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Possible nitric oxide mechanism in the protective effect of hesperidin against pentylenetetrazole (PTZ)-induced kindling and associated cognitive dysfunction in mice
Epilepsy & Behavior 29 (2013) 103–111
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Possible nitric oxide mechanism in the protective effect of hesperidin against pentylenetetrazole (PTZ)-induced kindling and associated cognitive dysfunction in mice Anil Kumar ⁎, Sree Lalitha, Jitendriya Mishra Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study (UGC-CAS), Panjab University, Chandigarh 160014, India
a r t i c l e
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Article history: Received 29 March 2013 Revised 5 June 2013 Accepted 7 June 2013 Available online 9 August 2013 Keywords: Generalized epilepsy Kindling Mitochondrial dysfunction Reactive oxygen species Seizure Hesperidin
a b s t r a c t Epilepsy is a complex neurological disorder manifested by recurrent episodes of convulsive seizures, loss of consciousness, and sensory disturbances. Pentylenetetrazole (PTZ)-induced kindling primarily represents a model of generalized epilepsy. The present study has been undertaken to evaluate the neuroprotective potential of hesperidin and its interaction with nitric oxide modulators against PTZ-induced kindling and associated cognitive dysfunction in mice. The experimental protocol comprised of eleven groups (n = 6), where a subconvulsive dose of PTZ (40 mg/kg, i.p.) had been administered every other day for a period of 12 days, and seizure episodes were noted after each PTZ injection over a period of 30 min. The memory performance tests were carried out on days 13 and 14 followed by the estimation of biochemical and mitochondrial parameters. Chronic administration of a subconvulsive dose of PTZ resulted in an increase in convulsive activity culminating in generalized clonic– tonic seizures, as revealed by a progressive increase in seizure score as well as alteration in antioxidant enzyme levels (lipid peroxidation, nitrite, glutathione, super oxide dismutase, and catalase) and mitochondrial complex (I, II, and IV) activities, whereas chronic treatment with hesperidin (200 mg/kg) significantly attenuated these behavioral, biochemical, and mitochondrial alterations. Further, treatment with L-arginine (100 mg/kg) or L-NAME (10 mg/kg) in combination with hesperidin significantly modulated the protective effect of hesperidin which was significant as compared to their effects per se in PTZ-treated animals. Thus, the present study suggests a possible involvement of the NO–cGMP pathway in the neuroprotective effect of hesperidin in PTZ-kindled mice. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Epilepsy is a complex neurological disorder often characterized by abnormal synchronous neural activity. Clinical manifestations of epilepsy include recurrent episodes of convulsive seizures, loss of consciousness, memory impairment, and sensory disturbances. Epilepsy is the most frequent neurological disorder after stroke with an incidence of 0.3–0.5% and a prevalence of 5 to 10 persons per thousand in populations worldwide [1]. Alterations in several classic neurotransmitter systems such as the glutamatergic [2] and GABAergic [3] systems have been implicated in the genesis of epileptic seizures. Further, nitric oxide (NO) has been reported to be a potential neurotransmitter or retrograde messenger linked to synaptic plasticity [4], regulation of brain excitability, and triggering of seizure activity [5,6]. Further, NO has been reported to be a controversial modulator of seizure susceptibility with either anticonvulsant [7] or proconvulsant effects [8]. Nitric oxide (NO) is an important brain messenger released upon the activation of the glutamate N-methyl-D-aspartate (NMDA) receptor and ⁎ Corresponding author. Fax: +91 172 2543101. E-mail address: [email protected] (A. Kumar). 1525-5050/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yebeh.2013.06.007
subsequent Ca2+ dependent activation of neuronal nitric oxide synthase (nNOS) [9,10]. However, NO is synthesized from L-arginine and has been widely implicated in several neuroinflammatory and neurodegenerative conditions due to its prooxidant and antioxidant actions [11]. Nitric oxide activates soluble guanyl acetylase leading to the formation of cyclic guanosine monophosphate (cGMP) in the central nervous system (CNS) [12]. The elevated cGMP formation in CNS stimulates N-methyl-D-aspartate (NMDA)-type glutamate receptors [13] which leads to the activation of Ca2+-calmodulin dependent systems and the overproduction of NO by nNOS activation [14,15]. Excessive NO produced during seizures affects oxidative phosphorylation by inhibiting the mitochondrial respiratory enzymes [16], and the resistant mitochondrial dysfunction induces apoptotic neuronal death [17,18]. The mitochondrial respiratory chain is sensitive to both NO and peroxynitrite. Nitric oxide is known to slow down mitochondrial respiratory functions, and its transnitrosylation product, S-nitrosothil, reduces mitochondrial complex activities [17]. In recent times, NO modulators like L-arginine (substrate for NO synthesis), L-NAME (G-nitroL-arginine-methyl ester) [nitric oxide synthase inhibitor (NOSi)] and 7-NI (7-nitroindazole) have been widely used to study the involvement of the NO–cGMP pathway in the protective effect of several bioactive
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compounds [10,19–23]. However, several studies conducted in our laboratory demonstrated that hesperidin possesses discernible neuroprotective effects through its NO mechanism in cerebral ischemia– reperfusion injury-induced cognitive dysfunction and 3-nitro propionic acid-induced Huntington's disease-like symptoms [10,19,23]. However, the neuroprotective mechanism of hesperidin in epileptic conditions is yet to be determined. Repeated administration of subconvulsive doses of pentylenetetrazole (PTZ) continuously downregulates the sensitivity of GABAA receptors, thus favoring glutamate-aggravated seizures [24]. However, memory impairment is common in patients with epilepsy [25] and has been attributed to the dysfunction of surviving hippocampal neurons, as well as the adverse effects of antiepileptic drugs [26,27]. The pentylenetetrazole-induced kindling model offers several unique opportunities to study the mechanisms of epileptogenesis and its associated cognitive deficits as well as the behavioral manifestations of seizures [28]. However, the mechanisms underlying such impairments remain unclear. Flavonoids, which are polyphenolic compounds, can directly quench free radicals and inhibit enzymes of oxygen reduction pathways [19]. Hesperidin, a bioflavonoid richly found in oranges and lemons, has been reported to be an effective supplement in the treatment of several neurodegenerative diseases [23,29]. Above all, hesperidin has been reported to contribute to the intracellular antioxidant defense systems as a powerful agent against superoxide, singlet oxygen, and hydroxyl radicals [30,31]. Various reports suggest the involvement of the NO– cGMP pathway in the neuroprotective effect of hesperidin [15,30,31] as well as in arresting seizure genesis [31]. Hence, keeping the above reports in mind, the present study has been designed to explore the possible involvement of the nitric oxide mechanism in the neuroprotective effect of hesperidin in PTZ-induced kindling in mice.
Table 1 Grouping of animals. Name
Treatment
Group 1 Group 2 Group 3 Groups 4–5 Groups 6–7 Group 8 Group 9 Group 10 Group 11
Naive (vehicle) HES (200 mg/kg) per se PTZ (40 mg/kg, i.p.) + vehicle (0.5% w/v sod., CMC) HES (100 and 200 mg/kg, p.o.) + PTZ (40 mg/kg, i.p.) L-Arg (100) and L-NAME (10) + PTZ (40 mg/kg, i.p.) L-Arg (100) + HES (100) + PTZ (40 mg/kg, i.p.) L-Arg (100) + HES (200) + PTZ (40 mg/kg, i.p.) L-NAME (10) + HES (100) + PTZ (40 mg/kg, i.p.) L-NAME (10) + HES (200) + PTZ (40 mg/kg, i.p.)
and conducted according to the National Science Academy guidelines for the use and care of animals. 2.2. Drugs and treatment schedule Pentylenetetrazole, hesperidin (HES), L-arginine (substrate for NO synthesis), and L-NAME [nitric oxide synthase inhibitor (NOSi)] were procured from Sigma-Aldrich (St. Louis, MO, USA), and doses were selected on the basis of our previous study reports [10,32]. Hesperidin was suspended in 0.5% w/v sodium carboxymethylcellulose (CMC) and administered per oral (p.o.) for a period of 12 days. Pentylenetetrazole, L-arginine, and L-NAME were dissolved in saline and administered intraperitoneally (i.p.). L-Arginine or L-NAME was administered daily 30 min before hesperidin treatment, whereas hesperidin was administered 60 min before PTZ administration (Fig. 1). The experimental protocol comprised of eleven experimental groups containing six animals in each group (Table 1). 2.3. Induction of kindled seizures and design of the experiment
2. Methods 2.1. Animals Male LACA mice (20–30 g) bred in the Central Animal House (CAH) facility of Panjab University, Chandigarh were used in the study. The animals were housed under standard laboratory conditions maintained under a natural light and dark cycle and had free access to food and water. All the experiments were carried out between 09.00 and 15.00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) [IAEC/170–175/UIPS-29/30-8-2011]
A subconvulsive dose of PTZ (40 mg/kg, i.p.) was administered on alternating days for a period of 12 days until the animal exhibited full motor seizures. After each injection of PTZ, animals were kept in a plexiglass chamber (30 × 24 × 22 cm) with partitions in between, and seizure intensity was observed over a cutoff period of 30 min. The intensity of seizures was evaluated using a four-point scoring system: 0 = no effect, 1 = jerks, 2 = Straub's tail, and 3 = clonus. Mean kindling score was plotted against the duration of treatment as described by [33,34]. Cognitive behavior was assessed 24 h after the last PTZ injection (13th and 14th days).
Fig. 1. Experimental protocol of PTZ-induced kindling.
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2.4. Behavioral parameters 2.4.1. Passive avoidance with negative (punishment) reinforcement: step-down Learning and memory processes were evaluated in a modified twotask learning paradigm that has been validated in our laboratory. The apparatus consists of a plexiglass chamber (27 × 27 × 27 cm) with metal grid floor containing a wooden platform (10 × 7 cm) fixed at its center, which served as the shock-free zone (SFZ). The animals were given training trials wherein the animals were placed individually on SFZ, and each descent of the animal to the grid floor was punished by electric foot shock for 2 s (50 V a.c.). The animals were allowed to remain on the SFZ for 60 s thereafter. The step-down latency in seconds was recorded after the last training session without giving shock [acquisition latency (AL)]. The animals were tested again after 24 h, without giving shock, and the time taken for the mouse to step down was measured [retention latency (RL)]. A cutoff time of 600 s was chosen for the animal which did not step down from the SFZ in this period [35]. 2.4.2. Elevated plus maze paradigm Elevated plus maze is another paradigm to assess memory dysfunction, which consists of two opposite open arms (16 cm × 5 cm) and two closed arms of equal dimensions connected with a central square of 5 cm × 5 cm. Each arm contains 10-cm-high walls and the apparatus is elevated to 25 cm above the floor. The mice were placed individually at the end of one open arm, facing away from the central platform, and the time taken by the animals to move from the open arm to either of the closed arms was recorded as initial transfer latency (ITL). If the animal(s) did not enter either of the closed arms within 90 s (cutoff time), it was gently pushed towards a closed arm. The animals were allowed to explore the maze for 10 s after reaching the closed arm, and then, they were returned to their home cage. After 24 h, the total procedure was repeated, and the time taken by the animals to enter the closed arm [retention transfer latency (RTL)] was recorded [36]. 2.5. Biochemical estimations Following behavioral assessments, animals were sacrificed by decapitation; brains were rinsed in isotonic saline, weighed, and divided into two equal halves. One half was used for biochemical estimations and the other one for mitochondrial complex enzyme estimations. For biochemical analysis, 10% (w/v) tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 ×g at 4 °C for 15 min. Aliquots of supernatants were separated and used for biochemical estimations. A double beam UV–VIS spectrophotometer [UV-Pharmaspec 1700, Shimadzu (Japan)] was used for biochemical and mitochondrial assessments. 2.5.1. Measurement of lipid peroxidation The quantitative measurement of lipid peroxidation in the brain was performed according to the method of Wills. The amount of malondialdehyde (MDA), a measure of lipid peroxidation, was measured by reaction with thiobarbituric acid at 532 nm using the double beam UV–VIS spectrophotometer [UV-Pharmaspec 1700, Shimadzu (Japan)]. The values were calculated using the molar extinction coefficient of chromophore (1.56 × 105 M−1 cm−1) and expressed as nanomoles of malondialdehyde per milligram of protein [37]. 2.5.2. Estimation of nitrite The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (NO), was determined with a colorimetric assay with Greiss reagent (0.1% N-(1-naphthyl) ethylenediaminedihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid) as described by Green and his coworkers. Equal volumes of the supernatant
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and the Greiss reagent were mixed; the mixture was incubated for 10 min at room temperature. The absorbance was determined at 540 nm with the double beam UV–VIS spectrophotometer [UVPharmaspec 1700, Shimadzu (Japan)]. The concentration of nitrite in the supernatant was determined from a sodium nitrite standard curve and was expressed as micromole per liter [38]. 2.5.3. Estimation of reduced glutathione (GSH) Reduced glutathione in the brain was estimated according to the method described by Ellman and his group. Briefly, 1 ml of supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold-digested at 4 °C for 1 h. The sample was centrifuged at 1200 rpm for 15 min at 4 °C. To 1 ml of this supernatant, 2.7 ml of 0.1 M phosphate buffer (pH 8) and 0.2 ml of 5,5-dithiobis 2-nitrobenzoic acid (DTNB) were added. The yellow color developed was read immediately at 412 nm using the double beam UV–VIS spectrophotometer [UV-Pharmaspec 1700, Shimadzu (Japan)]. Results were calculated using the molar extinction coefficient of chromophore (1.36 × l04 M−1 cm−1) and were expressed as micromole GSH per milligram protein [39]. 2.5.4. Catalase estimation Catalase activity was assayed by the method of Luck, wherein breakdown of hydrogen peroxides (H2O2) is measured at 240 nm. Briefly, the assay mixture consisted of 3 ml of H2O2 phosphate buffer and 0.05 ml of supernatant of tissue homogenate (10%), and change in absorbance was recorded at 240 nm using the double beam UV– VIS spectrophotometer [UV-Pharmaspec 1700 Shimadzu, (Japan)]. The results were expressed as micromole H2O2 decomposed per milligram of protein/min [40]. 2.5.5. Superoxide dismutase activity estimation Superoxide dismutase activity was accessed according to the method described by Kono, wherein the reduction of nitrobluetetrazolium (NBT) was inhibited by the superoxide dismutase and measured at 560 nm using the double beam UV–VIS spectrophotometer [UVPharmaspec 1700, Shimadzu (Japan)]. Briefly, the reaction was initiated by the addition of the hydroxylamine hydrochloride to the mixture containing NBT and the sample. The results were expressed as unit/mg protein, where one unit of enzyme is defined as the amount of enzyme inhibiting the rate of reaction by 100% [41]. 2.5.6. Protein estimation The protein was measured by the biuret method using bovine serum albumin as standard [42]. 2.6. Mitochondrial complex enzyme estimation 2.6.1. Isolation of rat brain mitochondria The second half of the brain was used for mitochondrial isolation as described in the method of Berman and Hastings. The brains were homogenized in isolated buffer, and the homogenates were centrifuged at 13,000 ×g for 5 min at 4 °C. Pellets were resuspended in isolation buffer with ethylene glycol tetraacetic acid (EGTA) and spun again at 13,000 ×g at 4 °C for 5 min. The resulting supernatants were transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13,000 ×g at 4 °C for 10 min. Pellets containing pure mitochondria were resuspended in isolation buffer without EGTA [43]. 2.6.2. NADH dehydrogenase (complex I) activity Nicotinamide adenine dinucleotide (NADH) dehydrogenase activity was measured spectrophotometrically using the method of King and Howard. The method involves catalytic oxidation of NADH to NAD+ with subsequent reduction of cytochrome c. The reaction mixture contained 0.2 M glycylglycine buffer (pH 8.5), 6 mM NADH in 2 mM glycylglycine buffer, and 10.5 mM cytochrome c. The reaction was
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initiated by the addition of a requisite amount of solubilized mitochondrial sample followed by an absorbance change at 550 nm for 2 min [44]. 2.6.3. Succinate dehydrogenase (SDH) (complex II) activity Succinate dehydrogenase was measured spectrophotometrically as described by king. The method involves the oxidation of succinate by an artificial electron acceptor, potassium ferricyanide. The reaction mixture contained 0.2 M phosphate buffer (pH 7.8), 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. The reaction was initiated by the addition of the mitochondrial sample followed by an absorbance change at 420 nm for 2 min [45]. 2.6.4. Cytochrome oxidase (complex IV) assay Cytochrome oxidase activity was assayed in brain mitochondria according to the method of Sottocasa and his coworkers. The assay mixture contained 0.3 mM of reduced cytochrome c in 75 mM phosphate buffer. The reaction was started by the addition of solubilized mitochondrial sample, and absorbance change was recorded at 550 nm for 2 min [46]. 2.7. Statistical analysis Results were expressed as mean ± SEM. The data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test. A p-value b 0.05 was considered to be statistically significant. 3. Results 3.1. Protective effect of hesperidin and its interaction with NO modulators against pentylenetetrazole-induced kindling score The repetitive administration of a subconvulsive dose of PTZ (40 mg/kg, i.p., every other day) for a period of 12 days resulted in a gradual increase in convulsive activity (seizure score) culminating in generalized clonic–tonic seizures, as compared to the naive group. Unlike hesperidin (100 mg/kg), treatment with hesperidin (200 mg/kg) for 12 days significantly reduced the course of kindling as evidenced by the decrease in seizure score as compared to the control (PTZ-kindled) group. On the other hand, L-arginine (100 mg/kg) pretreatment for 12 days significantly worsened convulsive activity as compared to the control group, whereas its combination with hesperidin attenuated the severity of seizure activity which was significant as compared to their effects per se in the PTZ-treated animals. Conversely, L-NAME (10 mg/kg) administration for 12 days significantly suppressed seizure activity (reduced cumulative kindling score) as compared to the control group, whereas its combination with 100-mg/kg hesperidin rather than 200-mg/kg hesperidin showed further potentiation in their protective effect (increased mean kindling score), which was significant as compared to their effects per se in the PTZ-treated animals, which could be attributed to their ceiling effects. However, treatment with hesperidin (200 mg/kg) per se did not show any significant effect as compared to the naive group (Fig. 2). 3.2. Modulatory effect of L-arginine and L-NAME on protective effect of hesperidin on passive avoidance response against PTZ-kindled mice Profound impairment in learning and memory was exhibited by chronic PTZ-treated animals as evident from the decrease in stepdown latency [AL and RL] which was significant as compared to naive animals. However, unlike hesperidin (100 mg/kg), treatment with hesperidin (200 mg/kg) for 12 days significantly delayed the step-down latencies (AL and RL) in the acquisition as well as in the retention trials as compared to the PTZ-kindled (control) group. In addition, treatment with L-arginine (100 mg/kg, i.p.) caused further decrease in AL and RL which was found to be significant as compared to the control group,
Fig. 2. Effect of hesperidin and its modification by L-arginine and L-NAME against pentylenetetrazole-induced kindling score. Data are expressed as mean ± SEM. (b) p b 0.05 as compared to PTZ, (c) p b 0.05 as compared to HES (100 mg/kg), (d) p b 0.05 as compared to HES (200 mg/kg), (e) p b 0.05 as compared to L-NAME (10) + PTZ (Kruskal–Wallis ANOVA followed by Dunn's test). HES: hesperidin; L-arg: L-arginine.
indicating severe learning and memory impairment. On the other hand, L-NAME (10 mg/kg) treatment for 12 days showed superior step-down latencies (AL and RL) during acquisition and retention trials as compared to the control group. Interestingly, pretreatment with L-arginine in combination with hesperidin further worsened the step-down latencies (AL and RL), which was significant as compared to their effect per se in the PTZ-treated animals. However, pretreatment with L-NAME in combination with hesperidin (100 mg/kg) showed significant improvement in step-down latency as compared to their effects per se, whereas pretreatment with L-NAME in combination with hesperidin (200 mg/kg) did not show any significant improvement in step-down latency as compared to their effects per se, which may be attributed to their ceiling effects (Fig. 3). However, treatment with hesperidin (200 mg/kg) per se did not show any significant effect as compared to the naive group. 3.3. Modulatory effect of L-arginine and L-NAME on protective effect of hesperidin in elevated plus maze task against PTZ-induced kindling in mice Naive animals exhibited a significant difference between initial transfer latency (ITL) and retention transfer latency (RTL), signifying the acquisition and retrieval of the task. The control animals exhibited a significant increase in initial transfer latency (ITL) and retention transfer latency (RTL) as compared to the naive group, indicating profound cognitive deficit. However, unlike treatment with 100-mg/kg hesperidin, treatment with 200-mg/kg hesperidin improved RTL along with a decrease in ITL which was significant as compared to the PTZ (control) group, showing its neuroprotective potential. In addition, treatment with L-arginine (100 mg/kg, i.p.) exhibited further delays in ITL and RTL which were found to be significant as compared to the control group, indicating severe learning and memory impairment. On the other hand, L-NAME (10 mg/kg) treatment for 12 days showed superior transfer latencies (ITL and RTL) during acquisition and retention trials as compared to the control group. Interestingly, pretreatment with L-arginine in combination with hesperidin further worsened the protective effect of hesperidin, which was significant as compared to their effect per se in PTZ-treated animals. However, pretreatment with L-NAME in combination with 100-mg/kg hesperidin showed significant improvement in escape latency as compared to
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Fig. 3. Modulatory effect of L-arginine and L-NAME on the protective effect of hesperidin in passive avoidance task against PTZ-kindled mice. Data are expressed as mean ± SEM. (a) p b 0.05 as compared to AL of naive, (a') p b 0.05 as compared to RL of naive, (b) p b 0.05 as compared to AL of PTZ, (b') p b 0.05 as compared to RL of PTZ, (c) p b 0.05 as compared to AL of HES (100) + PTZ, (c') p b 0.05 as compared to RL of HES (100) + PTZ, (d) p b 0.05 as compared to AL of HES (200) + PTZ, (d') p b 0.05 as compared to RL of HES (200) + PTZ, (e) p b 0.05 as compared to AL of L-NAME (10) + PTZ, (e') p b 0.05 as compared to RL of L-NAME (10) + PTZ (ANOVA followed by Tukey's test). HES: hesperidin; L-arg: L-arginine.
their effects per se in PTZ-treated animals, whereas pretreatment with L-NAME in combination with 200-mg/kg hesperidin did not show any significant improvement in escape latencies (ITL and RTL) as compared to their effects per se in PTZ-treated animals, which may be attributed to their ceiling effects. However, treatment with hesperidin (200 mg/kg) per se was found to be nonsignificant in altering the escape latencies as compared to the naive group of animals (Fig. 4). 3.4. Effect of hesperidin, L-NAME, L-arginine, and their combinations on brain lipid peroxidation and nitrite levels in PTZ-kindled mice Repeated administration of a subconvulsive dose of PTZ induced oxidative stress as indicated by a significant rise in brain MDA and nitrite levels as compared to the naive group. However, hesperidin (200 mg/kg) treatment significantly lowered MDA and nitrite levels as compared to the PTZ (control) group. Further, L-arginine (100 mg/kg) potentiated the PTZ-induced oxidative stress as indicated by the significant elevation in MDA and nitrite levels, whereas L-NAME (10 mg/kg)
treatment significantly attenuated the oxidative stress parameters as evident from the decreased MDA and nitrite levels as compared to the PTZ (control) group. Further, treatment with hesperidin in combination with L-arginine significantly reversed the protective effect of hesperidin as compared to the control group, whereas in combination with L-NAME, hesperidin (100 mg/kg) significantly potentiated the protective effects as compared to their effect per se in PTZ-treated animals (Figs. 5A and B). However, the effects of hesperidin (200 mg/kg), in combination with L-NAME, were found to be nonsignificant as compared to its effects per se, which may be attributed to its ceiling effect. The treatment with hesperidin (200 mg/kg) per se did not show any significant effect as compared to the naive group. 3.5. Effect of hesperidin, L-arginine, L-NAME, and their combinations on glutathione, superoxide dismutase, and catalase levels in PTZ-kindled mice Chronic PTZ (40 mg/kg) challenge caused significant depletion in brain SOD, GSH, and catalase levels as compared to the naive group.
Fig. 4. Modulatory effect of L-arginine and L-NAME on protective effect of hesperidin in elevated plus maze task against PTZ-kindled mice. Data are expressed as mean ± SEM. (a) p b 0.05 as compared to ITL of naive, (a') p b 0.05 as compared to RTL of naive, (b) p b 0.05 as compared to ITL of PTZ, (b') p b 0.05 as compared to RTL of PTZ, (c) p b 0.05 as compared to ITL of HES (100) + PTZ, (c') p b 0.05 as compared to RTL of HES (100) + PTZ, (d) p b 0.05 as compared to ITL of HES (200) + PTZ, (d') p b 0.05 as compared to RTL of HES (200) + PTZ, (e) p b 0.05 as compared to ITL of L-NAME (10) + PTZ, (e') p b 0.05 as compared to RTL of L-NAME (10) + PTZ (ANOVA followed by Tukey's test). HES: hesperidin; L-arg: L-arginine.
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Fig. 5. Effect of hesperidin, L-arginine, L-NAME, and their combinations on LPO (A), nitrite (B), GSH (C), SOD (D), and catalase (E) levels in the brain of PTZ-kindled mice. Values are expressed as mean ± SEM. ANOVA followed by Tukey test. (a) p b 0.05 as compared to naive, (b) p b 0.05 as compared to PTZ, (c) p b 0.05 as compared to HES (100) + PTZ, (d) p b 0.05 as compared to HES (200) + PTZ, (e) p b 0.05 as compared to L-NAME (10) + PTZ. HES: hesperidin; L-arg: L-arginine.
Unlike 100-mg/kg hesperidin, administration of 200-mg/kg hesperidin for 12 days showed significant protection against depletion in brain SOD, GSH, and catalase levels, whereas L-arginine (100 mg/kg) caused further damage to these antioxidant enzymes which was significant as compared to the PTZ (control) group. On the other hand, L-NAME (10 mg/kg) pretreatment for 12 days showed significant protection against the depletion of the above antioxidant enzymes. On the other hand, administration of hesperidin in combination with L-arginine (100 mg/kg) further reversed the protective effect of hesperidin which was significant as compared to their effects per se. However, administration of hesperidin (100 mg/kg) in combination with L-NAME (10 mg/kg) significantly restored all the three antioxidant enzyme levels as compared to their effect per se, suggesting a possible involvement of the NO pathway (Figs. 5C, D, and E). However, the effects of hesperidin (200 mg/kg), in combination with L-NAME, were found to be nonsignificant as compared to its effects per se, which may be attributed to its ceiling effect. The treatment with hesperidin (200 mg/kg) per se did not show any significant effect as compared to the naive group.
3.6. Effect of hesperidin, L-arginine, L-NAME, and their combinations on mitochondrial complex enzyme activities in PTZ-kindled mice Chronic PTZ administration significantly impaired mitochondrial complex enzyme (I, II, and IV) activities as compared to the naive animals. However, treatment with hesperidin (200 mg/kg) and L-NAME (10 mg/kg) restored these mitochondrial complex enzyme activities, whereas treatment with L-arginine further damaged the mitochondrial enzyme (I and IV) activities, which were significant as compared to the PTZ (control) group. Further, administration of hesperidin in combination with L-arginine (100 mg/kg) further reversed the protective effect of hesperidin, whereas its combination with L-NAME (10 mg/kg) significantly restored mitochondrial complex enzyme (I and IV) activities as compared to their effects per se. However, hesperidin (200 mg/kg) treatment in combination with L-NAME was found to be nonsignificant as compared to their effects per se, which may be attributed to the ceiling effect. Treatment with hesperidin (200 mg/kg) per se for 12 days was found to be nonsignificant as compared to the naive animals (Figs. 6A, B, and C).
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Fig. 6. Effect of hesperidin, L-arginine, L-NAME, and their combinations on mitochondrial complex I (A), II (B), and IV (C) in the brain of PTZ-kindled mice. Data are expressed as mean ± SEM. ANOVA followed by Tukey's test. (a) p b 0.05 as compared to naive, (b) p b 0.05 as compared to PTZ, (c) p b 0.05 as compared to HES (100) + PTZ, (d) p b 0.05 as compared to HES (200) + PTZ, (e) p b 0.05 as compared to L-NAME (10) + PTZ. HES: hesperidin; L-arg: L-arginine.
4. Discussion A growing body of evidence suggests that repetitive administration of a subconvulsive dose of PTZ (a blocker of the Cl− channel of GABAA receptors) [47] replicates the appearance and progressive intensification of convulsant activity, culminating in generalized seizure (i.e., chemical kindling) [48]. Consistent with the above reports, repeated administration of a subconvulsive dose of PTZ (40 mg/kg) every other day for twelve days resulted in kindling-like behavior in mice as evident from the seizure score, antioxidant enzyme levels, and mitochondrial complex activities. However, hesperidin (200 mg/kg) treatment significantly reduced the seizure score on the 7th, 9th, and 12th days as compared to the PTZ (control) group, showing anticonvulsant activity. Furthermore, treatment with the combination of hesperidin and L-arginine or L-NAME showed significant modulation in their neuroprotective effect suggesting NO-mediated neuroprotection. Hesperidin has been reported to contribute to the intracellular antioxidant defense systems as a powerful agent against superoxide, singlet oxygen, and hydroxyl radicals [29,30]. Consistent with the above reports, PTZ produced significant oxidative stress indicated by raised lipid peroxidation and nitrite as well as decreased glutathione, superoxide dismutase, and catalase levels. Further, hesperidin treatment for 12 days countered the PTZ-induced oxidative stress by attenuating the alteration in the antioxidant enzyme levels. These results could be attributed to the potent antioxidant action of hesperidin as a free radical scavenger, especially against superoxide, singlet oxygen, and hydroxyl radicals [29,30]. Moreover, hesperidin offers protection by terminating the lipid peroxidative side chain rather than scavenging extracellular nonlipid radicals that initiate lipid peroxidation [49]. On the other hand, treatment with combined hesperidin and L-arginine caused
further damage to the oxidative status of the animals, whereas the combination of hesperidin and L-NAME potentiated their protective effect, showing NO-dependent modulation of neuroprotective activity by hesperidin. Nitric oxide is considered to play a pivotal role in the genesis and the spread of epileptiform hyperactivity [50]. Various reports suggest that NO plays a major role in the development of kindling [6], and, recently, it has been reported that oxidative stress and nitrosative stress are the main pathological hallmarks of various neurodegenerative disorders including epilepsy [51,52]. Recent observations indicate that hyperactivation of NMDA receptors, which may be caused by NO overproduction, results in oxidative stress due to reactive species generation and mitochondrial dysfunction [18]. The superoxide in the mitochondria sometimes combines with nitric oxide to produce peroxynitrite, which further breaks down to release hydroxyl radicals, thus initiating the process of lipid peroxidation and formation of protein adducts leading to neuronal cell damage [53]. Peroxynitrite can also increase the levels of both nitric oxide and superoxide, which results in increased peroxynitrite formation. Hence, it is now an established fact that PTZ-induced kindling is associated with an increase in the amount of neuronal NOS [53,54] and mediated via the NO–cGMP pathway. Consistent with those reported in the literature, in the present study, L-arginine [substrate for NO synthesis] and L-NAME [nitric oxide synthase inhibitor (NOSi)] were employed to investigate the possible involvement of the NO mechanism in the protective effect of hesperidin against PTZ-induced kindling in mice. The study results revealed that L-arginine treatment potentiated the kindling score, whereas L-NAME treatment attenuated the rise in kindling score as compared to the PTZ-treated group (control). The neuronal activity and functional state of mitochondria are closely related [55,56]. Excess NO promotes an increase in O− 2 production
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by binding to the haem moiety of the cytochrome c oxidase, the complex IV of the electron transport chain in the mitochondrial membrane, resulting in transient inhibition of the electron flow yielding an increase in O− 2 synthesis by complexes I and III (which are relatively insensitive to NO), thus favoring the intracellular production of peroxynitrite [57]. Intense seizure activity causes massive opening of NMDA-dependent ion channels [58], which results in elevated intracellular and intramitochondrial Ca2+ leading to mitochondrial membrane depolarization, thereby resulting in energy failure and superoxide production [59]. The excessive NO and peroxynitrite produced during seizures affect oxidative phosphorylation by inhibiting the mitochondrial respiratory enzymes [17], and the resistant mitochondrial dysfunction induces apoptotic neuronal death [17,18]. Consistent with the above reports, chronic PTZ administration resulted in the reduction of the activities of mitochondrial complex as compared to the naive group, which was significantly restored following hesperidin treatment. Further, the neuroprotective effect of hesperidin was drastically augmented following its combination with L-arginine which potentiated the reduction in the mitochondrial enzyme activity induced by PTZ. On the other hand, combination of hesperidin with L-NAME potentiated their neuroprotective effect as evident from the restored mitochondrial enzyme complex activities. Hence, the significant potentiation of the protective effect of hesperidin by L-NAME and the marked reversal of the protective effect of hesperidin by L-arginine support our hypothesis about the possible involvement of the NO–cGMP pathway in the neuroprotective effect of hesperidin against PTZ-induced kindling in mice. From the passive avoidance test and the elevated plus maze task, it is evident that chronic PTZ treatment causes learning and memory impairments. These results are in conformity with those of other workers who also demonstrated cognitive impairment after administration of a subconvulsive dose of PTZ [57,60]. However, hesperidin treatment significantly protected against the impaired learning and memory performance in PTZ-kindled mice, showing its potential against PTZ-induced memory impairment. Further, administration of hesperidin in combination with L-arginine caused additional memory impairment, whereas L-NAME administration attenuated the memory impairment caused by the PTZ administration. Interestingly, administration of hesperidin with L-arginine reversed their protective effect, whereas L-NAME potentiated the memory-enhancing effect of hesperidin when they were coadministered, suggesting NO-mediated neuroprotection by hesperidin. Meanwhile, it is postulated that free radicals function as causative agents for oxidative stress and neuroinflammatory injury which may be responsible for the development of cognitive dysfunction. Therefore, agents that modulate reactive oxygen species may be useful against cognitive impairment. Thus, it is possible that hesperidin via its antioxidant potential might have ameliorated the cognitive impairment caused by chronic PTZ administration. Further, the modulation of the protective effect of hesperidin by NOS modulators supports the theory about the possible involvement of the NO–cGMP pathway in the neuroprotective effect of hesperidin. Taken altogether, chronic treatment with hesperidin showed protective effects against PTZ-induced oxidative stress, mitochondrial dysfunction, and cognitive impairment. It not only reversed the deleterious effect of L-arginine but also potentiated the protective effect of L-NAME on PTZ-induced kindling. The biochemical and mitochondrial enzyme complex activity observations clearly implicated the role of free radicals in PTZ-induced kindling and also explained the possible involvement of the nitric oxide mechanism in the protective effect of hesperidin. In conclusion, the present study demonstrates the therapeutic potential of hesperidin in epileptic conditions. The study further highlights the involvement of the nitric oxide mechanism in the neuroprotective effect of hesperidin which provides hope that hesperidin could be used as a supplement in the treatment of generalized epilepsy and related problems. However, further studies are warranted to establish its exact mechanism and clinical status in epileptic states before approaching any clinical implications.
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Possible nitric oxide mechanism in the protective effect of hesperidin against pentylenetetrazole (PTZ)-induced kindling and associated cognitive dysfunction in mice Anil Kumar ⁎, Sree Lalitha, Jitendriya Mishra Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study (UGC-CAS), Panjab University, Chandigarh 160014, India
a r t i c l e
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Article history: Received 29 March 2013 Revised 5 June 2013 Accepted 7 June 2013 Available online 9 August 2013 Keywords: Generalized epilepsy Kindling Mitochondrial dysfunction Reactive oxygen species Seizure Hesperidin
a b s t r a c t Epilepsy is a complex neurological disorder manifested by recurrent episodes of convulsive seizures, loss of consciousness, and sensory disturbances. Pentylenetetrazole (PTZ)-induced kindling primarily represents a model of generalized epilepsy. The present study has been undertaken to evaluate the neuroprotective potential of hesperidin and its interaction with nitric oxide modulators against PTZ-induced kindling and associated cognitive dysfunction in mice. The experimental protocol comprised of eleven groups (n = 6), where a subconvulsive dose of PTZ (40 mg/kg, i.p.) had been administered every other day for a period of 12 days, and seizure episodes were noted after each PTZ injection over a period of 30 min. The memory performance tests were carried out on days 13 and 14 followed by the estimation of biochemical and mitochondrial parameters. Chronic administration of a subconvulsive dose of PTZ resulted in an increase in convulsive activity culminating in generalized clonic– tonic seizures, as revealed by a progressive increase in seizure score as well as alteration in antioxidant enzyme levels (lipid peroxidation, nitrite, glutathione, super oxide dismutase, and catalase) and mitochondrial complex (I, II, and IV) activities, whereas chronic treatment with hesperidin (200 mg/kg) significantly attenuated these behavioral, biochemical, and mitochondrial alterations. Further, treatment with L-arginine (100 mg/kg) or L-NAME (10 mg/kg) in combination with hesperidin significantly modulated the protective effect of hesperidin which was significant as compared to their effects per se in PTZ-treated animals. Thus, the present study suggests a possible involvement of the NO–cGMP pathway in the neuroprotective effect of hesperidin in PTZ-kindled mice. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Epilepsy is a complex neurological disorder often characterized by abnormal synchronous neural activity. Clinical manifestations of epilepsy include recurrent episodes of convulsive seizures, loss of consciousness, memory impairment, and sensory disturbances. Epilepsy is the most frequent neurological disorder after stroke with an incidence of 0.3–0.5% and a prevalence of 5 to 10 persons per thousand in populations worldwide [1]. Alterations in several classic neurotransmitter systems such as the glutamatergic [2] and GABAergic [3] systems have been implicated in the genesis of epileptic seizures. Further, nitric oxide (NO) has been reported to be a potential neurotransmitter or retrograde messenger linked to synaptic plasticity [4], regulation of brain excitability, and triggering of seizure activity [5,6]. Further, NO has been reported to be a controversial modulator of seizure susceptibility with either anticonvulsant [7] or proconvulsant effects [8]. Nitric oxide (NO) is an important brain messenger released upon the activation of the glutamate N-methyl-D-aspartate (NMDA) receptor and ⁎ Corresponding author. Fax: +91 172 2543101. E-mail address: [email protected] (A. Kumar). 1525-5050/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yebeh.2013.06.007
subsequent Ca2+ dependent activation of neuronal nitric oxide synthase (nNOS) [9,10]. However, NO is synthesized from L-arginine and has been widely implicated in several neuroinflammatory and neurodegenerative conditions due to its prooxidant and antioxidant actions [11]. Nitric oxide activates soluble guanyl acetylase leading to the formation of cyclic guanosine monophosphate (cGMP) in the central nervous system (CNS) [12]. The elevated cGMP formation in CNS stimulates N-methyl-D-aspartate (NMDA)-type glutamate receptors [13] which leads to the activation of Ca2+-calmodulin dependent systems and the overproduction of NO by nNOS activation [14,15]. Excessive NO produced during seizures affects oxidative phosphorylation by inhibiting the mitochondrial respiratory enzymes [16], and the resistant mitochondrial dysfunction induces apoptotic neuronal death [17,18]. The mitochondrial respiratory chain is sensitive to both NO and peroxynitrite. Nitric oxide is known to slow down mitochondrial respiratory functions, and its transnitrosylation product, S-nitrosothil, reduces mitochondrial complex activities [17]. In recent times, NO modulators like L-arginine (substrate for NO synthesis), L-NAME (G-nitroL-arginine-methyl ester) [nitric oxide synthase inhibitor (NOSi)] and 7-NI (7-nitroindazole) have been widely used to study the involvement of the NO–cGMP pathway in the protective effect of several bioactive
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compounds [10,19–23]. However, several studies conducted in our laboratory demonstrated that hesperidin possesses discernible neuroprotective effects through its NO mechanism in cerebral ischemia– reperfusion injury-induced cognitive dysfunction and 3-nitro propionic acid-induced Huntington's disease-like symptoms [10,19,23]. However, the neuroprotective mechanism of hesperidin in epileptic conditions is yet to be determined. Repeated administration of subconvulsive doses of pentylenetetrazole (PTZ) continuously downregulates the sensitivity of GABAA receptors, thus favoring glutamate-aggravated seizures [24]. However, memory impairment is common in patients with epilepsy [25] and has been attributed to the dysfunction of surviving hippocampal neurons, as well as the adverse effects of antiepileptic drugs [26,27]. The pentylenetetrazole-induced kindling model offers several unique opportunities to study the mechanisms of epileptogenesis and its associated cognitive deficits as well as the behavioral manifestations of seizures [28]. However, the mechanisms underlying such impairments remain unclear. Flavonoids, which are polyphenolic compounds, can directly quench free radicals and inhibit enzymes of oxygen reduction pathways [19]. Hesperidin, a bioflavonoid richly found in oranges and lemons, has been reported to be an effective supplement in the treatment of several neurodegenerative diseases [23,29]. Above all, hesperidin has been reported to contribute to the intracellular antioxidant defense systems as a powerful agent against superoxide, singlet oxygen, and hydroxyl radicals [30,31]. Various reports suggest the involvement of the NO– cGMP pathway in the neuroprotective effect of hesperidin [15,30,31] as well as in arresting seizure genesis [31]. Hence, keeping the above reports in mind, the present study has been designed to explore the possible involvement of the nitric oxide mechanism in the neuroprotective effect of hesperidin in PTZ-induced kindling in mice.
Table 1 Grouping of animals. Name
Treatment
Group 1 Group 2 Group 3 Groups 4–5 Groups 6–7 Group 8 Group 9 Group 10 Group 11
Naive (vehicle) HES (200 mg/kg) per se PTZ (40 mg/kg, i.p.) + vehicle (0.5% w/v sod., CMC) HES (100 and 200 mg/kg, p.o.) + PTZ (40 mg/kg, i.p.) L-Arg (100) and L-NAME (10) + PTZ (40 mg/kg, i.p.) L-Arg (100) + HES (100) + PTZ (40 mg/kg, i.p.) L-Arg (100) + HES (200) + PTZ (40 mg/kg, i.p.) L-NAME (10) + HES (100) + PTZ (40 mg/kg, i.p.) L-NAME (10) + HES (200) + PTZ (40 mg/kg, i.p.)
and conducted according to the National Science Academy guidelines for the use and care of animals. 2.2. Drugs and treatment schedule Pentylenetetrazole, hesperidin (HES), L-arginine (substrate for NO synthesis), and L-NAME [nitric oxide synthase inhibitor (NOSi)] were procured from Sigma-Aldrich (St. Louis, MO, USA), and doses were selected on the basis of our previous study reports [10,32]. Hesperidin was suspended in 0.5% w/v sodium carboxymethylcellulose (CMC) and administered per oral (p.o.) for a period of 12 days. Pentylenetetrazole, L-arginine, and L-NAME were dissolved in saline and administered intraperitoneally (i.p.). L-Arginine or L-NAME was administered daily 30 min before hesperidin treatment, whereas hesperidin was administered 60 min before PTZ administration (Fig. 1). The experimental protocol comprised of eleven experimental groups containing six animals in each group (Table 1). 2.3. Induction of kindled seizures and design of the experiment
2. Methods 2.1. Animals Male LACA mice (20–30 g) bred in the Central Animal House (CAH) facility of Panjab University, Chandigarh were used in the study. The animals were housed under standard laboratory conditions maintained under a natural light and dark cycle and had free access to food and water. All the experiments were carried out between 09.00 and 15.00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) [IAEC/170–175/UIPS-29/30-8-2011]
A subconvulsive dose of PTZ (40 mg/kg, i.p.) was administered on alternating days for a period of 12 days until the animal exhibited full motor seizures. After each injection of PTZ, animals were kept in a plexiglass chamber (30 × 24 × 22 cm) with partitions in between, and seizure intensity was observed over a cutoff period of 30 min. The intensity of seizures was evaluated using a four-point scoring system: 0 = no effect, 1 = jerks, 2 = Straub's tail, and 3 = clonus. Mean kindling score was plotted against the duration of treatment as described by [33,34]. Cognitive behavior was assessed 24 h after the last PTZ injection (13th and 14th days).
Fig. 1. Experimental protocol of PTZ-induced kindling.
A. Kumar et al. / Epilepsy & Behavior 29 (2013) 103–111
2.4. Behavioral parameters 2.4.1. Passive avoidance with negative (punishment) reinforcement: step-down Learning and memory processes were evaluated in a modified twotask learning paradigm that has been validated in our laboratory. The apparatus consists of a plexiglass chamber (27 × 27 × 27 cm) with metal grid floor containing a wooden platform (10 × 7 cm) fixed at its center, which served as the shock-free zone (SFZ). The animals were given training trials wherein the animals were placed individually on SFZ, and each descent of the animal to the grid floor was punished by electric foot shock for 2 s (50 V a.c.). The animals were allowed to remain on the SFZ for 60 s thereafter. The step-down latency in seconds was recorded after the last training session without giving shock [acquisition latency (AL)]. The animals were tested again after 24 h, without giving shock, and the time taken for the mouse to step down was measured [retention latency (RL)]. A cutoff time of 600 s was chosen for the animal which did not step down from the SFZ in this period [35]. 2.4.2. Elevated plus maze paradigm Elevated plus maze is another paradigm to assess memory dysfunction, which consists of two opposite open arms (16 cm × 5 cm) and two closed arms of equal dimensions connected with a central square of 5 cm × 5 cm. Each arm contains 10-cm-high walls and the apparatus is elevated to 25 cm above the floor. The mice were placed individually at the end of one open arm, facing away from the central platform, and the time taken by the animals to move from the open arm to either of the closed arms was recorded as initial transfer latency (ITL). If the animal(s) did not enter either of the closed arms within 90 s (cutoff time), it was gently pushed towards a closed arm. The animals were allowed to explore the maze for 10 s after reaching the closed arm, and then, they were returned to their home cage. After 24 h, the total procedure was repeated, and the time taken by the animals to enter the closed arm [retention transfer latency (RTL)] was recorded [36]. 2.5. Biochemical estimations Following behavioral assessments, animals were sacrificed by decapitation; brains were rinsed in isotonic saline, weighed, and divided into two equal halves. One half was used for biochemical estimations and the other one for mitochondrial complex enzyme estimations. For biochemical analysis, 10% (w/v) tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 ×g at 4 °C for 15 min. Aliquots of supernatants were separated and used for biochemical estimations. A double beam UV–VIS spectrophotometer [UV-Pharmaspec 1700, Shimadzu (Japan)] was used for biochemical and mitochondrial assessments. 2.5.1. Measurement of lipid peroxidation The quantitative measurement of lipid peroxidation in the brain was performed according to the method of Wills. The amount of malondialdehyde (MDA), a measure of lipid peroxidation, was measured by reaction with thiobarbituric acid at 532 nm using the double beam UV–VIS spectrophotometer [UV-Pharmaspec 1700, Shimadzu (Japan)]. The values were calculated using the molar extinction coefficient of chromophore (1.56 × 105 M−1 cm−1) and expressed as nanomoles of malondialdehyde per milligram of protein [37]. 2.5.2. Estimation of nitrite The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (NO), was determined with a colorimetric assay with Greiss reagent (0.1% N-(1-naphthyl) ethylenediaminedihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid) as described by Green and his coworkers. Equal volumes of the supernatant
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and the Greiss reagent were mixed; the mixture was incubated for 10 min at room temperature. The absorbance was determined at 540 nm with the double beam UV–VIS spectrophotometer [UVPharmaspec 1700, Shimadzu (Japan)]. The concentration of nitrite in the supernatant was determined from a sodium nitrite standard curve and was expressed as micromole per liter [38]. 2.5.3. Estimation of reduced glutathione (GSH) Reduced glutathione in the brain was estimated according to the method described by Ellman and his group. Briefly, 1 ml of supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold-digested at 4 °C for 1 h. The sample was centrifuged at 1200 rpm for 15 min at 4 °C. To 1 ml of this supernatant, 2.7 ml of 0.1 M phosphate buffer (pH 8) and 0.2 ml of 5,5-dithiobis 2-nitrobenzoic acid (DTNB) were added. The yellow color developed was read immediately at 412 nm using the double beam UV–VIS spectrophotometer [UV-Pharmaspec 1700, Shimadzu (Japan)]. Results were calculated using the molar extinction coefficient of chromophore (1.36 × l04 M−1 cm−1) and were expressed as micromole GSH per milligram protein [39]. 2.5.4. Catalase estimation Catalase activity was assayed by the method of Luck, wherein breakdown of hydrogen peroxides (H2O2) is measured at 240 nm. Briefly, the assay mixture consisted of 3 ml of H2O2 phosphate buffer and 0.05 ml of supernatant of tissue homogenate (10%), and change in absorbance was recorded at 240 nm using the double beam UV– VIS spectrophotometer [UV-Pharmaspec 1700 Shimadzu, (Japan)]. The results were expressed as micromole H2O2 decomposed per milligram of protein/min [40]. 2.5.5. Superoxide dismutase activity estimation Superoxide dismutase activity was accessed according to the method described by Kono, wherein the reduction of nitrobluetetrazolium (NBT) was inhibited by the superoxide dismutase and measured at 560 nm using the double beam UV–VIS spectrophotometer [UVPharmaspec 1700, Shimadzu (Japan)]. Briefly, the reaction was initiated by the addition of the hydroxylamine hydrochloride to the mixture containing NBT and the sample. The results were expressed as unit/mg protein, where one unit of enzyme is defined as the amount of enzyme inhibiting the rate of reaction by 100% [41]. 2.5.6. Protein estimation The protein was measured by the biuret method using bovine serum albumin as standard [42]. 2.6. Mitochondrial complex enzyme estimation 2.6.1. Isolation of rat brain mitochondria The second half of the brain was used for mitochondrial isolation as described in the method of Berman and Hastings. The brains were homogenized in isolated buffer, and the homogenates were centrifuged at 13,000 ×g for 5 min at 4 °C. Pellets were resuspended in isolation buffer with ethylene glycol tetraacetic acid (EGTA) and spun again at 13,000 ×g at 4 °C for 5 min. The resulting supernatants were transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13,000 ×g at 4 °C for 10 min. Pellets containing pure mitochondria were resuspended in isolation buffer without EGTA [43]. 2.6.2. NADH dehydrogenase (complex I) activity Nicotinamide adenine dinucleotide (NADH) dehydrogenase activity was measured spectrophotometrically using the method of King and Howard. The method involves catalytic oxidation of NADH to NAD+ with subsequent reduction of cytochrome c. The reaction mixture contained 0.2 M glycylglycine buffer (pH 8.5), 6 mM NADH in 2 mM glycylglycine buffer, and 10.5 mM cytochrome c. The reaction was
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initiated by the addition of a requisite amount of solubilized mitochondrial sample followed by an absorbance change at 550 nm for 2 min [44]. 2.6.3. Succinate dehydrogenase (SDH) (complex II) activity Succinate dehydrogenase was measured spectrophotometrically as described by king. The method involves the oxidation of succinate by an artificial electron acceptor, potassium ferricyanide. The reaction mixture contained 0.2 M phosphate buffer (pH 7.8), 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. The reaction was initiated by the addition of the mitochondrial sample followed by an absorbance change at 420 nm for 2 min [45]. 2.6.4. Cytochrome oxidase (complex IV) assay Cytochrome oxidase activity was assayed in brain mitochondria according to the method of Sottocasa and his coworkers. The assay mixture contained 0.3 mM of reduced cytochrome c in 75 mM phosphate buffer. The reaction was started by the addition of solubilized mitochondrial sample, and absorbance change was recorded at 550 nm for 2 min [46]. 2.7. Statistical analysis Results were expressed as mean ± SEM. The data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test. A p-value b 0.05 was considered to be statistically significant. 3. Results 3.1. Protective effect of hesperidin and its interaction with NO modulators against pentylenetetrazole-induced kindling score The repetitive administration of a subconvulsive dose of PTZ (40 mg/kg, i.p., every other day) for a period of 12 days resulted in a gradual increase in convulsive activity (seizure score) culminating in generalized clonic–tonic seizures, as compared to the naive group. Unlike hesperidin (100 mg/kg), treatment with hesperidin (200 mg/kg) for 12 days significantly reduced the course of kindling as evidenced by the decrease in seizure score as compared to the control (PTZ-kindled) group. On the other hand, L-arginine (100 mg/kg) pretreatment for 12 days significantly worsened convulsive activity as compared to the control group, whereas its combination with hesperidin attenuated the severity of seizure activity which was significant as compared to their effects per se in the PTZ-treated animals. Conversely, L-NAME (10 mg/kg) administration for 12 days significantly suppressed seizure activity (reduced cumulative kindling score) as compared to the control group, whereas its combination with 100-mg/kg hesperidin rather than 200-mg/kg hesperidin showed further potentiation in their protective effect (increased mean kindling score), which was significant as compared to their effects per se in the PTZ-treated animals, which could be attributed to their ceiling effects. However, treatment with hesperidin (200 mg/kg) per se did not show any significant effect as compared to the naive group (Fig. 2). 3.2. Modulatory effect of L-arginine and L-NAME on protective effect of hesperidin on passive avoidance response against PTZ-kindled mice Profound impairment in learning and memory was exhibited by chronic PTZ-treated animals as evident from the decrease in stepdown latency [AL and RL] which was significant as compared to naive animals. However, unlike hesperidin (100 mg/kg), treatment with hesperidin (200 mg/kg) for 12 days significantly delayed the step-down latencies (AL and RL) in the acquisition as well as in the retention trials as compared to the PTZ-kindled (control) group. In addition, treatment with L-arginine (100 mg/kg, i.p.) caused further decrease in AL and RL which was found to be significant as compared to the control group,
Fig. 2. Effect of hesperidin and its modification by L-arginine and L-NAME against pentylenetetrazole-induced kindling score. Data are expressed as mean ± SEM. (b) p b 0.05 as compared to PTZ, (c) p b 0.05 as compared to HES (100 mg/kg), (d) p b 0.05 as compared to HES (200 mg/kg), (e) p b 0.05 as compared to L-NAME (10) + PTZ (Kruskal–Wallis ANOVA followed by Dunn's test). HES: hesperidin; L-arg: L-arginine.
indicating severe learning and memory impairment. On the other hand, L-NAME (10 mg/kg) treatment for 12 days showed superior step-down latencies (AL and RL) during acquisition and retention trials as compared to the control group. Interestingly, pretreatment with L-arginine in combination with hesperidin further worsened the step-down latencies (AL and RL), which was significant as compared to their effect per se in the PTZ-treated animals. However, pretreatment with L-NAME in combination with hesperidin (100 mg/kg) showed significant improvement in step-down latency as compared to their effects per se, whereas pretreatment with L-NAME in combination with hesperidin (200 mg/kg) did not show any significant improvement in step-down latency as compared to their effects per se, which may be attributed to their ceiling effects (Fig. 3). However, treatment with hesperidin (200 mg/kg) per se did not show any significant effect as compared to the naive group. 3.3. Modulatory effect of L-arginine and L-NAME on protective effect of hesperidin in elevated plus maze task against PTZ-induced kindling in mice Naive animals exhibited a significant difference between initial transfer latency (ITL) and retention transfer latency (RTL), signifying the acquisition and retrieval of the task. The control animals exhibited a significant increase in initial transfer latency (ITL) and retention transfer latency (RTL) as compared to the naive group, indicating profound cognitive deficit. However, unlike treatment with 100-mg/kg hesperidin, treatment with 200-mg/kg hesperidin improved RTL along with a decrease in ITL which was significant as compared to the PTZ (control) group, showing its neuroprotective potential. In addition, treatment with L-arginine (100 mg/kg, i.p.) exhibited further delays in ITL and RTL which were found to be significant as compared to the control group, indicating severe learning and memory impairment. On the other hand, L-NAME (10 mg/kg) treatment for 12 days showed superior transfer latencies (ITL and RTL) during acquisition and retention trials as compared to the control group. Interestingly, pretreatment with L-arginine in combination with hesperidin further worsened the protective effect of hesperidin, which was significant as compared to their effect per se in PTZ-treated animals. However, pretreatment with L-NAME in combination with 100-mg/kg hesperidin showed significant improvement in escape latency as compared to
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Fig. 3. Modulatory effect of L-arginine and L-NAME on the protective effect of hesperidin in passive avoidance task against PTZ-kindled mice. Data are expressed as mean ± SEM. (a) p b 0.05 as compared to AL of naive, (a') p b 0.05 as compared to RL of naive, (b) p b 0.05 as compared to AL of PTZ, (b') p b 0.05 as compared to RL of PTZ, (c) p b 0.05 as compared to AL of HES (100) + PTZ, (c') p b 0.05 as compared to RL of HES (100) + PTZ, (d) p b 0.05 as compared to AL of HES (200) + PTZ, (d') p b 0.05 as compared to RL of HES (200) + PTZ, (e) p b 0.05 as compared to AL of L-NAME (10) + PTZ, (e') p b 0.05 as compared to RL of L-NAME (10) + PTZ (ANOVA followed by Tukey's test). HES: hesperidin; L-arg: L-arginine.
their effects per se in PTZ-treated animals, whereas pretreatment with L-NAME in combination with 200-mg/kg hesperidin did not show any significant improvement in escape latencies (ITL and RTL) as compared to their effects per se in PTZ-treated animals, which may be attributed to their ceiling effects. However, treatment with hesperidin (200 mg/kg) per se was found to be nonsignificant in altering the escape latencies as compared to the naive group of animals (Fig. 4). 3.4. Effect of hesperidin, L-NAME, L-arginine, and their combinations on brain lipid peroxidation and nitrite levels in PTZ-kindled mice Repeated administration of a subconvulsive dose of PTZ induced oxidative stress as indicated by a significant rise in brain MDA and nitrite levels as compared to the naive group. However, hesperidin (200 mg/kg) treatment significantly lowered MDA and nitrite levels as compared to the PTZ (control) group. Further, L-arginine (100 mg/kg) potentiated the PTZ-induced oxidative stress as indicated by the significant elevation in MDA and nitrite levels, whereas L-NAME (10 mg/kg)
treatment significantly attenuated the oxidative stress parameters as evident from the decreased MDA and nitrite levels as compared to the PTZ (control) group. Further, treatment with hesperidin in combination with L-arginine significantly reversed the protective effect of hesperidin as compared to the control group, whereas in combination with L-NAME, hesperidin (100 mg/kg) significantly potentiated the protective effects as compared to their effect per se in PTZ-treated animals (Figs. 5A and B). However, the effects of hesperidin (200 mg/kg), in combination with L-NAME, were found to be nonsignificant as compared to its effects per se, which may be attributed to its ceiling effect. The treatment with hesperidin (200 mg/kg) per se did not show any significant effect as compared to the naive group. 3.5. Effect of hesperidin, L-arginine, L-NAME, and their combinations on glutathione, superoxide dismutase, and catalase levels in PTZ-kindled mice Chronic PTZ (40 mg/kg) challenge caused significant depletion in brain SOD, GSH, and catalase levels as compared to the naive group.
Fig. 4. Modulatory effect of L-arginine and L-NAME on protective effect of hesperidin in elevated plus maze task against PTZ-kindled mice. Data are expressed as mean ± SEM. (a) p b 0.05 as compared to ITL of naive, (a') p b 0.05 as compared to RTL of naive, (b) p b 0.05 as compared to ITL of PTZ, (b') p b 0.05 as compared to RTL of PTZ, (c) p b 0.05 as compared to ITL of HES (100) + PTZ, (c') p b 0.05 as compared to RTL of HES (100) + PTZ, (d) p b 0.05 as compared to ITL of HES (200) + PTZ, (d') p b 0.05 as compared to RTL of HES (200) + PTZ, (e) p b 0.05 as compared to ITL of L-NAME (10) + PTZ, (e') p b 0.05 as compared to RTL of L-NAME (10) + PTZ (ANOVA followed by Tukey's test). HES: hesperidin; L-arg: L-arginine.
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Fig. 5. Effect of hesperidin, L-arginine, L-NAME, and their combinations on LPO (A), nitrite (B), GSH (C), SOD (D), and catalase (E) levels in the brain of PTZ-kindled mice. Values are expressed as mean ± SEM. ANOVA followed by Tukey test. (a) p b 0.05 as compared to naive, (b) p b 0.05 as compared to PTZ, (c) p b 0.05 as compared to HES (100) + PTZ, (d) p b 0.05 as compared to HES (200) + PTZ, (e) p b 0.05 as compared to L-NAME (10) + PTZ. HES: hesperidin; L-arg: L-arginine.
Unlike 100-mg/kg hesperidin, administration of 200-mg/kg hesperidin for 12 days showed significant protection against depletion in brain SOD, GSH, and catalase levels, whereas L-arginine (100 mg/kg) caused further damage to these antioxidant enzymes which was significant as compared to the PTZ (control) group. On the other hand, L-NAME (10 mg/kg) pretreatment for 12 days showed significant protection against the depletion of the above antioxidant enzymes. On the other hand, administration of hesperidin in combination with L-arginine (100 mg/kg) further reversed the protective effect of hesperidin which was significant as compared to their effects per se. However, administration of hesperidin (100 mg/kg) in combination with L-NAME (10 mg/kg) significantly restored all the three antioxidant enzyme levels as compared to their effect per se, suggesting a possible involvement of the NO pathway (Figs. 5C, D, and E). However, the effects of hesperidin (200 mg/kg), in combination with L-NAME, were found to be nonsignificant as compared to its effects per se, which may be attributed to its ceiling effect. The treatment with hesperidin (200 mg/kg) per se did not show any significant effect as compared to the naive group.
3.6. Effect of hesperidin, L-arginine, L-NAME, and their combinations on mitochondrial complex enzyme activities in PTZ-kindled mice Chronic PTZ administration significantly impaired mitochondrial complex enzyme (I, II, and IV) activities as compared to the naive animals. However, treatment with hesperidin (200 mg/kg) and L-NAME (10 mg/kg) restored these mitochondrial complex enzyme activities, whereas treatment with L-arginine further damaged the mitochondrial enzyme (I and IV) activities, which were significant as compared to the PTZ (control) group. Further, administration of hesperidin in combination with L-arginine (100 mg/kg) further reversed the protective effect of hesperidin, whereas its combination with L-NAME (10 mg/kg) significantly restored mitochondrial complex enzyme (I and IV) activities as compared to their effects per se. However, hesperidin (200 mg/kg) treatment in combination with L-NAME was found to be nonsignificant as compared to their effects per se, which may be attributed to the ceiling effect. Treatment with hesperidin (200 mg/kg) per se for 12 days was found to be nonsignificant as compared to the naive animals (Figs. 6A, B, and C).
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Fig. 6. Effect of hesperidin, L-arginine, L-NAME, and their combinations on mitochondrial complex I (A), II (B), and IV (C) in the brain of PTZ-kindled mice. Data are expressed as mean ± SEM. ANOVA followed by Tukey's test. (a) p b 0.05 as compared to naive, (b) p b 0.05 as compared to PTZ, (c) p b 0.05 as compared to HES (100) + PTZ, (d) p b 0.05 as compared to HES (200) + PTZ, (e) p b 0.05 as compared to L-NAME (10) + PTZ. HES: hesperidin; L-arg: L-arginine.
4. Discussion A growing body of evidence suggests that repetitive administration of a subconvulsive dose of PTZ (a blocker of the Cl− channel of GABAA receptors) [47] replicates the appearance and progressive intensification of convulsant activity, culminating in generalized seizure (i.e., chemical kindling) [48]. Consistent with the above reports, repeated administration of a subconvulsive dose of PTZ (40 mg/kg) every other day for twelve days resulted in kindling-like behavior in mice as evident from the seizure score, antioxidant enzyme levels, and mitochondrial complex activities. However, hesperidin (200 mg/kg) treatment significantly reduced the seizure score on the 7th, 9th, and 12th days as compared to the PTZ (control) group, showing anticonvulsant activity. Furthermore, treatment with the combination of hesperidin and L-arginine or L-NAME showed significant modulation in their neuroprotective effect suggesting NO-mediated neuroprotection. Hesperidin has been reported to contribute to the intracellular antioxidant defense systems as a powerful agent against superoxide, singlet oxygen, and hydroxyl radicals [29,30]. Consistent with the above reports, PTZ produced significant oxidative stress indicated by raised lipid peroxidation and nitrite as well as decreased glutathione, superoxide dismutase, and catalase levels. Further, hesperidin treatment for 12 days countered the PTZ-induced oxidative stress by attenuating the alteration in the antioxidant enzyme levels. These results could be attributed to the potent antioxidant action of hesperidin as a free radical scavenger, especially against superoxide, singlet oxygen, and hydroxyl radicals [29,30]. Moreover, hesperidin offers protection by terminating the lipid peroxidative side chain rather than scavenging extracellular nonlipid radicals that initiate lipid peroxidation [49]. On the other hand, treatment with combined hesperidin and L-arginine caused
further damage to the oxidative status of the animals, whereas the combination of hesperidin and L-NAME potentiated their protective effect, showing NO-dependent modulation of neuroprotective activity by hesperidin. Nitric oxide is considered to play a pivotal role in the genesis and the spread of epileptiform hyperactivity [50]. Various reports suggest that NO plays a major role in the development of kindling [6], and, recently, it has been reported that oxidative stress and nitrosative stress are the main pathological hallmarks of various neurodegenerative disorders including epilepsy [51,52]. Recent observations indicate that hyperactivation of NMDA receptors, which may be caused by NO overproduction, results in oxidative stress due to reactive species generation and mitochondrial dysfunction [18]. The superoxide in the mitochondria sometimes combines with nitric oxide to produce peroxynitrite, which further breaks down to release hydroxyl radicals, thus initiating the process of lipid peroxidation and formation of protein adducts leading to neuronal cell damage [53]. Peroxynitrite can also increase the levels of both nitric oxide and superoxide, which results in increased peroxynitrite formation. Hence, it is now an established fact that PTZ-induced kindling is associated with an increase in the amount of neuronal NOS [53,54] and mediated via the NO–cGMP pathway. Consistent with those reported in the literature, in the present study, L-arginine [substrate for NO synthesis] and L-NAME [nitric oxide synthase inhibitor (NOSi)] were employed to investigate the possible involvement of the NO mechanism in the protective effect of hesperidin against PTZ-induced kindling in mice. The study results revealed that L-arginine treatment potentiated the kindling score, whereas L-NAME treatment attenuated the rise in kindling score as compared to the PTZ-treated group (control). The neuronal activity and functional state of mitochondria are closely related [55,56]. Excess NO promotes an increase in O− 2 production
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by binding to the haem moiety of the cytochrome c oxidase, the complex IV of the electron transport chain in the mitochondrial membrane, resulting in transient inhibition of the electron flow yielding an increase in O− 2 synthesis by complexes I and III (which are relatively insensitive to NO), thus favoring the intracellular production of peroxynitrite [57]. Intense seizure activity causes massive opening of NMDA-dependent ion channels [58], which results in elevated intracellular and intramitochondrial Ca2+ leading to mitochondrial membrane depolarization, thereby resulting in energy failure and superoxide production [59]. The excessive NO and peroxynitrite produced during seizures affect oxidative phosphorylation by inhibiting the mitochondrial respiratory enzymes [17], and the resistant mitochondrial dysfunction induces apoptotic neuronal death [17,18]. Consistent with the above reports, chronic PTZ administration resulted in the reduction of the activities of mitochondrial complex as compared to the naive group, which was significantly restored following hesperidin treatment. Further, the neuroprotective effect of hesperidin was drastically augmented following its combination with L-arginine which potentiated the reduction in the mitochondrial enzyme activity induced by PTZ. On the other hand, combination of hesperidin with L-NAME potentiated their neuroprotective effect as evident from the restored mitochondrial enzyme complex activities. Hence, the significant potentiation of the protective effect of hesperidin by L-NAME and the marked reversal of the protective effect of hesperidin by L-arginine support our hypothesis about the possible involvement of the NO–cGMP pathway in the neuroprotective effect of hesperidin against PTZ-induced kindling in mice. From the passive avoidance test and the elevated plus maze task, it is evident that chronic PTZ treatment causes learning and memory impairments. These results are in conformity with those of other workers who also demonstrated cognitive impairment after administration of a subconvulsive dose of PTZ [57,60]. However, hesperidin treatment significantly protected against the impaired learning and memory performance in PTZ-kindled mice, showing its potential against PTZ-induced memory impairment. Further, administration of hesperidin in combination with L-arginine caused additional memory impairment, whereas L-NAME administration attenuated the memory impairment caused by the PTZ administration. Interestingly, administration of hesperidin with L-arginine reversed their protective effect, whereas L-NAME potentiated the memory-enhancing effect of hesperidin when they were coadministered, suggesting NO-mediated neuroprotection by hesperidin. Meanwhile, it is postulated that free radicals function as causative agents for oxidative stress and neuroinflammatory injury which may be responsible for the development of cognitive dysfunction. Therefore, agents that modulate reactive oxygen species may be useful against cognitive impairment. Thus, it is possible that hesperidin via its antioxidant potential might have ameliorated the cognitive impairment caused by chronic PTZ administration. Further, the modulation of the protective effect of hesperidin by NOS modulators supports the theory about the possible involvement of the NO–cGMP pathway in the neuroprotective effect of hesperidin. Taken altogether, chronic treatment with hesperidin showed protective effects against PTZ-induced oxidative stress, mitochondrial dysfunction, and cognitive impairment. It not only reversed the deleterious effect of L-arginine but also potentiated the protective effect of L-NAME on PTZ-induced kindling. The biochemical and mitochondrial enzyme complex activity observations clearly implicated the role of free radicals in PTZ-induced kindling and also explained the possible involvement of the nitric oxide mechanism in the protective effect of hesperidin. In conclusion, the present study demonstrates the therapeutic potential of hesperidin in epileptic conditions. The study further highlights the involvement of the nitric oxide mechanism in the neuroprotective effect of hesperidin which provides hope that hesperidin could be used as a supplement in the treatment of generalized epilepsy and related problems. However, further studies are warranted to establish its exact mechanism and clinical status in epileptic states before approaching any clinical implications.
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