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Antioxidant propolis attenuates kainate-induced neurotoxicity via adenosine A1 receptor modulation in the rat
Neuroscience Letters 355 (2004) 231–235 www.elsevier.com/locate/neulet
Antioxidant propolis attenuates kainate-induced neurotoxicity via adenosine A1 receptor modulation in the rat Yong-Soo Kwona,1, Dae-Hun Parkb,1, Eun-Joo Shina, Myung Sang Kwonb, Kwang Ho Koc, Won-Ki Kimd, Jin Hyeong Jhooe, Wang-Kee Jhooa, Myung-Bok Wieb, Bae Dong Jungb, Hyoung-Chun Kima,* a
Neurotoxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, South Korea b Department of Veterinary Medicine, Kangwon National University, Chunchon 200-701, South Korea c Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, South Korea d Department of Pharmacology, Ewha Institute of Neuroscience, Ewha Women’s University Medical School, Seoul, South Korea e Clinical Research Institute, Seoul National University Hospital, Seoul, South Korea Received 6 June 2003; received in revised form 30 September 2003; accepted 28 October 2003
Abstract We examined the effects of the antioxidant propolis on seizures induced by kainic acid (KA). Sprague– Dawley rats received propolis (75 and 150 mg/kg, p.o.) five times at 12 h intervals. KA (10 mg/kg, i.p.) was injected 1 h after the last propolis treatment. Pretreatment with propolis significantly attenuated KA-induced seizures and KA-induced increases in hippocampal AP-1 DNA binding activity in a dosedependent manner. KA induced increases in the levels of malondialdehyde and protein carbonyl, and a decrease in the ratio of GSH/GSSG. These oxidative stresses and neuronal degenerations were significantly attenuated by pretreatment with propolis. The neuroprotective effects of propolis appeared to be counteracted by adenosine receptor antagonists [A1 antagonist, 8-cyclopentyl-1,3-dimethylxanthine (25 or 50 mg/kg); A2A antagonist, 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (0.5 or 1 mg/kg); and A2B antagonist, alloxazine (1.5 or 3.0 mg/kg)]. However, this counteraction was most pronounced in the presence of the A1 antagonist. Our results suggest that the protective effect of propolis against KA-induced neurotoxic oxidative damage is, at least in part, via adenosine A1 receptor modulation. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Propolis; Kainate; Oxidative stress; Ratio of GSH/GSSG; AP-1 DNA binding activity; Hippocampus; Adenosine receptors
Accumulating evidence indicates that oxidative stress may contribute to the development of the seizures induced by kainic acid (KA) [1,4,6,8 –10]. KA has been used as a model for both temporal lobe epilepsy and neurodegenerative disorders [19]. Honeybee propolis has been widely used as a folk medicine. It has been shown to have broad biological activities, which are principally attributed to the presence of flavonoids (major component; rutin, quercetin, galangin, etc.) [7] and caffeic acid phenethyl ester (CAPE) [13]. The prevailing opinion is that the broad biological activities of flavonoids and CAPE are related, in part, to their antioxidant actions [7]. A careful review of the literature indicates that * Corresponding author. Tel.: þ 82-33-250-6917; fax: þ82-33-255-7865. E-mail address: [email protected] (H.C. Kim). 1 Y.-S. Kwon and D.-H. Park contributed equally to this work.
the effects of propolis on neurodegenerative disorders have not yet been demonstrated. The purine nucleotide adenosine might act as a neuromodulator of the CNS and an endogenous neuroprotectant [2,18]. Adenosine exerts its effects on the neuronal activity via four G protein-coupled receptors, A1, A2A, A2B and A3 [2]. While adenosine A1 and A2B receptors are widely distributed in the brain, the A2A receptor distribution is restricted to the dopamine-innervated regions [2]. A3 receptors are expressed at low levels in the brain [2]. It has been demonstrated that adenosine A1 receptors are reduced in the temporal lobe of epileptic [5] and experimental epileptic brains [3]. Furthermore, the adenosine release evoked by KA may be involved in the production of free radicals [1]. In the present study, we examined the role of adenosine receptors in the propolis-mediated pharmacological action in response to KA insult.
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.10.075
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All animals were treated in strict accordance with the NIH Guide for the Humane Care and Use of Laboratory Animals. Male Sprague – Dawley rats (Bio Genomics, Charles River Technology, Gapyung-Gun, Gyeonggi-Do, South Korea) weighing about 250 g were maintained on a 12:12 light/dark cycle and fed ad libitum. They were adapted to these conditions for 2 weeks before KA (10 mg/kg, i.p.) administration. All rats were drug and seizure naive before the experiment. Ethanol-extracted propolis (75 or 150 mg/kg; Sigma, St. Louis, MO) suspended in vehicle (2% Tween 80 plus 2% sodium lauryl sulfate in the saline solution) was administered orally five times at 12 h intervals. Control rats received the same volume of vehicle. One hour after the last administration of propolis, KA (10 mg/kg) was administered intraperitoneally. Thirty minutes before KA, some rats received one of the following adenosine receptor antagonists: the A1 antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT, 25 or 50 mg/kg, i.p.; RBI, Natick, MA); the A2A antagonist, 1,3,7-trimethyl8-(3-chlorostyryl)xanthine (CSC, 0.5 or 1.0 mg/kg, i.p.; Sigma, St. Louis, MO); or the A2B antagonist, alloxazine (ALL, 1.5 or 3.0 mg/kg, i.p.; RBI, Natick, MA). Animals were sacrificed 4 and 48 h after KA administration. Brains were rapidly removed, and hippocampi were dissected and then stored at 2 80 8C. Using an automated video tracking system (Noldus Information Technology, Wageningen, The Netherlands) [10], seizure activity was rated according to the scale devised by Racine [16] during a 4 h period following KA challenge. We examined hippocampal AP-1 DNA binding activity following KA to confirm possible convulsant or anticonvulsant effects [15]. The AP-1 oligomer was purchased from Stratagene (La Jolla, CA). Double-stranded oligomer (25 ng) was labeled with [g-32P]ATP using 10 units of T4 polynucleotide kinase (U.S. Biochemicals, Cleveland, OH). The AP-1 oligomer (22-mer; 50 -CTAGTGATGAGTCAGCCGGATC-30 ) contained the consensus sequence (50 -TGAGTCA-30 ). To characterize AP-1 DNA binding activity, nuclear extracts were preincubated with a 100-fold excess of unlabeled AP-1 oligomer. A 100-fold excess of an unrelated oligomer (NFJ; 50 -TAGGGGTGGAGTCTCCATG-30 ; Research Genetics, Huntsville, AL), normal rabbit serum (NRS; Dako Corporation, Carpinteria, CA) or bovine serum albumin (BSA; Sigma, St. Louis, MO) were used as controls. The addition of cold AP-1 oligomer showed that the AP-1 DNA binding activity was specific. The addition of NRS or NFJ did not affect DNA binding. BSA also did not bind to the DNA probe. The amount of lipid peroxidation was determined [10] by measuring the accumulation of thiobarbituric acid-reactive substances in homogenates of hippocampus, and was expressed as the malondialdehyde (MDA) content. The extent of protein oxidation in the hippocampus was assessed by determining the quantity of protein carbonyl groups, which was measured spectrophotometrically using the 2,4dinitrophenylhydrazine (DNPH) labeling procedure as
described by Oliver et al. [14]. Protein was measured using a BCA protein assay reagent (Pierce, Rockford, IL). GSH and GSSG from dissected hippocampal tissues were measured immediately using a minor modification [10] of the method described by Reed et al. [17]. Cresyl violet stain was used to examine the degeneration of neurons [10]. The significance of the differences in the ratio of convulsing was assessed by chi-square test. Significant differences in seizures were analyzed using the nonparametric Wilcoxon signed rank test. The differences in the levels of MDA, protein carbonyl, GSH, GSSG and neuronal density were compared by Student’s t-test for paired data. Animals pretreated with saline (vehicle) and then given 10 mg/kg of KA showed robust behavioral seizures (3.4 ^ 0.6) that lasted 4 –5 h. Thirty-one of 36 animals (86.1%) exhibited seizures. Pretreatment with propolis significantly protected against the KA-induced convulsive behaviors in a dose-related manner. Although the A2A receptor antagonist CSC and the A2B receptor antagonist ALL both appeared to counteract the anticonvulsant effects of propolis slightly, the effect of the A1 receptor antagonist CPT was the most significant (Fig. 1A). There was a low level of AP-1 DNA binding activity in the absence of KA. A significant increase (P , 0:01) in AP1 DNA binding activity occurred 4 h after KA administration, and was still evident after 48 h. In contrast, the pretreatment with propolis caused significant reductions in AP-1 DNA binding activity (for propolis at 75 mg/kg, P , 0:05; for propolis at 150 mg/kg, P , 0:01) in a doserelated fashion. Consistently at each time-point, CPT most significantly reversed the propolis-induced reduction in AP1 DNA binding activity, although CSC and ALL both altered propolis-modulated AP-1 DNA binding activity (Fig. 1B,C). KA produced significant increases in the levels of MDA [unit ¼ nmol/g wet tissue; vehicle: 121.2 ^ 13.3 (n ¼ 6) vs. KA: 172.6 ^ 12.1 (n ¼ 6), P , 0:01] and carbonyl [unit ¼ nmol/mg protein; vehicle: 4.80 ^ 0.52 (n ¼ 6) vs. KA: 7.88 ^ 0.38 (n ¼ 6), P , 0:01] which were evident at 4 h after KA administration. The increases in lipid peroxidation and protein oxidation were significantly decreased in a dose-related fashion by pretreatment with propolis [MDA; KA: 172.6 ^ 12.1 (n ¼ 6) vs. propolis 75 or 150 mg/kg plus KA: 151.9 ^ 9.8 (n ¼ 6) or 133.1 ^ 6.8 (n ¼ 6), P , 0:05 or P , 0:01; carbonyl; KA: 7.88 ^ 0.38 (n ¼ 6) vs. propolis 75 or 150 mg/kg plus KA: 6.95 ^ 0.62 (n ¼ 6) or 5.33 ^ 0.28 (n ¼ 6), P , 0:05 or P , 0:01]. CPT dose-dependently reversed the significant propolismediated reductions in the levels of MDA [CPT 25 mg þ propolis 150 mg/kg plus KA: 146.3 ^ 14.1 (n ¼ 6); propolis 150 mg/kg plus KA: 133.1 ^ 6.8 (n ¼ 6) vs. CPT 50 mg þ propolis 150 mg/kg plus KA: 174.9 ^ 12.6 (n ¼ 6), P , 0:05] and carbonyl [CPT 25 mg þ propolis 150 mg/kg plus KA: 6.25 ^ 0.32 (n ¼ 6); propolis 150 mg/kg plus KA: 5.33 ^ 0.28 (n ¼ 6) vs. CPT 50 mg þ propolis 150 mg/kg plus KA: 7.80 ^ 0.43 (n ¼ 6),
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P , 0:05]. However, CSC [MDA; propolis 150 mg/kg plus KA: 133.1 ^ 6.8 (n ¼ 6) vs. CSC 0.5 or 1.0 mg/kg þ propolis 150 mg/kg plus KA: 147.2 ^ 10.4 (n ¼ 6) or 150.8 ^ 10.6 (n ¼ 6); carbonyl; 150 mg/kg plus KA: 5.33 ^ 0.28 (n ¼ 6) vs. CSC 0.5 or 1.0 mg/kg þ propolis 150 mg/kg plus KA: 6.42 ^ 0.62 (n ¼ 6) or 6.85 ^ 0.57 (n ¼ 6)], but not ALL, appeared to affect the significant decreases in these oxidative indexes caused by propolis. The KA-induced increase in the MDA and carbonyl value returned to near control (vehicle) levels by 48 h after KA treatment. No significant differences were observed in the ratio of GSH [unit ¼ nmol/g wet tissue; vehicle: 1.48 ^ 0.12 (4 h point) or 1.55 ^ 0.11 (2 day point)]/GSSG [unit ¼ nmol/g wet tissue; vehicle: 0.030 ^ 0.003 (4 h) or 0.029 ^ 0.004 (2 days)] in the absence of KA. After KA treatment, the GSH/GSSG ratio in the hippocampus time-dependently decreased (P , 0:01) [4 h after KA, GSH: 1.05 ^ 0.08 (n ¼ 6), GSSG: 0.044 ^ 0.003 (n ¼ 6); 2 days after KA, GSH: 0.81 ^ 0.14 (n ¼ 6), GSSG: 0.057 ^ 0.003 (n ¼ 6)]. However, propolis dose-dependently prevented in the GSH/GSSG ratio in the hippocampus (4 h point, propolis 150 mg/kg plus KA vs. KA, P , 0:05; 2 day point, propolis 75 or 150 mg/kg plus KA vs. KA, P , 0:05 or P , 0:01) after KA administration [4 h after KA, GSH: 1.25 ^ 0.10 (plus propolis 75 mg/kg) or 1.41 ^ 0.10 (plus propolis 150 mg/kg); 2 days after KA, GSH: 1.11 ^ 0.11 (plus propolis 75 mg/kg) or 1.24 ^ 0.13 (plus propolis 150 mg/kg), GSSG: 0.048 ^ 0.004 (plus propolis 75 mg/kg) or 0.042 ^ 0.003 (plus propolis 150 mg/kg)]. CPT was the most efficacious in counteracting the significant reductions in the GSH/GSSG ratios mediated by propolis, although both CSC and ALL also appeared to alter the propolis-mediated GSH status (Fig. 2). The neuronal layers of the hippocampus in the animal without KA were clearly visible with cresyl violet staining. At 2 days after KA injection, significant neuronal losses were observed in the CA1 and CA3 regions; these losses were significantly blocked by pretreatment with propolis at Fig. 1. Effects of propolis with or without adenosine receptor antagonists on kainate-induced seizures (A) and on increases in AP-1 DNA binding activity (B,C). §Number of convulsions (the number of animals that convulsed/total number of animals receiving each treatment). Hippocampal nuclear extracts were prepared at 4 h (B) and at 2 days (C) after KA treatment. The relative intensities of the bands were quantified by laser densitometry [8]. Veh, vehicle; P 75 or 150, propolis 75 or 150 mg/kg; CPT 25 or 50, 8-cyclopentyl-1,3-dimethylxanthine 25 or 50 mg/kg, i.p.; CSC 0.5 or 1.0, 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine 0.5 or 1.0 mg/kg, i.p.; ALL 1.5 or 3.0, alloxazine 1.5 or 3.0 mg/kg, i.p. Animal numbers used in each group for seizure score and convulsing are in parentheses. Four animals in each group for AP-1 DNA binding activity were used. Each value is the mean ^ SEM. *P , 0:002 vs. Veh þ KA, **P , 0:001 vs. Veh þ KA, #P , 0:02 vs. P150 þ KA, ##P , 0:001 vs. P150 þ KA (x2test for convulsing). aP , 0:01 vs. Veh þ Veh, bP , 0:05 vs. Veh þ P150, c P , 0:01 vs. Veh þ P150, dP , 0:05 vs. Veh þ KA, eP , 0:01 vs. Veh þ KA, fP , 0:05 vs. P150 þ KA, gP , 0:01 vs. P50 þ KA (seizure scores or AP-1 DNA binding activity were analyzed using the nonparametric Wilcoxon signed rank test or Student’s t-test for paired data).
Fig. 2. Effects of propolis with or without adenosine receptor antagonists on the kainate-induced alterations in the ratio of GSH/GSSG. Each value is the mean ^ SEM of six animals. aP , 0:01 vs. Veh þ Veh, bP , 0:05 vs. Veh þ KA, cP , 0:01 vs. Veh þ KA, d P , 0:05 vs. P150 þ KA, e P , 0:01 vs. P150 þ KA (Student’s t-test for paired data).
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Fig. 3. Photomicrographs of cresyl violet-stained sections 2 days after KA treatment. Vehicle- or propolis (150 mg/kg)-treated rat (A) disclosed that neuronal layers of the hippocampi were apparently visible after cresyl violet staining. KA-induced neuronal damage was very severe; areas containing coagulation necrosis in the CA1 and CA3 regions did not stain (B). Pretreatment with propolis significantly prevented neuronal losses (C). This neuroprotection was counteracted in the presence of CPT at 50 mg/kg (D), CSC at 1.0 mg/kg (E) or ALL at 3.0 mg/kg (F). Scale bar, 500 mm. Surviving neurons in the stratum pyramidale were calculated using an image analysis system [10]. The number of animals used in each group is in parentheses. Each value is the mean ^ SEM. aP , 0:02 vs. Veh þ P150, b P , 0:01 vs. Veh þ P150, cP , 0:05 vs. Veh þ KA, dP , 0:01 vs. Veh þ KA, eP , 0:05 vs. P150 þ KA, fP , 0:01 vs. P150 þ KA (Student’s t-test for paired data).
150 mg/kg. CPT reversed the neuroprotective effects of propolis most significantly, but CSC and ALL both influenced the pharmacological action of propolis (Fig. 3). The present study clearly demonstrated that the antioxidant propolis is an effective neuroprotective agent against KA, and acts, at least in part, via adenosine A1 receptor modulation. It has been reported that a high dose (much greater than the dosage used in this study) of A1 antagonist CPT (10 mg/kg, i.p.) exacerbates KA-induced neuronal degeneration and that the A1 agonist N 6cyclopenthyladenosine (CHA) attenuates the neuronal cell loss induced by KA [12]. Similarly, adenosine A1 receptor inhibition/loss may also constitute one of the key mechanisms underlying the phenomenon of the selective vulnerability of typical global ischemia [18]. Therefore, adenosine A1 receptor may play an important role in modulating neuronal damage induced by such pathological conditions as epilepsy and cerebral ischemia. Because our findings indicate that oxidative stress may play a role in the acute induction of AP-l DNA binding activity after KA-induced seizures, we cannot rule out the
possibility that propolis inhibits the induction of AP-1 transcription factors that respond within hours to seizureevoked oxidative stress. KA-induced losses in GSH homeostasis may represent an important index of cellular damage [4,10] because there is a correlation between neuronal loss induced by seizures and the impairment of GSH status [10]. Interestingly, we also observed that impairment of the GSH system occurred in the hippocampus before histological signs of neuronal death. Recently, we demonstrated that cytosolic Cu/Zn-superoxide dismutase (SOD-1) and mitochondrial Mn-superoxide dismutase (SOD-2) protein expressions disappeared within 12 h after KA injection, before significant pyramidal cell losses in the hippocampus [8,9]. Thus, it is possible that the impairment of GSH status in the early stage of a KA insult could be a sensitive index of oxidative stress [4,10]. Recently, it was recognized that a prolonged depletion of endogenous GSH may result in an accumulation of reactive oxygen radicals generated through activation of the KA receptor, which could facilitate the expression of the AP-1 transcription factor in murine hippocampus [11]. Thus, we postulate that propolis significantly blocks seizure-induced neuronal loss by attenuating the impairment of GSH metabolism via, in part, adenosine A1 receptor modulation. The novel antioxidant/anticonvulsant effect could be an important contribution to extending the neuroprotective potential of propolis.
Acknowledgements This study was supported by a grant of the Korea Health 21 R&D Project (02-PJ10-PG6-AG01-0003), Ministry of Health & Welfare, Republic of Korea.
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Antioxidant propolis attenuates kainate-induced neurotoxicity via adenosine A1 receptor modulation in the rat Yong-Soo Kwona,1, Dae-Hun Parkb,1, Eun-Joo Shina, Myung Sang Kwonb, Kwang Ho Koc, Won-Ki Kimd, Jin Hyeong Jhooe, Wang-Kee Jhooa, Myung-Bok Wieb, Bae Dong Jungb, Hyoung-Chun Kima,* a
Neurotoxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, South Korea b Department of Veterinary Medicine, Kangwon National University, Chunchon 200-701, South Korea c Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, South Korea d Department of Pharmacology, Ewha Institute of Neuroscience, Ewha Women’s University Medical School, Seoul, South Korea e Clinical Research Institute, Seoul National University Hospital, Seoul, South Korea Received 6 June 2003; received in revised form 30 September 2003; accepted 28 October 2003
Abstract We examined the effects of the antioxidant propolis on seizures induced by kainic acid (KA). Sprague– Dawley rats received propolis (75 and 150 mg/kg, p.o.) five times at 12 h intervals. KA (10 mg/kg, i.p.) was injected 1 h after the last propolis treatment. Pretreatment with propolis significantly attenuated KA-induced seizures and KA-induced increases in hippocampal AP-1 DNA binding activity in a dosedependent manner. KA induced increases in the levels of malondialdehyde and protein carbonyl, and a decrease in the ratio of GSH/GSSG. These oxidative stresses and neuronal degenerations were significantly attenuated by pretreatment with propolis. The neuroprotective effects of propolis appeared to be counteracted by adenosine receptor antagonists [A1 antagonist, 8-cyclopentyl-1,3-dimethylxanthine (25 or 50 mg/kg); A2A antagonist, 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (0.5 or 1 mg/kg); and A2B antagonist, alloxazine (1.5 or 3.0 mg/kg)]. However, this counteraction was most pronounced in the presence of the A1 antagonist. Our results suggest that the protective effect of propolis against KA-induced neurotoxic oxidative damage is, at least in part, via adenosine A1 receptor modulation. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Propolis; Kainate; Oxidative stress; Ratio of GSH/GSSG; AP-1 DNA binding activity; Hippocampus; Adenosine receptors
Accumulating evidence indicates that oxidative stress may contribute to the development of the seizures induced by kainic acid (KA) [1,4,6,8 –10]. KA has been used as a model for both temporal lobe epilepsy and neurodegenerative disorders [19]. Honeybee propolis has been widely used as a folk medicine. It has been shown to have broad biological activities, which are principally attributed to the presence of flavonoids (major component; rutin, quercetin, galangin, etc.) [7] and caffeic acid phenethyl ester (CAPE) [13]. The prevailing opinion is that the broad biological activities of flavonoids and CAPE are related, in part, to their antioxidant actions [7]. A careful review of the literature indicates that * Corresponding author. Tel.: þ 82-33-250-6917; fax: þ82-33-255-7865. E-mail address: [email protected] (H.C. Kim). 1 Y.-S. Kwon and D.-H. Park contributed equally to this work.
the effects of propolis on neurodegenerative disorders have not yet been demonstrated. The purine nucleotide adenosine might act as a neuromodulator of the CNS and an endogenous neuroprotectant [2,18]. Adenosine exerts its effects on the neuronal activity via four G protein-coupled receptors, A1, A2A, A2B and A3 [2]. While adenosine A1 and A2B receptors are widely distributed in the brain, the A2A receptor distribution is restricted to the dopamine-innervated regions [2]. A3 receptors are expressed at low levels in the brain [2]. It has been demonstrated that adenosine A1 receptors are reduced in the temporal lobe of epileptic [5] and experimental epileptic brains [3]. Furthermore, the adenosine release evoked by KA may be involved in the production of free radicals [1]. In the present study, we examined the role of adenosine receptors in the propolis-mediated pharmacological action in response to KA insult.
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.10.075
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All animals were treated in strict accordance with the NIH Guide for the Humane Care and Use of Laboratory Animals. Male Sprague – Dawley rats (Bio Genomics, Charles River Technology, Gapyung-Gun, Gyeonggi-Do, South Korea) weighing about 250 g were maintained on a 12:12 light/dark cycle and fed ad libitum. They were adapted to these conditions for 2 weeks before KA (10 mg/kg, i.p.) administration. All rats were drug and seizure naive before the experiment. Ethanol-extracted propolis (75 or 150 mg/kg; Sigma, St. Louis, MO) suspended in vehicle (2% Tween 80 plus 2% sodium lauryl sulfate in the saline solution) was administered orally five times at 12 h intervals. Control rats received the same volume of vehicle. One hour after the last administration of propolis, KA (10 mg/kg) was administered intraperitoneally. Thirty minutes before KA, some rats received one of the following adenosine receptor antagonists: the A1 antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT, 25 or 50 mg/kg, i.p.; RBI, Natick, MA); the A2A antagonist, 1,3,7-trimethyl8-(3-chlorostyryl)xanthine (CSC, 0.5 or 1.0 mg/kg, i.p.; Sigma, St. Louis, MO); or the A2B antagonist, alloxazine (ALL, 1.5 or 3.0 mg/kg, i.p.; RBI, Natick, MA). Animals were sacrificed 4 and 48 h after KA administration. Brains were rapidly removed, and hippocampi were dissected and then stored at 2 80 8C. Using an automated video tracking system (Noldus Information Technology, Wageningen, The Netherlands) [10], seizure activity was rated according to the scale devised by Racine [16] during a 4 h period following KA challenge. We examined hippocampal AP-1 DNA binding activity following KA to confirm possible convulsant or anticonvulsant effects [15]. The AP-1 oligomer was purchased from Stratagene (La Jolla, CA). Double-stranded oligomer (25 ng) was labeled with [g-32P]ATP using 10 units of T4 polynucleotide kinase (U.S. Biochemicals, Cleveland, OH). The AP-1 oligomer (22-mer; 50 -CTAGTGATGAGTCAGCCGGATC-30 ) contained the consensus sequence (50 -TGAGTCA-30 ). To characterize AP-1 DNA binding activity, nuclear extracts were preincubated with a 100-fold excess of unlabeled AP-1 oligomer. A 100-fold excess of an unrelated oligomer (NFJ; 50 -TAGGGGTGGAGTCTCCATG-30 ; Research Genetics, Huntsville, AL), normal rabbit serum (NRS; Dako Corporation, Carpinteria, CA) or bovine serum albumin (BSA; Sigma, St. Louis, MO) were used as controls. The addition of cold AP-1 oligomer showed that the AP-1 DNA binding activity was specific. The addition of NRS or NFJ did not affect DNA binding. BSA also did not bind to the DNA probe. The amount of lipid peroxidation was determined [10] by measuring the accumulation of thiobarbituric acid-reactive substances in homogenates of hippocampus, and was expressed as the malondialdehyde (MDA) content. The extent of protein oxidation in the hippocampus was assessed by determining the quantity of protein carbonyl groups, which was measured spectrophotometrically using the 2,4dinitrophenylhydrazine (DNPH) labeling procedure as
described by Oliver et al. [14]. Protein was measured using a BCA protein assay reagent (Pierce, Rockford, IL). GSH and GSSG from dissected hippocampal tissues were measured immediately using a minor modification [10] of the method described by Reed et al. [17]. Cresyl violet stain was used to examine the degeneration of neurons [10]. The significance of the differences in the ratio of convulsing was assessed by chi-square test. Significant differences in seizures were analyzed using the nonparametric Wilcoxon signed rank test. The differences in the levels of MDA, protein carbonyl, GSH, GSSG and neuronal density were compared by Student’s t-test for paired data. Animals pretreated with saline (vehicle) and then given 10 mg/kg of KA showed robust behavioral seizures (3.4 ^ 0.6) that lasted 4 –5 h. Thirty-one of 36 animals (86.1%) exhibited seizures. Pretreatment with propolis significantly protected against the KA-induced convulsive behaviors in a dose-related manner. Although the A2A receptor antagonist CSC and the A2B receptor antagonist ALL both appeared to counteract the anticonvulsant effects of propolis slightly, the effect of the A1 receptor antagonist CPT was the most significant (Fig. 1A). There was a low level of AP-1 DNA binding activity in the absence of KA. A significant increase (P , 0:01) in AP1 DNA binding activity occurred 4 h after KA administration, and was still evident after 48 h. In contrast, the pretreatment with propolis caused significant reductions in AP-1 DNA binding activity (for propolis at 75 mg/kg, P , 0:05; for propolis at 150 mg/kg, P , 0:01) in a doserelated fashion. Consistently at each time-point, CPT most significantly reversed the propolis-induced reduction in AP1 DNA binding activity, although CSC and ALL both altered propolis-modulated AP-1 DNA binding activity (Fig. 1B,C). KA produced significant increases in the levels of MDA [unit ¼ nmol/g wet tissue; vehicle: 121.2 ^ 13.3 (n ¼ 6) vs. KA: 172.6 ^ 12.1 (n ¼ 6), P , 0:01] and carbonyl [unit ¼ nmol/mg protein; vehicle: 4.80 ^ 0.52 (n ¼ 6) vs. KA: 7.88 ^ 0.38 (n ¼ 6), P , 0:01] which were evident at 4 h after KA administration. The increases in lipid peroxidation and protein oxidation were significantly decreased in a dose-related fashion by pretreatment with propolis [MDA; KA: 172.6 ^ 12.1 (n ¼ 6) vs. propolis 75 or 150 mg/kg plus KA: 151.9 ^ 9.8 (n ¼ 6) or 133.1 ^ 6.8 (n ¼ 6), P , 0:05 or P , 0:01; carbonyl; KA: 7.88 ^ 0.38 (n ¼ 6) vs. propolis 75 or 150 mg/kg plus KA: 6.95 ^ 0.62 (n ¼ 6) or 5.33 ^ 0.28 (n ¼ 6), P , 0:05 or P , 0:01]. CPT dose-dependently reversed the significant propolismediated reductions in the levels of MDA [CPT 25 mg þ propolis 150 mg/kg plus KA: 146.3 ^ 14.1 (n ¼ 6); propolis 150 mg/kg plus KA: 133.1 ^ 6.8 (n ¼ 6) vs. CPT 50 mg þ propolis 150 mg/kg plus KA: 174.9 ^ 12.6 (n ¼ 6), P , 0:05] and carbonyl [CPT 25 mg þ propolis 150 mg/kg plus KA: 6.25 ^ 0.32 (n ¼ 6); propolis 150 mg/kg plus KA: 5.33 ^ 0.28 (n ¼ 6) vs. CPT 50 mg þ propolis 150 mg/kg plus KA: 7.80 ^ 0.43 (n ¼ 6),
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P , 0:05]. However, CSC [MDA; propolis 150 mg/kg plus KA: 133.1 ^ 6.8 (n ¼ 6) vs. CSC 0.5 or 1.0 mg/kg þ propolis 150 mg/kg plus KA: 147.2 ^ 10.4 (n ¼ 6) or 150.8 ^ 10.6 (n ¼ 6); carbonyl; 150 mg/kg plus KA: 5.33 ^ 0.28 (n ¼ 6) vs. CSC 0.5 or 1.0 mg/kg þ propolis 150 mg/kg plus KA: 6.42 ^ 0.62 (n ¼ 6) or 6.85 ^ 0.57 (n ¼ 6)], but not ALL, appeared to affect the significant decreases in these oxidative indexes caused by propolis. The KA-induced increase in the MDA and carbonyl value returned to near control (vehicle) levels by 48 h after KA treatment. No significant differences were observed in the ratio of GSH [unit ¼ nmol/g wet tissue; vehicle: 1.48 ^ 0.12 (4 h point) or 1.55 ^ 0.11 (2 day point)]/GSSG [unit ¼ nmol/g wet tissue; vehicle: 0.030 ^ 0.003 (4 h) or 0.029 ^ 0.004 (2 days)] in the absence of KA. After KA treatment, the GSH/GSSG ratio in the hippocampus time-dependently decreased (P , 0:01) [4 h after KA, GSH: 1.05 ^ 0.08 (n ¼ 6), GSSG: 0.044 ^ 0.003 (n ¼ 6); 2 days after KA, GSH: 0.81 ^ 0.14 (n ¼ 6), GSSG: 0.057 ^ 0.003 (n ¼ 6)]. However, propolis dose-dependently prevented in the GSH/GSSG ratio in the hippocampus (4 h point, propolis 150 mg/kg plus KA vs. KA, P , 0:05; 2 day point, propolis 75 or 150 mg/kg plus KA vs. KA, P , 0:05 or P , 0:01) after KA administration [4 h after KA, GSH: 1.25 ^ 0.10 (plus propolis 75 mg/kg) or 1.41 ^ 0.10 (plus propolis 150 mg/kg); 2 days after KA, GSH: 1.11 ^ 0.11 (plus propolis 75 mg/kg) or 1.24 ^ 0.13 (plus propolis 150 mg/kg), GSSG: 0.048 ^ 0.004 (plus propolis 75 mg/kg) or 0.042 ^ 0.003 (plus propolis 150 mg/kg)]. CPT was the most efficacious in counteracting the significant reductions in the GSH/GSSG ratios mediated by propolis, although both CSC and ALL also appeared to alter the propolis-mediated GSH status (Fig. 2). The neuronal layers of the hippocampus in the animal without KA were clearly visible with cresyl violet staining. At 2 days after KA injection, significant neuronal losses were observed in the CA1 and CA3 regions; these losses were significantly blocked by pretreatment with propolis at Fig. 1. Effects of propolis with or without adenosine receptor antagonists on kainate-induced seizures (A) and on increases in AP-1 DNA binding activity (B,C). §Number of convulsions (the number of animals that convulsed/total number of animals receiving each treatment). Hippocampal nuclear extracts were prepared at 4 h (B) and at 2 days (C) after KA treatment. The relative intensities of the bands were quantified by laser densitometry [8]. Veh, vehicle; P 75 or 150, propolis 75 or 150 mg/kg; CPT 25 or 50, 8-cyclopentyl-1,3-dimethylxanthine 25 or 50 mg/kg, i.p.; CSC 0.5 or 1.0, 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine 0.5 or 1.0 mg/kg, i.p.; ALL 1.5 or 3.0, alloxazine 1.5 or 3.0 mg/kg, i.p. Animal numbers used in each group for seizure score and convulsing are in parentheses. Four animals in each group for AP-1 DNA binding activity were used. Each value is the mean ^ SEM. *P , 0:002 vs. Veh þ KA, **P , 0:001 vs. Veh þ KA, #P , 0:02 vs. P150 þ KA, ##P , 0:001 vs. P150 þ KA (x2test for convulsing). aP , 0:01 vs. Veh þ Veh, bP , 0:05 vs. Veh þ P150, c P , 0:01 vs. Veh þ P150, dP , 0:05 vs. Veh þ KA, eP , 0:01 vs. Veh þ KA, fP , 0:05 vs. P150 þ KA, gP , 0:01 vs. P50 þ KA (seizure scores or AP-1 DNA binding activity were analyzed using the nonparametric Wilcoxon signed rank test or Student’s t-test for paired data).
Fig. 2. Effects of propolis with or without adenosine receptor antagonists on the kainate-induced alterations in the ratio of GSH/GSSG. Each value is the mean ^ SEM of six animals. aP , 0:01 vs. Veh þ Veh, bP , 0:05 vs. Veh þ KA, cP , 0:01 vs. Veh þ KA, d P , 0:05 vs. P150 þ KA, e P , 0:01 vs. P150 þ KA (Student’s t-test for paired data).
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Fig. 3. Photomicrographs of cresyl violet-stained sections 2 days after KA treatment. Vehicle- or propolis (150 mg/kg)-treated rat (A) disclosed that neuronal layers of the hippocampi were apparently visible after cresyl violet staining. KA-induced neuronal damage was very severe; areas containing coagulation necrosis in the CA1 and CA3 regions did not stain (B). Pretreatment with propolis significantly prevented neuronal losses (C). This neuroprotection was counteracted in the presence of CPT at 50 mg/kg (D), CSC at 1.0 mg/kg (E) or ALL at 3.0 mg/kg (F). Scale bar, 500 mm. Surviving neurons in the stratum pyramidale were calculated using an image analysis system [10]. The number of animals used in each group is in parentheses. Each value is the mean ^ SEM. aP , 0:02 vs. Veh þ P150, b P , 0:01 vs. Veh þ P150, cP , 0:05 vs. Veh þ KA, dP , 0:01 vs. Veh þ KA, eP , 0:05 vs. P150 þ KA, fP , 0:01 vs. P150 þ KA (Student’s t-test for paired data).
150 mg/kg. CPT reversed the neuroprotective effects of propolis most significantly, but CSC and ALL both influenced the pharmacological action of propolis (Fig. 3). The present study clearly demonstrated that the antioxidant propolis is an effective neuroprotective agent against KA, and acts, at least in part, via adenosine A1 receptor modulation. It has been reported that a high dose (much greater than the dosage used in this study) of A1 antagonist CPT (10 mg/kg, i.p.) exacerbates KA-induced neuronal degeneration and that the A1 agonist N 6cyclopenthyladenosine (CHA) attenuates the neuronal cell loss induced by KA [12]. Similarly, adenosine A1 receptor inhibition/loss may also constitute one of the key mechanisms underlying the phenomenon of the selective vulnerability of typical global ischemia [18]. Therefore, adenosine A1 receptor may play an important role in modulating neuronal damage induced by such pathological conditions as epilepsy and cerebral ischemia. Because our findings indicate that oxidative stress may play a role in the acute induction of AP-l DNA binding activity after KA-induced seizures, we cannot rule out the
possibility that propolis inhibits the induction of AP-1 transcription factors that respond within hours to seizureevoked oxidative stress. KA-induced losses in GSH homeostasis may represent an important index of cellular damage [4,10] because there is a correlation between neuronal loss induced by seizures and the impairment of GSH status [10]. Interestingly, we also observed that impairment of the GSH system occurred in the hippocampus before histological signs of neuronal death. Recently, we demonstrated that cytosolic Cu/Zn-superoxide dismutase (SOD-1) and mitochondrial Mn-superoxide dismutase (SOD-2) protein expressions disappeared within 12 h after KA injection, before significant pyramidal cell losses in the hippocampus [8,9]. Thus, it is possible that the impairment of GSH status in the early stage of a KA insult could be a sensitive index of oxidative stress [4,10]. Recently, it was recognized that a prolonged depletion of endogenous GSH may result in an accumulation of reactive oxygen radicals generated through activation of the KA receptor, which could facilitate the expression of the AP-1 transcription factor in murine hippocampus [11]. Thus, we postulate that propolis significantly blocks seizure-induced neuronal loss by attenuating the impairment of GSH metabolism via, in part, adenosine A1 receptor modulation. The novel antioxidant/anticonvulsant effect could be an important contribution to extending the neuroprotective potential of propolis.
Acknowledgements This study was supported by a grant of the Korea Health 21 R&D Project (02-PJ10-PG6-AG01-0003), Ministry of Health & Welfare, Republic of Korea.
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