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Nicotine versus 6-hydroxy-l -nicotine against chlorisondamine induced memory impairment and oxidative stress in the rat hippocampus
Biomedicine & Pharmacotherapy 86 (2017) 102–108
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Original article
Nicotine versus 6-hydroxy-L-nicotine against chlorisondamine induced memory impairment and oxidative stress in the rat hippocampus Lucian Hritcu* , Radu Ionita, Diana Elena Motei, Cornelia Babii, Marius Stefan, Marius Mihasan* Department of Biology, Alexandru Ioan Cuza University of Iasi, Bd. Carol I, No. 11, 700506, Romania
A R T I C L E I N F O
Article history: Received 3 November 2016 Received in revised form 28 November 2016 Accepted 4 December 2016 Keywords: Arthrobacter nicotinovorans Nicotine 6-Hydroxy-L-nicotine Chlorisondamine Memory Oxidative stress
A B S T R A C T
6-Hydroxy-L-nicotine (6HLN), a nicotine derivative from nicotine degradation by Arthrobacter nicotinovorans pAO1 strain was found to improve behavioral deficits and to reverse oxidative stress in the rat hippocampus. Rats were given CHL (10 mg/kg, i.p.) were used as an Alzheimer’s disease-like model. The nicotine (0.3 mg/kg) and 6HLN (0.3 mg/kg) were administered alone or in combination in the CHL-treated rats. Memory-related behaviors were evaluated using Y-maze and radial arm-maze tests. The antioxidant enzymes activity and the levels of the biomarkers of oxidative stress were measured in the hippocampus. Statistical analyses were performed using two-way ANOVA and Tukey’s post hoc test. F values for which p < 0.05 were regarded as statistically significant. CHL-caused memory deficits and oxidative stress enhancing were observed. Both nicotine and 6HLN administration attenuated the cognitive deficits and recovered the antioxidant capacity in the rat hippocampus of the CHL rat model. Our results suggest that 6HLN versus nicotine confers anti-amnesic properties in the CHL-induced a rat model of memory impairment via reversing cholinergic function and decreasing brain oxidative stress, suggesting the use of this compound as an alternative agent in AD treatment. © 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Alzheimer disease (AD) is a neurodegenerative affliction characterized by memory impairment [1] associated with three major changes in the brain [2]: i) the extra- and intracellular plaques deposits of b-amyloid peptide, (ii) the neurofibrillary tangles and (iii) death of forebrain cholinergic neurons and a significant decrease in acetylcholine (ACh) levels [3]. The search for neuroprotective therapeutics for AD has been a long time dominated mainly by the amyloid and the tau hypothesis. Unfortunately, both approaches failed so far to provide any efficient therapeutic strategy [2]. The involving of nicotinic acetylcholine receptors (nAChR) subtypes a7 and a4b2 in AD pathogenesis [4] has led to the proposal of a new approach [5]. The memory deficits and non-cognitive symptoms could be improved through using of the nAChR modulators to increase the availability of receptors for ACh and to overcome the death of the forebrain cholinergic neurons.
* Corresponding authors. E-mail addresses: [email protected] (L. Hritcu), [email protected] (M. Mihasan). http://dx.doi.org/10.1016/j.biopha.2016.12.008 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
Nicotine is considered to be an agonist of nAChR. Its high potency as a cognition-enhancing agent and AD therapeutic strategy [6] has been explained not only by its ability to bind and modulate nAChRs but also through its anti-oxidant effects at low concentrations [7] and recently, through its ability to interact with b-amyloid peptide [8]. However, due to negative effects on human organs such as lungs [9] and bad publicity related to smoking[10], nicotine doesn’t impose as a feasible therapeutic agent for AD. The crystal structure determination of the nicotinic receptor binding domain homolog (nAchBP) with bound nicotine [11] followed by the recent solving of the a7 nAChRs structure [12] has spurred the interest of several academic and pharmaceutical laboratories in the possibility that new nicotinic drugs could be designed. From this point of view, nicotine derivatives are ideal candidates, offering a wide array of possibilities. The difficulty resides, firstly in the identification of molecules which have the beneficial effects of nicotine, but elude its side effects [13] and, secondly, in providing simple and reliable methods for production and isolation of the identified molecules. In this context, Arthrobacter nicotinovorans pAO1 strain through metabolizing nicotine could deliver new nicotine-derivatives with unexplored biotechnological potential.
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6-Hydroxy-L-nicotine (6HLN) is a metabolic intermediate found in the nicotine catabolic pathway encoded by Arthrobacter nicotinovorans pAO1 [14]. 6HLN resulted by a hydroxylation reaction mediated by nicotine-dehydrogenase (NDH, EC 1.5.99.4), a multimeric enzyme encoded by the ndhL, ndhM, and ndhS genes of pAO1. The compound is further dehydrogenated by 6-hydroxy-L-nicotine oxidase (6HLNO, EC 1.5.3.5) with the formation of 6-hydroxy-methylmyosmine [15]. 6HLN was repeatedly reported to temporarily accumulate in the growth medium of Arthrobacter nicotinovorans pAO1 [16,17]. Previously, we have shown that 6-hydroxy-L-nicotine (6HLN) a natural product obtained from nicotine degradation by Arthrobacter nicotinovorans enhanced behavioral response and consequently decrease oxidative stress generation in the rat hippocampus of the scopolamine treated rats [18]. Here, we further hypothesized that 6HLN versus nicotine ameliorates chlorisondamine (CHL) induced cognitive impairment through decreasing of the hippocampal oxidative stress. CHL is a known nAChR antagonist that can cross the blood-brain barrier and exerts a nicotinic blockade that lasts for months [19]. The pharmacological effects of both nicotine and 6HLN on CHL-induced memory impairment associated with brain oxidative stress were investigated, and also their possible mechanism of action underlying these effects. 2. Materials and methods 2.1. Strains and growth conditions Arthrobacter nicotinovorans pAO1+ (strain ATCC 4991) was grown on citrate medium supplemented with 3 mM nicotine [20] on a rotary shaker at 28 C/190 rpm. 100 ml medium in a 500 ml flask were inoculated with 1 ml 24 h old preculture and allowed to grow 10 h before harvest producing about 8 mg of 6HLN. 2.2. General analytical methods Quantitative data on nicotine metabolites were obtained by high-performance liquid chromatography (HPLC) analysis by comparing the retention times and absorption spectra with literature data [21]. Supplementary, chemical synthesized 6HLN (a kind gift from Prof. Dr. Roderich Brandsch – Institute of Biochemistry and Molecular Biology, Albert-Ludwigs-University of Freiburg, Germany) was used as standard. HPLC analysis was performed as described by Tang et al. [22] with slight modifications. A Shimadzu Prominence UPLC system equipped with a Machery-Nagel Nucleodur RP C18 ec column (150 4.6 mm, particle size 3 mm) was used for separation of nicotine metabolites from the growth medium. The mobile phase was a mixture of 1 mM H2SO4: methanol (75:25 v/v) at a flow rate of 1 ml/min. The separation was performed at 30 C using isocratic elution and 6HLN levels were monitored at 290 nm. 2.3. Animals A total number of thirty male Wistar rats (3–4-month-old) weighing 250 10 g at the start of the experiment was used. The animals were housed in a temperature and light-controlled room (22 C, a 12 h light/dark cycle starting at 08:00 h) and were fed and allowed to drink water ad libitum. The experiments were conducted in the quiet laboratory between hours of 10:00 h– 16:00 h. Rats were treated in accordance with the guidelines of animal bioethics from the Act on Animal Experimentation and Animal Health and Welfare from Romania and all procedures were in compliance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the
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protection of animals used for scientific purposes. This study was approved by the Committee on the Ethics of Animal Experiments of the Alexandru Ioan Cuza University of Iasi, Faculty of Biology (Permit Number: 2197) and also, efforts were made to minimize animal suffering and to reduce the number of animals used. 2.4. Drugs ()Nicotine (free base, Nic), 6HLN and chlorisondamine (CHL) were diluted in an isotonic solution (0.9% NaCl). All reagents, except 6HLN, were purchased from Sigma-Aldrich, Germany. 2.5. Drug administration The rats were divided into six different groups (five animals per group) as follows: (1) the Control group received saline treatment (0.9% NaCl); (2) the ()Nicotine (Nic, free base, 0.3 mg/kg)-alonetreated group, as positive control; (3) the Chlorisondamine (CHL, 10 mg/kg)-alone-treated group, as negative control; (4) the 6-hydroxy-L-nicotine (6HLN, 0.3 mg/kg)-alone-treated group, as positive control; (5) the CHL-treated group received Nic treatment (CHL + Nic) and (6) the CHL-treated group received 6HLN treatment (CHL + 6HLN). The drug doses used in this experiment were chosen since they have been demonstrated by our group to provide significant effects on memory formation and antioxidant profile [18,23]. CHL was injected 24 h before the experiments. Also, Nic and 6HLN were injected alone and in the CHL treated-groups, 30 min before the behavioral testing. 2.6. Y-maze task Spontaneous alternation was examined using a three-arm Ymaze (35 25 10 cm). Rats were individually placed at the end of one arm, facing the center, and allowed to move freely through the maze for 8 min. An arm entry was counted when the hind paws of the rat were completely within the arm. The alternation score (%) of each rat was defined as the ratio of the actual number of alternations to the possible number (defined as the total number of arm entries minus two) multiplied by 100 [24,25]. At the beginning of each trial the maze was thoroughly cleaned with 10% ethanol. 2.7. Radial arm-maze task The radial arm-maze used in the present study consisted of 8 arms, numbered from 1 to 8 (48 12 cm), extending radially from a central area (32 cm in diameter). The apparatus was placed 40 cm above the floor and surrounded by various extra-maze visual cues placed at the same position during the study. Prior to the performance of the maze task, the animals were kept on restricted diet and body weight was maintained of 85% of their free-feeding weight over a week period, with water being available ad libitum. During the pre-training session (4 days), rats were individually placed in the center of the maze and were allowed to explore to habituate them to the maze for 5 min. Initially, the food pellets (50 mg, Bioserve Inc.) were placed throughout the maze but gradually restricted to the end of each arm in the food cup. Five target baited arms (nos. 1, 2, 4, 5, and 7) were selected which remained constant for a given rat throughout training. The other three arms (nos. 3, 6, 8) were never baited. In the training session, starting with the day fifth to day twelfth, the animals were trained by performing one trial per day for seven consecutive days. Each rat was individually placed in the center of the maze and subjected to working and reference memory tasks. An arm entry was counted when a rat ate the bait or reached the end of an arm. Measures were made of the number of working memory errors (entering a baited arm, but previously entered) and
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reference memory errors (entering an arm that was never baited). Criterion performance was defined as consumption of all 5 baits or until 5 min had elapsed. Reference memory is regarded as a longterm memory for information that remains constant over repeated trials (memory for the positions of reinforced arms), whereas working memory is considered a short-time memory in which the information to be remembered changes in every trial (memory for the positions of arms that had already been visited in each trial) [18,26]. 2.8. Oxidative stress assay Shortly after completion of the behavioral tests, all rats were deeply anesthetized (using sodium pentobarbital, 100 mg/kg b.w., i.p., Sigma-Aldrich, Germany), decapitated and whole brains were removed. The hippocampi were carefully excised. Each of the hippocampal samples was weighted and homogenized (1:10) with Potter Homogenizer coupled with Cole-Parmer Servodyne Mixer in ice-cold 0.1 M potassium phosphate buffer (pH 7.4), 1.15% KCl. The homogenate was centrifuged (15 min at 960 g) and the supernatant (200 ml) was used for assays of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) specific activities, the total content of reduced glutathione (GSH) and malondialdehyde (MDA) level. 2.8.1. Determination of hippocampal SOD activity The activity of superoxide dismutase (SOD, EC 1.15.1.1) was assayed by monitoring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). Each 1.5 ml reaction mixture contained 100 mM TRIS/HCl (pH 7.8), 75 mM NBT, 2 mM riboflavin, 6 mM EDTA, and 200 ml of supernatant. Monitoring the increase in absorbance at 560 nm followed the production of blue formazan. One unit of SOD is defined as the quantity required to inhibit the rate of NBT reduction by 50% as previously described by Winterbourn et al. [27]. 2.8.2. Determination of hippocampal GPX activity Glutathione peroxidase (GPX, E.C. 1.11.1.9) activity was analyzed by a spectrophotometric assay. A reaction mixture consisting of 1 ml of 0.4 M phosphate buffer (pH 7.0) containing 0.4 mM EDTA, 1 ml of 5 mM NaN3, 1 ml of 4 mM GSH, and 200 ml of supernatant was preincubated at 37 C for 5 min. Then 1 ml of 4 mM H2O2 was added and incubated at 37 C for further 5 min. The excess amount of GSH was quantified by the DTNB method as previously described by Sharma and Gupta [28]. One unit of GPX is defined as the amount of enzyme required to oxidize 1 nmol GSH/min. 2.8.3. Determination of hippocampal CAT activity Catalase (CAT, EC 1.11.1.6) activity was assayed following the method of Sinha [29]. The reaction mixture consisted of 150 ml phosphate buffer (0.01 M, pH 7.0), 200 ml supernatant. The reaction was started by adding 250 ml H2O2 0.16 M, incubated at 37 C for 1 min and the reaction was stopped by addition of 1 ml of dichromate: acetic acid reagent. The tubes were immediately kept in a boiling water bath for 15 min and the green colour developed during the reaction was read at 570 nm on a spectrophotometer. Control tubes, devoid of the enzyme, were also processed in parallel. The enzyme activity is expressed as mmol of H2O2 consumed/min/mg protein.
a final volume of 1.5 ml with distilled water and absorbance was read in a spectrophotometer at 412 nm and results were expressed as mg GSH/mg protein. 2.8.5. Determination of hippocampal MDA level Malondialdehyde (MDA), which is an indicator of lipid peroxidation, was spectrophotometrically measured by using the thiobarbituric acid assay as previously described by Ohkawa et al. [31]. 200 ml of supernatant was added and briefly mixed with 1 ml of 50% trichloroacetic acid in 0.1 M HCl and 1 ml of 26 mM thiobarbituric acid. After vortex mixing, samples were maintained at 95 C for 20 min. Afterward, samples were centrifuged at 960 g for 10 min and supernatants were read at 532 nm. A calibration curve was constructed using MDA as standard and the results were expressed as nmol/mg protein. 2.8.6. Estimation of protein concentration Estimation of protein was done using a bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich, Germany). The BCA protein assay is a detergent-compatible formulation based on BCA for the colorimetric detection and quantification of total protein, as previously described by Smith et al. [32]. 2.9. Statistical analysis The group differences in the Y-maze and the radial armmaze tasks and the results for biochemical parameter assay were statistically analyzed by two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using GraphPad Prism 6 software for Windows, La Jolla, CA, USA. In order to evaluate differences between groups in the radial arm-maze task, separate repeated-measures ANOVA were calculated on the number of working memory errors and the number of reference memory errors with group (Control, Nic, CHL, 6HLN, CHL + Nic and CHL + 6HLN) as between-subject factor and days (1–7) as within-subjects factors. Data are expressed as mean standard errors of mean (S.E.M). F values for which p < 0.05 were regarded as statistically significant. Pearson’s correlation coefficient and regression analysis were used in order to evaluate the relation between behavioral measures, the antioxidant defense and lipid peroxidation. 3. Results 3.1. HPLC analyses The HPLC method described by Tang et al. [22] and modified as stated in the methods section was successfully used to separate the growth medium of Arthrobacter nicotinovorans. A typical chromatogram of the growth medium from which nicotine was completely consumed is shown in Fig. 1a. Three peaks could be observed at 290 nm and were identified based on their absorption spectra. The peak with RT = 5.5 is nicotine-blue (4,5,40 ,50 -tetrahydroxy-3,30 -diazadiphenoquinone-(2,20 )), the main product of the nicotine catabolic pathway in this bacteria. The peak with RT = 3.5 is unknown as we were unable to find any similar spectra in the literature. The peak with RT = 2.3 is 6HLN. Fig. 1b depicts the purity and spectrum of the 6HLN preparations used for animal treatments. 3.2. Effect of nicotine and 6HLN on memory performance
2.8.4. Total hippocampal content of reduced GSH Glutathione (GSH) was measured following the method of Fukuzawa and Tokumura [30]. 200 ml of supernatant was added to 1.1 ml of 0.25 M sodium phosphate buffer (pH 7.4) followed by the addition of 130 ml DTNB 0.04%. Finally, the mixture was brought to
In the Y-maze test, significant overall differences between all groups (F(5, 24) = 28.88, p < 0.0001) on the spontaneous alternation percentage were evidenced (Fig. 2a). CHL significantly decrease the spontaneous alternation percentage as compared
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3.3. Effect of nicotine and 6HLN on oxidative stress
Fig. 1. HPLC analysis of (a) Arthrobacter nicotinovorans growth medium upon nicotine depletion and (b) 6HLN preparations used for animal treatments. RT = 5.5 – nicotine-blue (4,5,40 ,50 -tetrahydroxy-3,30 -diazadiphenoquinone-(2,20 )); RT = 3.5 – unknown metabolite; RT = 2.3–6 HLN. Inlay – 6HLN spectrum.
to control group (p < 0.0001). Rats were given CHL and received nicotine and 6HLN, but especially 6HLN displayed significant differences (p < 0.0001) for the spontaneous alternation percentage as compared to the CHL-alone-treated group. The results suggest that both nicotine and 6HLN, but especially 6HLN, are capable of improving spontaneous alternation in the CHL-induced cholinergic dysfunction. In the radial arm-maze test, significant overall differences between all groups (F(5, 24) = 7.11, p < 0.0001) for the working memory errors were evidenced (Fig. 2b). CHL significantly increased working memory errors as compared to control group (p < 0.01). The CHL rats with both nicotine and 6HLN, but especially 6HLN, showed decreased working memory errors (p < 0.001) as compared to the CHL-alone-treated group. Moreover, repeated-measures ANOVA revealed a significant time difference (F(6168) = 18.03, p < 0.0001), a significant group difference (F(5168) = 8.68, p < 0.0001) and a significant time and group interaction (F(30,168) = 5.42, p < 0.0001) for working memory errors. ANOVA revealed significant overall differences between all groups (F (5, 24) = 10.86, p < 0.0001) for reference memory errors (Fig. 2c). CHL significantly increased reference memory errors as compared to control group (p < 0.01). The CHL rats with both nicotine and 6HLN, but especially 6HLN, showed decreased reference memory errors (p < 0.00001) as compared to CHL-alone-treated rats. Additionally, repeated-measures ANOVA revealed a significant time difference (F(6168) = 17.47, p < 0.0001), a significant group difference (F(5168) = 13.45, p < 0.0001) and a significant group difference (F(30,168) = 20.47, p < 0.0001) for reference memory errors.
Biochemical analyses of SOD, GPX and CAT specific activities along with reduced GSH and MDA levels estimated in the hippocampal homogenates showed significant overall differences between all groups for SOD activity (F(5,24) = 7.56, p < 0.001) (Fig. 3a), for GPX activity (F(5,24) = 6.21, p < 0.001) (Fig. 3b), for CAT activity (F(5,24) = 22.21, p < 0.0001) (Fig. 3c), for the total content of reduced GSH (F(5,24) = 43.49, p < 0.0001) (Fig. 3d) and for MDA level (F(5,24) = 5.98, p < 0.001) (Fig. 3e). CHL treatment significantly decreased (p < 0.01) the antioxidant enzymes activity (SOD, GPX, and CAT), and GSH reduced level while the MDA level was over normal levels. Rats were given CHL and received nicotine and 6HLN, but especially 6HLN, exhibited a significant increase of the SOD, CAT, and GPX specific activities, and the total content of reduced GSH together with a decreased MDA level as compared to the CHL-alone treated group (p < 0.01). Importantly, when linear regression was determined, significant correlations between the spontaneous alternation percentage vs. SOD (n = 30, r = 0.717, p < 0.001), spontaneous alternation percentage vs. GPX (n = 30, r = 0.507, p < 0.01), spontaneous alternation percentage vs. CAT (n = 30, r = 0.758, p < 0.0001), spontaneous alternation percentage vs. MDA (n = 30, r = 0.795, p < 0.0001), working memory errors vs. SOD (n = 30, r = 0.799, p < 0.0001), working memory errors vs. GPX (n = 30, r = 0.754, p < 0.0001), working memory errors vs. CAT (n = 30, r = 0.815, p < 0.0001), working memory errors vs. MDA (n = 30, r = 0.754, p < 0.0001), reference memory errors vs. SOD (n = 30, r = 0.537, p < 0.01), reference memory errors vs. CAT (n = 30, r = 0.525, p < 0.01), reference memory errors vs. MDA (n = 30, r = 0.598, p < 0.001) were observed. Additionally, significant correlations were observed between the SOD vs. MDA (n = 30, r = 0.683, p < 0.001), GPX vs. MDA (n = 30, r = 0.508, p < 0.01) and CAT vs. MDA (n = 30, r = 0.656, p < 0.001) when linear regression was determined. 4. Discussion In this study, both nicotine and 6HLN administered alone or in combination with CHL-treated rats causes behavioral effects as memory-enhancing based on specific behavioral tests such as Ymaze and radial arm-maze were investigated. It has been documented that when administered intracerebroventricularly or systemic, CHL persistent block for a period of several weeks the central nicotinic responses, examined either behaviorally or in vitro [33,34]. In addition, the main mechanism by which CHL exerts its long-lasting blockade of nAChR in the central nervous system remains elusive [35]. Previously, we used in our studies CHL as an experimental tool to study the involvement of nAChR in memory processes [23]. Numerous studies reported that nicotine and nicotinic agonists improved memory performance in experimental rats [23,36–39]. It has been suggested that using of nAChR antagonists in an animal model of memory impairment has clinical relevance because it models the functional effect of nicotinic receptor loss [40]. Studies using a mouse model of AD have shown that early AD stage is marked by cholinergic hypofunction, neuronal marker loss, and decreased nAChR density from the cortex and hippocampus [41]. It has been reported that nicotine can modulate several different types of hippocampus-dependent learning [42], such as Y-maze and radial arm-maze tasks used in the present study. The current study found that both nicotine and 6HLN, but especially 6HLN alone treatment, enhanced spatial memory formation as compared to control group in Y-maze task. However, administration of 6HLN as compared to nicotine in the CHL-treated rats significantly increased the spontaneous alternation
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Fig. 2. Effects of the nicotine (Nic, 0.3 mg/kg b.w., i.p.) and 6HLN (0.3 mg/kg b.w., i.p.) administration in the Y-maze task on the spontaneous alternation% (a), on the working memory errors (b) and the reference memory errors (c) during 7 days training in radial arm-maze task in the chlorisondamine (CHL)-treated rats. Values are mean SEM (n = 5 animals per group). For Turkey’s post-hoc analyses- #CHL vs. CHL + Nic: p < 0.001 and ##CHL vs. CHL + 6HLN: p < 0.0001 (a), #CHL vs. CHL + Nic: p < 0.01 and ##CHL vs. CHL + 6HLN: p < 0.001 (b) and #CHL vs. CHL + Nic: p < 0.0001 and ##CHL vs. CHL + 6HLN: p < 0.00001 (c).
percentage as compared to CHL alone treated rats, suggesting that nicotine and 6HLN selectively improved memory formation of CHL-treated rats within the Y-maze task. Furthermore, both nicotine and 6HLN, but especially 6HLN, significantly enhanced working memory, as evidenced by decreasing the number of working memory errors in the radial arm-maze task. CHL-treated rats injected with 6HLN showed a significant improvement of working memory as compared to CHL-alone treated rats. Moreover, in the CHL-treated rats, 6HLN significantly improved reference memory, as evidenced by decreasing the number of reference memory errors in the radial arm-maze task. Consisting of
our previous study [18], 6HLN plays an important role in the restoration of spatial memory, especially in the rats with cholinergic deficits. There is strong evidence that antioxidants exhibit the neuroprotective effects on the brain function [43]. In this study, the antioxidative status in the rat was evaluated using the SOD, CAT, and GPX specific activities, as well as the total content of reduced GSH and MDA level in rat hippocampus as indices. Our results revealed that both nicotine and 6HLN, but especially 6HLN-alone treatment, exhibited antioxidant effects as evidenced by significant increase of SOD, GPX, and CAT specific activities along with decreased
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Fig. 3. Effects of the nicotine (Nic, 0.3 mg/kg b.w., i.p.) and 6HLN (0.3 mg/kg b.w., i.p.) administration on SOD (a), GPX (b), and CAT (c) specific activities, on reduced GSH (d) and MDA (e) levels in the chlorisondamine (CHL)-treated rats. Values are mean SEM (n = 5 animals per group). For Turkey’s post-hoc analyses- #CHL vs. CHL + Nic: p < 0.01 and ## CHL vs. CHL + 6HLN: p < 0.001 (a), #CHL vs. CHL + Nic: p < 0.01 and ##CHL vs. CHL + 6HLN: p < 0.001 (b), #CHL vs. CHL + Nic: p < 0.001 and ##CHL vs. CHL + 6HLN: p < 0.0001 (c), #CHL vs. CHL + Nic: p < 0.01 and ##CHL vs. CHL + 6HLN: p < 0.0001 (d) and #CHL vs. CHL + Nic: p < 0.001 and ##CHL vs. CHL + 6HLN: p < 0.0001 (e).
content of reduced GSH and MDA level. In addition, treatment of the CHL-induced amnesic rats with nicotine and 6HLN, but especially with 6HLN, exhibited inhibitory effects on the MDA level with a concomitant increase of the SOD, CAT, GPX specific activities and the total content of reduced GSH when compared with CHL-alone treated rats. Thus, we can suggest that 6HLN as compared to nicotine exhibited a high protective effect on the rat hippocampus through its antioxidant proprieties as evidenced by its ability to inhibit lipid peroxidation and increase antioxidant enzymes activity in the hippocampus of the CHL-treated rats.
Moreover, we found significant correlations after calculation of linear regression between the spontaneous alternation percentage vs. SOD, spontaneous alternation percentage vs. GPX, spontaneous alternation percentage vs. CAT, spontaneous alternation percentage vs. MDA, working memory errors vs. SOD, working memory errors vs. GPX, working memory errors vs. CAT, working memory errors vs. MDA, reference memory errors vs. SOD, reference memory errors vs. CAT, reference memory errors vs. MDA, SOD vs. MDA, GPX vs. MDA, and CAT vs. MDA. Therefore, we could suggest that the enhancing of behavioral parameters in the Y-maze and the radial arm-maze tasks and the antioxidant
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defense together with the decrease of lipid peroxidation could be related to the involvement of nicotine and 6HLN in neuroprotection against CHL-induced oxidative stress generation in the rat hippocampus. 5. Conclusion Taken together, administration of nicotine and 6HLN improved spatial memory in the Y-maze and radial arm-maze tasks decreased lipid peroxidation and enhanced antioxidant status in the hippocampus of the CHL-treated rats. This observed effect could suggest possible neuroprotective effects of both nicotine and 6HLN. However, the effects of 6HLN are significantly increased as compared with nicotine in the CHL-treated rats. From the observation of this study, we can suggest that 6HLN, may have a beneficial role in the treatment and/or management of memory disorders such as AD, and indicate the important significance of the nicotinic acetylcholine system in brain dysfunction. Conflict of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by a grant from the Romanian National Authority for Scientific Research and Innovation, CNCS – UEFISCDI, project number PN-II-RU-TE-2014-4-0106. References [1] S. Maria Cecilia Rodrigues, V. Flavia Pereira Dias, M. Marcella Soares, S. Matheus de Freitas, R. Mariana Maximo, R. Patricia Mattos da, C. Maísa Rosa, S. Marcelo Henrique dos, S. Marisi Gomes, V. Claudio, Donepezil: an important prototype to the design of new drug candidates for Alzheimer’s Disease, Mini Rev. Med. Chem. 14 (1) (2014) 2–19. [2] A.M. Palmer, Neuroprotective therapeutics for Alzheimer’s disease: progress and prospects, Trends Pharmacol. Sci. 32 (3) (2011) 141–147. [3] D.S. Auld, T.J. Kornecook, S. Bastianetto, R. Quirion, Alzheimer’s disease and the basal forebrain cholinergic system: relations to b-amyloid peptides, cognition, and treatment strategies, Prog. Neurobiol. 68 (3) (2002) 209–245. [4] H.R. Parri, C.M. Hernandez, K.T. Dineley, Research update: alpha7 nicotinic acetylcholine receptor mechanisms in Alzheimer's disease, Biochem. Pharmacol. 82 (8) (2011) 931–942. [5] R. Medeiros, N.A. Castello, D. Cheng, M. Kitazawa, D. Baglietto-Vargas, K.N. Green, T.A. Esbenshade, R.S. Bitner, M.W. Decker, F.M. LaFerla, a7 nicotinic receptor agonist enhances cognition in aged 3xTg-AD mice with robust plaques and tangles, Am. J. Pathol. 184 (2) (2014) 520–529. [6] K.N. Murray, N. Abeles, Nicotine’s effect on neural and cognitive functioning in an aging population, Aging Ment. Health 6 (2) (2002) 129–138. [7] M.B. Newman, G.W. Arendash, R.D. Shytle, P.C. Bickford, T. Tighe, P.R. Sanberg, Nicotine’s oxidative and antioxidant properties in CNS, Life Sci. 71 (24) (2002) 2807–2820. [8] S. Boopathi, P. Kolandaivel, Targeted studies on the interaction of nicotine and morin molecules with amyloid b-protein, J. Mol. Model. 20 (3) (2014) 1–15. [9] S.S. Hecht, J.B. Hochalter, P.W. Villalta, S.E. Murphy, 20 -Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: formation of a lung carcinogen precursor, Proc. Natl. Acad. Sci. U. S. A. 97 (23) (2000) 12493– 12497. [10] J. Buccafusco, Neuronal nicotinic receptor subtypes: defining therapeutic targets, Mol. Interv. 4 (2004) 285–295. [11] P.H.N. Celie, S.E. van Rossum-Fikkert, W.J. van Dijk, K. Brejc, A.B. Smit, T.K. Sixma, Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures, Neuron 41 (6) (2004) 907– 914. [12] D.D. Mowrey, Q. Liu, V. Bondarenko, Q. Chen, E. Seyoum, Y. Xu, J. Wu, P. Tang, Insights into distinct modulation of a7 and a7b2 nicotinic acetylcholine receptors by the volatile anesthetic isoflurane, J. Biol. Chem. 288 (50) (2013) 35793–35800. [13] D. Pogocki, T. Ruman, M. Danilczuk, M. Danilczuk, M. Celuch, E. Wałajtys-Rode, Application of nicotine enantiomers, derivatives and analogues in therapy of neurodegenerative disorders, Eur. J. Pharmacol. 563 (1–3) (2007) 18–39.
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Original article
Nicotine versus 6-hydroxy-L-nicotine against chlorisondamine induced memory impairment and oxidative stress in the rat hippocampus Lucian Hritcu* , Radu Ionita, Diana Elena Motei, Cornelia Babii, Marius Stefan, Marius Mihasan* Department of Biology, Alexandru Ioan Cuza University of Iasi, Bd. Carol I, No. 11, 700506, Romania
A R T I C L E I N F O
Article history: Received 3 November 2016 Received in revised form 28 November 2016 Accepted 4 December 2016 Keywords: Arthrobacter nicotinovorans Nicotine 6-Hydroxy-L-nicotine Chlorisondamine Memory Oxidative stress
A B S T R A C T
6-Hydroxy-L-nicotine (6HLN), a nicotine derivative from nicotine degradation by Arthrobacter nicotinovorans pAO1 strain was found to improve behavioral deficits and to reverse oxidative stress in the rat hippocampus. Rats were given CHL (10 mg/kg, i.p.) were used as an Alzheimer’s disease-like model. The nicotine (0.3 mg/kg) and 6HLN (0.3 mg/kg) were administered alone or in combination in the CHL-treated rats. Memory-related behaviors were evaluated using Y-maze and radial arm-maze tests. The antioxidant enzymes activity and the levels of the biomarkers of oxidative stress were measured in the hippocampus. Statistical analyses were performed using two-way ANOVA and Tukey’s post hoc test. F values for which p < 0.05 were regarded as statistically significant. CHL-caused memory deficits and oxidative stress enhancing were observed. Both nicotine and 6HLN administration attenuated the cognitive deficits and recovered the antioxidant capacity in the rat hippocampus of the CHL rat model. Our results suggest that 6HLN versus nicotine confers anti-amnesic properties in the CHL-induced a rat model of memory impairment via reversing cholinergic function and decreasing brain oxidative stress, suggesting the use of this compound as an alternative agent in AD treatment. © 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Alzheimer disease (AD) is a neurodegenerative affliction characterized by memory impairment [1] associated with three major changes in the brain [2]: i) the extra- and intracellular plaques deposits of b-amyloid peptide, (ii) the neurofibrillary tangles and (iii) death of forebrain cholinergic neurons and a significant decrease in acetylcholine (ACh) levels [3]. The search for neuroprotective therapeutics for AD has been a long time dominated mainly by the amyloid and the tau hypothesis. Unfortunately, both approaches failed so far to provide any efficient therapeutic strategy [2]. The involving of nicotinic acetylcholine receptors (nAChR) subtypes a7 and a4b2 in AD pathogenesis [4] has led to the proposal of a new approach [5]. The memory deficits and non-cognitive symptoms could be improved through using of the nAChR modulators to increase the availability of receptors for ACh and to overcome the death of the forebrain cholinergic neurons.
* Corresponding authors. E-mail addresses: [email protected] (L. Hritcu), [email protected] (M. Mihasan). http://dx.doi.org/10.1016/j.biopha.2016.12.008 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
Nicotine is considered to be an agonist of nAChR. Its high potency as a cognition-enhancing agent and AD therapeutic strategy [6] has been explained not only by its ability to bind and modulate nAChRs but also through its anti-oxidant effects at low concentrations [7] and recently, through its ability to interact with b-amyloid peptide [8]. However, due to negative effects on human organs such as lungs [9] and bad publicity related to smoking[10], nicotine doesn’t impose as a feasible therapeutic agent for AD. The crystal structure determination of the nicotinic receptor binding domain homolog (nAchBP) with bound nicotine [11] followed by the recent solving of the a7 nAChRs structure [12] has spurred the interest of several academic and pharmaceutical laboratories in the possibility that new nicotinic drugs could be designed. From this point of view, nicotine derivatives are ideal candidates, offering a wide array of possibilities. The difficulty resides, firstly in the identification of molecules which have the beneficial effects of nicotine, but elude its side effects [13] and, secondly, in providing simple and reliable methods for production and isolation of the identified molecules. In this context, Arthrobacter nicotinovorans pAO1 strain through metabolizing nicotine could deliver new nicotine-derivatives with unexplored biotechnological potential.
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6-Hydroxy-L-nicotine (6HLN) is a metabolic intermediate found in the nicotine catabolic pathway encoded by Arthrobacter nicotinovorans pAO1 [14]. 6HLN resulted by a hydroxylation reaction mediated by nicotine-dehydrogenase (NDH, EC 1.5.99.4), a multimeric enzyme encoded by the ndhL, ndhM, and ndhS genes of pAO1. The compound is further dehydrogenated by 6-hydroxy-L-nicotine oxidase (6HLNO, EC 1.5.3.5) with the formation of 6-hydroxy-methylmyosmine [15]. 6HLN was repeatedly reported to temporarily accumulate in the growth medium of Arthrobacter nicotinovorans pAO1 [16,17]. Previously, we have shown that 6-hydroxy-L-nicotine (6HLN) a natural product obtained from nicotine degradation by Arthrobacter nicotinovorans enhanced behavioral response and consequently decrease oxidative stress generation in the rat hippocampus of the scopolamine treated rats [18]. Here, we further hypothesized that 6HLN versus nicotine ameliorates chlorisondamine (CHL) induced cognitive impairment through decreasing of the hippocampal oxidative stress. CHL is a known nAChR antagonist that can cross the blood-brain barrier and exerts a nicotinic blockade that lasts for months [19]. The pharmacological effects of both nicotine and 6HLN on CHL-induced memory impairment associated with brain oxidative stress were investigated, and also their possible mechanism of action underlying these effects. 2. Materials and methods 2.1. Strains and growth conditions Arthrobacter nicotinovorans pAO1+ (strain ATCC 4991) was grown on citrate medium supplemented with 3 mM nicotine [20] on a rotary shaker at 28 C/190 rpm. 100 ml medium in a 500 ml flask were inoculated with 1 ml 24 h old preculture and allowed to grow 10 h before harvest producing about 8 mg of 6HLN. 2.2. General analytical methods Quantitative data on nicotine metabolites were obtained by high-performance liquid chromatography (HPLC) analysis by comparing the retention times and absorption spectra with literature data [21]. Supplementary, chemical synthesized 6HLN (a kind gift from Prof. Dr. Roderich Brandsch – Institute of Biochemistry and Molecular Biology, Albert-Ludwigs-University of Freiburg, Germany) was used as standard. HPLC analysis was performed as described by Tang et al. [22] with slight modifications. A Shimadzu Prominence UPLC system equipped with a Machery-Nagel Nucleodur RP C18 ec column (150 4.6 mm, particle size 3 mm) was used for separation of nicotine metabolites from the growth medium. The mobile phase was a mixture of 1 mM H2SO4: methanol (75:25 v/v) at a flow rate of 1 ml/min. The separation was performed at 30 C using isocratic elution and 6HLN levels were monitored at 290 nm. 2.3. Animals A total number of thirty male Wistar rats (3–4-month-old) weighing 250 10 g at the start of the experiment was used. The animals were housed in a temperature and light-controlled room (22 C, a 12 h light/dark cycle starting at 08:00 h) and were fed and allowed to drink water ad libitum. The experiments were conducted in the quiet laboratory between hours of 10:00 h– 16:00 h. Rats were treated in accordance with the guidelines of animal bioethics from the Act on Animal Experimentation and Animal Health and Welfare from Romania and all procedures were in compliance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the
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protection of animals used for scientific purposes. This study was approved by the Committee on the Ethics of Animal Experiments of the Alexandru Ioan Cuza University of Iasi, Faculty of Biology (Permit Number: 2197) and also, efforts were made to minimize animal suffering and to reduce the number of animals used. 2.4. Drugs ()Nicotine (free base, Nic), 6HLN and chlorisondamine (CHL) were diluted in an isotonic solution (0.9% NaCl). All reagents, except 6HLN, were purchased from Sigma-Aldrich, Germany. 2.5. Drug administration The rats were divided into six different groups (five animals per group) as follows: (1) the Control group received saline treatment (0.9% NaCl); (2) the ()Nicotine (Nic, free base, 0.3 mg/kg)-alonetreated group, as positive control; (3) the Chlorisondamine (CHL, 10 mg/kg)-alone-treated group, as negative control; (4) the 6-hydroxy-L-nicotine (6HLN, 0.3 mg/kg)-alone-treated group, as positive control; (5) the CHL-treated group received Nic treatment (CHL + Nic) and (6) the CHL-treated group received 6HLN treatment (CHL + 6HLN). The drug doses used in this experiment were chosen since they have been demonstrated by our group to provide significant effects on memory formation and antioxidant profile [18,23]. CHL was injected 24 h before the experiments. Also, Nic and 6HLN were injected alone and in the CHL treated-groups, 30 min before the behavioral testing. 2.6. Y-maze task Spontaneous alternation was examined using a three-arm Ymaze (35 25 10 cm). Rats were individually placed at the end of one arm, facing the center, and allowed to move freely through the maze for 8 min. An arm entry was counted when the hind paws of the rat were completely within the arm. The alternation score (%) of each rat was defined as the ratio of the actual number of alternations to the possible number (defined as the total number of arm entries minus two) multiplied by 100 [24,25]. At the beginning of each trial the maze was thoroughly cleaned with 10% ethanol. 2.7. Radial arm-maze task The radial arm-maze used in the present study consisted of 8 arms, numbered from 1 to 8 (48 12 cm), extending radially from a central area (32 cm in diameter). The apparatus was placed 40 cm above the floor and surrounded by various extra-maze visual cues placed at the same position during the study. Prior to the performance of the maze task, the animals were kept on restricted diet and body weight was maintained of 85% of their free-feeding weight over a week period, with water being available ad libitum. During the pre-training session (4 days), rats were individually placed in the center of the maze and were allowed to explore to habituate them to the maze for 5 min. Initially, the food pellets (50 mg, Bioserve Inc.) were placed throughout the maze but gradually restricted to the end of each arm in the food cup. Five target baited arms (nos. 1, 2, 4, 5, and 7) were selected which remained constant for a given rat throughout training. The other three arms (nos. 3, 6, 8) were never baited. In the training session, starting with the day fifth to day twelfth, the animals were trained by performing one trial per day for seven consecutive days. Each rat was individually placed in the center of the maze and subjected to working and reference memory tasks. An arm entry was counted when a rat ate the bait or reached the end of an arm. Measures were made of the number of working memory errors (entering a baited arm, but previously entered) and
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reference memory errors (entering an arm that was never baited). Criterion performance was defined as consumption of all 5 baits or until 5 min had elapsed. Reference memory is regarded as a longterm memory for information that remains constant over repeated trials (memory for the positions of reinforced arms), whereas working memory is considered a short-time memory in which the information to be remembered changes in every trial (memory for the positions of arms that had already been visited in each trial) [18,26]. 2.8. Oxidative stress assay Shortly after completion of the behavioral tests, all rats were deeply anesthetized (using sodium pentobarbital, 100 mg/kg b.w., i.p., Sigma-Aldrich, Germany), decapitated and whole brains were removed. The hippocampi were carefully excised. Each of the hippocampal samples was weighted and homogenized (1:10) with Potter Homogenizer coupled with Cole-Parmer Servodyne Mixer in ice-cold 0.1 M potassium phosphate buffer (pH 7.4), 1.15% KCl. The homogenate was centrifuged (15 min at 960 g) and the supernatant (200 ml) was used for assays of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) specific activities, the total content of reduced glutathione (GSH) and malondialdehyde (MDA) level. 2.8.1. Determination of hippocampal SOD activity The activity of superoxide dismutase (SOD, EC 1.15.1.1) was assayed by monitoring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). Each 1.5 ml reaction mixture contained 100 mM TRIS/HCl (pH 7.8), 75 mM NBT, 2 mM riboflavin, 6 mM EDTA, and 200 ml of supernatant. Monitoring the increase in absorbance at 560 nm followed the production of blue formazan. One unit of SOD is defined as the quantity required to inhibit the rate of NBT reduction by 50% as previously described by Winterbourn et al. [27]. 2.8.2. Determination of hippocampal GPX activity Glutathione peroxidase (GPX, E.C. 1.11.1.9) activity was analyzed by a spectrophotometric assay. A reaction mixture consisting of 1 ml of 0.4 M phosphate buffer (pH 7.0) containing 0.4 mM EDTA, 1 ml of 5 mM NaN3, 1 ml of 4 mM GSH, and 200 ml of supernatant was preincubated at 37 C for 5 min. Then 1 ml of 4 mM H2O2 was added and incubated at 37 C for further 5 min. The excess amount of GSH was quantified by the DTNB method as previously described by Sharma and Gupta [28]. One unit of GPX is defined as the amount of enzyme required to oxidize 1 nmol GSH/min. 2.8.3. Determination of hippocampal CAT activity Catalase (CAT, EC 1.11.1.6) activity was assayed following the method of Sinha [29]. The reaction mixture consisted of 150 ml phosphate buffer (0.01 M, pH 7.0), 200 ml supernatant. The reaction was started by adding 250 ml H2O2 0.16 M, incubated at 37 C for 1 min and the reaction was stopped by addition of 1 ml of dichromate: acetic acid reagent. The tubes were immediately kept in a boiling water bath for 15 min and the green colour developed during the reaction was read at 570 nm on a spectrophotometer. Control tubes, devoid of the enzyme, were also processed in parallel. The enzyme activity is expressed as mmol of H2O2 consumed/min/mg protein.
a final volume of 1.5 ml with distilled water and absorbance was read in a spectrophotometer at 412 nm and results were expressed as mg GSH/mg protein. 2.8.5. Determination of hippocampal MDA level Malondialdehyde (MDA), which is an indicator of lipid peroxidation, was spectrophotometrically measured by using the thiobarbituric acid assay as previously described by Ohkawa et al. [31]. 200 ml of supernatant was added and briefly mixed with 1 ml of 50% trichloroacetic acid in 0.1 M HCl and 1 ml of 26 mM thiobarbituric acid. After vortex mixing, samples were maintained at 95 C for 20 min. Afterward, samples were centrifuged at 960 g for 10 min and supernatants were read at 532 nm. A calibration curve was constructed using MDA as standard and the results were expressed as nmol/mg protein. 2.8.6. Estimation of protein concentration Estimation of protein was done using a bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich, Germany). The BCA protein assay is a detergent-compatible formulation based on BCA for the colorimetric detection and quantification of total protein, as previously described by Smith et al. [32]. 2.9. Statistical analysis The group differences in the Y-maze and the radial armmaze tasks and the results for biochemical parameter assay were statistically analyzed by two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using GraphPad Prism 6 software for Windows, La Jolla, CA, USA. In order to evaluate differences between groups in the radial arm-maze task, separate repeated-measures ANOVA were calculated on the number of working memory errors and the number of reference memory errors with group (Control, Nic, CHL, 6HLN, CHL + Nic and CHL + 6HLN) as between-subject factor and days (1–7) as within-subjects factors. Data are expressed as mean standard errors of mean (S.E.M). F values for which p < 0.05 were regarded as statistically significant. Pearson’s correlation coefficient and regression analysis were used in order to evaluate the relation between behavioral measures, the antioxidant defense and lipid peroxidation. 3. Results 3.1. HPLC analyses The HPLC method described by Tang et al. [22] and modified as stated in the methods section was successfully used to separate the growth medium of Arthrobacter nicotinovorans. A typical chromatogram of the growth medium from which nicotine was completely consumed is shown in Fig. 1a. Three peaks could be observed at 290 nm and were identified based on their absorption spectra. The peak with RT = 5.5 is nicotine-blue (4,5,40 ,50 -tetrahydroxy-3,30 -diazadiphenoquinone-(2,20 )), the main product of the nicotine catabolic pathway in this bacteria. The peak with RT = 3.5 is unknown as we were unable to find any similar spectra in the literature. The peak with RT = 2.3 is 6HLN. Fig. 1b depicts the purity and spectrum of the 6HLN preparations used for animal treatments. 3.2. Effect of nicotine and 6HLN on memory performance
2.8.4. Total hippocampal content of reduced GSH Glutathione (GSH) was measured following the method of Fukuzawa and Tokumura [30]. 200 ml of supernatant was added to 1.1 ml of 0.25 M sodium phosphate buffer (pH 7.4) followed by the addition of 130 ml DTNB 0.04%. Finally, the mixture was brought to
In the Y-maze test, significant overall differences between all groups (F(5, 24) = 28.88, p < 0.0001) on the spontaneous alternation percentage were evidenced (Fig. 2a). CHL significantly decrease the spontaneous alternation percentage as compared
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3.3. Effect of nicotine and 6HLN on oxidative stress
Fig. 1. HPLC analysis of (a) Arthrobacter nicotinovorans growth medium upon nicotine depletion and (b) 6HLN preparations used for animal treatments. RT = 5.5 – nicotine-blue (4,5,40 ,50 -tetrahydroxy-3,30 -diazadiphenoquinone-(2,20 )); RT = 3.5 – unknown metabolite; RT = 2.3–6 HLN. Inlay – 6HLN spectrum.
to control group (p < 0.0001). Rats were given CHL and received nicotine and 6HLN, but especially 6HLN displayed significant differences (p < 0.0001) for the spontaneous alternation percentage as compared to the CHL-alone-treated group. The results suggest that both nicotine and 6HLN, but especially 6HLN, are capable of improving spontaneous alternation in the CHL-induced cholinergic dysfunction. In the radial arm-maze test, significant overall differences between all groups (F(5, 24) = 7.11, p < 0.0001) for the working memory errors were evidenced (Fig. 2b). CHL significantly increased working memory errors as compared to control group (p < 0.01). The CHL rats with both nicotine and 6HLN, but especially 6HLN, showed decreased working memory errors (p < 0.001) as compared to the CHL-alone-treated group. Moreover, repeated-measures ANOVA revealed a significant time difference (F(6168) = 18.03, p < 0.0001), a significant group difference (F(5168) = 8.68, p < 0.0001) and a significant time and group interaction (F(30,168) = 5.42, p < 0.0001) for working memory errors. ANOVA revealed significant overall differences between all groups (F (5, 24) = 10.86, p < 0.0001) for reference memory errors (Fig. 2c). CHL significantly increased reference memory errors as compared to control group (p < 0.01). The CHL rats with both nicotine and 6HLN, but especially 6HLN, showed decreased reference memory errors (p < 0.00001) as compared to CHL-alone-treated rats. Additionally, repeated-measures ANOVA revealed a significant time difference (F(6168) = 17.47, p < 0.0001), a significant group difference (F(5168) = 13.45, p < 0.0001) and a significant group difference (F(30,168) = 20.47, p < 0.0001) for reference memory errors.
Biochemical analyses of SOD, GPX and CAT specific activities along with reduced GSH and MDA levels estimated in the hippocampal homogenates showed significant overall differences between all groups for SOD activity (F(5,24) = 7.56, p < 0.001) (Fig. 3a), for GPX activity (F(5,24) = 6.21, p < 0.001) (Fig. 3b), for CAT activity (F(5,24) = 22.21, p < 0.0001) (Fig. 3c), for the total content of reduced GSH (F(5,24) = 43.49, p < 0.0001) (Fig. 3d) and for MDA level (F(5,24) = 5.98, p < 0.001) (Fig. 3e). CHL treatment significantly decreased (p < 0.01) the antioxidant enzymes activity (SOD, GPX, and CAT), and GSH reduced level while the MDA level was over normal levels. Rats were given CHL and received nicotine and 6HLN, but especially 6HLN, exhibited a significant increase of the SOD, CAT, and GPX specific activities, and the total content of reduced GSH together with a decreased MDA level as compared to the CHL-alone treated group (p < 0.01). Importantly, when linear regression was determined, significant correlations between the spontaneous alternation percentage vs. SOD (n = 30, r = 0.717, p < 0.001), spontaneous alternation percentage vs. GPX (n = 30, r = 0.507, p < 0.01), spontaneous alternation percentage vs. CAT (n = 30, r = 0.758, p < 0.0001), spontaneous alternation percentage vs. MDA (n = 30, r = 0.795, p < 0.0001), working memory errors vs. SOD (n = 30, r = 0.799, p < 0.0001), working memory errors vs. GPX (n = 30, r = 0.754, p < 0.0001), working memory errors vs. CAT (n = 30, r = 0.815, p < 0.0001), working memory errors vs. MDA (n = 30, r = 0.754, p < 0.0001), reference memory errors vs. SOD (n = 30, r = 0.537, p < 0.01), reference memory errors vs. CAT (n = 30, r = 0.525, p < 0.01), reference memory errors vs. MDA (n = 30, r = 0.598, p < 0.001) were observed. Additionally, significant correlations were observed between the SOD vs. MDA (n = 30, r = 0.683, p < 0.001), GPX vs. MDA (n = 30, r = 0.508, p < 0.01) and CAT vs. MDA (n = 30, r = 0.656, p < 0.001) when linear regression was determined. 4. Discussion In this study, both nicotine and 6HLN administered alone or in combination with CHL-treated rats causes behavioral effects as memory-enhancing based on specific behavioral tests such as Ymaze and radial arm-maze were investigated. It has been documented that when administered intracerebroventricularly or systemic, CHL persistent block for a period of several weeks the central nicotinic responses, examined either behaviorally or in vitro [33,34]. In addition, the main mechanism by which CHL exerts its long-lasting blockade of nAChR in the central nervous system remains elusive [35]. Previously, we used in our studies CHL as an experimental tool to study the involvement of nAChR in memory processes [23]. Numerous studies reported that nicotine and nicotinic agonists improved memory performance in experimental rats [23,36–39]. It has been suggested that using of nAChR antagonists in an animal model of memory impairment has clinical relevance because it models the functional effect of nicotinic receptor loss [40]. Studies using a mouse model of AD have shown that early AD stage is marked by cholinergic hypofunction, neuronal marker loss, and decreased nAChR density from the cortex and hippocampus [41]. It has been reported that nicotine can modulate several different types of hippocampus-dependent learning [42], such as Y-maze and radial arm-maze tasks used in the present study. The current study found that both nicotine and 6HLN, but especially 6HLN alone treatment, enhanced spatial memory formation as compared to control group in Y-maze task. However, administration of 6HLN as compared to nicotine in the CHL-treated rats significantly increased the spontaneous alternation
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Fig. 2. Effects of the nicotine (Nic, 0.3 mg/kg b.w., i.p.) and 6HLN (0.3 mg/kg b.w., i.p.) administration in the Y-maze task on the spontaneous alternation% (a), on the working memory errors (b) and the reference memory errors (c) during 7 days training in radial arm-maze task in the chlorisondamine (CHL)-treated rats. Values are mean SEM (n = 5 animals per group). For Turkey’s post-hoc analyses- #CHL vs. CHL + Nic: p < 0.001 and ##CHL vs. CHL + 6HLN: p < 0.0001 (a), #CHL vs. CHL + Nic: p < 0.01 and ##CHL vs. CHL + 6HLN: p < 0.001 (b) and #CHL vs. CHL + Nic: p < 0.0001 and ##CHL vs. CHL + 6HLN: p < 0.00001 (c).
percentage as compared to CHL alone treated rats, suggesting that nicotine and 6HLN selectively improved memory formation of CHL-treated rats within the Y-maze task. Furthermore, both nicotine and 6HLN, but especially 6HLN, significantly enhanced working memory, as evidenced by decreasing the number of working memory errors in the radial arm-maze task. CHL-treated rats injected with 6HLN showed a significant improvement of working memory as compared to CHL-alone treated rats. Moreover, in the CHL-treated rats, 6HLN significantly improved reference memory, as evidenced by decreasing the number of reference memory errors in the radial arm-maze task. Consisting of
our previous study [18], 6HLN plays an important role in the restoration of spatial memory, especially in the rats with cholinergic deficits. There is strong evidence that antioxidants exhibit the neuroprotective effects on the brain function [43]. In this study, the antioxidative status in the rat was evaluated using the SOD, CAT, and GPX specific activities, as well as the total content of reduced GSH and MDA level in rat hippocampus as indices. Our results revealed that both nicotine and 6HLN, but especially 6HLN-alone treatment, exhibited antioxidant effects as evidenced by significant increase of SOD, GPX, and CAT specific activities along with decreased
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Fig. 3. Effects of the nicotine (Nic, 0.3 mg/kg b.w., i.p.) and 6HLN (0.3 mg/kg b.w., i.p.) administration on SOD (a), GPX (b), and CAT (c) specific activities, on reduced GSH (d) and MDA (e) levels in the chlorisondamine (CHL)-treated rats. Values are mean SEM (n = 5 animals per group). For Turkey’s post-hoc analyses- #CHL vs. CHL + Nic: p < 0.01 and ## CHL vs. CHL + 6HLN: p < 0.001 (a), #CHL vs. CHL + Nic: p < 0.01 and ##CHL vs. CHL + 6HLN: p < 0.001 (b), #CHL vs. CHL + Nic: p < 0.001 and ##CHL vs. CHL + 6HLN: p < 0.0001 (c), #CHL vs. CHL + Nic: p < 0.01 and ##CHL vs. CHL + 6HLN: p < 0.0001 (d) and #CHL vs. CHL + Nic: p < 0.001 and ##CHL vs. CHL + 6HLN: p < 0.0001 (e).
content of reduced GSH and MDA level. In addition, treatment of the CHL-induced amnesic rats with nicotine and 6HLN, but especially with 6HLN, exhibited inhibitory effects on the MDA level with a concomitant increase of the SOD, CAT, GPX specific activities and the total content of reduced GSH when compared with CHL-alone treated rats. Thus, we can suggest that 6HLN as compared to nicotine exhibited a high protective effect on the rat hippocampus through its antioxidant proprieties as evidenced by its ability to inhibit lipid peroxidation and increase antioxidant enzymes activity in the hippocampus of the CHL-treated rats.
Moreover, we found significant correlations after calculation of linear regression between the spontaneous alternation percentage vs. SOD, spontaneous alternation percentage vs. GPX, spontaneous alternation percentage vs. CAT, spontaneous alternation percentage vs. MDA, working memory errors vs. SOD, working memory errors vs. GPX, working memory errors vs. CAT, working memory errors vs. MDA, reference memory errors vs. SOD, reference memory errors vs. CAT, reference memory errors vs. MDA, SOD vs. MDA, GPX vs. MDA, and CAT vs. MDA. Therefore, we could suggest that the enhancing of behavioral parameters in the Y-maze and the radial arm-maze tasks and the antioxidant
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defense together with the decrease of lipid peroxidation could be related to the involvement of nicotine and 6HLN in neuroprotection against CHL-induced oxidative stress generation in the rat hippocampus. 5. Conclusion Taken together, administration of nicotine and 6HLN improved spatial memory in the Y-maze and radial arm-maze tasks decreased lipid peroxidation and enhanced antioxidant status in the hippocampus of the CHL-treated rats. This observed effect could suggest possible neuroprotective effects of both nicotine and 6HLN. However, the effects of 6HLN are significantly increased as compared with nicotine in the CHL-treated rats. From the observation of this study, we can suggest that 6HLN, may have a beneficial role in the treatment and/or management of memory disorders such as AD, and indicate the important significance of the nicotinic acetylcholine system in brain dysfunction. Conflict of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by a grant from the Romanian National Authority for Scientific Research and Innovation, CNCS – UEFISCDI, project number PN-II-RU-TE-2014-4-0106. References [1] S. Maria Cecilia Rodrigues, V. Flavia Pereira Dias, M. Marcella Soares, S. Matheus de Freitas, R. Mariana Maximo, R. Patricia Mattos da, C. Maísa Rosa, S. Marcelo Henrique dos, S. Marisi Gomes, V. Claudio, Donepezil: an important prototype to the design of new drug candidates for Alzheimer’s Disease, Mini Rev. Med. Chem. 14 (1) (2014) 2–19. [2] A.M. Palmer, Neuroprotective therapeutics for Alzheimer’s disease: progress and prospects, Trends Pharmacol. Sci. 32 (3) (2011) 141–147. [3] D.S. Auld, T.J. Kornecook, S. Bastianetto, R. Quirion, Alzheimer’s disease and the basal forebrain cholinergic system: relations to b-amyloid peptides, cognition, and treatment strategies, Prog. Neurobiol. 68 (3) (2002) 209–245. [4] H.R. Parri, C.M. Hernandez, K.T. Dineley, Research update: alpha7 nicotinic acetylcholine receptor mechanisms in Alzheimer's disease, Biochem. Pharmacol. 82 (8) (2011) 931–942. [5] R. Medeiros, N.A. Castello, D. Cheng, M. Kitazawa, D. Baglietto-Vargas, K.N. Green, T.A. Esbenshade, R.S. Bitner, M.W. Decker, F.M. LaFerla, a7 nicotinic receptor agonist enhances cognition in aged 3xTg-AD mice with robust plaques and tangles, Am. J. Pathol. 184 (2) (2014) 520–529. [6] K.N. Murray, N. Abeles, Nicotine’s effect on neural and cognitive functioning in an aging population, Aging Ment. Health 6 (2) (2002) 129–138. [7] M.B. Newman, G.W. Arendash, R.D. Shytle, P.C. Bickford, T. Tighe, P.R. Sanberg, Nicotine’s oxidative and antioxidant properties in CNS, Life Sci. 71 (24) (2002) 2807–2820. [8] S. Boopathi, P. Kolandaivel, Targeted studies on the interaction of nicotine and morin molecules with amyloid b-protein, J. Mol. Model. 20 (3) (2014) 1–15. [9] S.S. Hecht, J.B. Hochalter, P.W. Villalta, S.E. Murphy, 20 -Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: formation of a lung carcinogen precursor, Proc. Natl. Acad. Sci. U. S. A. 97 (23) (2000) 12493– 12497. [10] J. Buccafusco, Neuronal nicotinic receptor subtypes: defining therapeutic targets, Mol. Interv. 4 (2004) 285–295. [11] P.H.N. Celie, S.E. van Rossum-Fikkert, W.J. van Dijk, K. Brejc, A.B. Smit, T.K. Sixma, Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures, Neuron 41 (6) (2004) 907– 914. [12] D.D. Mowrey, Q. Liu, V. Bondarenko, Q. Chen, E. Seyoum, Y. Xu, J. Wu, P. Tang, Insights into distinct modulation of a7 and a7b2 nicotinic acetylcholine receptors by the volatile anesthetic isoflurane, J. Biol. Chem. 288 (50) (2013) 35793–35800. [13] D. Pogocki, T. Ruman, M. Danilczuk, M. Danilczuk, M. Celuch, E. Wałajtys-Rode, Application of nicotine enantiomers, derivatives and analogues in therapy of neurodegenerative disorders, Eur. J. Pharmacol. 563 (1–3) (2007) 18–39.
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