Protective effects of flavonol isoquercitrin, against 6-hydroxy dopamine (6-OHDA) - induced toxicity in PC12 cells
© Magalingam et al.; licensee BioMed Central Ltd. 2014
Received: 17 August 2013
Accepted: 16 January 2014
Published: 21 January 2014
Free radicals-induced neurodegeneration is one of the many causes of Parkinson’s disease (PD). This study investigated the neuroprotective effects of flavonol isoquercitrin against toxicity induced by 6-hydroxy-dopamine (6-OHDA) in rat pheochromocytoma (PC12) cells.
PC12 cells were pretreated with different concentrations of isoquercitrin for 4, 8 and 12 hours and incubated with 6-OHDA for 24 hours to induce oxidative cell damage.
A significant cytoprotective activity was observed in isoquercitrin pre-treated cells in a dose-dependent manner. There was a significant increase (P < 0.01) in the antioxidant enzymes namely superoxide dismutase, catalase, glutathione peroxidase, and glutathione in isoquercitrin pretreated cells compared to cells incubated with 6-OHDA alone. Isoquercitrin significantly reduced (P < 0.01) lipid peroxidation in 6-OHDA treated cells. These results suggested that isoquercitrin protects PC 12 cells against 6-OHDA–induced oxidative stress.
The present study suggests the protective role of isoquercitrin on 6-hydroxydopamine-induced toxicity by virtue of its antioxidant potential. Isoquercitrin could be a potential therapeutic agent against neurodegeneration in Parkinson’s disease.
KeywordsAntioxidant Flavonoids 6-hydroxydopamine Parkinson’s disease Oxidative stress
Parkinson’s disease (PD) is a neurodegenerative disease involving the degeneration of dopaminergic neurons in the striatum. The selective loss of dopaminergic neurons in the substantia nigra is the primary neuropathology in PD[1–3]. One of the many causes of PD is the accumulation of free radicals and oxidative stress which leads to the selective neuronal loss[4–6]. Though great advances have been made in the development of novel drugs to treat this disease, the appropriate pharmacological agent for PD is still elusive[3, 7]. Alternative therapy in PD is aimed to effectively prevent the progression of neurodegeneration process hence improving the clinical presentation of PD including tremor, bradykinesia, rigor and hypokinesia. Free radical scavengers such as antioxidant agents may be helpful in prolonging the survival of dopaminergic neurons. Flavonoids are naturally occurring polyphenol compounds widely distributed throughout the plant kingdom[9, 10]. They exhibit a variety of biological activities, such as anti-oxidation, anti-inflammation, anti-bacteria, and anti-allergy[11–14]. Many of these flavonoids act as neuroprotective agents in many neurological disorders[4, 15].
Isoquercitrin, also known as quercetin 3-glucoside, is a glucose-bound derivative of quercetin, and is reported to have anti-inflammatory and antioxidant activities[5, 6]. Isoquercitrin is widely found in mangoes, rheum nobile, apples, onions and in many other fruits and vegetables[16, 17]. It is considered to be a “bio-quercetin” without the potential adverse effect of quercetin. Although several studies have demonstrated the antioxidant property of isoquercitrin[5, 6, 16, 17], but the neuroprotective potential of isoquercitrin is not well explored. Hence, the objectives of our study are to establish the neuroprotective role of isoquercitrin as well as to elucidate the antioxidant mechanisms of isoquercitrin in 6-OHDA-induced neurotoxicity in PC12 neuronal cells. PC12 cells are commonly used in the investigation of neurotherapeutics study for Parkinson’s disease. These cells are known to secrete dopamine neurotransmitter and contain high amounts of dopamine transporters. The cell line is derived from the rat pheochromocytoma and is frequently used as an in vitro model to study neuronal toxicities of drugs on central dopaminergic neurons[19, 20].
Dose response of 6-OHDA toxicity
Evidence of isoquercitrin in protecting 6-OHDA- induced oxidative stress
Superoxide dismutase (SOD)
Many lines of evidence have proven the oxidative stress due to imbalance in the free radical generation and endogenous antioxidant defense system may lead to the selective neuronal loss in Parkinson’s disease[21–23]. Hence, flavonoid polyphenols, particularly isoquercitrin can be an ideal candidate as neuroprotective agent to cease or delay the degeneration of dopaminergic cells[24, 25]. In our study, the neuroprotective role of isoquercitrin, a flavonoid glycoside was investigated using a 6-OHDA induced PC12 cellular model of Parkinson’s disease. In the cell viability assay, isoquercitrin pretreatment has demonstrated a remarkable increase of cell viability at 12 hours of incubation with 10 μM of isoquercitrin. The isoquercitrin pretreated prior to 6-OHDA exposure resulted in stimulation of antioxidant enzyme activity in the neuronal cells as the cells are more resilient in coping with upcoming oxidative stress[26, 27]. The antioxidant enzyme defense system functions in eliminating the free radical induced cellular damage during the defense against microorganisms, toxic chemicals and other conditions of cellular stress[20, 21, 28].
To further validate the neuroprotective role of isoquercitrin, the antioxidant enzyme status of isoquercitrin pretreated PC12 cells was assessed to confirm the antioxidant capability of this flavonol. The antioxidant enzymes which were studied are superoxide dismutase (SOD), catalase (CAT), glutathione and glutathione peroxidase (GPx). SOD occur in significantly higher amounts in the brain and these enzymes, which are of three types (Cu-Zn SOD, Mn-SOD and EC-SOD) readily catalyze the dismutation of the superoxide anion to oxygen molecules and hydrogen peroxide, a less toxic molecule[21, 29]. There was a significant increase in SOD in all the isoquercitrin pretreated cells in a dose dependent manner. This increase in SOD activity proved that there was a direct activation of SOD by isoquercitrin to catalyze the superoxide anions produced by 6-OHDA. Catalase, a tetrameric structure with four indistinguishable tetrahedrally arranged residues with a single ferri-protoporphyrin subunit is ubiquitously found in the liver, kidney and erythrocytes. Catalase accounts for detoxifying H2O2 molecules, whereby it is converted to oxygen and water molecule and this reaction through a reaction known as the catalytic reaction. The CAT activity in isoquercitrin pretreated 6-OHDA induced PC12 cells showed a statistically significant effect in all the treatment groups. The CAT activity in the 6-OHDA treated group was significantly reduced compared to control group. The increased CAT activity could be due to two mechanisms, (i) increased in hydrogen peroxide molecules by SOD triggered the CAT enzyme, (ii) isoquercitrin caused direct activation of CAT enzyme which catalyzed the toxic hydrogen peroxide to water and oxygen molecules[29, 32].
Glutathione peroxidase, which is largely found in the cytoplasm and mitochondria of eukaryotic cells is a vital antioxidant enzyme that catalyzes the reduction of hydroperoxides. In this study, the GPx/Glutathione activity was also increased by antioxidant treatment. Isoquercitrin probably interacted with GPx and glutathione to enhance their antioxidant activity in PC12 cells[21, 34]. Malondialdehyde (MDA) is a naturally occurring product of lipid peroxidation that will react with the thiobarbituric acid (TBA) and form MDA-TBA adducts. Malondialdehyde produced due to lipid peroxidation accumulated in the cells and cause cell damage. MDA increased in 6-OHDA treated cells and isoquercitrin effectively reduced the levels of MDA in the pre-treated cells[27–29, 32].
Free radicals generated by neurotoxin, 6-hydroxydopamine caused neuronal cell loss via DNA defects, lipid peroxidation and cytoskeletal disorganization. Moreover, studies also demonstrated that 6-OHDA induced neuronal loss was due to inhibition of the mitochondria respiratory chain complexes I and IV, oxidative phosphorylation uncoupling, mitochondrial membrane potential collapse[4, 22, 35, 36]. In this study, we have proved that 6-hydoxydopamine induced oxidative stress and cell death by decreasing the scavenging enzymes (SOD, catalase and GPx) in PC 12 cells. Isoquercitrin pretreatment caused a significant elevation in these scavenging enzyme levels and attenuate oxidative damage to the cells[22, 37, 38]. Isoquercitrin helped to maintain the levels of these antioxidant enzymes and suppress lipid peroxidation as well as protect the neuronal cell from undergoing cell death.
The results of this study confirmed that neurotoxin 6-hydroxy dopamine causes suppression of antioxidant enzyme levels in PC 12 cells, which could be a reason for the progressive neuronal death. Isoquercitrin’s role in protecting against 6-OHDA induced oxidative stress, suggests that this flavonol may serve as a potential neuroprotective agent against the underlying pathology associated with neurodegenerative diseases like Parkinson’s disease.
PC12 cells were purchased from ATCC (#CRL-1721.1 PC12 ADH, RattusNorvegicus). 6-hydroxydopamine, isoquercitrin, poly-L-Lysine, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide),3,4 – dihydroxy-L-phenylalanine (Levodopa) and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich (USA). Dulbecco’s modified eagle’s medium (DMEM), pen-strep, horse serum, and fetal bovine serum was purchased from GIBCO Inc. (USA). Kits for glutathione peroxidase, superoxide dismutase, catalase, thiobarbiturate assay, and glutathione were purchased from the Cayman Chemical Company (USA).
PC12 cells were grown in a humidified incubator with 5% CO2 at a temperature of 37°C in DMEM medium supplemented with 5% horse serum and 5% fetal bovine serum and pen-step (100 U/ml). The cells were cultured in poly-L-lysine coated T-75 culture flasks. The cells used in the experiments were taken between passage 2 and 8 as cells tend to get clumpy and difficult to isolate after passage 10. When the cells were 80% confluent, they were dislodged from the flask using a cell scraper. The dispersed cells were grown on poly-L-lysine coated 96-well microplate at a density of 1 × 105 cells/ml and allowed overnight incubation to facilitate cell adhesion to the substrate. The cells were treated for 4, 8 and 12 hours in the presence of isoquercitrin at three different concentrations, 10 μM, 50 μM and 100 μM. Subsequently, the pre-treated cells were induced using 6-OHDA for 24 hours and assayed for its antioxidant activities. Control cells were cultured in complete DMEM alone and positive control cells were treated only with 6-OHDA.
Determination of cell viability
The cytotoxicity effect of isoquercitrin pretreatment on 6-OHDA induced PC12 was determined using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. MTT, a yellow tetrazole, is reduced to insoluble purple formazan in the mitochondria of viable cells and appears purple. The insoluble purple formazan was dissolved using a solubilizing solvent and the colored solution was measured at 570 nm using a microplate-reader. Data on cell viability were expressed as percentage of the surviving control cell in the study.
Glutathione assay kit utilizes an optimized enzymatic recycling method that uses glutathione reductase for quantification of GSH. Rate of production of yellow colored 5-Thio-2-nitrobenzoic acid (TNB) is directly proportional to the recycling reaction and concentration of glutathione in the sample. Lipid peroxides, derived from polyunsaturated fatty acids, are unstable and decompose to form a complex series of compounds which include reactive carbonyl compounds, such as MDA. MDA-TBA adducts formed by the reaction of MDA and TBA under high temperature and acidic conditions is measured colorimetrically at 550 nm. Super oxide dismutase assay kit utilizes a tetrazolium salt for the detection of superoxide radicals (O2–) generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of an enzyme necessary to exhibit 50% dismutation of superoxide radical. Superoxide dismutase levels were determined from a standard curve and expressed as U/ml of protein. Glutathione peroxidase assay kit measures GPx activity indirectly by a coupled reaction with glutathione reductase. Oxidized glutathione, produced upon reduction of an organic hydroperoxide by GPx, is recycled to its reduced state by glutathione reductase and NADPH. The oxidation of NADPH to NADP + is accompanied by a decrease in absorbance at 340 nm. Glutathione peroxidase levels were determined from a standard curve and expressed as nmol/min/ml of protein. The Cayman chemical catalase kit utilizes the peroxidatic function of catalase for determination of enzyme activity. The assay is based on the reaction of catalase with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced is measured spectrophotometrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole as the chromogen.
Data were expressed as mean ± SEM. The results were analyzed using analysis of variance (ANOVA) using SPSS Inc. software (SPSS Statistics, V20.0.0). Differences between treatment groups were determined using the Bonferonni post-hoc test. A value of P < 0.05 was considered statistically significant.
- Covy JP, Giasson BI: Alpha-Synuclein, leucine-rich repeat kinase-2, and manganese in the pathogenesis of Parkinson disease. Neurotoxicology. 2011, 2011 (32): 622-629.View ArticleGoogle Scholar
- Garcia Ruiz PJ, Catalan MJ, Fernandez Carril JM: Initial motor symptoms of Parkinson disease. Neurologist. 2011, 17: 18-S20.View ArticleGoogle Scholar
- Ybot-Gorrin I, Vivancos-Matellano F, Chacón-Peña JR, Alonso-Navarro H, Jiménez-Jiménez FJ: Assessment of Parkinson disease: what do we need to show neuroprotection?. Neurologist. 2011, 17: S21-S29.PubMedView ArticleGoogle Scholar
- Glinka Y, Gassen M, Youdim MB: Mechanism of 6-hydroxydopamine neurotoxicity. J Neural Transm. 1997, 50: 55-66. 10.1007/978-3-7091-6842-4_7.View ArticleGoogle Scholar
- Rogerio AP, Kanashiro A, Fontanari C, da Silva EV, Lucisano-Valim YM, Soares EG, Faccioli LH: Anti-inflammatory activity of quercetin and isoquercitrin in experimental murine allergic asthma. Inflamm Res. 2007, 56: 402-408. 10.1007/s00011-007-7005-6.PubMedView ArticleGoogle Scholar
- Jung SH, Kim BJ, Lee EH, Osborne NN: Isoquercitrin is the most effective antioxidant in the plant Thujaorientalis and able to counteract oxidative-induced damage to a transformed cell line (RGC-5 cells). Neurochem Int. 2010, 57: 713-721. 10.1016/j.neuint.2010.08.005.PubMedView ArticleGoogle Scholar
- Chen S, Le W: Neuroprotective therapy in Parkinson disease. Am J Ther. 2006, 13: 450-457.View ArticleGoogle Scholar
- Shimohama S, Sawada H, Kitamura Y, Taniguchi T: Disease model: Parkinson’s disease. Trends Mol Med. 2003, 9: 360-365. 10.1016/S1471-4914(03)00117-5.PubMedView ArticleGoogle Scholar
- Hertog MGL, Hollman PCH, van de Putte B: Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. J Agric Food Chem. 1992, 40: 2379-2383. 10.1021/jf00024a011.View ArticleGoogle Scholar
- Mu X, He GY, Cheng Y, Li X, Xu B, Du G: Baicalein exerts neuroprotective effects in 6-hydroxydopamine-induced experimental parkinsonism in vivo and in vitro. Pharmacol Biochem Behav. 2009, 92: 642-648. 10.1016/j.pbb.2009.03.008.PubMedView ArticleGoogle Scholar
- Pietta PG: Flavonoids as antioxidants. J Nat Prod. 2000, 63: 1035-1042. 10.1021/np9904509.PubMedView ArticleGoogle Scholar
- García-Lafuente A, Guillamón E, Villares A, Rostagno MA, Martínez JA: Flavonoids as anti-inflammatory agents: implications in cancer and cardiovascular disease. Inflamm Res. 2009, 58: 537-552. 10.1007/s00011-009-0037-3.PubMedView ArticleGoogle Scholar
- Alcaráz LE, Blanco SE, Puig ON, Tomás F, Ferretti FH: Antibacterial activity of flavonoids against methicillin-resistant Staphylococcus aureus strains. J Theor Biol. 2000, 205: 231-240. 10.1006/jtbi.2000.2062.PubMedView ArticleGoogle Scholar
- Cheong H, Ryu SY, Oak MH, Cheon SH, Yoo GS, Kim KM: Studies of structure activity relationship of flavonoids for the anti-allergic actions. Arch Pharm Res. 1998, 21: 478-480. 10.1007/BF02974647.PubMedView ArticleGoogle Scholar
- Agati G, Azzarello E, Pollastri S, Tattini M: Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 2012, 196: 67-76.PubMedView ArticleGoogle Scholar
- Cornard JP, Boudet AC, Merlin JC: Theoretical investigation of the molecular structure of the isoquercitrin molecule. J Mol Struct. 1999, 508: 37-49. 10.1016/S0022-2860(99)00004-6.View ArticleGoogle Scholar
- Lee S, Park HS, Notsu Y, Ban HS, Kim YP, Ishihara K, Hirasawa N, Jung SH, Lee YS, Lim SS, Park EH, Shin KH, Seyama T, Hong J, Ohuchi K: Effects of hyperin, isoquercitrin and quercetin on lipopolysaccharide induced nitrite production in rat peritoneal macrophages. Phytother Res. 2008, 22: 1552-1556. 10.1002/ptr.2529.PubMedView ArticleGoogle Scholar
- Gasparotto A, Gasparotto FM, Boffo MA, Lourenço EL, Stefanello ME, Salvador MJ, da Silva-Santos JE, Marques MC, Kassuya CA: Diuretic and potassium-sparing effect of isoquercitrin—An active flavonoid of Tropaeolummajus L. J Ethnopharmacol. 2011, 134: 210-215. 10.1016/j.jep.2010.12.009.PubMedView ArticleGoogle Scholar
- Jenner P: Oxidative damage in neurodegenerative disease. Lancet. 1994, 344: 796-798. 10.1016/S0140-6736(94)92347-7.PubMedView ArticleGoogle Scholar
- Walkinshaw G, Waters CM: Neurotoxin-induced cell death in neuronal PC12 cells is mediated by induction of apoptosis. Neuroscience. 1994, 63: 975-987. 10.1016/0306-4522(94)90566-5.PubMedView ArticleGoogle Scholar
- Datta K, Sinha S, Chattopadhyay P: Reactive oxygen species in health and disease. Natl Med J India. 2000, 13 (6): 304-310.PubMedGoogle Scholar
- Dauer W, Przedborski S: Parkinson’s disease: mechanisms and models. Neuron. 2003, 39: 889-909. 10.1016/S0896-6273(03)00568-3.PubMedView ArticleGoogle Scholar
- Gilgun-Sherki Y, Melamed E, Offen D: Oxidative stress induced - neurodegenerative diseases: the need for antioxidants that penetrate the blood brain barrier. Neuropharmacol. 2001, 40: 959-975. 10.1016/S0028-3908(01)00019-3.View ArticleGoogle Scholar
- Vauzour D, Vafeiadou K, Rodriguez-Mateos A, Rendeiro C, Spencer JP: The neuroprotective potential of flavonoids: a multiplicity of effects. Genes Nutr. 2008, 3: 115-126. 10.1007/s12263-008-0091-4.PubMedPubMed CentralView ArticleGoogle Scholar
- Chanh PH, Ifansyah N, Chahine R, Mounayar-Chalfoun A, Gleye J, Moulis C: Comparative effects of total flavonoids extracted from Ribesnigrum leaves, rutin and isoquercitrin on biosynthesis and release of prostaglandins in the ex vivo rabbit heart. Prostaglandins Leukot Med. 1986, 22: 295-300. 10.1016/0262-1746(86)90140-X.PubMedView ArticleGoogle Scholar
- Lobo V, Patil A, Phatak A, Chandra N: Free radicals, antioxidants and functional foods: Impact on human health. Pharma Rev. 2010, 4: 118-126. 10.4103/0973-7847.70902.View ArticleGoogle Scholar
- Wei Z, Bai OJ, Richardson S, Mousseau DD, Li XM: Olanzapine protects PC12 cells from oxidative stress induced by hydrogen peroxide. J Neurosci Res. 2003, 73: 364-368. 10.1002/jnr.10668.PubMedView ArticleGoogle Scholar
- Allen RG, Tresini M: Oxidative stress and gene regulation. Free Radic Biol Med. 2000, 28: 463-499. 10.1016/S0891-5849(99)00242-7.PubMedView ArticleGoogle Scholar
- Reiter RJ: Oxidative damage in the central nervous system: protection by melatonin. Prog Neurobiol. 1998, 56: 359-384. 10.1016/S0301-0082(98)00052-5.PubMedView ArticleGoogle Scholar
- Yoshimoto M, Sakamoto H, Yoshimoto N, Kuboi R, Nakao K: Stabilization of quaternary structure and activity of bovine liver catalase through encapsulation in liposomes. Enzyme Microb Technol. 2007, 41 (6): 849-858.View ArticleGoogle Scholar
- Singh R, Wiseman B, Deemagarn T, Jha V, Switala J, Loewen PC: Comparative study of catalase-peroxidases (KatGs). Arch Biochem Biophys. 2008, 471: 207-214. 10.1016/j.abb.2007.12.008.PubMedView ArticleGoogle Scholar
- Nehlig A: The neuroprotective effects of cocoa flavanol and its influence on cognitive performance. Br J Clin Pharmacol. 2013, 75: 716-727.PubMedPubMed CentralGoogle Scholar
- José MM, Cristina PM, Ignacio ND: Antioxidant enzymes and human diseases. Clin Biochem. 1999, 32: 595-603. 10.1016/S0009-9120(99)00075-2.View ArticleGoogle Scholar
- Blum D, Torch S, Lambeng N, Nissou M, Alim-Louis B, Sadoul R, Verna J: Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s disease. Prog Neurobiol. 2001, 65: 135-172. 10.1016/S0301-0082(01)00003-X.PubMedView ArticleGoogle Scholar
- Niedernhofer LJ, Daniels JS, Rouzer CA, Greene RE, Marnett LJ: Malondialdehyde, a product of lipid peroxidation, is mutagenic in human cells. J Biol Chem. 2003, 278: 31426-31433. 10.1074/jbc.M212549200.PubMedView ArticleGoogle Scholar
- Qu CP, Xu ZR, Liu GJ, Liu C, Li Y, Wei ZG, Liu GF: Differential expression of copper-zinc superoxide dismutase gene of Polygonumsibiricum leaves, stems and underground stems, subjected to high-salt stress. Int J Mol Sci. 2010, 11: 5234-5245. 10.3390/ijms11125234.PubMedPubMed CentralView ArticleGoogle Scholar
- Haleagrahara N, Siew CJ, Kumar P: Effect of quercetin and desferrioxamine on 6-hydroxydopamine (6-OHDA) induced neurotoxicity in striatum of rats. J Toxicol Sci. 2013, 38: 25-33. 10.2131/jts.38.25.PubMedView ArticleGoogle Scholar
- Yamada K, Umegaki H, Iguchi A, Kameyama T, Nabeshima T: Possible involvement of catalase in the protective effects of interleukin-6 against 6-hydroxydopamine toxicity in PC12 cells. Brain Res Bull. 1997, 46: 573-577.View ArticleGoogle Scholar
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