 35, 43]. Some other studies indicated brain plexing ability and can protect against Fe2+-citrate in- histaminergic neuronal system involvement in the duced lipid peroxidation [3]. The neurotoxin 6-OHDA pathogenesis of schizophrenia based on the protection is well-known to cause neuronal death by a free offered by histamine 3-receptor antagonists/inverse radical-mediated mechanism as well as by inhibiting agonists in the established animal models of schizo- the mitochondrial complex I and IV [18]. As MGF phrenia[29,30]. has a wide spectrum of pharmacological activity in- Drugs used to treat schizophrenia include the older cluding antioxidant and anti-inflammatory activity generation medications such as chlorpromazine, [26, 32] and accumulating evidence suggests that haloperidol, perphenazine and fluphenazine, which MGF is neuroprotective, the current study is aimed are known to cause extrapyramidal side effects. On to examine the cytoprotective role of MGF against the other side, the newer generation drugs including 6-OHDA-induced neurotoxicity in cultured rat pri- the atypical antipsychotics like clozapine, risperidone, mary mesencephalic cells and its beneficial effects in olanzapine, quetiapine, and ziprasidone have little or the mouse model of ketamine-induced schizhophrenia. no side effects. Due to the low acceptability of these synthetics, the search has been shifted to the natural products having antioxidant and anti-inflammatory properties, which can afford better health promoting effects,comparedtosynthetics. MaterialsandMethods Mangiferin (MGF), a naturally occurring gluco- sylxanthone (Fig. 1) commonly encountered in sev- Animals Male Swiss mice (7–8 weeks old, 20–25 g) and adult male/female Wistar rats (4–5 months old) obtained from the Central Animal House of Federal University of Ceará, were used. They were housed in environ- mentally controlled conditions (23 ± 2°C, 12-h light- dark cycle), with free access to standard diet (Purina Chow) and water ad libitum. Rats were mated to ob- tain rats on pregnancy and to collect embryonic mes- encephalic cells, for in vitro studies. Mice were used for in vivo experiments aimed to study the effects of Fig. 1. Chemical structure of mangiferin (1,3,6,7-tetrahydroxy- drugs in ketamine model of schizophrenia. The xanthoneC2-D-glucoside) experimental protocols were approved by the Animal Pharmacological Reports, 2012, 64, 848856 849 Care and Use Committee of the Federal University of ProtectiveeffectofMGFagainstcelldeath Ceara in accordance with the ethical guidelines of the inducedby6-OHDA International Association for the Study of Pain Mesencephalic cell culture plates having a cell den- (IASP). 4 sity of 5 × 10 cells per well were pre-incubated with MGF in concentrations of 10, 30, and 100 µM for 15 min, prior to addition of 6-OHDA (40 µM). Each Chemicals concentration of MGF was tested in six replicates and repeated three times in separate experiments. Cell Mangiferin (MGF) was extracted and isolated from viability after exposure to 6-OHDA or MGF (10, 30, the bark of Mangifera indica L. (Anacardiaceae) as and 100 µM) alone, or in combination was deter- per procedures reported earlier [10]. The isolated mined in culture plates by MTT as described by Mos- MGF was approximately of 95% purity [4] having the mann [36]. This method measures mitochondrial molecular weight (MW) 422.5 and melting point activity based on the reductive cleavage of yellow (m.p.) 271°C. Minimum essential medium (MEM), tetrazolium salt to a purple formazan compound by trypsin, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di- the dehydrogenase activity of intact mitochondria. phenyltetrazolium bromide], cytochalasin-B, propid- Briefly, cells were washed once with PBS before the ium iodide (PI), ethidium bromide (EtBr) and fetal addition of 0.1 ml of serum free medium containing bovine serum (FBS) were purchased from Sigma MTT (1 mg/ml) to each well. After incubation for 3 h, Chemical Co. (St. Louis, MO, USA). Acridine orange the supernatant was removed and the formazan prod- uct obtained was dissolved in 1 ml dimethyl sulfoxide (AO) was purchased from BDH Chemicals Ltd., (DMSO) with stirring for 15 min on a microtiter plate Poole, England and ketamine from Sigma-Aldrich shaker and the absorbance was read at 550 nm. The (MO, USA). MGF was dissolved in 0.02% DMSO