Neuroscience and Behavioral Physiology, Vol. 41, No. 7, September, 2011
Effects of Quercetin on the Severity of Chemically Induced Convulsions and 70-kDal Heat Shock Protein Content in Brain Structures in Rats L. E. Nitsinskaya,1 I. V. Ekimova,1 I. V. Guzhova,2 B. A. Feizulaev,3 and Yu. F. Pastukhov1
Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 96, No. 3, pp. 283–292, March, 2010. Original article submitted July 23, 2009. Behavioral methods were used to study the effects of the 70-kDal heat shock protein (Hsp70) expression inhibitor quercetin on convulsions and motor disorders evoked by N-methyl-D-aspartic acid (N-methylD-aspartate (NMDA)) or corazol in adult Wistar rats. These experiments showed that intraperitoneal administration of quercetin 4 h before microinjection of NMDA into the third ventricle of the brain increased the duration of the toxic component of convulsive seizures and the severity of convulsions and ataxic symptoms. The same dose and route of quercetin increased the duration of clonic and tonic convulsions but did not alter the severity of convulsive seizures or ataxia symptoms induced by intraperitoneal injection of corazol. Immunoblotting studies showed that administration of quercetin decreased the content of the inducible form of Hsp70 in the hippocampus, thalamus, and corpus callosum. These data provide evidence that the Hsp70 expression inhibitor quercetin has anticonvulsant properties. It is suggested that Hsp70 has a role in the central mechanisms regulating behavioral convulsions and motor disorders induced by NMDA and corazol in rats. KEYWORDS: heat shock protein 70, convulsions, NMDA, corazol, quercetin, rats.
Contemporary anticonvulsants produce a variety of side effects and improve patient’s status in only 30% of cases [26]. Thus, the question of extending studies directed not only to creating a new generation of anticonvulsants, but also addressing the molecular mechanisms underlying convulsive activity is currently acute. Chaperone compounds of the heat shock protein family with molecular weight 70 kDal (heat shock protein 70, Hsp70)) have potential in this regard. Hsp70 is a cellular protein and is one of the main
systems controlling the quality of proteins. Thanks to its chaperone activity, both forms of Hsp70 – the constitutive (Hsc70) and the inducible (Hsp70i) – take part in polypeptide folding and refolding processes, accelerate the translocation of proteins through membranes, and have roles in the proteolytic degradation of unstable proteins, the assembly and disassembly of protein complexes, and suppression of protein aggregation [5, 7]. In the mammalian CNS, high levels of Hsp70 are seen in normal conditions, while Hsp70i is seen on exposure to a variety of stress factors [17, 28]. The functions of these proteins in nervous tissues have received insufficient study. The location of both forms of Hsp70 in brain synapses, synaptic vesicles, neuron bodies, and dendrites suggests that it has a role in the processes of synaptic plasticity and the modification and renewal of synaptic proteins [12, 17, 28]. A number of publications have reported increased expression of Hsp70i in the hippocampus after generalized
1 I.
M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 M. Torez Prospekt, 194223 St. Petersburg, Russia; e-mail:
[email protected]. 2 Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretskii Prospekt, 194064 St. Petersburg, Russia. 3 Dagestan State University, 43-a Gadzhiev Street, 367000 Makhachkala, Russia.
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Effects of Quercetin on the Severity of Chemically Induced Convulsions convulsions provoked chemically with kainic [35] or ibotenic [33] acids, as well as on development of temporalfrontal epilepsy in humans [37]. However, the significance of increased Hsp70i expression in the brain in the mechanisms of epileptogenesis remains incompletely understood. Some authors believe that Hsp70i expression and content in different parts of the limbic system on development of epileptiform activity is linked with the neuroprotective function of this protein, as those neurons in which Hsp70i content increases remain undamaged after convulsions evoked by kainic acid or bicuculline [11, 35, 36]. Increased Hsp70i expression in transgenic mice also leads to significantly less damage to hippocampal neurons after convulsive seizures induced by kainic acid [39]. At the same tie, another study showed that increased Hsp70i expression in the hippocampus in epilepsy in humans and after kainate convulsions in rats does not have the expected neuroprotective effect in this structure [37]. The finding of such opposite effects make it difficult to come to any definitive conclusion regarding the neuroprotective potential of Hsp70 in epilepsy. On the other hand, the literature contains data providing indirect evidence that on development of epileptiform activity in the brain, not only can the Hsp70 chaperone have a neuroprotective effect, but it may be involved in the processes underlying the operation of neural networks and the regulation of convulsive activity. Studies in rats showed that thermal preconditioning, which induces massive expression of heat shock proteins in the CNS, including Hsp70, can decrease brain epileptiform activity and motor convulsions induced by chemical convulsants and audiogenic stimulation [7, 35]. Increases in Hsp70 in the rat brain obtained by injection into the CSF system have a hypnosedative effect [7–9] and decrease the severity of convulsions induced by N-methyl-D-aspartic acid (N-methyl-D-aspartate, (NMDA)) and post-convulsion motor disorders [2, 3]. Thermal preconditioning of mouse medulla oblongata slices [22] and exogenous application of Hsp70 to living rat olfactory cortex slices [6] was able to protect, at the synaptic level, the synaptic transmission of glycine, glutamate, and γ-aminobutyric acid (GABA) impaired by heat stress and to modulate NMDA-associated excitatory postsynaptic processes. Overall, these data provide grounds for further exploration of the fundamental significance of the molecular chaperones Hsp70 in controlling convulsive activity. There are therefore grounds for studying the effects of an Hsp70 blocker on the development of convulsive activity and motor disorders. The flavonoid quercetin is such a substance; this compound blocks the expression of endogenous Hsp70 at the transcriptional level [19]. In vitro experiments on prostate tumor [30] and myeloma and lymphoma [24] cell lines, as well as vascular endothelium and ascitic carcinoma [1] cells, have demonstrated the ability of quercetin to block Hsp70 expression and decrease the protective mechanisms of cells in response to various types of stress. In vivo experiments have shown that quercetin can
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completely prevent the accumulation of Hsp70 in lens epithelial cells [38] and cardiac, brain, and liver cells [27] and block the antihypotensive effect of adaptation to heat stress [27]. The study consisted of two tasks: 1) investigation of the effects of the Hsp70 expression inhibitor quercetin on NMDA- and cortisol-induced convulsions and resultant motor disorders; 2) investigation of the effects of quercetin on Hsp70i content in brain structures.
METHODS Studies were performed on adult male Wistar rats weighing 170–220 g (n = 41). All animals were kept in the animal house at a constant environmental temperature of 23 ± 1°C with free access to food and water. In the first series of experiments, convulsive seizures were induced by microinjections of the NMDA glutamate receptor agonist N-methyl-D-aspartate (NMDA, Sigma, USA) into the third ventricle via previously implanted guide cannulae at stereotaxic coordinates: 0.8 mm caudal to the bregma and 6.0 mm beneath the skull surface [31]. Surgery for implantation of guide cannulae into the third ventricle was performed under Nembutal anesthesia (i.p., 50 mg/kg). Experiments were started at least 7–10 days after cannula implantation. NMDA was given at doses of 100–150 ng in 2 μl of 0.01 M phosphate buffer (pH 7.4) using a Hamilton syringe (Hamilton, USA, volume 10 μl). Administration of these doses led to the development of convulsive seizures in only 50–60% of rats. It is known from published data that 5–15% [4] of Wistar rats develop convulsive seizures in response to sound stimuli. A uniform group of animals with increased sensitivity to NMDA was obtained by selecting rats which responded to sound stimuli (sinusoidal tones at 8 kHz and 8–100 dB) with convulsive seizures. Administration of NMDA (100 and 150 ng) to these animals elicited full convulsive seizures in 100% of cases. The experiments showed that the smallest NMDA dose eliciting complete convulsive seizures in 90% of rats was 80 ng. This NMDA dose was used in our experiments. Experimental rats of this series received the Hsp70 expression blocker quercetin (ICN, USA), a bioflavonoid, at a dose of 5 mg/kg 4 h before initiation of NMDA-induced convulsions (n = 11). Quercetin was dissolved in physiological saline supplemented with 1% Tween-20 and given i.p. in 0.2 ml. Control rats (n = 8) in this series of experiments received i.p. injections of 0.2 ml of physiological saline supplemented with 1% Tween-20 (control solution) 4 h before NMDA. Motor convulsions were recorded in terms of the following behavioral measures: the latent period of convulsions, assessed as the onset of hyperlocomotor activity of the “wild” running type, the duration of “wild” running, the duration of generalized clonic and tonic convulsions, and
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Fig. 1. Effects of quercetin on components of convulsive seizures evoked by microinjections of N-methyl-D-aspartic acid (NMDA) into the third ventricle of the brain in Wistar rats predisposed to audiogenic convulsions. Rats given i.p. injections of control solution 4 h before NMDA (n = 8) are termed control i.p. + NMDA; rats given i.p. quercetin 4 h before NMDA injections (n = 11) are identified as quercetin + NMDA. Here and in Fig. 2: *statistically significant differences between the experimental and control groups, p < 0.05. Data are given as arithmetic means ± error of the mean.
total convulsion duration. The severity of convulsive seizures was assessed in points on a modified Racine scale [34]: 0 points corresponded to no reaction, 1 to freezing, increased heart rate and respiratory rate, and orofacial convulsions, 2 to powerful psychomotor arousal, headlong “wild” running, 3 to minimal clonic convulsions without loss of posture, 4 to generalized clonic convulsions with loss of posture, 5 to tonic convulsions, and 6 to death. In the second series of experiments, convulsions were initiated by i.p. injections of corazol (pentylenetetrazol, Sigma, USA). In contrast to NMDA-induced convulsions, behavioral convulsions after corazol developed in 85–90% of Wistar rats, so selection of rats predisposed to audiogenic convulsions was not performed. Experimental rats in this series (n = 11) were given quercetin as in the first series of experiments 4 h before administration of corazol (75–80 mg/kg), while controls (n = 9) received control solution. The following parameters were recorded: the latent period of convulsive seizures, assessed in terms of the first myoclonic twitch of the skeletal musculature of the trunk, and the duration of the generalized clonic and tonic components of convulsions. Seizure severity was assessed in points using the following modified Racine scale: 0 points corresponded to no reaction, 1 to freezing, 2 to isolated myoclonic twitches, 3 to minimal convulsions with twitching of the head and forelimb muscles, 4 to generalized clonic convulsions with or without loss of posture, 5 to tonic convulsions with loss of righting reflexes and falling onto the side, and 6 to death. In the first and second series of experiments, the duration of motor disorders was determined after cessation of convulsive seizures, these consisting mainly of the symptoms of ataxia. The periods during which convulsive activity and subsequent motor disorders were followed were 50 min after NMDA and 30 min after corazol.
In the third series of experiments, the effects of quercetin on Hsp70i content in brain structures were studied by immunoblotting. Rats were decapitated 4 h after i.p. quercetin or control solution, and brains were then extracted. Brains were placed on a cold support and the following structures were extracted: the amygdala, hypothalamus, thalamus, hippocampus, piriform cortex, corpus callosum, sensorimotor cortex, cerebellum, and midbrain. Tissues were homogenized and then lysed in buffer (20 mM NaCl, 150 mM Tris-HCl pH 7.5, 2 mM EDTA, and 0.5% Triton X-100), followed by centrifugation (13,000 rpm, 10 min). The total protein concentration in the supernatant was estimated using the Bradford method [13]. Equal quantities of protein were separated using the Laemmli method [23]. After transfer of proteins to nitrocellulose membranes (Immobilon, Sigma, USA), membranes were incubated with primary monoclonal antibodies to Hsp70 (clone 2E4, diluted 1:2000) and then with secondary goat anti-mouse IgG antibodies labeled with horseradish peroxidase (Sigma, USA, diluted 1:10,000). Peroxidase reactions were visualized by amplified chemiluminescence. The consistency of total protein loading in each sample was verified (endogenous controls required for quantitative determination of Hsp70i expression) by re-incubating membranes with antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Abcam, UK, diluted 1:5000). Photographic films were dried and scanned. Immunoblotting data were evaluated in terms of optical density using the program VideoTest. Behavioral results with normal distributions were analyzed using Student’s t test, while results with non-normal distributions were analyzed using the Mann–Whitney U test. Differences in results were regarded as statistically significant at a significance level of p < 0.05. Data were analyzed using Statistica 6.0.
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Fig. 2. Effects of quercetin on components of convulsive seizures evoked by i.p. corazol in Wistar rats. Rats receiving i.p. injections of control solution (n = 9) 4 h before corazol are designated i.p. control + corazol; rats given i.p. injections of quercetin (n = 11) 4 h before injections of corazol are designated quercetin + control.
RESULTS Our studies showed that administration of NMDA to rats of the control group led to the development of “wild” running with a latent period of 7 ± 3.9 sec. The “wild” running phase ended in 87% of animals with generalized clonic convulsions, which in 25% of rats developed into a phase of tonic convulsions. The severity of convulsive seizures was 4 points. Death occurred in 12% of rats with convulsions. After convulsive seizures, animals showed motor disorders with symptoms of ataxia consisting mainly of unusual, clumsy, and sudden movements, some animals also displaying loss of balance while rising on the hindlimbs and falling onto their sides. Administration of quercetin was found to increase the total duration of NMDA-induced convulsions (Fig. 1) and their severity (severity increased to 5 points). Increases in the total duration of seizures occurred predominantly as a result of a 4.3-fold (p < 0.05; Fig. 1) increase in the duration of the tonic component of convulsions as compared with controls. The severity of convulsive seizures increased because of a 30% increase in the proportion of rats with life-threatening tonic convulsions. However, lethality among the animals of this group did not change. After cessation of convulsive seizures, there was a 62% increase in the proportion of rats with symptoms of ataxia and a 7-fold increase in their duration (p < 0.05). In most cases, administration of quercetin increased the symptoms of ataxia as compared with controls. Almost all the animals showed barreling movements or were completely unable to move. Administration of corazol to rats of the control group led to the development of convulsive seizures with a latent period of 43 ± 1.6 sec, starting with orofacial convulsions and developing into minimal convulsions. Minimal convulsions were apparent in the animals as individual twitches of the skeletal musculature of the trunk. In 100% of cases this was then followed by a clonic convulsion phase which in
80–90% of cases progressed to a tonic convulsion phase. Seizure severity on the scale was 6 points. After the end of convulsive seizures, the animals showed motor disorders in the form of ataxia which, in contrast to NMDA-induced convulsions, was characterized by the complete inability to move. Death occurred in 90% of rats of this group 20 ± 2 min from the moment at which the convulsant was given. After corazol convulsions, as after NMDA-induced convulsions, quercetin led to a 1.4-fold increase in the duration of tonic convulsions (p < 0.05) (Fig. 2) but had no effect on the latent period of convulsive seizures. In contract to NMDA-induced convulsions, quercetin affected the duration of clonic convulsions, increasing this by a factor of 2 (p < 0.05, Fig. 2). Lethality was not altered among the animals of this group, though it occurred 7–8 min earlier than in the control group. Studies of brain Hsp70i content showed that in controls, the Hsp70i level in the sensorimotor cortex was lower than in the other structures investigated (Fig. 3). The most marked changes in Hsp70i levels 4 h after i.p. quercetin were seen in the hippocampus, corpus callosum, and thalamus. Hsp70i content in these structures decreased or became undetectable as compared with controls (Fig. 3). In the amygdala, hypothalamus, piriform cortex, sensorimotor cortex, cerebellum, and midbrain, Hsp70i contents did not change as compared with controls.
DISCUSSION The results obtained here showed that the Hsp70 expression inhibitor quercetin decreased Hsp70i levels in the hippocampus, thalamus, and corpus callosum and had proconvulsant effects in models of convulsions evoked by hyperactivation of NMDA glutamate receptors (administration of NMDA) and blockade of types A and B γ-aminobutyric acid receptors (administration of corazol) in rats.
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Fig. 3. Effects of quercetin on contents of the inducible form of 70-kDal heat shock protein in brain structures in Wistar rats. Rats receiving i.p. injections of control solution 4 h before decapitation are identified as Q. The endogenous control protein glyceraldehyde-3-phosphate dehydrogenase is designated GAPDH.
NMDA-induced convulsions are known to provide an experimental analog of generalized clonic-tonic epilepsy, while corazol convulsions provide a model of absences and generalized tonic-clonic epilepsy in humans [32]. In the NMDA-induced convulsions model, quercetin increased the overall duration and severity of convulsive seizures, mainly affecting the tonic component of convulsions. The duration of tonic convulsions and the proportion of animals with the tonic component of convulsions increased significantly after quercetin. In the corazol convulsions model, quercetin affected both clonic and tonic convulsions, increasing their duration but not significantly altering the proportion of animals with the clonic and tonic components of convulsions. In addition, blockade of Hsp70 expression with quercetin led to increases in the motor dysfunction (ataxia) seen after the cessation of NMDA-induced convulsions. The decreases in Hsp70i content in the brain structures studied here 4 h after administration of quercetin appear to be associated with the fact that this agent selectively suppresses the transcription of heat shock protein genes, which is mediated by activation of transcription factor HSF1 [19]. This property is also characteristic of the inhibitory effect of quercetin on the induction of Hsp70i in the brain. Increases in the dura-
tions of the components of convulsive seizures and postconvulsive motor disorders seen after inhibition of Hsp70 expression by quercetin provide evidence that the chaperone Hsp70 may be involved in the central mechanisms regulating convulsive states. The significant decreases in the Hsp70i levels in the hippocampus and thalamus, whose neurons and neural networks are involved in the mechanisms initiating and maintaining epileptiform activity in the brain [10, 14], lead to increases in the impairments to synaptic processes in these structures induced by NMDA or corazol and the consequent increase in convulsive seizures and ataxia. This suggestion is based on studies reported by a number of authors [6, 22] showing that Hsp70, which has chaperone activity, is able to modulate synaptic transmission and influence the neurotropic and plastic properties of neural structures. Thermal preconditioning, which evokes massive Hsp70 expression, had a protective effect on synaptic processes in subsequent heat shock in Drosophila larvae [21] and in mouse medulla oblongata slices [22]. Application of recombinant Hsp72 protected the synaptic transmission of GABA, glycine, and glutamate in acute heat stress induced by modulating the presynaptic mechanisms of release of these transmitters [22]. Another study per-
Effects of Quercetin on the Severity of Chemically Induced Convulsions formed on rat olfactory cortex slices demonstrated the ability of exogenous Hsp70 to produce dose-dependent increases or, conversely, decreases in the amplitude of NMDA-associated excitatory postsynaptic potentials [6]. Furthermore, the literature contains data providing evidence that Hsp70 has a role in the modification and release of synaptic proteins, clathrin-dependent exocytosis of synaptic vesicles [12], and postsynaptic receptor internalization [28]. On the other hand, inhibition of Hsp70 synthesis by quercetin can influence not only synaptic processes in the brain, but also the induction of the proinflammatory cytokine interleukin-6 (IL-6), which published data show is involved in the mechanisms of epileptogenesis. A number of studies have shown that IL-6 levels increase in the cerebrospinal fluid after convulsive seizures not associated with infection or brain trauma in patients [20]. Increases in IL-6 levels have been observed in the hippocampus in rodents after limbic convulsions [25]. Increases in IL-6 contents in the brain in transgenic animals due to intranasal administration of IL-6 to mice led to the development of spontaneous convulsions [16] and increased the severity of corazol convulsions [20]. At the same time, intracellular Hsp70 is known to be involved in regulating the synthesis of proinflammatory cytokines. Studies using immunocompetent cells (monocytes and macrophages) and in vivo experiments have shown that increases in the production of inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, IL-6) induced by lipopolysaccharide were significantly suppressed by increases in endogenous Hsp70i expression and after use of highly purified (to remove contaminants) recombinant Hsp70i [18, 29]. It is possible that the decrease in Hsp70i content in the brain after administration of quercetin leads to activation of IL-6 and nuclear factor κB synthesis, which are responsible for triggering the apoptosis cascade [15]. These properties may also explain the proconvulsant effects of quercetin. The data obtained here on the proconvulsant effects of the chaperone heat shock protein expression inhibitor quercetin in experimental models of epilepsy provide evidence of the possible involvement of Hsp70 in the central mechanisms regulating behavioral convulsions and postconvulsion motor disorders. This study was supported by the Russian Foundation for Basic Research (Grant No. 05-04-49356) and the “Basic Sciences – Medicine” Program of the Presidium of the Russian Academy of Sciences. REFERENCES 1.
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